anti-MUC 1 antibodies

文档序号：939420 发布日期：2021-03-05 浏览：13次中文

阅读说明：本技术 抗-muc1抗体 (anti-MUC 1 antibodies ) 是由 J·盖莱尔特 A·弗莱什内 D·魏格尔特安特杰·丹尼尔茲克于 2019-05-17 设计创作，主要内容包括：本发明涉及针对癌症抗原MUC1的新抗体。具体地,通过在已知的抗-MUC1抗体的CDR-H2中缺失糖基化位点,得到具有改善的抗原结合的抗体。(The present invention relates to novel antibodies directed against the cancer antigen MUC 1. Specifically, by deleting glycosylation sites in the CDR-H2 of the known anti-MUC 1 antibody, an antibody with improved antigen binding is obtained.)

1. An antibody capable of binding to MUC1, comprising

(i) A heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 2, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) a light chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO. 4, CDR-L2 having the amino acid sequence of SEQ ID NO. 5, and CDR-L3 having the amino acid sequence of SEQ ID NO. 6.

2. The antibody of claim 1, wherein the amino acid at position 8 of SEQ ID NO 2 is selected from the group consisting of glutamine, histidine, tryptophan, tyrosine, lysine and arginine, or wherein the CDR-H2 has the amino acid sequence of SEQ ID NO 7.

3. The antibody of claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO 9 or an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO 9.

4. The antibody of any one of claims 1-3, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO 10 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO 10.

5. The antibody of any one of claims 1-4, wherein the light chain variable region has the amino acid sequence of SEQ ID NO 12 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO 12.

6. The antibody of any one of claims 1-5, wherein the heavy chain variable region of the antibody has the amino acid sequence of SEQ ID No. 10 and the light chain variable region of the antibody has the amino acid sequence of SEQ ID No. 12.

7. The antibody of any one of claims 1-6, wherein the antibody comprises an Fc region and is preferably an IgG1, IgG2, or IgG 4-type antibody.

8. The antibody of any one of claims 1-7, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO 15, in particular SEQ ID NO 22, and a light chain having the amino acid sequence of SEQ ID NO 16.

9. The antibody of claim 7 or 8, wherein the antibody comprises a glycosylation pattern having one or more of the following characteristics:

(i) a detectable amount of glycans carrying bisecting GlcNAc residues;

(ii) the relative amount of glycans carrying at least one galactose residue is at least 25% of the total amount of glycans attached to the Fc glycosylation site of the antibody in the composition.

10. The antibody of any one of claims 1-9, wherein the antibody is obtainable by production in a mammalian cell.

11. The antibody of any one of claims 1-10, wherein the antibody is obtainable by production in a human cell line selected from the group consisting of NM-H9D8 (DSM ACC2806), NM-H9D8-E6 (DSM ACC 2807), NM-H9D8-E6Q12 (DSM ACC2856), and cell lines derived therefrom.

12. The antibody of any one of claims 1-10, wherein the antibody is producible in a CHO cell line or a cell line derived therefrom.

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

14. A nucleic acid encoding the antibody of any one of claims 1-13.

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

16. A host cell comprising the nucleic acid of claim 14 or the expression cassette or vector of claim 15.

17. A conjugate comprising an antibody according to any one of claims 1-13 conjugated to a further agent, wherein the further agent is a polypeptide or protein.

18. The conjugate according to claim 17, wherein the further agent is a cytokine, an immunomodulatory compound, a tumor-specific antibody or an immunodetection site blocking or activating antibody.

19. A composition comprising the antibody of any one of claims 1-13, the nucleic acid of claim 14, the expression cassette or vector of claim 15, the host cell of claim 16, or the conjugate of claim 17 or 18.

20. An antibody according to any one of claims 1 to 13, a conjugate according to claim 17 or 18 or a composition according to claim 19 for use in medicine.

21. An antibody, conjugate or composition according to claim 20 for use in the treatment of cancer, infection, autoimmune disease or immunodeficiency disorder.

22. The antibody, conjugate or composition according to claim 20 for use in the diagnosis, detection and/or monitoring of cancer, infection, autoimmune disease or immunodeficiency disorders.

23. The antibody, conjugate or composition of claim 21 or 22, wherein the cancer is characterized by expression of TA-MUC 1.

24. The antibody, conjugate or composition of any one of claims 21-23, 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, blood cancer, endometrial cancer, thyroid cancer, leukemia, seminoma, melanoma, cancer, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal 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 metastases thereof.

25. The antibody, conjugate or composition of any one of claims 20-24, wherein the antibody is used in combination with other agents.

26. A method of increasing MUC1 binding affinity of an antibody, said antibody comprising

(i) A heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

the method comprises the following steps: the amino acid residue at position 8 of CDR-H2 was replaced with any amino acid residue other than asparagine, thereby generating CDR-H2 having the amino acid sequence of SEQ ID NO. 2.

27. The method of claim 26, wherein replacing the amino acid residue at position 8 of CDR-H2 is achieved by introducing a mutation into the nucleic acid encoding the antibody, wherein the mutation is introduced into the codon encoding the amino acid residue.

28. A method of producing an antibody having increased MUC1 binding affinity, the method comprising

(a) Providing a nucleic acid encoding an antibody comprising

(i) A heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(b) introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding the amino acid residue at position 8 of CDR-H2 such that the codon encodes any amino acid residue other than asparagine; and

29. The method of claim 28, wherein the antibody with increased MUC1 binding affinity comprises

Technical Field

The present invention relates to the field of antibodies. Mutant anti-MUC 1 antibodies having increased antigen binding affinity are provided. Specifically, a mutant form of the humanized antibody PankoMab is provided in which asparagine 57 of the heavy chain variable region is replaced with another amino acid. Thus, the glycosylation sites in the CDR2 region were deleted and antigen binding affinity was increased. In particular embodiments, the invention relates to therapeutic and diagnostic uses of the antibodies and methods of producing such antibodies.

Background

Antibodies against tumor-associated antigens are widely used therapeutic agents against cancer. Today, many anti-cancer antibodies are approved for human therapy. Some of these antibodies act by blocking certain signaling pathways critical to the survival or proliferation of a particular cancer cell. Other anti-cancer antibodies activate the patient's immune response against the targeted cancer cells, such as initiating antibody-dependent cellular cytotoxicity (ADCC) by natural killer cells. This mechanism is induced by the binding of the Fc portion of the antibody to Fc receptors on immune cells.

An interesting and important group of antibodies are antibodies against mucins. Mucins are a family of high molecular weight, highly glycosylated proteins produced by many epithelial tissues in vertebrates. They can be subdivided into membrane-bound mucins due to the presence of hydrophobic transmembrane domains, which facilitate retention in the plasma membrane, and mucins that are secreted to mucosal surfaces or secreted to become components of saliva. The human mucin family consists of many family members, including membrane-bound MUC 1.

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

In view of this, there is a need in the art to provide therapeutic anti-MUC 1 antibodies with improved properties.

Disclosure of Invention

The inventors of the present invention have found that the deletion of the glycosylation site in the heavy chain variable region of the PankoMab anti-MUC 1 antibody does not abrogate antigen binding, but rather unexpectedly increases the antigen affinity of the antibody. This is particularly surprising given that the glycosylation site is located in the second complementarity determining region (CDR-H2) of the heavy chain variable region. CDRs are those regions of an antibody that are directly involved in antigen binding and provide contact with an epitope. Thus, it is generally expected that amino acids that modify the CDRs will be detrimental to antigen binding affinity. The humanized PankoMab antibody additionally contains a glycosylation site in CDR-H2, which has a large carbohydrate structure. The carbohydrate structure is present directly at the binding interface with the antigen and is therefore thought to be involved in antigen binding. However, as demonstrated in the examples, the PankoMab variant (PM-N54Q) in which the glycosylation site is deleted by substitution of an amino acid with a carbohydrate structure, shows increased antigen binding affinity.

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

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

In a fifth aspect, the invention provides a conjugate comprising an antibody according to the invention conjugated to a further agent.

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

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

In an eighth aspect, the invention provides a method of increasing the MUC1 binding affinity of an antibody comprising

(i) A heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

In a ninth aspect, the invention provides a method of producing an antibody with increased MUC1 binding affinity, the method comprising

(a) Providing a nucleic acid encoding an antibody comprising

(i) A heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

In a tenth aspect, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject having cancer a therapeutically effective amount of an antibody according to the invention, a nucleic acid according to the invention, an expression cassette or vector according to the invention, or a host cell according to the invention.

In an eleventh aspect, the invention provides kits or devices comprising antibodies according to the invention and related methods, which are useful for the diagnosis, detection or monitoring of MUC 1-associated disorders such as cancer.

Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

Definition of

The following expressions used herein are generally intended to preferably have the meanings set forth below, unless the context in which they are used indicates otherwise.

The expression "comprising" as used herein also includes and specifically refers to the expressions "consisting essentially of and" consisting of, in addition to their literal meaning. Thus, the expression "comprising" refers to embodiments in which the subject matter that "comprises" the specifically listed elements does not comprise other elements, as well as embodiments in which the subject matter that "comprises" the specifically listed elements may and/or does comprise other elements. Likewise, the expression "having" is to be understood as meaning the expression "comprising" and also including and in particular referring to the expressions "consisting essentially of and" consisting of. The term "consisting essentially of" particularly refers to embodiments in which the subject matter comprises 20% or less, particularly 15% or less, 10% or less, or especially 5% or less of the other elements, in addition to the specifically listed elements that substantially make up the subject matter, where possible.

The term "antibody" particularly refers to a protein comprising at least two heavy chains and two light chains linked by disulfide bonds. Each heavy chain is composed of 1 heavy chain variable region (V)_H) And 1 heavy chain constant region (C)_H) And (4) forming. Each light chain is composed of 1 light chain variable region (V)_L) And 1 light chain constant region (C)_L) And (4) forming. The heavy chain constant region comprises three or (in the case of IgM-or IgE-type antibodies) four heavy chain constant domains (C)_H1、C_H2、C_H3And C_H4) Wherein the first constant domain C_H1Adjacent to the variable region and may be connected to a second constant domain C by a hinge region_H2. The light chain constant region consists of only one constant domain. The variable regions may be further subdivided into hypervariable regions (referred to as Complementarity Determining Regions (CDRs)) interspersed with more conserved regions (referred to as Framework Regions (FRs)), where each variable region comprises three CDRs and four FRs. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The heavy chain constant region can be of any type, such as a gamma-, delta-, alpha-, mu-, or epsilon-type heavy chain. Preferably, the heavy chain of the antibody is a gamma chain. In addition, the light chain constant region can also be of any type, such as kappa-or lambda-type light chains. Preferably, the light chain of the antibody is a kappa chain. The terms "heavy chain of the γ - (δ -, α -, μ -or e-) type" and "light chain of the κ - (λ -) type" refer to an antibody heavy chain or an antibody light chain, respectively: it has a constant region amino acid sequence derived from a naturally occurring heavy or light chain constant region amino acid sequence, particularly a human heavy or light chain constant region amino acid sequence. In particular, the amino acid sequence of the constant domain of a gamma-type (especially gamma-type 1) heavy chain is at least 95%, especially at least 98% identical to the amino acid sequence of the constant domain of a human gamma (especially human gamma 1) antibody heavy chain. Furthermore, the amino acids of the constant domain of the kappa-type light chainThe sequence is in particular at least 95%, in particular at least 98% identical to the amino acid sequence of the constant domain of the human kappa antibody light chain. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The antibody may be, for example, a humanized, human or chimeric antibody.

An antigen-binding portion of an antibody generally refers to the full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments of antibodies include Fab fragments, which are composed of V_L、V_H、C_LAnd C_H1Monovalent fragments consisting of domains; f (ab)₂A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region, each Fab fragment binding to the same antigen; from V_HAnd C_H1Domain-forming Fd fragments; v with one arm consisting of antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; and is composed of V_HDomain-composed dAb fragments.

The "Fab part" of an antibody is in particular meant to comprise the heavy and light chain variable regions (V)_HAnd V_L) And the first domain of the heavy chain and the light chain constant region (C)_H1And C_L) A portion of the antibody of (1). In the case of antibodies not comprising all these regions, the term "Fab part" refers only to the region V_H、V_L、C_H1And C_LThose present in antibodies. Preferably, the "Fab portion" refers to a portion of an antibody that corresponds to a fragment containing the antigen binding activity of the antibody obtained by digestion of a native antibody with papain. In particular, the Fab portion of the antibody includes an antigen binding site or its antigen binding ability. Preferably, the Fab part comprises at least the V of the antibody_HAnd (4) a region.

The "Fc portion" of an antibody is intended in particular to comprise the heavy chain constant regions 2,3 and, where applicable, 4 (C)_H2、C_H3And C_H4) A portion of the antibody of (1). In particular, the Fc part includes those in these regionsTwo of each. In the case of antibodies that do not contain all of these regions, then the term "Fc portion" refers only to region C_H2、C_H3And C_H4Of (a) are those present in the antibody. Preferably, the Fc portion comprises at least C of the antibody_H2And (4) a region. Preferably, the "Fc portion" refers to a portion of an antibody that corresponds to a fragment obtained by digesting a native antibody with papain that does not contain the antigen binding activity of the antibody. In particular, the Fc portion of an antibody is capable of binding to an Fc receptor and thus, for example, comprises an Fc receptor binding site or Fc receptor binding capacity.

The terms "antibody" and "antibody construct" as used herein refer in certain embodiments to the same class of antibody or population of antibody constructs, respectively. In particular, all antibodies or antibody constructs of the population show the characteristics used to define the antibody or antibody construct. In certain embodiments, all antibodies or antibody constructs in a population have the same amino acid sequence. Reference to a particular class of antibody (such as an antibody capable of specifically binding MUC1) is particularly directed to a population of such antibodies.

The term "antibody" as used herein also includes fragments and derivatives of said antibody. A "fragment or derivative" of an antibody is in particular a protein or glycoprotein derived from said antibody and capable of binding to the same antigen, in particular to the same epitope, as said antibody. Thus, a fragment or derivative of an antibody herein generally refers to a functional fragment or derivative. In a particularly preferred embodiment, the fragment or derivative of the antibody comprises a heavy chain variable region. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody or a derivative thereof. Examples of antibody fragments include: (i) a Fab fragment, which is a monovalent fragment consisting of the variable region and the first constant region of each of the heavy and light chains; (ii) f (ab)₂A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a variable region and a first constant region C of a heavy chain_H1A composed Fd fragment; (iv) (ii) an Fv fragment consisting of the variable regions of the heavy and light chains of a single arm of an antibody; (v) scFv fragment consisting of a single polypeptide(iii) an Fv fragment consisting of a chain; (vi) consisting of two Fv fragments covalently linked together (Fv)₂A fragment; (vii) a heavy chain variable domain; and (viii) a multimer consisting of a heavy chain variable region and a light chain variable region that are covalently linked together in such a way that association of the heavy chain and light chain variable regions can occur only intermolecularly, not intramolecularly. Derivatives of an antibody specifically include antibodies that bind to or compete for the same antigen as the parent antibody but have an amino acid sequence that is different from the parent antibody from which they are derived. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.

A target amino acid sequence is "derived from" or "corresponds to" a reference amino acid sequence if it has at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity over its entire length to the corresponding portion of the reference amino acid sequence. By "corresponding portion" is meant, for example, that framework region 1(FRH1) of the heavy chain variable region of the target antibody corresponds to framework region 1 of the heavy chain variable region of the reference antibody. In particular embodiments, a target amino acid sequence "derived from" or "corresponding to" a reference amino acid sequence is 100% homologous, or in particular 100% identical, over its entire length to the corresponding portion of the reference amino acid sequence. "homology" or "identity" of amino acid sequences or nucleotide sequences is preferably determined according to the invention over the entire length of the reference sequence or over the entire length of the corresponding part of the reference sequence (which corresponds to the sequence for which homology or identity is defined). An antibody derived from a parent antibody, which is defined by one or more amino acid sequences (e.g. a specific CDR sequence or a specific variable region sequence), in particular an antibody having an amino acid sequence, e.g. a CDR sequence or a variable region sequence, which is at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homologous or identical, in particular identical, to the respective amino acid sequence of the parent antibody. In certain embodiments, an antibody derived from a parent antibody (i.e., a derivative thereof) comprises the same CDR sequences as the parent antibody, but differs in the remaining sequences of the variable region.

The term "antibody" as used herein also refers to multivalent and multispecific antibodies, i.e. antibody constructs having more than two binding sites each binding to the same epitope, and antibody constructs having one or more binding sites that bind a first epitope and one or more binding sites that bind a second epitope, and optionally other binding sites that even bind to other epitopes.

By "specifically binds" is preferably meant that an agent, e.g., an antibody, binds more strongly to its specific target, e.g., an epitope, than to another target. Dissociation constant (K) if the agent binds to the first target_d) Below the dissociation constant for the second target, it binds more strongly to the first target than to the second target. Preferably, the dissociation constant of a target to which an agent specifically binds is more than 100-fold, 200-fold, 500-fold, or more than 1000-fold lower than the dissociation constant of a target to which the agent does not specifically bind. Furthermore, the term "specific binding" especially indicates that the binding affinity between the binding partners has at least 10⁶ M^-1Preferably at least 10⁷ M^-1More preferably at least 10⁸ M^-1Affinity constant K of_a. An antibody specific for an antigen is in particular capable of having at least 10⁶ M^-1Preferably at least 10⁷ M^-1More preferably at least 10⁸ M^-1K of_aAn antibody that binds to the antigen. For example, the term "anti-MUC 1 antibody" particularly refers to an antibody that specifically binds MUC1, and which preferably can have at least 10⁶M^-1Preferably at least 10⁷ M^-1More preferably at least 10⁸ M^-1K of_aBinds to MUC 1.

The term "MUC 1" denotes the protein MUC1, also known as mucin-1, Polymorphic Epithelial Mucin (PEM) or cancer antigen 15-3, in particular human MUC1 (accession number P15941). MUC1 is a member of the mucin family and encodes a membrane-bound glycosylated phosphoprotein. MUC1 has a core protein mass of 120-225 kDa, which is increased to 250-500 kDa by glycosylation. It extends 200-500 nm beyond the cell surface. The protein is anchored on the apical surface of many epithelial cells by a transmembrane domain. The extracellular domain comprises a Variable Number Tandem Repeat (VNTR) domain of 20 amino acids, wherein the number of repeats varies from 20 to 120 in different individuals. These repeats are rich in serine, threonine and proline residues, which allow for severe O-glycosylation. In certain embodiments, the term "MUC 1" refers to tumor-associated MUC1 ("TA-MUC 1"). TA-MUC1 is MUC1 present on cancer cells. This MUC1 differs from MUC1 present on non-cancer cells in its much higher expression level, its localization and its glycosylation. In particular, TA-MUC1 is present apolely on the entire cell surface of cancer cells, whereas in non-cancer cells, MUC1 has strict apical expression and therefore cannot be used for systemically administered antibodies. In addition, TA-MUC1 has aberrant O-glycosylation, which exposes a novel peptide epitope on the MUC1 protein backbone and a novel carbohydrate tumor antigen, such as Thomsen-Friedenreich antigen α (TF α).

"TF α", also known as Thomsen-Friedenreich antigen α or Core-1, refers to the disaccharide Gal- β 1,3-GalNAc, which is O-glycosidically linked in the cancer cell in an α -anomeric configuration to the protein's hydroxyl amino acid serine or threonine.

The term "sialic acid" especially refers to any N-or O-substituted derivative of neuraminic acid. It may refer to 5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid, but preferably only 5-N-acetylneuraminic acid. Sialic acids, in particular 5-N-acetylneuraminic acid, are preferably attached to the carbohydrate chain via a2, 3-or 2, 6-linkage. Preferably, in the antibodies described herein, 2, 3-as well as 2, 6-conjugated sialic acids are present.

"relative amount of glycans" according to the invention refers to a specific percentage or range of percentages of glycans attached to an antibody in an antibody preparation or composition comprising an antibody, respectively. In particular, the relative amount of glycans refers to a specific percentage or range of percentages of all glycans comprised in the antibody and thus attached to the polypeptide chain of the antibody in the antibody preparation or composition comprising the antibody. 100% glycans refer to all glycans attached to the antibody in the antibody preparation or composition comprising the antibody, respectively. For example, a relative amount of 10% glycans carrying bisecting GlcNAc refers to such antibody-containing compositions: wherein 10% of all glycans comprised in the antibody and thus attached to the antibody polypeptide chains in said composition comprise bisecting GlcNAc residues, and 90% of all glycans comprised in the antibody and thus attached to the antibody polypeptide chains in said composition do not comprise bisecting GlcNAc residues. The corresponding reference amount representing 100% glycans can be all glycan structures attached to the antibody in the composition, or all N-glycans, i.e. all glycan structures attached to asparagine residues of the antibody in the composition, or all complex glycans. The reference set of glycan structures is usually explicitly indicated by the skilled person or derived directly from the environment.

The term "N-glycosylation" refers to all glycans attached to asparagine residues of a protein polypeptide chain. These asparagine residues are typically part of an N-glycosylation site having the amino acid sequence Asn-Xaa-Ser/Thr, where Xaa can be any amino acid except proline. Likewise, an "N-glycan" is a glycan attached to an asparagine residue of a polypeptide chain. The terms "glycan", "glycan structure", "carbohydrate chain" and "carbohydrate structure" are generally used synonymously herein. N-glycans typically have a common core structure consisting of two N-acetylglucosamine (GlcNAc) residues and three mannose residues, with the structure Man α 1,6- (Man α 1,3-) Man β 1,4-GlcNAc β 1,4-GlcNAc β 1-Asn, where Asn is an asparagine residue in a polypeptide chain. N-glycans are subdivided into three distinct types, complex glycans, hybrid glycans, and high mannose glycans.

The numbers given herein, in particular the relative amounts of specific glycosylation properties, are preferably understood as approximate numbers. In particular, the number may preferably be up to 10% higher and/or lower, in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% higher and/or lower.

In a "conjugate," two or more compounds are linked together. In certain embodiments, at least some of the properties from each compound are retained in the conjugate. Attachment may be achieved by covalent or non-covalent bonds. Preferably, the compounds of the conjugate are linked by a covalent bond. The different compounds of the conjugate may be directly bound to each other by one or more covalent bonds between the atoms of the compounds. Alternatively, the compounds may be bound to each other by a chemical moiety such as a linker molecule, wherein the linker is covalently attached to an atom of the compound. If the conjugate consists of more than two compounds, these compounds may be linked, for example, in a chain conformation, one compound linked to the next, or several compounds each linked to a central compound.

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

The term "expression cassette" particularly refers to a nucleic acid construct capable of effecting and regulating the expression of the coding nucleic acid sequence introduced therein. The expression cassette may contain a promoter, ribosome binding site, enhancer and other control elements which regulate gene transcription or mRNA translation. The exact structure of the expression cassette may vary depending on the species or cell type, but typically includes 5' -nontranscribed and 5' -and 3' -untranslated sequences, such as TATA boxes, capping sequences, CAAT sequences, and the like, that are involved in initiation of transcription and translation, respectively. More specifically, the 5' -non-transcribed expression control sequence comprises a promoter region that includes a promoter sequence for transcriptional control of an operably linked nucleic acid. The expression cassette may also comprise an enhancer sequence or an upstream activator sequence.

According to the invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of the nucleic acid sequence to be expressed and controlling the expression of the sequence by providing recognition and binding sites for RNA polymerase. "promoters" may include other recognition and binding sites for other factors involved in the regulation of gene transcription. Promoters may control the transcription of prokaryotic or eukaryotic genes. In addition, a promoter may be "inducible," i.e., transcription is initiated in response to an inducing agent, or may be "constitutive" if transcription is not controlled by an inducing agent. If no inducer is present, the gene under the control of the inducible promoter is not expressed or is expressed only to a small extent. In the presence of an inducer, the gene is opened or the level of transcription is increased. Typically, this is mediated by the binding of specific transcription factors.

The term "vector" is used herein in its most general sense and includes any intermediate vehicle for a nucleic acid that enables the nucleic acid to be introduced, for example, into prokaryotic and/or eukaryotic cells and, where appropriate, integrated into the genome. Vectors of this type preferably replicate and/or express in cells. Vectors include plasmids, phagemids, bacteriophages or viral genomes. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, typically a circular DNA duplex, which can replicate independently of chromosomal DNA.

According to the present invention, the term "host cell" relates to any cell which can be transformed or transfected with an exogenous nucleic acid. According to the present invention, the term "host cell" includes prokaryotic (e.g.E.coli) or eukaryotic cells (e.g.mammalian cells, in particular human cells, yeast cells and insect cells). Mammalian cells, such as those from humans, mice, hamsters, pigs, goats or primates, are particularly preferred. Cells can be derived from a variety of tissue types and include primary cells and cell lines. The nucleic acid may be present in the host cell in a single copy or in two or more copies, and in one embodiment, is expressed in the host cell.

The term "patient" according to the present invention refers to a human, a non-human primate or other animal, in particular a mammal, such as a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent, e.g. a mouse and a rat. In a particularly preferred embodiment, the patient is a human.

The term "cancer" according to the present invention especially includes leukemia, seminoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer and lung cancer and metastases thereof. The term cancer according to the present invention also includes cancer metastases.

"tumor" refers to a group of cells or tissues formed by the proliferation of misregulated cells. Tumors may exhibit a partial or complete lack of structural organization and functional coordination with normal tissue, and often form distinct tissue masses, which may be benign or malignant.

"metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastases is a very complex process and typically involves the in-growth of cancer cells in normal tissues that detach from the primary tumor, enter the systemic circulation, and settle elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, whose cells are often similar to the original tumor. This means, for example, that if breast cancer metastasizes to the lungs, the secondary tumor consists of abnormal breast cells, not abnormal lung cells. Tumors in the lung were then termed metastatic breast cancer, but not lung cancer.

The term "pharmaceutical composition" especially refers to a composition suitable for administration to a human or animal, i.e. a composition comprising pharmaceutically acceptable components. Preferably, the pharmaceutical composition comprises the active compound or a salt or prodrug thereof and a carrier, diluent or pharmaceutical excipient, such as a buffer, preservative and tonicity modifier.

Numerical ranges described herein include the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be read with reference to the specification as a whole. According to one embodiment, the subject matter described herein as comprising certain steps in the case of a method or certain ingredients in the case of a composition means a subject matter consisting of the individual steps or ingredients. Preferably, the preferred aspects and embodiments described herein are selected and combined, and the specific subject matter resulting from the respective combinations of the preferred embodiments also belongs to the present disclosure.

Drawings

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

FIG. 2 shows surface plasmon resonance (Biacore) binding of anti-MUC 1 antibodies PM and PM-N54Q to Tn-glycosylated PDTR-MUC1 peptide. The maximum binding signals for different concentrations of PankoMab N54Q and PankoMab were plotted against antibody concentration.

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

Fig. 4 shows SDS acrylamide gels for electrophoretic separation of PankoMab N54Q and PankoMab under non-reducing (left) and reducing (right) conditions. Lane 1: PankoMab N54Q after the capture step; lane 2: PankoMab N54Q after the polishing step; lane 3: PankoMab after the capture step; lane 4: PankoMab after the polishing step; lane 5: and (4) marking molecular weight.

Fig. 5 shows coomassie blue stained gels with isoelectric focusing assay, in which PankoMab N54Q lacks Fab glycosylation and PankoMab is Fab glycosylated. Lane 1: PankoMab with Fab glycosylation; lane 2: PankoMab N54Q without Fab glycosylation.

Figure 6 shows that anti-MUC 1 antibody binds Fc γ receptor IIIa. Increasing concentrations of the antibodies PankoMab N54Q or PankoMab replaced the rabbit-anti-mouse coupled acceptor beads from Fc γ RIIIa loaded donor beads, thereby reducing the detected chemiluminescence. Low fucosylated antibodies in fig. 6A and high fucosylated antibodies in fig. 6B were applied in the assay.

FIG. 7 shows the binding of anti-MUC 1 antibodies PM-N54Q, PM-N54D, and PM with Fab glycosylation to tumor cell lines (A) CaOV-3 and (B) HSC-4 as analyzed by flow cytometry.

FIG. 8 shows the amino acid sequence of the heavy chain of humanized antibody PM N54Q (SEQ ID No: 15, wherein the amino acid at position 57 is Gln, SEQ ID No: 22).

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

FIG. 10 shows the amino acid sequence of the heavy chain of the humanized antibody PankoMab (SEQ ID No: 19).

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

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

Examples

Example 1:production of anti-MUC 1 antibody

The nucleic acid sequence of the heavy chain of the humanized PankoMab antibody was modified by mutating the codon for Asn54 (amino acid position 57 in SEQ ID NO: 11) according to the Kabat/EU numbering system to a codon for any amino acid other than Asn, particularly Gln (see, e.g., WO 2011/012309).

1) Production of anti-MUC 1 antibodies in human myeloid leukemia derived cell lines

A vector comprising coding sequences for the gamma 1-type heavy chain and the kappa-type light chain of the mutated antibody was transfected into the human myeloid leukemia derived cell line NM-H9D8 (DSM ACC 2806). Different α MUC 1-antibodies (PankoMab N54X/PM-N54X, wherein X is any amino acid other than N/Asn) comprising an N54X mutation or an amino acid mutation in the framework sequences of VH and VL were expressed in the resulting clones, thereby producing constructs with human glycosylation patterns. The concentration of alpha MUC 1-antibody in the supernatant was determined by Octet measurement using a protein A-coated needle, or by UV28 after protein A chromatographic purificationAbsorbance of 0 to quantify. The binding characteristics of the different alpha MUC 1-antibodies were determined by antigen-ELISA (see example 2) and also by Scatchard analysis (see example 3), by Biacore (see example 4a), by DRX²selected purified antibodies were analyzed by switchSENSE technology (see example 4b) or by flow cytometry (example 7).

In addition, PM-N54Q and non-mutated PankoMab with Fab-glycosylation were also expressed in the human myeloid leukemia derived cell line NM-H9D8-E6Q12 (DSM ACC2856) expressing antibodies with reduced fucose. Together with the same antibodies expressed in NM-H9D8, these antibodies were purified and analyzed in example 6 for their binding behavior to Fc γ receptor III a.

2) Production of anti-MUC 1 antibodies in CHO cell lines

The PM-N54Q coding sequence (the nucleotide sequence of the heavy chain of PM-N54Q represented by SEQ ID NO: 17 and the nucleotide sequence of the light chain of PM-N54Q represented by SEQ ID NO: 18) synthesized by GeneArtTM of ThermoFisher scientific was cloned into an expression vector and the resulting plasmid was electroporated into CHO cells. The pooled cells grown under selective pressure were used to make a PM-N54Q mutant antibody using general procedures.

Example 2:antigen ELISA

The antigen binding characteristics of PankoMab N54X (in which the N-glycosylation site in the Fab portion was knocked out) were compared to PankoMab having an N-glycosylation site in its Fab portion.

The Fab-deglycosylated form of the MUC 1-specific antibody PankoMab (PM-N54Q) was analyzed for binding characteristics relative to (glycosylated) PankoMab-GEX @, in an ELISA study using differently glycosylated and unglycosylated MUC 1-derived tandem repeat peptides. In general, both antibodies showed the same rank by virtue of binding to a glycosylated PDTR peptide (APPAHGVTSAPD-T (x) -RPAPGSTAPPAHGVTSA) with different glycosylation at T: gal beta 1-3GalNAc is observed and carried_α(TF) PDTR peptide with strongest binding, followed by sialylated TF and GalNAc_α(Tn) O-glycosylation. With sialylated GalNAc_α(sTn) O-glycosylThe binding of chemosynthesis is significantly lower. As PankoMab-GEX, PM-N54Q showed only very weak binding activity to the unglycosylated MUC1 PDTR peptide, indicating appropriate tumor specificity (FIG. 1C).

However, compared with PankoMab-GEX, the rubber products carrying GalNAc_αA4-fold higher binding to PM-N54Q was found in the TA-MUC1 antigen ELISA of biotinylated glycopeptides of (Tn) O-glycans. GalNAc when sialylated_α(sTn) glycosylation, PM-N54Q bound approximately 7-fold better to the same MUC1 peptide. At threonine of the PDTR-sequence with Gal beta 1-3GalNAc_α(TF) and sialylated TF (sTF) bound 2-fold better (FIG. 1A) than PM-N54Q.

Both antibodies showed strongly reduced binding to the MUC1 peptide variant APPAHGVTSAPE-s (Tn) -RPAPGSTAPPAHGVTSA with Tn glycosylation at serine compared to the counterpart at the pdt (Tn) R-peptide. However, here too, Fab-deglycosylated PM-N54Q bound significantly more strongly than PankoMab-GEX (FIG. 1B).

Various other Fab-deglycosylated PM-N54X variants were compared to pankomabs with N-glycosylation in their Fab portion. First, all variants were compared directly from the supernatant without purification. The concentration was determined by Octet. All PM-N54X variants bound better than Fab-glycosylated PM. In addition, a clear trend is visible depending on the chemical properties of the amino acid side chains. The carboxylic acid groups at the side chains showed the lowest binding enhancement. For amino acids with 1 or 2 nitrogens (as primary or secondary amines), best binding was observed (fig. 1D).

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

Furthermore, two different framework variants of PM-N54Q were analyzed for binding to the Tn-glycosylated PDTR-MUC1 peptide in an ELISA (see figure 1F). The framework variant mf-a carries 9 amino acid mutations in the VH and 3 amino acid mutations in the VL framework; the variant mf-b also carries 9 amino acid mutations in the VH and 4 amino acid mutations in the VL framework. Both mutated variants showed similar binding compared to the PM-N54Q antibody.

Example 3:saturation binding assay of anti-MUC 1 antibodies to MCF-7 and ZR-75-1 cells

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

In contrast to Fab-glycosylated PankoMab-GEX, the binding of the Fab-deglycosylated form of the MUC 1-specific antibody PankoMab (PM-N54Q) on a TA-MUC-1 positive human tumor cell line was evaluated by saturation binding analysis on human milk cancer cell lines ZR-75-1 and MCF-7 using radiolabeled antibody. The antibody was chelated with 12-fold molar excess of p-SCN-benzyl-DTPA in 50 mM sodium carbonate, 150 mM NaCl (pH 8.7) for 2 h at 37 ℃ followed by overnight incubation at 2-8 ℃. Free chelator was removed via desalting column and dead-end filtration (50 kDa cut-off, 6 Xbuffer exchange to PBS). Chelating the antibody with carrier-free in 6 mM phosphate, 1.6 mM KCl, 80 mM NaCl, 0.2M sodium acetate, 0.1M HCl¹¹¹In (2 μ Ci/μ g antibody) was radiolabeled for 1 h at 37 ℃. The preparation was neutralized by adding 8-9 volumes of 10-fold concentrated PBS. About 1/50 volumes of fetal bovine serum were added to the neutralized labeled antibody preparation. 1 x 10 was used per cell binding protocol⁶And (4) cells. Several concentrations of labeled antibody were added to the pelleted cells (30-1000 ng/200 μ L in 1% BSA/PBS). The resuspended cell-antibody mixture was measured in a gamma-counter and incubated for 1 h at 4 ℃. Cells with bound antibody were separated by centrifugation and washed with 1% BSA/PBS for an additional 1 hour at 4 ℃. Then in a gamma counter for binding¹¹¹The In-labeled antibody measures the cell pellet. Evaluation was performed by "single site specific ka" in GraphPad Prism. The data obtained are summarized in table 1. The data indicate high affinity and very high number of binding sites for PM-N54Q on these tumor cells. The binding was more than 2.5 times higher than PankoMab-GEX and the number of binding sites slightly increased.

TABLE 1 in MUC1⁺Swelling and swelling treating medicineAssociation constants and antigen binding sites on neoplastic cells

Example 4a surface plasmon resonance(BiaCore) analysis

The binding of the Fab-deglycosylated form of the MUC 1-specific antibody PankoMab (PM-N54Q) on TA-MUC-1 derived glycosylated peptides was evaluated by surface plasmon resonance analysis (Biacore). Streptavidin sensor chips were coated with biotinylated TA-MUC1 peptide (Tn glycosylated or not glycosylated). PankoMab and PM-N54Q were sequentially diluted 1:3 in HPS-EP from 3,600 nM to 4.9 nM. The diluent was injected at 50 μ L/min. The maximum binding at each concentration was determined as the Response Unit (RU) and evaluated using GraphPad Prism using "single site specific binding". FIG. 2 shows the binding curves obtained with PM-N54Q in comparison with PankoMab-GEX ®. Calculate the affinities (K) of 388 nM and 652 nM for PM-N54Q and PankoMab-GEX, respectively_D). Thus, in this experimental scenario, a nearly 2-fold increase in affinity was detectable.

Example 4b:fluorescent proximity sensing (by DRX) ² , Dynamic Biosensors)

One new method to determine the binding constant and affinity is to use the fluorescent proximity sensing of single stranded DNA (96-mer) imprinted on a chip and complementary DNA coupled to a ligand. In this study, streptavidin was used as a ligand to capture the biotinylated TA-MUC1 peptide. Binding of PankoMab to the peptide results in a change in fluorescence. Association and dissociation rates can be calculated during association and dissociation. Due to the higher sensitivity, faster interaction compared to surface plasmon resonance can be monitored. This results in binding kinetics that are different from SPR, but more comparable to the "gold standard" method KinExA (measured in liquid systems).

PankoMab and PM-N54Q were diluted from 300 nM to 3.67 nM in PE140 buffer in a 1:9 ladder and applied to the chip-bound peptides. Binding curves were evaluated by single-exponential ensemble fitting (instrument software). The binding curves for PM and PM-N54Q are shown in FIGS. 3A and B for example. The calculated affinities of PankoMab variants are shown in table 2:

TABLE 2 dissociation constants of PankoMab variants for antigen peptides

Example 5:biochemical characterization

Non-reducing and reducing SDS-PAGE was used to analyze the purity and identity of the antibodies. The band pattern in the non-reducing gel showed a major band of approximately 160 kDa, and systematic artifacts of heavy and light chains and combinations thereof (-25, 50-55, 75, 110, 135 kDa). The reducing gel showed unique light and heavy chain bands at 25 and 50-55 kDa. As expected, PM-N54Q has a smaller heavy chain due to the lack of Fab glycosylation (see figure 4 right).

The charge distributions are significantly different as shown by isoelectric focusing (IEF; see fig. 5). Fab glycosylation is significantly sialylated, whereas Fc glycosylation is only minimally sialylated. Thus, PankoMab-GEX ® has more charged isoforms than PM-N54Q, reflecting a higher level of negatively charged sialic acid in the Fab part.

Example 6:fc gamma receptor binding

Fc γ R binding assay for Fc γ RIIIa (CD16a) was based on the AlphaScreen @ technology of Perkinelmer. The AlphaScreen @ platform relies on the simple bead-based technology of Perkinelmer and is a more efficient alternative to traditional ELISA because no washing step is required.

For the receptor binding assay, a Ni-chelate donor bead was used to capture His-tagged Fc γ riiia (glycotope gmbh). The anti-MUC 1 antibody and rabbit-anti-mouse coupled receptor beads compete for binding to Fc γ R. In the case of Fc γ R interaction with rabbit-anti-mouse-bound acceptor beads, the donor and acceptor beads are in close proximity, which results in luminescence under 680nm laser excitation. Maximum signal (signal) is obtained without competitor_max). In case of competition, when the test antibody is reacted with Fc gammaR combined, signal_maxDecrease in a concentration-dependent manner. Chemiluminescence was quantified by measurement at 520-. All results are expressed as mean ± standard deviation of replicate samples. Data were evaluated and calculated using nonlinear curve fitting (sigmoidal dose-response variable slope) with GraphPad Prism 5 software. As a result, a concentration-dependent sigmoidal curve is obtained consisting of an upper plateau, a lower plateau, a slope and an EC₅₀And (4) defining.

As shown in fig. 6A and B, Fc γ RIIIa binding affinity was comparable for PankoMab N54Q and PankoMab, thus low fucosylated antibodies in panel a and high fucosylated antibodies in panel B were used in the assay. Thus, removal of Fab glycosylation does not affect the receptor interaction of the antibody.

Example 7 binding to the cell TA-MUC1

N54Q and N54D were transiently expressed and purified by protein a chromatography. Binding of the two variants to cell surface TA-MUC1 was compared to PM with Fab glycosylation using two different cancer cell lines. TA-MUC1 was expressed to an equal extent in the tongue squamous cell carcinoma line HSC-4, while the ovarian cancer cell line CaOV-3 was highly expressed. Tumor cells were incubated with serial dilutions of the antibodies and bound antibodies were detected using phycoerythrin-conjugated goat anti-human IgG (heavy and light chain) antibodies. Human IgG controls were included to control background staining. Binding was analyzed by flow cytometry.

Compared to the human IgG1 control, the constructs analyzed, PM-N54Q, and PM-N54D, showed strong and specific binding to HSC-4 and CaOV-3 cells expressing TA-MUC1 (FIG. 7). PM-N54D and TA-MUC1^{Height of}CaOV-3 cells bound comparable to PM with Fab glycosylation, while PM-N54Q showed slightly better binding (FIG. 7A). Using HSC-4 cancer cells expressing TA-MUC1 at moderate levels, the variant PM-N54Q bound significantly better to the cellular TA-MUC1 compared to PM, while PM-N54D showed weaker binding compared to PM with Fab glycosylation (fig. 7B).

Identification of deposited biological materials

On the date indicated in the table below, the cell lines DSM ACC2806, DSM ACC2807 and DSM ACC2856 were deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenttra e 7B, 38124 Braunschweig (DE) on the cell lines Glycotope GmbH, Robert-R ribbon-Str.10, 13125 Berlin (DE).

Cell line name	Login number	Depositor	Date of storage
				NM-H9D8	DSM ACC 2806	Glycotope GmbH	2006, 9 and 15 days
NM-H9D8-E6	DSM ACC 2807	Glycotope GmbH	2006, 10 months and 5 days
				NM-H9D8-E6Q12	DSM ACC 2856	Glycotope GmbH	8/2007

Detailed Description

The present invention is based on the development of a variant of the humanized anti-MUC 1 antibody PankoMab in which the glycosylation site in CDR-H2 has been deleted. Deletion of the glycosylation site is achieved by replacing the amino acid Asn (asparagine) 57 of the heavy chain variable region 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. Replacement of the asparagine residue with another residue will eliminate glycosylation because the carbohydrate structure can only be transferred to the asparagine residue by the enzyme of the host cell. It was surprisingly found that the deletion of the glycosylation site in CDR-H2 of PankoMab increases the antigen binding affinity of the antibody.

In view of these findings, the present invention provides an antibody capable of binding to MUC1, comprising

Incorporating MUC1

The antibody specifically binds to an epitope of MUC 1. "specific binding" refers to binding that is not non-specific adsorption. Examples of criteria for determining whether binding is specific may include the dissociation constant (referred to herein as "K_D"). This epitope is in the extracellular tandem repeat of MUC 1. In certain embodiments, the antibody binds MUC1 in a glycosylation dependent manner. In particular, if the tandem repeat is substituted at the threonine residue by N-acetylgalactosamine (Tn), sialyl α 2-6N-acetylgalactosamine (sTn), galactosyl 1-3N-acetylgalactosamine (TF) or galactosyl 1-3 (sialyl β 1-3)Group α 2-6) N-acetylgalactosamine (sTF), preferably Tn or TF, the antibody binds more strongly. Preferably, the carbohydrate moiety is bound to the threonine residue via an alpha-O-glycosidic bond. The epitope in the tandem repeat domain of MUC1 comprises in particular the amino acid sequence PDTR (SEQ ID NO: 13) or PESR (SEQ ID NO: 14). As mentioned above, the binding to this epitope is preferably glycosylation dependent, wherein the binding is increased in particular if the above carbohydrate moiety is attached to a threonine residue of the sequence PDTR or PESR (SEQ ID NO: 13 and 14), respectively.

This epitope is the tumor-associated MUC1 epitope (TA-MUC 1). The TA-MUC1 epitope refers in particular to an epitope of MUC1 which is present on tumor cells but not on normal cells and/or is accessible to antibodies in the host circulation only when present on tumor cells but not on normal cells. In certain embodiments, the antibody binds more strongly to cells expressing the TA-MUC1 epitope than to cells expressing normal, non-tumor MUC 1. Preferably, the binding is at least 1.5 times stronger, preferably at least 2 times stronger, at least 5 times stronger, at least 10 times stronger or at least 100 times stronger. For TA-MUC1 binding, the antibody preferably specifically binds to a glycosylated MUC1 tumor epitope such that the strength of the bond is increased at least 2-fold, preferably 4-fold or 10-fold, most preferably 20-fold, compared to the bond of an unglycosylated peptide of the same length and the same peptide sequence. The binding can be measured or determined by ELISA, RIA, surface plasmon resonance (hereinafter, referred to as "SPR") analysis, or the like. Examples of devices used in SPR analysis may include BlAcore (TM) (manufactured by GE Healthcare Bio-Sciences crop, ProteOn (TM) (manufactured by Bio-Rad Laboratories, Inc.), DRX2 Biosensor (manufactured by dynamics biosensers GmbH), SPR-Navi (TM) (manufactured by BioNavis Oy Ltd.), Spreeeta (TM) (manufactured by Texas Instruments Inc.), SPRi-PlexII (TM) (manufactured by Horiba, Ltd.), and Autolab SPR (TM) (manufactured by Metrohm). The binding of the antibody to the antigen expressed on the cell surface can be measured by flow cytometry or the like.

In addition, the antibody may exhibit antigen binding properties similar to a reference antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO. 11 or SEQ ID NO. 10 and a light chain variable region having the amino acid sequence of SEQ ID NO. 12. Preferably, the reference antibody is a humanized antibody PankoMab. In particular, the antibody according to the invention specifically binds to the same antigen as the reference antibody, and preferably binds to said antigen with a higher affinity. That is, the antibody preferably binds to the antigen with an affinity that is lower than the dissociation constant of the reference antibody, more preferably at least 10% lower, at least 20% lower, at least 30% lower or at least 50% lower. Furthermore, the antibody preferably shows cross-specificity with a reference antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO. 11 and a light chain variable region having the amino acid sequence of SEQ ID NO. 12. In particular, the humanized antibody is capable of blocking the binding of the reference antibody to MUC1 if present at a sufficiently high concentration. This is possible if the binding of the reference antibody to MUC1 is blocked when the antibody has bound to the antigen MUC 1.

anti-MUC 1 antibodies

The antibody of the invention capable of binding MUC1 comprises a heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO:1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, said light chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO. 4, CDR-L2 having the amino acid sequence of SEQ ID NO. 5, and CDR-L3 having the amino acid sequence of SEQ ID NO. 6.

In certain embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID No. 9. In particular, the heavy chain variable region comprises an amino acid sequence having at least 95%, in particular at least 98%, identity with the amino acid sequence of SEQ ID NO 9. In these embodiments, the heavy chain variable region still comprises the CDRs having the amino acid sequences of SEQ ID NOs 1,2 and 3. Thus, any sequence deviation from SEQ ID NO 9 is located in the framework regions, but not in the CDRs. Specifically, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 9.

In certain embodiments, CDR-H2 has the amino acid sequence of SEQ ID NO. 2, wherein the amino acid at position 8 of SEQ ID NO. 2 is selected from 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. Specifically, CDR-H2 has the amino acid sequence of SEQ ID NO. 7.

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

In certain embodiments, the light chain variable region comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 12. In particular, the light chain variable region comprises an amino acid sequence having at least 95%, in particular at least 98%, identity with the amino acid sequence of SEQ ID NO 12. In these embodiments, the light chain variable region still comprises the CDRs having the amino acid sequences of SEQ ID NOs 4, 5 and 6. Thus, any sequence deviation from SEQ ID NO 12 is located in the framework regions, but not in the CDRs. Specifically, the light chain variable region comprises the amino acid sequence of SEQ ID NO 12.

In specific embodiments, the heavy chain variable region has an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO 9, wherein 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 with at least 90% identity to the amino acid sequence of SEQ ID NO 12, wherein the CDRs still have the amino acid sequences of SEQ ID NO 4, 5 and 6. Specifically, the heavy chain variable region has an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO 9, wherein 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 with at least 95% identity to the amino acid sequence of SEQ ID NO 12, wherein the CDRs still have the amino acid sequences of SEQ ID NO 4, 5 and 6.

In specific embodiments, the heavy chain variable region has an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO 10, wherein 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 with at least 90% identity to the amino acid sequence of SEQ ID NO 12, wherein the CDRs still have the amino acid sequences of SEQ ID NO 4, 5 and 6. Specifically, the heavy chain variable region has an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO 10, wherein 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 with at least 95% identity to the amino acid sequence of SEQ ID NO 12, wherein the CDRs still have the amino acid sequences of SEQ ID NO 4, 5 and 6.

In a specific embodiment, the heavy chain variable region comprises the amino acid sequence: which has at least 90% identity with the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO 20. In particular, the heavy chain variable region comprises the amino acid sequence: it has at least 95%, in particular at least 98% identity with the amino acid sequence represented by amino acid numbers 20 to 136 of SEQ ID NO 20. In these embodiments, the heavy chain variable region comprises CDR-H1 having the amino acid sequence of SEQ ID NO:1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Thus, any sequence deviations from the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO 20 are located in the framework regions, but not in the CDRs. Specifically, the heavy chain variable region comprises the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO 20. In certain embodiments, the amino acid at position 76 of SEQ ID NO 20 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; 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. Specifically, CDR-H2 has the amino acid sequence of SEQ ID NO. 7 and/or the heavy chain variable region comprises the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO. 23.

In a specific embodiment, the light chain variable region comprises the amino acid sequence: which has at least 90% identity with the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO 21. In particular, the light chain variable region comprises the amino acid sequence: it has at least 95%, in particular at least 98% identity with the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO 21. In these embodiments, the light chain variable region still comprises the CDRs having the amino acid sequences of SEQ ID NOs 4, 5 and 6. Thus, any sequence deviations from the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO 21 are located in the framework regions, but not in the CDRs. Specifically, the light chain variable region comprises an amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO 21.

In particular embodiments, the heavy chain variable region has the amino acid sequence: which has at least 90% identity to the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO. 20, wherein the CDRs still have the amino acid sequences of SEQ ID NO. 1, 7 and 3, and the light chain variable region has the amino acid sequence: which has at least 90% identity with the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO 21, wherein the CDRs still have the amino acid sequences of SEQ ID NO 4, 5 and 6. Specifically, the heavy chain variable region has an amino acid sequence with at least 95% identity to the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO 20, wherein 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 with at least 95% identity to the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO 21, wherein the CDRs still have the amino acid sequences of SEQ ID NO 4, 5 and 6.

In a specific embodiment, the heavy chain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 15. In particular, the heavy chain comprises an amino acid sequence having at least 95%, in particular at least 98%, identity with the amino acid sequence of SEQ ID NO. 15. In these embodiments, the heavy chain comprises CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 2, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3. Thus, any sequence deviation from SEQ ID NO 15 is located in the framework regions, but not in the CDRs. Specifically, the heavy chain comprises the amino acid sequence of SEQ ID NO. 15. In certain embodiments, the amino acid at position 57 of SEQ ID NO. 15 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; 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. Specifically, CDR-H2 has the amino acid sequence of SEQ ID NO. 7 and/or the heavy chain variable region comprises the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO. 22.

In specific embodiments, the light chain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 16. In particular, the light chain comprises an amino acid sequence having at least 95%, in particular at least 98%, identity with the amino acid sequence of SEQ ID NO 16. In these embodiments, the light chain still comprises CDRs having the amino acid sequences of SEQ ID NOs 4, 5, and 6. Thus, any sequence deviation from SEQ ID NO 16 is located in the framework regions, but not in the CDRs. Specifically, the light chain comprises the amino acid sequence of SEQ ID NO 16.

In a specific embodiment, the heavy chain has an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO. 15, wherein 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 with at least 90% identity to the amino acid sequence of SEQ ID NO. 16, wherein the CDRs still have the amino acid sequences of SEQ ID NO. 4, 5 and 6. Specifically, the heavy chain has an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO. 15, wherein 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 with at least 95% identity to the amino acid sequence of SEQ ID NO. 16, wherein the CDRs still have the amino acid sequences of SEQ ID NO. 4, 5 and 6.

The antibodies according to the invention include and encompass modified forms thereof. Modified forms of the antibodies of the invention refer to antibodies of the invention having chemical or biological modifications. Chemically modified forms include forms having an amino acid backbone conjugated to a chemical moiety, forms having a chemically modified N-linked or O-linked carbohydrate chain, and the like. The chemical moiety or form may be toxic or cytotoxic. Biologically modified forms include forms that have undergone post-translational modification (e.g., N-linked or O-linked glycosylation, N-or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), forms that contain a methionine residue added to the N-terminus by expression using a prokaryotic host cell, and the like. Such modified forms are also intended to include forms that are labeled to allow detection or isolation of the antibodies or antigens of the invention, e.g., enzyme-labeled forms, fluorescently labeled forms, or affinity-labeled forms. Such modified forms of the antibodies of the invention may be used to improve the stability or blood retention of the original antibody of the invention, reduce antigenicity, detect or isolate the antibody or antigen, and the like.

In particular, the antibody may comprise one or more modifications selected from: defucosylation, reduced fucose, N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, substitution of 2 leucine (L) residues at positions 234 and 235 of the heavy chain to alanine (a) (LALA), amidation of proline residues, and deletion or absence of 1,2 or 3 amino acids at the carboxy terminus. In particular embodiments, the antibody lacks 1,2 or 3 carboxy-terminal amino acids in one or both heavy chains, or it lacks 1 carboxy-terminal amino acid and the carboxy-terminal proline residue is amidated at one or both heavy chains.

Such modifications may be made at any position or desired position of the antibody thereof. Alternatively, the same or 2 or more different modifications may be made at one or two or more positions therein.

For example, it is known that antibodies produced by cultured mammalian cells lack the carboxyl-terminal lysine residue in their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that occasionally 2 carboxy-terminal amino acid residues of the heavy chain (i.e., glycine and lysine) are lost, and that the proline residue newly located at the carboxy-terminal end is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such lack or modification in these heavy chain sequences does not affect either the ability of the antibody to bind its antigen or the effector functions of the antibody (complement activation, antibody-dependent cytotoxicity, etc.).

In certain embodiments, the antibody comprises a deletion or absence of 1 or 2 amino acids in the carboxy-terminus of the heavy chain and has an amidated residue (e.g., an amidated proline residue at the carboxy-terminus position of the heavy chain). However, the antibody according to the present invention is not limited to the above type as long as the deletion mutant retains the ability to bind to an antigen.

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

In particular embodiments, an antibody according to the invention may comprise two heavy chains, both of which lack one carboxy-terminal amino acid residue.

In a specific embodiment, the antibody comprises a heavy chain having an amino acid sequence represented by amino acid numbers 1-446 of SEQ ID NO 15 or 22 and a light chain having an amino acid sequence represented by amino acid numbers 1-219 of SEQ ID NO 16. In certain embodiments, the amino acid at position 57 of SEQ ID NO. 15 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; 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 certain embodiments, an antibody according to the 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 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO. 11 and a light chain variable region having the amino acid sequence of SEQ ID NO. 12.

In certain embodiments, the antibody has the following properties: (a) specifically binds to MUC1, and/or (b) has the activity of being internalized into a cell expressing MUC1 by binding to MUC 1.

In certain embodiments, the antibody comprises at least one antibody heavy chain. In particular, the antibody comprises two antibody heavy chains. The antibody heavy chain comprises in particular a VH domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain. In certain other embodiments, the antibody heavy chain comprises a CH2 domain and a CH3 domain, but does not comprise a CH1 domain. In other embodiments, one or more constant domains of the heavy chain may be replaced by other domains, in particular similar domains, such as albumin. Antibody heavy chains may be of any type, including gamma-, alpha-, epsilon-, delta-, and mu-chains, and are preferably gamma-chains, including gamma 1-, gamma 2-, gamma 3-, and gamma 4-chains, especially gamma 1-chains. Thus, the antibody is preferably an IgG-type antibody, such as an IgG1-, IgG 3-or IgG 4-type antibody, in particular an IgG 1-type antibody.

In particular, the antibody further comprises at least one antibody light chain, in particular two antibody light chains. The antibody light chain comprises in particular a VL domain and a CL domain. The antibody light chain may be a kappa-chain or a lambda-chain, especially a kappa-chain.

In certain embodiments, the antibody comprises two antibody heavy chains and two antibody light chains. In particular, the antibody comprises two gamma 1-type antibody heavy chains each comprising a VH domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain and two kappa-type antibody light chains each comprising a VL domain and a CL domain.

In an alternative embodiment, the antibody does not comprise an antibody light chain. In these embodiments, the light chain variable region may be fused to the N-terminus of the heavy chain variable region or inserted into the C-terminus of the heavy chain variable region. A peptide linker may be present to link the light chain variable region to the remainder of the heavy chain.

In a preferred embodiment, the antibody comprises an Fc region. The antibody may in particular be a whole antibody comprising two heavy chains each comprising the domains VH, CH1, hinge region, CH2 and CH3 and two light chains each comprising the domains VL and CL. The antibodies are particularly capable of binding to one or more human Fc γ receptors, particularly human Fc γ receptor IIIA. In an alternative embodiment, the antibody does not or does not significantly bind to human Fc γ receptor IIIA, in particular does not or does not significantly bind to any human Fc γ receptor. In these embodiments, the antibody specifically does not comprise a glycosylation site in the CH2 domain.

In alternative embodiments, the antibody does not comprise an Fc region. In these embodiments, the antibody is in particular a single chain variable fragment (scFv) or another antibody fragment which does not comprise an Fc region.

Glycosylation of anti-MUC 1 antibodies

The anti-MUC 1 antibody may comprise one or more CH2 domains in the heavy chain of the antibody. Natural human antibodies of the IgG class contain an N-glycosylation site in the CH2 domain. The CH2 domain present in an antibody may or may not contain an N-glycosylation site. In certain embodiments, the antibody does not comprise a glycosylation site in the CH2 domain. In particular, according to the IMGT/Eu numbering system, the antibody does not comprise an asparagine residue at a position in the heavy chain corresponding to position 297. For example, the antibody may comprise an Ala297 mutation in the heavy chain. In these embodiments, the antibody preferably has a strongly reduced ability or is completely devoid of the ability to induce antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) by binding to Fc γ receptors. A strongly reduced capacity in this respect particularly means an activity which is reduced to 10% or less, in particular 3% or less, 1% or less or 0.1% or less, compared to the same antibody comprising N-glycosylation sites in its CH2 domain and having a common mammalian glycosylation pattern, such as those obtainable by production in a human cell line or CHO cell line, e.g. the glycosylation pattern described herein. In these embodiments, the antibody is specifically an IgG 1-type antibody.

In an alternative embodiment, the CH2 domain present in the antibody comprises an N-glycosylation site. The glycosylation site is in particular at an amino acid position corresponding to amino acid position 297 of the heavy chain according to the IMGT/Eu numbering system and has the amino acid sequence motif Asn Xaa Ser/Thr, wherein Xaa may be any amino acid except proline. N-linked glycosylation at Asn297 is conserved in the homologous regions of mammalian IgG and other antibody isotypes. The actual position of this conserved glycosylation site may vary in the amino acid sequence of the antibody due to optional additional amino acid or other sequence modifications that may be present in the variable region. Preferably, the glycan to which the antibody is attached is a bi-antennary complex N-linked carbohydrate structure, preferably comprising at least the following structure:

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

wherein Asn is an asparagine residue in the polypeptide portion of the antibody; GlcNAc is N-acetylglucosamine, and Man is mannose. The terminal GlcNAc residue may further carry a galactose residue, which optionally may carry a sialic acid residue. Another GlcNAc residue (referred to as a bisecting GlcNAc) may be attached to the Man of the closest polypeptide. Fucose can bind to GlcNAc attached to Asn. In these embodiments, the antibody is specifically an IgG 1-type antibody.

In preferred embodiments, the antibody does not comprise N-glycolyl neuraminic acid (NeuGc) or a detectable amount of NeuGc. Furthermore, the antibody preferably also does not comprise a Galili epitope (Gal α 1,3-Gal structure) or a detectable amount of a Galili epitope. In particular, the relative amount of glycans carrying NeuGc and/or Gal α 1,3-Gal structures is less than 0.1% or even less than 0.02% of the total amount of glycans attached to the CH2 domain of the antibody in the population of antibodies.

In particular, the antibodies have a human glycosylation pattern. Due to these glycosylation properties, there are no foreign immunogenic non-human structures inducing side effects, which means that undesired side effects or disadvantages known to be caused by certain foreign sugar structures, such as immunogenic non-human sialic acids (NeuGc) or Galili epitopes (Gal-Gal structures) known for rodent production systems, or other structures such as immunogenic high mannose structures known from e.g. yeast systems, are avoided.

In particular embodiments, the antibody comprises a glycosylation pattern of glycans carrying bisecting GlcNAc residues in a detectable amount. In particular, the relative amount of glycans carrying bisecting GlcNAc residues is at least 0.5%, especially at least 1%, of the total amount of glycans attached to the glycosylation sites of the antibody in the composition. Further, in certain embodiments, the glycosylation pattern comprises a relative amount of glycans carrying at least one galactose residue of at least 25% of the total amount of glycans attached to the antibody in the composition. In particular, the relative amount of glycans carrying 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 a specific embodiment, the glycosylation pattern comprises glycans carrying at least one sialic acid residue in a relative amount of at least 1% of the total amount of glycans attached to the antibody in the composition. In particular, the relative amount of glycans carrying 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 pattern such that: it has a large amount of core fucose or a small amount of core fucose. A reduced amount of fucosylation increases the antibody's ability to induce ADCC. In certain embodiments, the relative amount of glycans carrying core fucose residues 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 carrying core fucose residues 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 the anti-MUC 1 antibody and the presence or absence of fucose in the glycan structure at said glycosylation site. ADCC activity is increased by glycosylation of the Fc portion of the antibody, and further by decreasing the amount of fucosylation in the glycosylation. In certain applications, fine-tuning of ADCC activity is important. Thus, in certain instances, antibodies without glycosylation sites in the CH2 domain, antibodies with glycosylation sites in the CH2 domain and with a high amount of fucosylation, or antibodies with glycosylation sites in the CH2 domain and with a low amount of fucosylation may be most advantageous.

Production of anti-MUC 1 antibodies

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

In certain embodiments, the antibodies are recombinantly produced in a human blood cell line, particularly in a human myeloid leukemia cell line. Preferred human cell lines useful for the production of antibodies and suitable production procedures are described in WO 2008/028686 a 2. In a specific embodiment, the antibody is obtained by expression in a human myeloid leukemia cell line selected from NM-H9D8, NM-H9D8-E6 and NM-H9D8-E6Q12 and cell lines derived therefrom. These cell lines were deposited under the Budapest treaty under the accession numbers DSM ACC2806 (NM-H9D 8; deposited at 15.9.2006), DSM ACC2807 (NM-H9D 8-E6; deposited at 5.10.2006) and DSM ACC2856 (NM-H9D8-E6Q 12; deposited at 8.8.2007) from Glycotope GmbH, Robert-R ssle-Str. 10, 13125 Berlin (DE) at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenttra E7B, 38124 Braunschweig (DE). NM-H9D8 cells provide glycosylation patterns with high sialylation, high bisection of GlycNAc, high galactosylation, and high fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells provide similar glycosylation patterns as NM-H9D8 cells, except that the degree of fucosylation is very low. Other suitable cell lines include K562, a human myeloid leukemia cell line (ATCC CCL-243) found in the American type culture Collection, and cell lines derived from the above cell lines.

In other embodiments, the antibody is recombinantly produced in CHO cells. Specifically, the antibodies can be produced recombinantly in a CHO dhfr-cell line such as the cell line of ATCC number CRL-9096.

Conjugates of anti-MUC 1 antibodies

In certain embodiments, the antibody comprises one or more additional agents conjugated thereto. The other agent may be any agent suitable for conjugation to an antibody. If more than one other agent is present in the antibody, these other agents may be the same or different, and in particular all the same. Conjugation of other agents to the antibody can be accomplished using any method known in the art. Other agents may be covalently attached (particularly by fusion or chemical coupling) or non-covalently attached to the antibody. In certain embodiments, the additional agent is covalently attached to the antibody, particularly via a linker moiety. The linker moiety may be any chemical entity suitable for attaching other reagents to the antibody.

The further agent is preferably used for the treatment, diagnosis, prognosis, detection and/or monitoring of diseases, in particular cancer. For example, the additional agent may be selected from radionuclides, chemotherapeutic agents, antibodies or antibody fragments, particularly those with different specificities from the anti-MUC 1 antibody, such as checkpoint antibodies that block or activate immunomodulatory targets, enzymes, interacting domains, detectable labels, toxins, cytolytic components, immunomodulatory agents, immune effectors, MHC class I or class II antigens, and liposomes. One particularly preferred additional agent is a radionuclide or cytotoxic agent capable of killing cancer cells, such as a chemotherapeutic agent. In certain preferred embodiments, the chemotherapeutic agent is linked to the anti-MUC 1 antibody to form a conjugate.

Specific examples of chemotherapeutic agents that may be conjugated as other agents include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxins, taxanes such as taxol, topoisomerase inhibitors such as irinotecan and topotecan, antineoplastic agents such as doxorubicin, or microtubule inhibitors such as auristatins and maytansine/maytansine.

The chemotherapeutic agent may be chosen in particular from: v-atpase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizing agents, microtubule destabilizing agents, auristatins, dolastatin, maytansine, maytansinoids, amanitins, methionine aminopeptidase, inhibitors of nuclear export protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, DHFR inhibitors, inhibitors of microtubule formation, stabilizing agents of microtubules, stabilizing agents of actin, topoisomerase II inhibitors, platinum compounds, ribosome inhibitors, RNA polymerase II inhibitors, and bacterial toxins. In particular embodiments, the chemotherapeutic agent is attached to an anti-MUC 1 antibody selected from the group consisting of: auristatins, microtubule inhibitors such as maytansinoids, DNA damaging agents, DNA alkylating agents, and DNA minor groove binders.

In certain embodiments, the chemotherapeutic agent is maytansine or a maytansinoid. Specific examples of maytansinoids useful for conjugation include maytansinol, and the like,N ^2'Deacetylation-N ^2'- (3-mercapto-1-oxopropyl) -maytansine (DM1),N ^2'Deacetylation-N ^2'- (4-mercapto-1-oxopentyl) -maytansine (DM3) andN ^2'deacetylation-N ^2'- (4-methyl-4-mercapto-1-oxopentyl) -maytansine (DM 4). Specifically, DM1 or DM4 was attached to an anti-MUC 1 antibody. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is an auristatin, particularly monomethyl auristatin f (mmaf), monomethyl auristatin e (mmae), or auristatin T. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a DNA minor groove binding agent, in particular Pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer), dactinomycin-hydroxybenzamide-azaindole (DUBA), seco-dactinomycin-hydroxybenzamide-azaindole (duca), or doxorubicin. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a DNA alkylating agent, particularly an indolinobenzodiazepine (indolinobenzodiazepine) or oxazolidinobenzodiazepine (oxazobisazepine). In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a DNA damaging agent, particularly calicheamicin. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is an inhibitor of microtubule formation, in particular tubulysin, ansamitocin, podophyllotoxin or vinblastine. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a stabilizer of microtubulesEspecially paclitaxel or epothilone. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a stabilizer of actin, particularly phallotoxin. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a topoisomerase II inhibitor, particularly teniposide, XK469, razoxane, amsacrine, idarubicin, or mebarone. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a platinum compound, particularly cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenonthriplatin, picoplatin, or satraplatin. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a ribosome inhibitor, particularly ricin, saporin, abrin, diphtheria toxin, or exotoxin a. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is an RNA polymerase II inhibitor, particularly an amatoxin, e.g., amanitin. In certain embodiments, the chemotherapeutic agent attached to the anti-MUC 1 antibody is a bacterial toxin, particularly anthrax toxin. Suitable antibody drug conjugates are also described in EP 16151774.3 and LU 92659, to which reference is expressly made herein.

In certain embodiments, the additional agent is a polypeptide or protein. The polypeptide or protein may in particular be fused to a polypeptide chain of an antibody. In certain embodiments, the additional agent, which is a polypeptide or protein, is fused to the C-terminus of the antibody light chain of the antibody. In embodiments where the antibody comprises two antibody light chains, additional agents, which are polypeptides or proteins, may be fused to the C-terminus of each of the two antibody light chains. In other embodiments, other agents that are polypeptides or proteins are fused to the C-terminus of the antibody heavy chain of the antibody. In embodiments where the antibody comprises two antibody heavy chains, additional agents, which are polypeptides or proteins, may be fused to the C-terminus of each of the two antibody heavy chains. The other agents may be the same or different and in particular have the same amino acid sequence. Suitable examples of such other agents as polypeptides or proteins may be selected from cytokines, chemokines, antibodies, antigen binding fragments, enzymes and interacting domains.

In certain embodiments, the additional agent that is a polypeptide or protein is a checkpoint antibody that blocks and/or triggers an activation signal. Examples of various targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM) and CD258 (LIGHT) as activation targets, CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA and phosphatidylserine as inhibition targets, and their corresponding ligands such as PDL 1. In particular examples, the anti-MUC 1 antibody comprises two heavy chains and two light chains as described herein, wherein an scFv fragment that specifically binds CD3 is fused to the C-terminus of each heavy chain; or wherein an scFv fragment that specifically binds PDL1 is fused to the C-terminus of each light chain.

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

Nucleic acids, expression cassettes, vectors, cell lines and compositions

In another aspect, the invention provides nucleic acids encoding antibodies. The nucleic acid sequence of the nucleic acid may have any nucleotide sequence suitable for encoding an antibody. Preferably, however, the nucleic acid sequence is adapted at least in part to the particular codon usage, in particular human codon usage, of the host cell or organism in which the nucleic acid is to be expressed. 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 continuous nucleic acid molecule, or it may consist of several nucleic acid molecules, each encoding a different part of the antibody. In a preferred embodiment, the present invention provides the nucleotide sequence of the heavy chain of the PankoMab variant (PM-N54Q) represented by SEQ ID NO: 17 and the nucleotide sequence of the light chain of the PankoMab variant (PM-N54Q) represented by SEQ ID NO: 18.

If the antibody is composed of more than one distinct amino acid chain, e.g., a light chain and a heavy chain of an antibody, the nucleic acid may, for example, be a single nucleic acid molecule containing several coding regions (each encoding one of the amino acid chains of the antibody, preferably separated by a regulatory element such as an IRES element) to produce separate amino acid chains, or the nucleic acid may be composed of several nucleic acid molecules, wherein each nucleic acid molecule contains one or more coding regions, each coding region encoding one of the amino acid chains of the antibody. In addition to the coding region encoding the antibody, the nucleic acid may also comprise other nucleic acid sequences or other modifications, for example, it may encode other proteins, may affect the transcription and/or translation of the coding region, may affect the stability or other physical or chemical properties of the nucleic acid, or may not function at all.

In another aspect, the invention provides an expression cassette or vector comprising a nucleic acid according to the invention and a promoter operably linked to said nucleic acid. In addition, the expression cassette or vector may comprise further elements, in particular elements capable of influencing and/or regulating the transcription and/or translation of the nucleic acid, the amplification and/or replication of the expression cassette or vector, the integration of the expression cassette or vector into the genome of the host cell, and/or the copy number of the expression cassette or vector in the host cell. Suitable expression cassettes and vectors comprising the various expression cassettes for expression of the antibody are well known in the art and need not be described further herein.

Furthermore, the present invention provides a host cell comprising 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 an established cell line, preferably a tumor-derived cell. Preferably, it is a bacterial cell such as e.coli, a yeast cell such as saccharomyces cell, in particular saccharomyces cerevisiae, an insect cell such as Sf9 cell, or a mammalian cell, in particular a human cell such as a tumor-derived human cell, a hamster cell such as CHO or a primate cell. In a preferred embodiment of the invention, the host cell is derived from a human myeloid leukemia cell. Preferably, it is selected from the following cells or cell lines: k562, KG1, MUTZ-3 or a cell or cell line derived therefrom, or a cell or cell line mixture comprising at least one of the aforementioned cells. The host cell is preferably selected from NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, and a cell or cell line derived from any one of said host cells. These cell lines and their properties are described in detail in PCT application WO 2008/028686A 2. In other embodiments, the host cell is a CHO dhfr-cell line such as that of ATCC number CRL-9096. In a preferred embodiment, the host cell is optimized for expression of glycoproteins having a particular glycosylation pattern, in particular antibodies. Preferably, the codon usage and/or the promoter in the coding region of the nucleic acid according to the invention and other elements of the expression cassette or vector are compatible with 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 particular aspect, the invention provides a method of producing an antibody in a host cell as described herein. The method comprises in particular the following steps: providing a host cell comprising a nucleic acid encoding an antibody, culturing the host cell under conditions suitable for expression of the antibody, and obtaining the antibody expressed by the host cell. The antibodies according to the invention can be obtained or obtainable by said method.

In another aspect, the invention provides a composition comprising said antibody, said nucleic acid, said expression cassette or vector or said host cell. The composition may also contain more than one of these components. In addition, the composition may comprise one or more further components selected from solvents, diluents and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, the components of the composition are preferably all pharmaceutically acceptable. The composition may be a solid or fluid composition, in particular a solution (preferably an aqueous solution), an emulsion or suspension or a lyophilized powder.

Use in medicine

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

Thus, in another aspect, the invention provides an antibody, nucleic acid, expression cassette or vector, host cell or composition for use in medicine. Preferably, the use in medicine is for the treatment, prognosis, diagnosis, detection and/or monitoring of diseases, for example, diseases associated with abnormal cell growth, e.g. 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 immunodeficiency. In a preferred embodiment, the disease is cancer.

Preferably, the cancer has detectable expression of MUC1 or TA-MUC1, preferably detectable by immunohistochemistry, ELISA, RIA, Enzyme Linked Immunospot (ELISPOT) assay, dot blot, Ouchterlony test or Convection Immunoelectrophoresis (CIE) or in situ hybridization. It includes, inter alia, cells having MUC1 or TA-MUC1 expression, which cells can be detected by immunohistochemistry or in situ hybridization. Prior to administration of the anti-MUC 1 antibody, the cancer may be tested at MUC1 or TA-MUC1 levels.

The invention further provides kits and devices comprising antibodies according to the invention, and related methods useful in the diagnosis, detection or monitoring of MUC 1-related disorders, such as cancer. In certain embodiments, a sandwich ELISA kit for testing, detection or diagnosis is provided comprising an antibody of the invention. The kit may further comprise one or more of the following: a solution of MUC1 or TA-MUC1 protein standard, a colorant, a buffer solution for dilution, an antibody for solid phase, an antibody for detection, a washing solution, and the like. Preferably, the amount of antibody bound to the antigen can be measured by applying methods such as absorbance, fluorescence, luminescence, or Radioisotope (RI) methods. Preferably, an absorbance plate reader, a fluorescence plate reader, a luminescence plate reader, an RI liquid scintillation counter, or the like is used in the measurement.

In certain embodiments, the invention provides antibodies according to the invention and compositions comprising them for use in Immunohistochemical (IHC) assays.

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

Different forms of cancer, including metastases, can be treated with antibodies according to the invention. The cancer may be selected from colon cancer, lung cancer, ovarian cancer, breast cancer such as triple negative breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, gastrointestinal cancer, renal cancer, head and neck cancer, thyroid cancer and urothelial cancer, among others. The cancer may further be selected in particular from the group consisting of gastric cancer, liver cancer, bladder cancer, skin cancer, prostate cancer and blood cancer. In certain embodiments, the cancer is a metastatic cancer. The cancer may include any type of metastasis, such as skin metastases, lymph node metastases, lung metastases, liver metastases, peritoneal metastases, pleural metastases, and/or brain metastases. In certain embodiments, the cancer has an inflammatory phenotype. In these embodiments, any of the above cancer types can be an inflammatory cancer.

In certain 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 may 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 celiac disease, type I diabetes, graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriatic arthritis, atopic dermatitis, scleroderma, sarcoidosis, primary biliary cirrhosis, guillain-barre syndrome, autoimmune hepatitis, and ankylosing spondylitis. In certain embodiments, the disease comprises or is associated with a cell expressing MUC1, particularly TA-MUC 1. For example, the cancer to be treated is MUC1 positive, in particular TA-MUC1 positive, i.e. comprises cancer cells expressing MUC1, in particular TA-MUC 1.

In particular embodiments, the antibodies are used in combination therapy with another therapeutic agent, particularly in combination therapy with another anti-cancer agent to treat cancer. The additional therapeutic agent may be any known anti-cancer agent. Suitable anti-cancer therapeutic agents that can be combined with the antibodies according to the invention can be chemotherapeutic agents, other antibodies, immunostimulants, cytokines, chemokines and vaccines. In addition, therapy with antibodies may be combined with radiation therapy, surgery, and/or traditional chinese medicine.

Anti-cancer agents that may be used in combination with the anti-MUC 1 antibody may be selected from any chemotherapeutic agent, particularly chemotherapeutic agents known to be effective in treating MUC 1-positive cancers. The type of chemotherapeutic agent also depends on the cancer to be treated. The combination partner may be selected from: taxanes such as paclitaxel (taxol), docetaxel (taxotere) and SB-T-1214; cyclophosphamide; imatinib; pazopanib; capecitabine; cytarabine; vinorelbine; gemcitabine; anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone; aromatase inhibitors such as aminoglutethimide, testolactone (Teslac), anastrozole (runing), letrozole (frolone), exemestane (anoxin), vorozole (rivzor), formestane (lantalone), fadrozole (Afema), 4-hydroxyandrostenedione, 1,4, 6-androstatriene-3, 17-dione (ATD) and 4-androstene-3, 6, 17-trione (6-oxo); topoisomerase inhibitors such as irinotecan, topotecan, camptothecin, lamellarin D, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, erietidine, aurintricarboxylic acid, and HU-331; platinum-based chemotherapeutic agents such as cis-diamminebischloroplatinum (II) (cisplatin), cis-diamminedicarboxylate (carboxylato) platinum (II) (carboplatin) and [ (1R,2R) -cyclohexane-1, 2-diamine ] (ethanediato-O, O') platinum (II) (oxaliplatin); PARP inhibitors such as olaparib, rucaparib and niraprib; TLR agonists such as imiquimod and resiquimod; and antimetabolites, especially antifolates such as methotrexate, pemetrexed, raltitrexed and pralatrexate, pyrimidine analogs such as fluorouracil, gemcitabine, fluorouridine, 5-fluorouracil and tegafur-uracil, and purine analogs, selective estrogen receptor modulators and estrogen receptor down-modulators.

In addition, therapeutic antibodies may also be used as further combination partners. It may be any antibody useful in cancer therapy other than the anti-MUC 1 antibody. In particular, other antibodies have been approved for cancer therapy by regulatory agencies such as the U.S. Food and Drug Administration (FDA), the european drug administration (EMA, predecessor EMEA), and the bundesist fur Arzneimittel und Medizinproduke (BfArM). Examples of other antibodies that may be used in combination therapy are anti-EGFR antibodies such as cetuximab, tomuzuximab, parlimumab, zalutumumab, nimotuzumab, matuzumab and nefirumab; anti-HER 2 antibodies such as trastuzumab, Timigutuzumab, and pertuzumab; anti-VEGF antibodies such as bevacizumab (atorvastatin); anti-CD 52 antibodies such as alemtuzumab (Campath); anti-CD 30 antibodies such as brentuximab (additris); anti-CD 33 antibodies such as gemtuzumab ozogamicin (Mylotarg); and anti-CD 20 antibodies such as rituximab (Rituxan ), tositumomab (Bexxar), and ibritumomab (zeuglin). Other exemplary antibodies suitable for use in the cancer therapy combinations described herein include antibodies to antigens selected from the group consisting of: Thomsen-Friedenreich antigens (TF. alpha., TF. beta.), Tn, Lewis Y, CD44, folate receptor. alpha., NeuGc-GM3 ganglioside, DLL-3, RANKL, PTK7, Notch-3, Ephrin A4, insulin-like growth factor receptor 1, activin receptor-like kinase-1, blocker-6, disialoganglioside GD2, endoglin, transmembrane NMglycoprotein B, CD56, tumor-associated calcium signal transduction protein 2, tissue factor, ectonucleotide pyrophosphatase/phosphodiesterase 3, CD70, P-cadherin, mesothelin, six transmembrane epithelial antigen 1 of prostate (STEAP1), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), connexin 4, guanylate cyclase C, solute carrier family member 44 (SLC44A4), prostate-specific membrane antigen (PSMA), zinc transporter ZIP6 (LIV1 (ZIP6)), SLIT and NTRK-like protein 6 (SLITRK6), trophoblast glycoprotein (TPBG; 5T4), Fyn3, carbonic anhydrase 9, NaPi2B, fibronectin ectodomain B, endothelin receptor ETB, VEGFR2 (CD309), tenascin c, collagen IV and periostin.

The anti-MUC 1 antibody may be further combined with a checkpoint antibody (i.e., an antibody that blocks or activates an immunomodulatory target). Thereby, the inhibitory signal against the immune response may be blocked and/or the activating signal may be triggered. Examples of various targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM) and CD258 (LIGHT) as activation targets, CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA and phosphatidylserine as inhibition targets, and their corresponding ligands such as PDL 1.

In other embodiments, the anti-MUC 1 antibody may be combined with treatment with immunomodulatory compounds such as chemokines, cytokines, growth factors, and vaccines. In this regard, 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-MUC 1 antibody is preferably used for the treatment of primary tumors, recurrent tumors and/or metastases of such tumors, and in particular for the treatment before, during or after surgery, and for the prevention or treatment of metastases. The anti-MUC 1 antibody is particularly useful as an adjunct therapy for treating patients. In certain embodiments, the anti-MUC 1 antibody is used to treat a patient as neoadjuvant therapy or as combined neoadjuvant-adjuvant therapy. In addition, the anti-MUC 1 antibody is used to treat patients as a palliative therapy.

Cancer treatment with anti-MUC 1 antibodies preferably results in inhibition of tumor growth, particularly a reduction in tumor size. In addition, further metastases are prevented and/or their number is reduced by the treatment. The treatment preferably results in an increase in progression-free survival; and/or increased longevity, thereby improving overall survival.

The invention further provides methods of treating, diagnosing, prognosing, detecting and/or monitoring a disease using the antibodies according to the invention. The embodiments and examples of the use of antibodies in medicine are equally applicable to medical methods. In particular, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody according to the invention.

For example, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject having cancer a therapeutically effective amount of an antibody according to the invention. In a specific embodiment, the cancer is characterized by expression of TA-MUC 1. The cancer may be selected from ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, liver cancer, kidney cancer, blood cancer, endometrial cancer, thyroid cancer, leukemia, seminoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, 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 metastases thereof.

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

Method of increasing MUC1 binding affinity

In another aspect, the invention provides a method of increasing the MUC1 binding affinity of an antibody comprising

(i) A heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

The antibody whose MUC1 binding affinity is to be increased is in particular an antibody capable of binding MUC1 as described herein, with the exception that it comprises an asparagine at position 8 of the CDR-H2 sequence.

In certain embodiments, the heavy chain variable region of an antibody whose MUC1 binding affinity is to be increased comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 11. In particular, the heavy chain variable region comprises an amino acid sequence having at least 95%, in particular at least 98%, identity with the amino acid sequence of SEQ ID NO. 11. In these embodiments, the heavy chain variable region still comprises the CDRs having the amino acid sequences of SEQ ID NOs 1, 8 and 3. Thus, any sequence deviation from SEQ ID NO 11 is located in the framework regions, but not in the CDRs. Specifically, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO 11.

In certain embodiments, the light chain variable region of the antibody whose MUC1 binding affinity is to be increased comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 12. In particular, the light chain variable region comprises an amino acid sequence having at least 95%, in particular at least 98%, identity with the amino acid sequence of SEQ ID NO 12. In these embodiments, the light chain variable region still comprises the CDRs having the amino acid sequences of SEQ ID NOs 4, 5 and 6. Thus, any sequence deviation from SEQ ID NO 12 is located in the framework regions, but not in the CDRs. Specifically, the light chain variable region comprises the amino acid sequence of SEQ ID NO 12.

In particular embodiments, the heavy chain variable region of an antibody whose MUC1 binding affinity is to be increased has an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO. 11, wherein 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 90% identical to the amino acid sequence of SEQ ID NO. 12, wherein the CDRs still have the amino acid sequences of SEQ ID NO. 4, 5 and 6. Specifically, the heavy chain variable region has an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO. 11, wherein 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 with at least 95% identity to the amino acid sequence of SEQ ID NO. 12, wherein the CDRs still have the amino acid sequences of SEQ ID NO. 4, 5 and 6.

For example, the antibody whose MUC1 binding affinity is to be increased is an anti-MUC 1 antibody disclosed in WO 2004/065423 a2 or WO 2011/012309 a 1. Specifically, the antibody whose MUC1 binding affinity is to be increased is gatipotuzumab or PankoMab.

The antibody whose MUC1 binding affinity is to be increased is in particular an antibody capable of binding MUC1 as described herein.

In certain embodiments, MUC1 is incorporated as described herein. Increasing the binding affinity of MUC1 or TA-MUC1 specifically indicates an increase of at least 10%, at least 20%, at least 33% or at least 50%. In preferred embodiments, MUC1 binding affinity is increased by at least 50%. MUC1 binding affinities may be determined as described in the examples, in particular using surface plasmon resonance analysis or switchSENSE Technology (DRX2 BIOSENSOR, manufactured by Dynamic BIOSENSORs GmbH), for example as described in examples 4a and b.

In certain embodiments, the step of replacing the amino acid residue at position 8 of CDR-H2 is achieved by introducing a mutation into the nucleic acid encoding the antibody, wherein the mutation is introduced into the codon encoding said amino acid residue. Introduction of the mutation may be accomplished by any method. Several suitable methods are known in the art and the skilled person is able to perform the necessary tasks to introduce mutations. The mutated nucleic acid may then be expressed, for example, in a host cell, resulting in an antibody with increased MUC1 binding affinity. Nucleic acids, host cells, and methods for producing antibodies are described herein and may be used in methods of increasing MUC1 binding affinity.

In particular embodiments, a method of increasing the MUC1 binding affinity of an antibody comprises the steps of:

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

(b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding the amino acid residue at position 8 of CDR-H2 such that the codon encodes any amino acid residue other than asparagine; and

The invention further provides a method of producing an antibody having increased MUC1 binding affinity, the method comprising

(a) Providing a nucleic acid encoding an antibody comprising

(i) A heavy chain variable region comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

With respect to other aspects, particularly with respect to methods of increasing MUC1 binding affinity of an antibody, the embodiments, features and examples described herein are equally applicable to methods of producing an antibody having increased MUC1 binding affinity.

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

For example, processing an antibody with increased MUC1 binding affinity may comprise isolating the antibody from a cell culture. Isolation of the antibody specifically means that the antibody is separated from the rest of the components of the cell culture. Isolation of the antibody from the cell culture medium can be carried out, for example, by chromatography. Suitable methods and means for isolating antibodies are known in the art and can be readily applied by those skilled in the art.

The obtained antibody may optionally be subjected to further processing steps, e.g., modification steps, such as chemical or enzymatic coupling of other reagents to the antibody, and/or formulation steps, to produce an antibody of desired quality and composition. Such further processing steps and methods are generally known in the art.

In other embodiments, step (d) further comprises the step of providing a pharmaceutical formulation comprising the antibody. Providing a pharmaceutical formulation comprising an antibody or formulating an antibody into a pharmaceutical composition particularly comprises exchanging the buffer solution or buffer solution components of the composition comprising the antibody. In addition, this step may include lyophilization of the antibody. In particular, the antibody is transferred to a composition comprising only pharmaceutically acceptable ingredients.

Detailed description of the preferred embodiments

In the following, specific embodiments of the present invention are described.

Embodiment 1. an antibody capable of binding to MUC1 comprising

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

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

Embodiment 4. the antibody according to 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, comprising

(i) Heavy chain variable region of

(a) Having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO 9, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 2, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO 12, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO. 4, CDR-L2 having the amino acid sequence of SEQ ID NO. 5, and CDR-L3 having the amino acid sequence of SEQ ID NO. 6.

Embodiment 7 an antibody capable of binding to MUC1 comprising

(i) Heavy chain variable region of

(a) Having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO 9, and

(ii) light chain variable region of

(a) Has an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO 12, and

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

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

Embodiment 10 an antibody capable of binding to MUC1, comprising

(i) Heavy chain variable region of

(a) Having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO 10, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 7 and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO 12, and

Embodiment 11 an antibody capable of binding MUC1, comprising

(i) Heavy chain variable region of

(a) Having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO 10, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 7 and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Has an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO 12, and

Embodiment 12 an antibody capable of binding MUC1, comprising

(i) A heavy chain variable region having the amino acid sequence of SEQ ID NO 9, and

(ii) the light chain variable region having the amino acid sequence of SEQ ID NO 12.

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

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

Embodiment 15 an antibody capable of binding MUC1, comprising

(i) A heavy chain variable region having the amino acid sequence of SEQ ID NO 10, and

(ii) the light chain variable region having the amino acid sequence of SEQ ID NO 12.

Embodiment 16 an antibody capable of binding MUC1, comprising

(i) Heavy chain variable region of

(a) Has an amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence represented by amino acid numbers 20 to 136 of SEQ ID NO 20, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 2 or 7, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Has an amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO 21, and

Embodiment 17 an antibody capable of binding MUC1, comprising

(i) A heavy chain variable region having an amino acid sequence represented by amino acid numbers 20 to 136 of SEQ ID NO 20 or 23, and

(ii) a light chain variable region having an amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 21.

Embodiment 18 an antibody capable of binding MUC1, comprising

(i) Heavy chain of

(a) Having an amino acid sequence which has at least 90% or at least 95% identity with the amino acid sequence of SEQ ID NO. 15, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 2 or 7, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Has an amino acid sequence having at least 90% or 95% identity to the amino acid sequence of SEQ ID NO 16, and

Embodiment 19 an antibody capable of binding MUC1, comprising

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

(ii) the light chain variable region having the amino acid sequence of SEQ ID NO 16.

Embodiment 20 an antibody capable of binding MUC1, comprising

(i) Heavy chain variable region of

(a) Has an amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence represented by amino acid numbers 20 to 460 of SEQ ID NO 20, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 2 or 7, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Has an amino acid sequence having at least 90% or 95% identity to the amino acid sequence represented by amino acid numbers 21 to 239 of SEQ ID NO 21, and

Embodiment 21 an antibody capable of binding MUC1, comprising

(i) A heavy chain having an amino acid sequence represented by amino acid numbers 20 to 460 of SEQ ID NO 20 or 23, and

(ii) a light chain having an amino acid sequence represented by amino acid numbers 21 to 239 of SEQ ID NO 21.

Embodiment 22 the antibody according to 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 the antibody according to any one of embodiments 1-21, wherein the antibody comprises two heavy chains, each heavy chain comprising a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

Embodiment 24. the antibody according to embodiment 22 or 23, wherein the antibody is an IgG-type antibody, in particular an IgG1, IgG2 or IgG 4-type antibody.

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

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

Embodiment 274 the antibody according to embodiment 25 or 26, wherein the light chain is a kappa-type light chain.

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

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

Embodiment 30 the antibody according to embodiment 29, wherein the antibody has a glycosylation pattern with one or more of the following characteristics:

(i) (ii) the relative amount of glycans carrying bisecting GlcNAc residues is at least 0.5% of the total amount of glycans attached to the glycosylation sites of the antibody in the composition;

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

(iii) the relative amount of glycans carrying core fucose residues is at least 60% of the total amount of glycans attached to the glycosylation sites of the antibody in the composition.

Embodiment 31 the antibody according to embodiment 29, wherein the antibody has a glycosylation pattern with one or more of the following characteristics:

(i) (ii) the relative amount of glycans carrying bisecting GlcNAc residues is at least 0.5% of the total amount of glycans attached to the glycosylation sites of the antibody in the composition;

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

(iii) the relative amount of glycans carrying core fucose residues is 40% or less of the total amount of glycans attached to glycosylation sites of the antibody in the composition.

Embodiment 32. the antibody according to any one of embodiments 1 to 31, comprising a further agent conjugated thereto.

Embodiment 33 the antibody according to embodiment 32, wherein the additional agent is a chemotherapeutic agent conjugated to the antibody.

Embodiment 34 the antibody according to embodiment 33, wherein the chemotherapeutic agent is selected from the group consisting of maytansine, a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binder.

Embodiment 35 the antibody according to embodiment 33, wherein the chemotherapeutic agent is selected from the group consisting of maytansinol, methadol,N ^2'deacetylation-N ^2'- (3-mercapto-1-oxopropyl) -maytansine (DM1),N ^2'Deacetylation-N ^2'- (4-mercapto-1-oxopentyl) -maytansine (DM3) andN ^2'deacetylation-N ^2'- (4-methyl-4-mercapto-1-oxopentyl) -maytansine (DM 4).

Embodiment 36 the antibody according to embodiment 33, wherein the chemotherapeutic agent is selected from monomethyl auristatin f (mmaf), monomethyl auristatin e (mmae), and auristatin T.

Embodiment 37 the antibody according to embodiment 33, wherein the chemotherapeutic agent is selected from Pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer), dacomicin-hydroxybenzamide-azaindole (DUBA), seco-dacomicin-hydroxybenzamide-azaindole (seco-DUBA), and doxorubicin.

Embodiment 38 the antibody according to embodiment 33, wherein the chemotherapeutic agent is selected from the group consisting of indolinobenzodiazepine (indolinobenzodiazepine) and oxazolidinobenzodiazepine (oxazolidinobenzodiazepine).

Embodiment 39 the antibody according to embodiment 33, wherein the chemotherapeutic agent is calicheamicin.

Embodiment 40 the antibody according to embodiment 32, wherein the further agent is a polypeptide or protein fused to the polypeptide chain of the antibody.

Embodiment 41. the antibody according to embodiment 40, wherein said antibody comprises two antibody heavy chains and two antibody light chains, and is fused as a polypeptide or protein to each C-terminus of said antibody heavy chains or to each C-terminus of said antibody light chains.

Embodiment 42. the antibody according to embodiment 40 or 41, wherein the further agent is selected from the group consisting of cytokines, chemokines, further antibodies, antigen binding fragments, enzymes and binding domains.

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

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

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

Embodiment 46. an expression cassette or vector comprising a nucleic acid according to embodiment 75 and a promoter operably linked to the nucleic acid.

Embodiment 47 a host cell comprising a nucleic acid according to embodiment 45 or an expression cassette or vector according to embodiment 46.

Embodiment 48. a pharmaceutical composition comprising an antibody or conjugate according to any one of embodiments 1-44 and one or more other components selected from solvents, diluents and excipients.

Embodiment 49. the antibody according to any one of embodiments 1 to 44 or the pharmaceutical composition according to embodiment 48 for use in medicine.

Embodiment 50. an antibody according to any one of embodiments 1 to 44 or a pharmaceutical composition according to embodiment 48 for 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 decreased immune activity such as immunodeficiency.

Embodiment 51. the antibody or pharmaceutical composition according to embodiment 50 for use in the treatment of cancer, in particular a TA-MUC 1-expressing cancer, 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, blood cancer, endometrial cancer, thyroid cancer, leukemia, seminoma, melanoma, cancer, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal 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 metastases thereof.

Embodiment 52. the antibody or pharmaceutical composition according to embodiment 50 for use in the treatment of an infection, wherein said infection is selected from the group consisting of a bacterial infection, a viral infection, a fungal infection and a parasitic infection.

Embodiment 53 the antibody or pharmaceutical composition according to embodiment 50 for use in the treatment of an autoimmune disease, wherein said autoimmune disease is selected from the group consisting of celiac disease, type I diabetes, graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus.

Embodiment 54 a method of increasing the MUC1 binding affinity of an antibody comprising

(i) Heavy chain variable region of

(a) Having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO 11, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO 12, and

Embodiment 55 a method of increasing the MUC1 binding affinity of an antibody comprising

(i) Heavy chain variable region of

(a) Having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO 11, and

(b) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(a) Has an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO 12, and

Embodiment 56. the method according to embodiment 54 or 55, wherein the substitution of the amino acid residue at position 8 of CDR-H2 is effected by introducing a mutation into the nucleic acid encoding the antibody, wherein the mutation is introduced into the codon encoding said amino acid residue.

Embodiment 57. the method according to any one of embodiments 54-56, comprising the steps of:

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

Embodiment 58 a method of producing an antibody with increased MUC1 binding affinity, the method comprising

(a) Providing a nucleic acid encoding an antibody comprising

(i) Heavy chain variable region of

(i1) Having an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO 11, and

(i2) comprising the following Complementarity Determining Regions (CDRs): CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3, and

(ii) light chain variable region of

(ii1) an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12, and

(ii2) comprising the following Complementarity Determining Regions (CDRs): CDR-L1 having the amino acid sequence of SEQ ID NO. 4, CDR-L2 having the amino acid sequence of SEQ ID NO. 5, and CDR-L3 having the amino acid sequence of SEQ ID NO. 6;

Embodiment 59 a method of producing an antibody with increased MUC1 binding affinity, the method comprising

(a) Providing a nucleic acid encoding an antibody comprising

(i) Heavy chain variable region of

(i1) Having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO 11, and

(ii) light chain variable region of

(ii1) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 12, and

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

Embodiment 61 the method according to embodiment 60, wherein the antibody is an IgG-type antibody, in particular an IgG1, IgG2 or IgG 4-type antibody.

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

Embodiment 63. the method according to embodiment 62, wherein the light chain is a kappa-type light chain.

Embodiment 64 the method according to any one of embodiments 54 to 63, wherein said antibody with increased MUC1 binding affinity is an antibody as defined in any one of embodiments 1 to 44.

Embodiment 65. a method of treating cancer in a subject in need thereof, the method comprising administering to a subject having cancer, in particular a TA-MUC 1-expressing cancer, a therapeutically effective amount of an antibody according to any one of embodiments 1-44 or a composition according to embodiment 48.

Embodiment 66. the method for treating cancer according to embodiment 65, wherein the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, blood cancer, endometrial cancer, thyroid cancer, leukemia, seminoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, 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 metastases thereof.

68页详细技术资料下载

anti-MUC 1 antibodies

相关技术

网友询问留言