Humanized antibodies to lewis y

Abstract

The present invention relates to humanized anti-Lewis Y antibodies that specifically bind Lewis Y and do not show any cross-reactivity. In particular, the humanized anti-Lewis Y antibodies do not bind to Lewis b or any other blood group carbohydrate antigen. In particular, the present invention relates to humanized anti-Lewis Y antibodies for use in the treatment of cancer.

Description

Invention Field

[0001] This invention relates to the field of antibodies. In particular, it provides a humanized anti-Lewis Y antibody that exhibits specific target specificity and affinity for a glycan antigen. In certain embodiments, this invention relates to a humanized anti-Lewis Y antibody for therapeutic and diagnostic purposes. Background of the Invention

[0003] Today, antibodies are widely used in the medical and research fields. In medicine, they have applications in many different areas. For example, antibodies are used as therapeutic agents to treat and prevent a variety of diseases, such as cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infections. In these therapies, antibodies can possess therapeutic activity themselves, for example, by blocking receptors or messenger molecules, thereby inhibiting their disease-related functions, or by recruiting and activating components of a patient's immune system.

[0004] Specific antibodies are produced by injecting an antigen into a mammal (such as a mouse, rat, rabbit, goat, sheep, or horse). Blood isolated from these animals contains polyclonal antibodies against the antigen in the serum. To obtain antibodies specific to a single epitope of the antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line, thereby generating hybridoma cells. Individual hybridoma cells are then isolated by dilution clones to produce cell clones that all produce the same monoclonal antibody.

[0005] However, in therapeutic applications, these monoclonal antibodies present a problem: they are derived from animal organisms, and their amino acid sequences differ from those of human antibodies. Therefore, the human immune system recognizes these animal antibodies as foreign and rapidly clears them from circulation. Furthermore, this can potentially trigger a systemic inflammatory response. The solution to this problem is to replace certain constant regions of the monoclonal antibody with corresponding portions of the human antibody. Replacing only the heavy and light chain constant regions yields chimeric antibodies, while additionally replacing the framework regions of the heavy and light chain variable regions produces so-called humanized antibodies.

[0006] In research, purified antibodies are used in many applications. They are most commonly used to identify and localize biomolecules, such as specific proteins. Biomolecules can be detected after isolation, for example, to determine their presence, concentration, integrity, or size. On the other hand, they can be detected in cell or tissue samples, for example, to determine their presence or location. Furthermore, antibodies are used in the isolation process of specific biological substances, particularly proteins, where antibodies specifically separate the biological substance of interest from the sample containing it.

[0007] In all these applications, tight binding and specific recognition of the antigen are crucial for the antibodies used. This results in higher activity and less cross-reactivity, particularly fewer adverse side effects in therapeutic applications. However, during the humanization of monoclonal antibodies, the affinity and specificity of the engineered antibodies are often reduced.

[0008] A fascinating and important group of antibodies targets carbohydrate moieties. In particular, tissue blood group carbohydrate chains are interesting targets for antibodies because they are often tumor-specific or tumor-associated antigens. For example, the carbohydrate structure Lewis Y [Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-] (also known as LeY) expressed by several different epithelial cancer cells, including breast cancer, bladder cancer, colon cancer, gastric cancer, pancreatic cancer, prostate cancer, ovarian cancer, and small cell lung cancer, is more abundant than any other cell surface protein antigen. A high percentage of tumor cells in their respective tumors are LeY-positive, even including cancer stem cells. Therefore, these carbohydrate antigens are potential targets for tumor imaging and active or passive immunotherapy.

[0009] Antibodies targeting these carbohydrate structures are often associated with several problems. In particular, undesirable side effects can arise due to the presence of identical or closely related carbohydrate structures in normal tissues, such as H2 expression on erythrocytes or Lewis X on human myeloid cells (e.g., mature granulocytes). For example, some antibodies targeting Lewis Y have failed in clinical trials because they are cross-reactive to Lewis X and have shown unfavorable efficacy and / or safety. Furthermore, cross-reactivity with other glycan epitopes is a common problem. For instance, most existing antibodies recognizing Lewis Y exhibit cross-reactivity with other carbohydrate structures such as Lewis b[Fucα1-4(Fucα1-2Galβ1-3)GlcNAcβ-], which reduces their therapeutic potential. Additionally, carbohydrate antigens typically elicit IgM-type immune responses, which are considered unsuitable antibody forms for treatment. Using recombinant antibody technology, class switching from IgM to IgG can be achieved. However, a considerable loss of functional affinity occurs due to the low intrinsic affinity of carbohydrate-binding antibodies.

[0010] Known specific antibodies against Lewis Y are the monoclonal antibodies A70-A / A9 and A70-C / C8, as well as the combined A / A9-C / C8 antibody obtained through chain rearrangement (this antibody contains the heavy chain of A70-A / A9 and the light chain of A70-C / C8). However, these are chimeric antibodies with mouse variable regions, which may lead to the problems associated with the aforementioned non-human antibodies. Therefore, humanization of these antibodies would be beneficial. Unfortunately, humanized antibodies generally exhibit lower affinity and specificity for their target antigens than their corresponding non-human or chimeric antibodies. This is because the overall three-dimensional structure of the variable region, particularly the conformation and orientation of the complementarity-determining region (CDR), can be altered by substitution of the framework region. This is particularly problematic for antibodies against carbohydrate antigens, which typically have low antigen affinity and specificity. For example, antibody A70-A / A9 exhibits cross-reactivity with Lewis b.

[0011] Therefore, there is a need in the art to provide humanized anti-Lewis Y antibodies with specific high antigen binding affinity and antigen specificity. Invention Overview

[0013] The inventors have discovered that humanized anti-Lewis Y antibodies possess antigen-binding affinity comparable to that of the parental chimeric antibodies from which they are derived. Furthermore, these humanized antibodies exhibit enhanced antigen specificity because cross-reactivity with the Lewis b of the parental chimeric antibodies is prevented in their humanized form.

[0014] Therefore, in a first aspect, the present invention relates to a humanized antibody capable of binding to Lewis Y and comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:10 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:22 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:22.

[0015] In a second aspect, the present invention provides a nucleic acid encoding an antibody according to the present invention. Furthermore, in a third aspect, an expression cassette or vector comprising a nucleic acid according to the present invention and a promoter operatively linked to said nucleic acid is provided; and in a fourth aspect, a host cell comprising the nucleic acid or expression cassette or vector according to the present invention is provided.

[0016] In a fifth aspect, the present invention provides a conjugate comprising the antibody of the present invention conjugated with another reagent.

[0017] In a sixth aspect, the present invention relates to compositions comprising the antibodies of the present invention, the nucleic acids of the present invention, the expression cassettes or vectors of the present invention, the host cells of the present invention, or the conjugates of the present invention.

[0018] According to a seventh aspect, the present invention provides antibodies, nucleic acids, expression cassettes or vectors, host cells, compositions or conjugates of the present invention for medical use, particularly for the treatment of cancer.

[0019] Other objects, features, advantages, and aspects of the invention will become apparent to those skilled in the art from the following description and appended claims. However, it should be understood that the following description, appended claims, and specific examples (which indicate preferred embodiments of the present application) are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will be readily apparent to those skilled in the art upon reading the following.

[0020] definition

[0021] As used herein, the following expressions are generally preferred to have the meanings described below, unless the context in which they are used indicates otherwise.

[0022] The term “comprising” as used herein, in addition to its literal meaning, also includes and specifically refers to the expressions “substantially composed of” and “consisting of”. Thus, the expression “comprising” means an embodiment in which the subject matter specifically lists the elements “comprising” does not contain other elements, and that a subject matter specifically lists the elements “including” may and / or does contain other elements. Similarly, the expression “having” should be understood to mean “comprising”, and also includes and specifically refers to the expressions “substantially composed of” and “consisting of”. Where possible, the term “substantially composed of” specifically refers to an embodiment in which, in addition to the specifically listed elements in which the subject matter substantially constitutes, the subject matter includes 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less of other elements.

[0023] The term "antibody" specifically refers to a protein containing at least two heavy chains and two light chains (linked by disulfide bonds). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain constant region includes three or (in the case of IgM or IgE antibodies) four heavy chain constant domains (CH1, CH2, CH3, and CH4), wherein the first constant domain CH1 is adjacent to the variable region and can be connected to the second constant domain CH2 via a hinge region. The light chain constant region consists of only one constant domain. The variable region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs), wherein each variable region includes three CDRs and four FRs. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen. The heavy chain constant region can be of any type, such as γ-, δ-, α-, μ-, or ε-type heavy chains. Preferably, the heavy chain of the antibody is a γ chain. Furthermore, the light chain constant region can also be of any type, such as a κ or λ type light chain. Preferably, the light chain of the antibody is a κ chain. The terms "γ-(δ-, α-, μ-, or ε-) type heavy chain" and "κ-(λ-) type light chain" refer to the antibody heavy chain or antibody light chain, respectively, having constant region amino acids derived from the amino acid sequence of the constant region of a naturally occurring heavy chain or light chain, particularly the amino acid sequence of the constant region of a human heavy chain or light chain. In particular, the amino acid sequence of the constant domain of the γ-type (especially γ1-type) heavy chain is at least 95%, particularly at least 98%, identical to the sequence of the constant domain of the human γ (especially one of the allotypes of human γ1) antibody heavy chain. Furthermore, the amino acid sequence of the constant domain of the κ-type light chain is, particularly at least 95%, particularly at least 98%, identical to the amino acid sequence of the constant domain of one of the allotypes of the human κ antibody light chain. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be, for example, humanized, human, or chimeric antibodies.

[0024] The antigen-binding portion of an antibody typically refers to the full-length or one or more fragments of the antibody that retain the ability to specifically bind to antigens. It has been shown that the antigen-binding function of an antibody can be achieved through fragments of the full-length antibody. Examples of antibody-binding fragments include Fab fragments; monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments containing two Fab fragments, each binding the same antigen, linked by disulfide bonds in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; and dAb fragments consisting of a VH domain.

[0025] The term "Fab moiety" of an antibody specifically refers to a portion of the antibody comprising the variable regions (VH and VL) of the heavy and light chains, as well as the first domains (CH1 and CL) of the constant regions of the heavy and light chains. In cases where the antibody does not contain all of these regions, the term "Fab moiety" refers only to the VH, VL, CH1, and CL regions present in the antibody. Preferably, "Fab moiety" refers to an antibody portion corresponding to a fragment obtained by digesting a natural antibody with papain, which contains the antibody's antigen-binding activity. Specifically, the Fab moiety of an antibody contains its antigen-binding site or antigen-binding capacity. Preferably, the Fab moiety contains at least the VH, VL, CH1, and CL regions of the antibody. H district.

[0026] The term "Fc moiety" of an antibody specifically refers to the antibody moiety containing the heavy chain constant regions 2, 3, and -4 (CH2, CH3, and CH4). In particular, the Fc moiety includes two of these regions. In cases where the antibody does not contain all of these regions, the term "Fc moiety" refers only to the regions CH2, CH3, and CH4 present in the antibody. Preferably, the Fc moiety contains at least the CH2 region of the antibody. Preferably, "Fc moiety" refers to an antibody moiety corresponding to a fragment obtained by digesting a natural antibody with papain, which does not contain the antigen-binding activity of the antibody. In particular, the Fc moiety of an antibody is capable of binding to an Fc receptor and therefore includes, for example, an Fc receptor binding site or Fc receptor binding capacity.

[0027] According to the present invention, the term "chimeric antibody" specifically refers to an antibody in which the constant region is derived from a human antibody or a human antibody common sequence, and in which at least one and preferably two variable regions are derived from a non-human antibody, such as a rodent antibody, for example a mouse antibody.

[0028] According to the present invention, the term "humanized antibody" specifically refers to a non-human antibody comprising a human constant region and a variable region, the amino acid sequence of which is modified to reduce the immunogenicity of the antibody when administered to a human body. An exemplary method for constructing a humanized antibody is CDR transplantation, in which the CDR or specificity-determining residues (SDRs) of the non-human antibody bind to a human frame region. Optionally, some residues of the human frame region may be reverse-mutated to residues of the parental non-human antibody, for example, to increase or restore antigen-binding affinity. Other humanization methods include, for example, resurfacing, hyperhumanization, and human string content optimization. In the resurfacing method, only those residues of the non-human frame region located on the antibody surface are replaced by residues in the corresponding human antibody sequence present at said location. Hyperhumanization essentially corresponds to CDR transplantation. However, while in CDR transplantation, the human frame region is typically selected based on its homology with the non-human frame region, in hyperhumanization, the selection of the human frame region is based on the similarity of the CDR. In human string content optimization, the differences between non-human antibody sequences and human germline sequences are scored, and then the antibody is mutated to minimize the score. Alternatively, humanized antibodies can also be obtained empirically, where multiple antibody humanization candidates are generated using human framework regions or a large library of human antibodies, and then the most promising candidates are identified through screening methods. Similarly, using the above-described reasonable methods, several humanized antibody candidates can be generated and then screened, for example, for their antigen binding.

[0029] As used herein, the term "human antibody" is intended to include antibodies with variable regions, where both the frame region and the CDR region are derived from human sequences.

[0030] As used herein, the term "antibody" may refer in some embodiments to a population of the same type of antibody. Specifically, all antibodies in the antibody population exhibit the characteristics used to define an antibody. In some embodiments, all antibodies in the antibody population have the same amino acid sequence. References to a particular type of antibody, such as anti-Lewis Y antibody, specifically refer to a population of such antibodies.

[0031] As used herein, the term "antibody" also includes fragments and derivatives of said antibody. A "fragment or derivative" of an antibody is particularly a protein or glycoprotein derived from said antibody and capable of binding to the same antigen, particularly to the same epitope of the antibody. Therefore, as used herein, a fragment or derivative of an antibody 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 shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody or a derivative thereof. Examples of antibody fragments include (i) Fab fragments, monovalent fragments consisting of variable regions and a first constant region of each heavy and light chain; (ii) F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in their hinge regions; (iii) Fd fragments consisting of variable regions of the heavy chain and a first constant domain CH1; (iv) Fv fragments consisting of variable regions of the heavy and light chains of a single antibody arm; (v) Fv fragments and scFv fragments consisting of a single polypeptide chain; (vi) (Fv)2 fragments consisting of two Fv fragments covalently linked together; (vii) a heavy chain variable domain; and (viii) multiply fragments consisting of covalently linked heavy and light chain variable regions in a manner such that binding of the heavy and light chain variable regions can only occur intermolecularly and not intramolecularly. Antibody derivatives particularly include antibodies that bind to the same antigen as the parent antibody, but whose amino acid sequence differs from that of the parent antibody from which the antibody is derived. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.

[0032] If a target amino acid sequence has at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the corresponding portion of a reference amino acid sequence over its entire length, then the target amino acid sequence is "derived from" or "corresponds to" the reference amino acid sequence. A "corresponding portion" means, for example, that frame region 1 of the heavy chain variable region (FRH1) of the target antibody corresponds to frame region 1 of the heavy chain variable region of the reference antibody. In a particular embodiment, the target amino acid sequence "derived from" or "corresponds to" the reference amino acid sequence is 100% homologous, or particularly 100% identical, to the corresponding portion of the reference amino acid sequence over its entire length. The "homology" or "identity" of the amino acid sequence or nucleotide sequence is preferably determined according to the invention over the entire length of the reference sequence or over the entire length of the corresponding portion of the reference sequence corresponding to the sequence defining homology or identity. Antibodies derived from parent antibodies defined by one or more amino acid sequences (e.g., a specific CDR sequence or a specific variable region sequence), particularly antibodies having 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% homology or identity (e.g., CDR sequence or variable region sequence) with the corresponding amino acid sequence of the parent antibody, especially identical amino acid sequences. In some embodiments, antibodies derived from parent antibodies (i.e., their derivatives) contain the same CDR sequence as the parent antibody but differ in the remaining sequence of the variable region.

[0033] As used in this article, the term "antibody" also refers to multivalent and multispecific antibodies, i.e., antibody constructs having two or more binding sites, each binding to the same epitope, and antibody constructs having one or more binding sites binding to a first epitope and one or more binding sites binding to a second epitope, and optionally, even further binding sites binding to further epitopes.

[0034] "Specific binding" preferably refers to a stronger binding of a reagent (such as an antibody) to a specific target (such as an epitope) than to another target. This occurs when the reagent binds at a lower dissociation constant (K) than the second target. d If the dissociation constant of the reagent binds to the first target, then the binding of the reagent to the first target is stronger than that to the second target. Preferably, the dissociation constant of the target specifically bound by the reagent is 100 times, 200 times, 500 times, or 1000 times lower than the dissociation constant of the target not specifically bound by the reagent. Furthermore, the term "specific binding" specifically refers to the binding affinity between the binding partners, where the affinity constant K is... a For at least 10 5 M -1 Preferably at least 10 6 M -1More preferably at least 10 7 M -1 For example, at least 10 8 M -1 Antibodies specific to a particular antigen, especially those capable of using K... a For at least 10 5 M -1 Preferably at least 10 6 M -1 More preferably at least 10 7 M -1 Antibodies that bind to the antigen with affinity. For example, the term "anti-Lewis Y antibody" refers to an antibody that specifically binds to Lewis Y, and preferably one that can bind to K. a For at least 10 5 M -1 Preferably at least 10 6 M -1 More preferably at least 10 7 M -1 The affinity of Lewis Y.

[0035] As used herein, the term "A70-A / A9" specifically refers to human / mouse chimeric antibodies having the heavy and light chain variable region amino acid sequences of SEQ ID NO:30 and 31, respectively.

[0036] According to the invention, the term "Lewis Y" or "LeY" specifically refers to the carbohydrate structure (or oligosaccharide) Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-, which can be linked to a supporting structure or carrier molecule, such as a peptide, protein, lipid, or carbohydrate structure. In the above structure, Fuc represents a fucose residue, Gal represents a galactose residue, and GlcNAc represents an N-acetylglucosamine residue. "α1-2", "β1-4", and "α1-3" represent the connection between two adjacent monosaccharide residues, particularly the respective connection between carbon atom C1 of the left monosaccharide and carbon atom C2, C4, or C3 of the right monosaccharide, wherein the connection at carbon atom C1...

[0037] The term "GlcNAcβ-" indicates that the GlcNAc residues at the reducing end of the Lewis Y oligosaccharide are linked to the supporting structure in a β-configuration. Figure 1 It also shows a schematic diagram of the structure of Lewis Y and related blood group antigens.

[0038] Lewis Y is a carbohydrate antigen in the human blood group system. It is expressed on many tumors and can be used as a tumor-associated or tumor-specific antigen for targeted cancer therapy.

[0039] The term "sialic acid" specifically refers to any N- or O-substituted derivative of neuraminic acid. It can refer to 5-N-acetylneuraminic acid and 5-N-ethanolylneuraminic acid, but preferably only to 5-N-acetylneuraminic acid.

[0040] The terms “glycan,” “glycan structure,” “carbohydrate,” “glycan chain,” and “carbohydrate structure” are generally used as synonyms in this article.

[0041] In a "conjugate," two or more compounds are linked together. In some embodiments, at least some properties of each compound are retained in the conjugate. The linking can be achieved by covalent or non-covalent bonds. Preferably, the compounds of the conjugate are linked by covalent bonds. The different compounds of the conjugate can be directly linked to each other by one or more covalent bonds between the atoms of the compounds. Alternatively, the compounds can be linked to each other by chemical parts, such as linking molecules, which are covalently attached to the atoms of the compounds. If the conjugate consists of two or more compounds, these compounds can be linked, for example, in a chain conformation, with one compound linked to the next compound, or several compounds can each be linked to a central compound.

[0042] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, ribonucleic acid, and deoxyribonucleic acid. It can include naturally occurring and synthetic nucleotides and can be modified naturally or synthetically, for example by methylation, 5'- and / or 3'-capping.

[0043] The term "expression cassette" specifically refers to a nucleic acid construct capable of enabling and regulating the expression of a coding nucleic acid sequence introduced therein. An expression cassette may include a promoter, a ribosome binding site, an enhancer, and other control elements regulating gene transcription or mRNA translation. The exact structure of an expression cassette can vary depending on species or cell type function, but typically includes 5'-untranscribed and 5'- and 3'-untranslated sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, cap sequences, CAAT sequences, etc. More specifically, the 5'-untranscribed expression control sequence contains a promoter region containing a promoter sequence for operatively linking the nucleic acid to the transcriptional control. Expression cassettes may also include enhancer sequences or upstream activator sequences.

[0044] According to the present invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of the nucleic acid sequence to be expressed, and controls the expression of that sequence by providing a recognition and binding site for RNA polymerase. A "promoter" may include further recognition and binding sites for other factors involved in the regulation of gene transcription. A promoter can control the transcription of prokaryotic or eukaryotic genes. Furthermore, a promoter may be "inducible," i.e., initiating transcription in response to an inducer, or it may be "constitutive" if transcription is not controlled by an inducer. Genes controlled by inducible promoters are not expressed or are expressed only at low levels in the absence of an inducer. In the presence of an inducer, the gene is turned on or the transcriptional level increases. Generally, this is mediated by the binding of specific transcription factors.

[0045] The term "vector" is used herein in its most general sense and includes any intermediate vector for nucleic acids, such as those capable of introducing said nucleic acids into prokaryotic and / or eukaryotic cells and, where appropriate, integrating them into the genome. Such vectors preferably replicate and / or are expressed in cells. Vectors include plasmids, bacteriophages, bacteriophage genomes, or viral genomes. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, typically a circular DNA double helix, which can replicate independently of chromosomal DNA.

[0046] According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the present invention, the term "host cell" includes prokaryotic (e.g., *Escherichia coli*) or eukaryotic cells (e.g., mammalian cells, particularly human or hamster cells, yeast cells, and insect cells). Mammalian cells, such as cells derived 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. Nucleic acids may be present in the host cell in a single copy or two or more copies, and in one embodiment, are expressed in the host cell.

[0047] According to the present invention, the term "patient" refers to a human, a non-human primate, or another animal, particularly a mammal such as a cow, horse, pig, sheep, goat, dog, cat, or rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human.

[0048] The term "cancer" according to the present invention specifically includes leukemia, seminoma, melanoma, teratoma, lymphoma, 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, bladder cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer and their metastases. The term "cancer" according to the present invention also includes cancer metastases. The term "cancer" also refers to and / or includes cancer stem cells, particularly cancer stem cells of the specific types of cancer described above.

[0049] A tumor is a group of cells or tissues that form due to disordered cell proliferation. Tumors may present as a partial or complete lack of structural organization and functional coordination (compared to normal tissue), usually forming a distinct mass of tissue, and can be benign or malignant.

[0050] Metastasis refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process, typically involving cancer cells separating from the primary tumor, entering the circulation, and residing and growing in normal tissues elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells are usually similar to those in the original tumor. For example, this means that if breast cancer metastasizes to the lungs, the secondary tumor is composed of abnormal breast cells, not abnormal lung cells. The tumor in the lungs is called metastatic breast cancer, not lung cancer.

[0051] The term "pharmaceutical composition" specifically refers to a composition suitable for administration to humans or animals, i.e., a composition containing pharmaceutically acceptable components. Preferably, the pharmaceutical composition comprises an active compound or a salt or prodrug thereof, as well as a carrier, diluent, or pharmaceutical excipient, such as a buffer, preservative, and tension modifier. Invention Details

[0053] This invention is based on the development of a humanized anti-Lewis Y antibody with antigen-binding affinity similar to that of the corresponding chimeric antibody. The inventors have further demonstrated that the humanized antibody unexpectedly exhibits improved antigen specificity. In addition to its primary specificity for Lewis Y, the parental chimeric antibody also shows significant binding to Lewis b. This cross-reactivity is common for antibodies binding to carbohydrate antigens, as these antigens are relatively small and can be very similar. For example, the only difference between Lewis Y and Lewis b is the switching of the connection point between the fucose arm and the fuco-galactose arm at the central residue of GlcNAc (see...). Figure 1However, the humanized anti-Lewis Y antibody provided by this invention surprisingly does not show any cross-reactivity with other carbohydrate antigens, especially with Lewis b.

[0054] In view of these findings, the present invention provides a humanized antibody capable of binding to Lewis Y and comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:10 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:22 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:22.

[0055] Furthermore, the humanized antibody can exhibit antigen-binding properties similar to a reference antibody, which comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:30 and a light chain variable region having the amino acid sequence of SEQ ID NO:31. Preferably, the reference antibody is a human / mouse chimeric antibody A70-A / A9. Specifically, the humanized antibody according to the invention can specifically bind to the same antigen as the reference antibody, and can preferably bind to the antigen with comparable affinity. That is, the humanized antibody preferably binds to the antigen with an affinity up to 1000 times higher, more preferably up to 200 times higher, up to 100 times higher, up to 20 times higher, or up to 10 times higher than that of the reference antibody. Most preferably, the dissociation constant is substantially the same as that of the reference antibody, particularly not more than 2 times higher. Furthermore, the humanized antibody preferably exhibits cross-specificity with the reference antibody, which comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:30 and a light chain variable region having the amino acid sequence of SEQ ID NO:31. In particular, if present at a sufficiently high concentration, the humanized antibody can block the binding of the reference antibody to Lewis Y. It is possible that the binding of the reference antibody to Lewis Y is blocked when the humanized antibody according to the invention has already bound to Lewis Y.

[0056] In some embodiments, the humanized antibody specifically binds to Lewis Y. Specifically, the humanized antibody does not exhibit significant binding affinity for Lewis b and / or binds to Lewis b nonspecifically. In some embodiments, the dissociation constant of the humanized antibody binding to Lewis Y is at least 10-fold lower than that of the humanized antibody binding to Lewis b. Specifically, the dissociation constant of the humanized antibody binding to Lewis Y is at least 20-fold, at least 50-fold, or at least 100-fold lower than that of the humanized antibody binding to Lewis b.

[0057] In some embodiments, the heavy chain variable region contains at least 93% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:10. In particular, the heavy chain variable region contains at least 95%, especially at least 98%, of the same amino acid sequence as the amino acid sequence of SEQ ID NO:10.

[0058] In a particular embodiment, the heavy chain variable region of the humanized antibody comprises a complementarity-determining region CDR-H1 having the amino acid sequence of SEQ ID NO:12 or 13, a CDR-H2 having the amino acid sequence of SEQ ID NO:14 or 15, and a CDR-H3 having the amino acid sequence of SEQ ID NO:16. Specifically, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10, and additionally comprises three specific CDRs having the amino acid sequences of SEQ ID NO:12, 14, and 16 or SEQ ID NO:13, 15, and 16. Therefore, any sequence deviations from SEQ ID NO:10 are located in the frame region, not in the CDRs.

[0059] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:11 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:11. Specifically, the heavy chain variable region comprises an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO:11. More specifically, the heavy chain variable region comprises an amino acid sequence that is at least 95%, particularly at least 98%, identical to the amino acid sequence of SEQ ID NO:11. In a particular embodiment, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:11, and additionally has three specific CDRs having the amino acid sequences of SEQ ID NO:12, 14, and 16.

[0060] Specifically, the humanized antibody may comprise a heavy chain variable region having an amino acid sequence according to any one of SEQ ID NO:1 to 9. In particular, the heavy chain variable region has an amino acid sequence according to any one of SEQ ID NO:1 to 6, especially SEQ ID NO:1 or 4, preferably SEQ ID NO:1. In a particular embodiment, the heavy chain variable region comprises at least 90%, particularly at least 93%, and at least 95% of the amino acid sequence of SEQ ID NO:1, and especially at least 98% of the amino acid sequence of SEQ ID NO:1. In these embodiments, the heavy chain variable region preferably has three specific CDRs having amino acid sequences of SEQ ID NO:12, 14, and 16.

[0061] In some embodiments, the light chain variable region comprises an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO:22. Specifically, the light chain variable region comprises an amino acid sequence that is at least 95%, particularly at least 98%, identical to the amino acid sequence of SEQ ID NO:22.

[0062] In a particular embodiment, the light chain variable region of the humanized antibody comprises a complementarity-determining region (CDR-L1) having the amino acid sequence of SEQ ID NO:24 or 25, a CDR-L2 having the amino acid sequence of SEQ ID NO:26 or 27, and a CDR-L3 having the amino acid sequence of SEQ ID NO:28 or 29. Specifically, the light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:18, and additionally comprises three specific CDRs having the amino acid sequences of SEQ ID NO:24, 26, and 28 or SEQ ID NO:25, 27, and 29. Therefore, any sequence deviation from SEQ ID NO:22 is located in the frame region, not in the CDRs.

[0063] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:23 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:23. Specifically, the light chain variable region comprises an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO:23. Specifically, the light chain variable region comprises an amino acid sequence that is at least 95%, particularly at least 98%, identical to the amino acid sequence of SEQ ID NO:23. In a particular embodiment, the light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:23, and additionally has three specific CDRs having the amino acid sequences of SEQ ID NO:24, 26, and 28.

[0064] Specifically, the humanized antibody may comprise a light chain variable region having an amino acid sequence according to any one of SEQ ID NO:17 to 21. In particular, the light chain variable region has an amino acid sequence according to SEQ ID NO:17 or 18, especially SEQ ID NO:17. In a particular embodiment, the light chain variable region comprises an amino acid sequence that is at least 90% identical, particularly at least 93%, at least 95% identical, or particularly at least 98% identical to the amino acid sequence of SEQ ID NO:17.

[0065] In a particular embodiment, the humanized antibody has a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10, and additionally has three specific CDRs having amino acid sequences of SEQ ID NO:12, 14, and 16 or SEQ ID NO:13, 15, and 16; and a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:22, and additionally has three specific CDRs having amino acid sequences of SEQ ID NO:24, 26, and 28 or SEQ ID NO:25, 27, and 29. In some preferred embodiments, the humanized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22.

[0066] In a particular embodiment, the humanized antibody has a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:11, and additionally has three specific CDRs having amino acid sequences of SEQ ID NO:12, 14, and 16; and a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:23, and additionally has three specific CDRs having amino acid sequences of SEQ ID NO:24, 26, and 28. In some preferred embodiments, the humanized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23.

[0067] In some embodiments, the humanized antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:1 to 7 and 9, particularly 1 to 6, and the light chain variable region comprising an amino acid sequence of SEQ ID NO:17 or 18, particularly 17. Specifically, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1 or 4 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:17. Specifically, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:17. In other embodiments, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:19.

[0068] In a preferred embodiment, the humanized antibody comprises an Fc region. The humanized antibody can be, in particular, a whole antibody. The humanized antibody can be any isotype, especially an IgG antibody, particularly IgG1, IgG2, or IgG4. In a specific embodiment, the humanized antibody is an IgG1 antibody. The humanized antibody is particularly capable of binding to one or more human Fc receptors, especially human Fcγ receptors, such as Fcγ receptor IIIa.

[0069] In a further embodiment, the humanized anti-Lewis Y antibody is a fragment of an antibody. Specifically, the fragment is selected from (i) the Fab fragment; (ii) the F(ab)2 fragment; (iii) the Fd fragment; (iv) the Fv fragment; (v) the scFv fragment; and (vi) the (Fv)2 fragment. In some embodiments, the humanized anti-Lewis Y antibody does not contain an Fc region.

[0070] In some embodiments, the humanized anti-Lewis Y antibody is glycosylated, particularly N-glycosylated. Specifically, the humanized antibody has a glycosylation site in the second constant domain (CH2) of the heavy chain. Antibodies typically have two heavy chains with the same amino acid sequence. Therefore, the humanized antibody preferably has at least two glycosylation sites, one in each of its two CH2 domains. This glycosylation site is particularly located at amino acid position 297 of the heavy chain according to the Kabat number and has the amino acid sequence motif Asn-Xaa-Ser / Thr, where Xaa can be any amino acid except proline. The N-linked glycosylation at Asn297 is conserved in the homologous regions of mammalian IgG and other antibody isotypes. The actual location of this conserved glycosylation site may vary in the amino acid sequence of the antibody due to optional additional amino acids or other sequence modifications that may be present in the variable region.

[0071] In a preferred embodiment, the humanized anti-Lewis Y antibody does not contain N-glycosidic neuraminic acid (NeuGc) or a detectable amount of NeuGc. Furthermore, the humanized antibody preferably also does not contain a Galili epitope (Galα1,3-Gal structure) or a detectable amount of Galili epitope. In particular, the relative amount of glycans carrying NeuGc and / or Galα1,3-Gal structures is less than 0.1% or even less than 0.02% of the total amount of glycans attached to the Fc moiety of the humanized antibody in the antibody population.

[0072] In other embodiments, the humanized anti-Lewis Y antibody is not glycosylated at its CH2 domain. In these embodiments, the CH2 domain of the antibody can be mutated, for example by replacing the asparagine residue at position 297 (or the corresponding position) of the heavy chain with any other amino acid, such as alanine or glutamine. The lack of glycosylation in the CH2 domain reduces the binding of the antibody to the Fcγ receptor, thus reducing effector function. In further embodiments, the humanized anti-Lewis Y antibody may have other or additional amino acid substitutions that reduce Fc receptor binding, including, for example, Leu235Glu (“LE mutation”), Leu234Ala / Leu235Ala (“LALA” mutation), Ser228Pro / Leu235Glu (“SPLE” mutation), Leu234Ala / Leu235Ala / Pro329Gly (“LALA-PG” mutation), and combinations thereof.

[0073] Humanized anti-Lewis Y antibodies are preferably produced recombinantly in host cells. Therefore, humanized antibodies are particularly monoclonal antibodies. The host cells used to produce humanized antibodies can be any host cell suitable for antibody production. Suitable host cells are particularly 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 the EB66 duck cell line, rodent cells such as CHO, NSO, SP2 / 0, and YB2 / 0 cell lines, and human cells such as HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz-3, and KG1 cell lines.

[0074] In some embodiments, humanized anti-Lewis Y antibodies are recombinantly generated in human cell lines, particularly human myeloid leukemia cell lines. Preferred human cell lines and suitable methods for preparing anti-Lewis Y antibodies are described in WO 2008 / 028686 A2. In a particular embodiment, humanized anti-Lewis Y antibodies are obtained by expression in human myeloid leukemia cell lines selected from NM-H9D8, NM-H9D6-E6, and NM-H9D9-E6Q12. In accordance with the requirements of the Budapest Treaty, these cell lines are deposited with accessions DSM ACC2806 (NM-H9D8; deposited September 15, 2006), DSM ACC2807 (NM-H9D8-E6; deposited October 5, 2006), and DSM ACC2856 (NM-H9D8-E6Q12; deposited August 8, 2007) at the German Center for Microbial and Cell Culture Collection (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE), Glycotope GmbH, Robert- -Str. 10, 13125 Berlin (DE). NM-H9D8 cells provide a glycosylation pattern with high sialylation, high bipartite GlycNAc, high galactosylation, and high fucosylation. NM-H9D8-E6 and NM-H9D6-E6Q12 cells provide a similar glycosylation pattern to NM-H9D8 cells, except with very low fucosylation. Other suitable cell lines include K562, a human myeloid leukemia cell line residing at the American Center for Type Culture Collection (ATCC CCL-243), CHO cells, and cell lines derived from the above. In a particular embodiment, a humanized anti-Lewis Y antibody is recombinantly generated in CHO cells, particularly in CHO-dhfr- cells.

[0075] In certain embodiments, the humanized anti-Lewis Y antibody is provided as a conjugate comprising an antibody conjugated to another reagent, such as a detectable marker or a therapeutically active substance. The humanized antibody may be conjugated to one or more other reagents. If more than one other reagent is present in the conjugate, these other reagents may be the same or different, particularly all the same. The conjugation of the additional reagent to the humanized antibody can be achieved using any method known in the art. The additional reagent may be covalently linked, particularly by fusion or chemical conjugation, or non-covalently linked to the antibody. In some embodiments, the additional reagent is covalently linked to the humanized antibody, particularly via a linker portion. The linker portion may be any chemical entity suitable for attaching the additional reagent to the humanized antibody.

[0076] Another reagent is preferably used for the treatment, diagnosis, prognosis, and / or monitoring of diseases, particularly cancer. For example, this other reagent may be selected from radionuclides, chemotherapeutic agents, antibodies, bispecific antibodies or antibody fragments, particularly those that are different from humanized anti-Lewis Y antibodies, enzymes, interacting domains, detectable markers, toxins, cytolytic components, immunomodulators, immune effectors, immunosuppressants, cytokines, chemokines, MHC class I or II antigens, and liposomes in species and / or specificity.

[0077] In some embodiments, the other reagent is a peptide or protein. This peptide or protein can be fused to the peptide chain of the humanized antibody. In some embodiments, the other reagent, as a peptide or protein, is fused to the C-terminus of the antibody light chain of the humanized antibody. In embodiments where the humanized antibody comprises two antibody light chains, the other reagent, as a peptide or protein, can be fused to the C-terminus of each of the two antibody heavy chains. In further embodiments, an additional reagent, as a peptide or protein, is fused to the C-terminus of the antibody heavy chain of the humanized antibody. In embodiments where the humanized antibody comprises two antibody heavy chains, the other reagent, as a peptide or protein, can be fused to the C-terminus of each of the two antibody light chains. The other reagents can be the same or different, particularly having the same amino acid sequence. In embodiments where the humanized antibody does not comprise one or more light chains and one or more heavy chains, for example, where the humanized antibody is an antibody fragment, the other reagent, as a peptide or protein, can be fused to the C-terminus or N-terminus of the peptide chain of the humanized antibody. Suitable examples of such other reagents as peptides or proteins may be selected from cytokines, chemokines, antibodies, antigen-binding fragments, enzymes, and interacting domains.

[0078] In some embodiments, other agents as peptides or proteins are checkpoint antibodies that block and / or trigger activation signals. Examples of various targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM), and CD258 (LIGHT) as activation targets, and CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA, and phosphatidylserine as inhibition targets, and their respective ligands such as PDL1. In further embodiments, additional agents as peptides or proteins are anticancer antibodies against tumor-associated antigens. Exemplary suitable tumor targets and anticancer antibodies that can be used as fusion partners are described below with respect to combination therapies.

[0079] In a further embodiment, the additional agent as a peptide 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, such as IL-15. Suitable growth factors include G-CSF and GM-CSF.

[0080] In certain embodiments, other agents, such as peptides or proteins, are antigen receptors, particularly T-cell receptors or T-cell co-receptors, or portions thereof and / or chimeras. Specifically, a humanized anti-Lewis Y antibody is fused with a transmembrane domain and an intracellular T-cell signaling domain to form a chimeric antigen receptor (CAR). The intracellular domain is specifically derived from one or more T-cell receptors or co-receptors. Optionally, the CAR also includes a hinge region between the humanized antibody and the transmembrane domain.

[0081] In these embodiments, the humanized anti-Lewis Y antibody is particularly a single-chain antibody fragment containing a heavy chain variable region and a light chain variable region within a single polypeptide chain, particularly the scFv fragment. The hinge region can be, for example, a hinge region based on an immunoglobulin superfamily member or a proximal membrane region. Exemplary hinge regions include hinge regions derived from IgG, CD8, and CD28. The transmembrane domain can be a hydrophobic α-helix spanning the cell membrane. For example, it is derived from CD28. The intracellular T cell signaling domain particularly includes the cytoplasmic domain of the T cell receptor ζ chain. Furthermore, the intracellular T cell signaling domain may include other domains of T cell co-stimulatory proteins. Exemplary other domains include signaling domains derived from CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0082] An exemplary CAR may include, from the N-terminus to the C-terminus, (i) a humanized anti-Lewis Y antibody in the form of an scFv fragment, (ii) an extracellular hinge region derived from CD8, (iii) a transmembrane domain derived from CD28, (iv) a cytoplasmic signaling domain derived from CD28, and (v) a signaling domain derived from the T cell receptor ζ chain.

[0083] Alternatively, humanized anti-Lewis Y antibodies, particularly single-chain forms such as scFv, can be fused at the N-terminus to the CD3 chain, especially the CD3ε chain, of the T-cell receptor complex to form a chimeric antigen receptor. Or, humanized anti-Lewis Y antibodies, particularly single-chain forms such as scFv, can be fused to binding domains capable of specifically binding to naturally occurring or engineered receptors on T cells or NK cells.

[0084] In some implementations, the other agents are cytotoxic or chemotherapeutic agents, particularly cytotoxins. Specific examples of chemotherapeutic agents that can be conjugated as other agents include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenes, vinca alkaloids, podophyllotoxin, taxanes such as paclitaxel, topoisomerase inhibitors such as irinotecan and topotecan, antitumor drugs such as doxorubicin, or microtubule inhibitors such as auristatin and maytansine / maytanyl alkaloids.

[0085] Chemotherapy agents can be specifically selected from V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, oliguria, dovastatin, maytansine, maytansine alkaloids, amatoxins, methionine aminopeptidase, protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphotransferase inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinin inhibitors, HDAC inhibitors, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, DNA inserting agents, DNA small groove binding agents, DHFR inhibitors, microtubule formation inhibitors, microtubule stabilizers, actin stabilizers, topoisomerase II inhibitors, platinum compounds, ribosome inhibitors, RNA polymerase II inhibitors, and bacterial toxins. In a particular embodiment, the chemotherapeutic agent attached to the anti-LeY antibody is selected from olipattin, maytansine alkaloids, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, and DNA small groove binding agents.

[0086] In some implementations, the chemotherapeutic agent is maytansine or maytanyl alkaloids. Specific examples of maytanyl alkaloids that can be used for conjugation include maytanyl alkaloids, N... 2' -Deacetylated-N 2 '-(3-mercapto-1-oxopropyl)-matansine (DM1), N 2' -Deacetylated-N 2' -(4-Mercapto-1-oxypentyl)-Maytansine (DM3) and N 2' -Deacetyl-N 2'-(4-methyl-4-mercapto-1-oxypentyl)-maytansin (DM4). Specifically, DM1 or DM4 is attached to the anti-LeY antibody. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is olistatin, particularly monomethylolistatin F (MMAF), monomethylolistatin E (MMAE), or olistatin T. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a DNA small groove binding agent, particularly pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer), docamycin, docamycin-hydroxybenzamide-azaindole (DUBA), seco-docamycin-hydroxybenzamide-azaindole (seco-DUBA), or doxorubicin. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a DNA alkylating agent, particularly indolinobenzodiazepine or oxazolidinobenzodiazepine. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a DNA damaging agent, particularly kazidromycin. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a topoisomerase I inhibitor, particularly camptothecin and its derivatives, such as 7-ethyl-10-hydroxycamptothecin (SN-38), (S)-9-dimethylaminomethyl-10-hydroxycamphor (topotecan), (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indoleaza[1,2-b]quinoline-10,13-dione (Exatecan (DX-8951f)) and DXd. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a microtubule formation inhibitor, particularly tubulolysin, anserine, podophyllotoxin, or vincristine. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a microtubule stabilizer, particularly paclitaxel or epothilone. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is an actin stabilizer, particularly phalloidin. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a topoisomerase II inhibitor, particularly teniposide, XK469, zosidine, azoxystrobin, idarubicin, or mebarone. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a platinum compound, particularly cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthreneplatin, picoplatin, or sattraplatin. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a ribosome inhibitor, particularly ricin, saponins, abrin, diphtheria toxin, or exotoxin A.In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is an RNA polymerase II inhibitor, particularly amatoxins, such as amatoxins. In some embodiments, the chemotherapeutic agent attached to the anti-LeY antibody is a bacterial toxin, particularly anthrax toxin. Suitable antibody-pharmaceutical conjugates are also described in EP 16 151 774.3 and LU 92659, and are explicitly mentioned herein.

[0087] The following section on combination therapy describes other suitable toxins that can be conjugated to humanized anti-Lewis Y antibodies.

[0088] In another aspect, the present invention provides a nucleic acid encoding a humanized anti-Lewis Y antibody. The nucleic acid sequence can have any nucleotide sequence suitable for encoding an antibody. However, preferably, the nucleic acid sequence is at least partially adapted to the specific codon usage of the host cell or organism to which the nucleic acid is to be expressed, particularly human codon usage. The nucleic acid can be double-stranded or single-stranded DNA or RNA, preferably double-stranded DNA such as cDNA or single-stranded RNA such as mRNA. It can be a single continuous nucleic acid molecule or composed of several nucleic acid molecules, each encoding a different portion of the humanized antibody.

[0089] If the humanized antibody consists of more than one distinct amino acid chain (e.g., a light chain and a heavy chain), the nucleic acid can be, for example, a single nucleic acid molecule containing multiple coding regions, each coding region encoding one amino acid chain of the antibody, preferably separated by a regulatory element such as an IRES element to produce separated amino acid chains. Alternatively, the nucleic acid can consist of multiple nucleic acid molecules, each comprising one or more coding regions, each coding region encoding one amino acid chain of the antibody. Alternatively, the nucleic acid can be a single nucleic acid molecule containing a coding region encoding both the heavy and light chains, said coding region being separated by a self-cleaving peptide (e.g., a 2A peptide) and / or a linker peptide containing a protease recognition site (e.g., a furin recognition site). In addition to the coding region encoding the humanized antibody, the nucleic acid can also include additional nucleic acid sequences or other modifications, which may, for example, encode other proteins, affect the transcription and / or translation of the coding region, affect the stability or other physical or chemical properties of the nucleic acid, or may have no function at all.

[0090] In some implementations, nucleic acids are viral vectors that can be used to infect human cells. For example, these viral vectors can be used to treat humans, such as by infecting and / or replicating diseased cells like tumor cells with a virus, or by modifying T cells to obtain CAR T cells in implementations where humanized antibodies are in the form of chimeric antigen receptors.

[0091] In another aspect, the present invention provides an expression cassette or vector comprising a nucleic acid according to the invention and a promoter operatively linked to said nucleic acid. Furthermore, the expression cassette or vector may include other elements, particularly those capable of influencing and / or regulating the transcription and / or translation of the nucleic acid, the amplification and / or replication of the expression cassette or vector, the integration of the expression cassette or vector into the host cell genome, and / or the copy number of the expression cassette or vector in the host cell. Suitable expression cassettes and vectors containing corresponding expression cassettes for expressing antibodies are well known in the art and therefore do not require further description herein.

[0092] Furthermore, the present invention provides a host cell comprising the nucleic acid according to the invention or the expression cassette or vector according to the invention. The host cell can be any host cell. It can be an isolated cell or a cell contained in a tissue. Preferably, the host cell is a cultured cell, particularly a primary cell or a cell line of an established cell line, preferably a tumor-derived cell. Preferably, it is a bacterial cell, such as *Escherichia coli*, a yeast cell, such as *Saccharomyces cerevisiae*, particularly *Saccharomyces cerevisiae*, an insect cell, such as Sf9 cells, or a mammalian cell, particularly a human cell, such as a tumor-derived human cell, a hamster cell, such as CHO, or a primate cell. In a preferred embodiment of the invention, the host cell is derived from human myeloid leukemia cells. Preferably, it is selected from the following cells or cell lines: K562, KG1, MUTZ-3, CHO, or cells or cell lines derived therefrom, or cells or mixtures containing at least one of the above cells. The host cells are preferably selected from NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, and cells or cell lines derived from any of the aforementioned hosts, or cells or mixtures containing at least one of the above-mentioned cells. These cell lines and their properties are described in detail in PCT application WO 2008 / 028686 A2. In a preferred embodiment, the host cells are optimized to express glycoproteins, particularly antibodies, with a specific glycosylation pattern. Preferably, the use of codons in the coding region and / or promoter of the nucleic acid according to the invention, as well as other elements of the expression cassette or vector, are compatible with and more preferably optimized for the type of host cells used. Preferably, the humanized antibody is produced from the host cells or cell lines described above.

[0093] In another aspect, the present invention provides compositions comprising humanized antibodies, nucleic acids, expression cassettes or vectors, host cells, or conjugates. The compositions may also contain more than one of these components. Furthermore, the compositions may contain one or more other components selected from solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, all components of the composition are preferably pharmaceutically acceptable. The composition may be a solid or liquid composition, particularly preferably an aqueous solution, emulsion, suspension, or lyophilized powder.

[0094] Humanized anti-Lewis Y antibodies or conjugates thereof are particularly useful in medicine, especially for the treatment, diagnosis, prognosis, and / or monitoring of diseases, particularly those described herein, such as cancer and infections, preferably cancer. Therefore, in another aspect, the present invention provides humanized antibodies, nucleic acids, expression cassettes or vectors, host cells, conjugates, or compositions for medical use. Preferably, the medicinal use is for the treatment, prognosis, diagnosis, and / or monitoring of diseases, such as diseases associated with abnormal cell growth, such as cancer or infectious diseases. Infectious diseases particularly include viral and bacterial infections, especially infections caused by viruses or bacteria with Lewis Y on their surface. Exemplary infections include Helicobacter pylori infection.

[0095] In a preferred embodiment, the disease is cancer, particularly epithelial cancer, especially advanced epithelial cancer. Preferably, the cancer is selected from lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, stomach cancer, leukemia such as acute myeloid leukemia, lymphoma such as multiple myeloma, head and neck cancer, pancreatic cancer, liver cancer, prostate cancer, and bladder cancer, especially non-small cell lung cancer, colon cancer, breast cancer, and ovarian cancer.

[0096] In some embodiments, the disease to be treated is a disease associated with abnormal cell growth, such as cancer. This cancer is Lewis Y positive and specifically includes cancer cells that carry Lewis Y on their cell surface. In a particular embodiment, a humanized anti-Lewis Y antibody is used in combination with another anticancer therapeutic agent. The other therapeutic agent can be any known anticancer drug, and in particular, can be an antibody targeting a cancer antigen. [The text then repeats itself, so the translation will only include the first instance.] Suitable antibodies for Y antibody combinations include anti-EGFR antibodies such as cetuximab (Erbitux), toltuximab, panitumumab (Vectibix), and nimotuzumab (Theraloc); anti-HER2 antibodies such as trastuzumab (Herceptin), telmitutuzumab, and pertuzumab; anti-VEGF antibodies such as bevacizumab (Avastin) and vanouzumab; anti-CD52 antibodies such as alemtuzumab (Campath); anti-CD30 antibodies such as brentuximab vedotin (Adcetris); anti-CD33 antibodies such as gemtuzumab (Mylotarg); and anti-CD20 antibodies such as levofloxacin (Betastatin). Toxicitumb (Rituxan, Mabthera), tosimomab (Bexxar), and teimozab (Zevalin); anti-CTLA4 antibodies, such as ipilimumab and teimozab; anti-PD1 / PD-L1 antibodies, such as nivolumab, atezolizumab, and avelumab; antibodies against TNF and TNFR superfamily members, such as uricoxib, MEDI6469, TRX518, and varilumab; CSF1R antibodies, such as imetuzumab; anti-B7-H3 antibodies, such as enotozumab; anti-LAG3 antibodies; anti-4-1BB antibodies; anti-ICOS antibodies; and anti-OX-40 antibodies.

[0097] Other anticancer agents that can be combined with humanized anti-Lewis Y antibodies and optionally one or more other antibodies may be selected from paclitaxel, such as paclitaxel (paclitaxel), docetaxel (docetaxel), and SB-T-1214; cyclophosphamide; lapatinib; erlotinib; imatinib; pazopanib; capecitabine; cytarabine; vinorelbine; gemcitabine; anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, rubicin, and mitoxantrone; aromatase inhibitors such as amino... Glutamate, Teslac, Arimidex, Femara, Aromasin, Rivizior, Lentaron, Afema, 4-hydroxyandrostenedione, 1,4,6-androstriene-3,17-dione (ATD), and 4-androsten-3,6,17-trione (6-OXO); Topoisomerase inhibitors, such as irinotecan, topotecan, camptothecin, spirotinic acid D, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, acridine, rose alkaloids, ginsenosides, and HU-331; platinum-based chemotherapeutic agents, such as cis-diamine dichloroplatin(II) (cisplatin), cis-dimethylamine (1,1-cyclobutanedicarboxy)platin(II) (carboplatin), and [(1R,2R)-cyclohexane-1,2-dichloroplatinum]. [Amine] (ethylenediamino-O,O′)platin(II) (oxaliplatin); antimetabolites, particularly folic acid antagonists such as methotrexate, pemetrexed, raltitrexed, and pralatrexate; pyrimidine analogs such as fluorouracil, gemcitabine, fluorouridine, 5-fluorouracil, and uridine-uracil, as well as purine analogs; and inhibitors of poly(ADP-ribose) polymerase (PARP inhibitors) such as olaparib, rucaparib, niraparib, and taraparib. Other suitable toxins that can be used in combination with humanized anti-Lewis Y antibodies are described above in the section on reagents that can be conjugated to humanized antibodies.

[0098] Treatment with humanized anti-Lewis Y antibodies can be further combined with immunostimulants, cytokines, chemokines, radiotherapy, vaccines such as protein, peptide or RNA vaccines, B-Raf inhibitors such as vemurafenib, dexamethasone, and protease inhibitors such as bortezomib and lenalidomide.

[0099] For use in treating cancers with cells expressing Lewis Y, humanized antibodies can be conjugated to another agent as described above, wherein the other agent is preferably a cytotoxic agent, such as a radionuclide or cytotoxin. Exemplary cytotoxic agents have been described above. Cytotoxic agents also include prodrug compounds that produce cytotoxic activity only upon activation, such as by light irradiation or an enzymatic reaction in vivo. One or more of the aforementioned anticancer therapeutic agents can also be used as another agent conjugated to humanized anti-Lewis Y antibodies. Furthermore, humanized antibodies can be engineered to enhance their ability to activate a patient's immune response, particularly their ability to activate ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity). This can be achieved, for example, by optimizing the amino acid sequence and / or glycosylation pattern of the antibody, particularly its constant regions.

[0100] For use as a diagnostic reagent in disease diagnosis, prognosis, and / or monitoring, humanized antibodies are preferably conjugated to a labeling reagent capable of generating a detectable signal. Specifically, the labeling reagent may be a radionuclide, a fluorophore, or an enzyme. Brief description of the attached diagram

[0102] Figure 1 A schematic diagram of the Lewis carbohydrate antigen family is shown. Lewis antigens are a group of related glycans that carry fucose linked to GlcNAc monosaccharides in α1-3 (Lewis X, Y) or α1-4 (Lewis A, B).

[0103] Figure 2 The results of the antigen ELISA assay are shown. Binding of different humanized anti-Lewis Y antibody variants to Lewis Y, Lewis b, and Globo H was tested. High OD signal indicates strong antibody-antigen binding. A: Binding of 25 ng / mL antibody variants to Lewis Y (LeY), Lewis b (Leb), and Globo H. B: Binding of 25 ng / mL and 12.5 ng / mL variants to Lewis Y. C: Binding of 100 ng / mL, 50 ng / mL, and 25 ng / mL antibody variants to Lewis b. D: Binding of 100 ng / mL, 50 ng / mL, and 25 ng / mL antibody variants to Globo H. Control (ctrl.+): Binding of parental / mouse chimeric antibody AA9.

[0104] Figure 3The results of the antigen ELISA assay are shown. Binding curves of different humanized anti-Lewis Y antibody variants with Lewis Y(A) and Lewis b(B) were measured. Control 1 (ctrl. + AA9): Parental / mouse chimeric antibody AA9. Control 2 (ctrl. + recombinant chain): Recombinant antibody containing the heavy chain of human / mouse chimeric antibody AA9 and the light chain of human / mouse chimeric antibody CC8.

[0105] Figure 4 The results of the antigen ELISA assay are shown. Binding curves of humanized anti-Lewis Y antibody AA9-3-10.1 with Lewis Y and Lewis b were measured. Control (ctrl.): Parental / mouse chimeric antibody AA9.

[0106] Figure 5 The results of antigen ELISA assays are shown. The binding of humanized anti-Lewis Y antibody AA9-3-10.1 (humanized AA9) and parental / mouse chimeric antibody AA9 (chimeric AA9) to different carbohydrate antigens was tested at an antibody concentration of 50 ng / mL.

[0107] Figure 6 The results of the antigen ELISA assay are shown. The binding of different sequence-optimized versions of the humanized anti-Lewis Y antibody AA9-3-10 to Lewis Y and Lewis b was tested. High OD signals indicate strong antibody-antigen binding. A: Binding of 50 ng / mL antibody variants to Lewis Y (LeY) and Lewis b (Leb). B: Binding of 50 ng / mL and 25 ng / mL antibody variants to Lewis Y. C: Binding of 100 ng / mL and 50 ng / mL antibody variants to Lewis b. Control (pos.-ctrl.-Set2 128+129): Binding of parental / mouse chimeric antibody AA9.

[0108] Figure 7 The results of antigen ELISA assays are shown. The specificity of different anti-Lewis Y antibodies was tested by analyzing their binding to Lewis Y and other related carbohydrate antigens. A: Humanized anti-Lewis Y antibody AA9-3-10.1; B: Anti-Lewis Y antibody h3S193; C: Anti-Lewis Y antibody BR96.

[0109] Figure 8 The results of the antigen ELISA assay are shown. The binding curves of anti-Lewis Y antibodies AA9-3-10.1, h3S193, and BR96 with Lewis Y were measured.

[0110] Figure 9The binding of humanized anti-Lewis Y antibody variants AA9-3-10.1, BR96, and h3S193 (as hIgG1) to tumor cell lines Ls-174T, T-47D, H9D8, and Colo-205 was demonstrated. Unrelated hIgG1 was used as a negative control. Binding was reported as the percentage of antibody-positive cells out of total viable cells.

[0111] Figure 10 The binding of humanized anti-Lewis Y antibody variants AA9-3-10.1, BR96, and h3S193 (as mIgG1) to leukocytes isolated from five healthy donors is shown. Unrelated hIgG1 was used as a reference. Binding to granulocytes, lymphocytes, and monocytes was reported as median fluorescence intensity (MFI).

[0112] Figure 11 The proliferation inhibition assays of toxin-conjugated AA9-3-10.1 (as hIgG1) and isotype controls from different tumor cell lines are shown. Proliferation is reported as a percentage of proliferation relative to the culture medium control. Example

[0113] Example 1: Humanization of the mouse heavy and light chain variable regions of anti-LeY antibody

[0114] Nucleic acid sequences encoding the variable regions of the mouse heavy and light chains of two monoclonal anti-LeY antibodies (AA9: SEQ ID NO: 30 and 31; CC8: SEQ ID NO: 32) were linked to the genomic sequences of the human constant γ1 region (CH) and the human constant κ region (CL), respectively.

[0115] Based on these chimeric clones, humanized antibodies were constructed. For this purpose, point mutations were introduced into the nucleic acid sequences of the frame regions of mouse VH and VL to generate corresponding human frame regions. Target human frame regions were selected from a human germline antibody library. Specifically, the most relevant frame regions were selected from the library based on their overall sequence similarity and CDR loop classification. All obtained data were considered to design a set of different parental mouse antibodies with humanized variable light and variable heavy chain sequences. Some variants contained inverse mutations of the mouse sequence at key positions. Humanized variants of the light chain variable region were cloned into a κ-chain vector, and humanized variants of the heavy chain variable region were cloned into a γ1-chain vector.

[0116] Antibodies containing different combinations of the obtained heavy and light chains were generated, and their expression and LeY binding profiles were screened. The variable regions of the heavy and light chains of the following humanized antibodies were selected for further analysis.

[0117] Table 1

[0118]

[0119]

[0120] Example 2: Binding of humanized antibody variants to different carbohydrate antigens

[0121] After expressing different constructs in NM-H9D8 cells, the titers of humanized antibody variants were determined and their concentrations adjusted. The binding of the humanized antibodies to Lewis Y, Lewis b, and Globo H was then analyzed in an antigen ELISA. Briefly, the antigen (conjugated to polyacrylamide) was coated onto 96-well MaxiSorp plates (Nunc ThermoScientific) and incubated overnight. Non-specific binding was blocked, and test samples were added at different concentrations. Anti-human IgG Fc POD secondary antibody was then added, followed by a TMB substrate reaction. Binding antibodies were measured at 450 / 630 nm using an EnSpire 2300 multilabel reader (PerkinElmer) or at 450 / 620 nm using a Tecan SPARK plate reader.

[0122] All variants showed significant binding to Lewis Y, similar to the parental chimeric antibody. Furthermore, all antibodies were negative for Globo H. Surprisingly, however, the selected humanized antibody variants exhibited significantly reduced cross-reactivity to Lewis b (see [link to relevant documentation]). Figure 2 , 3 and 4).

[0123] Humanized antibody variants AA9-3-6 (containing VL3 and VH6), AA9-3-9 (containing VL3 and VH9), AA9-3-10 (containing VL3 and VH10), and CC8-1-11 (containing VL1 and VH11) were further analyzed using antigen ELISA for their subtle specificity with Lewis Y, Galα1, 2-Gal, and β-N-acetyl-D-glucosamine-6-sulfate. The analyzed humanized antibodies showed strong, highly specific binding with Lewis Y, but no significant binding with other carbohydrate antigens. Binding with the following carbohydrate antigens was tested at an antibody concentration of 50 ng / mL (see also...). Figure 5 ):

[0124] Table 2

[0125]

[0126]

[0127] Example 3: Generating further humanized variants based on antibody AA9-3-10

[0128] The humanized antibody variant AA9-3-10 (including VL3 and VH10) was selected as the best candidate for further optimization of the humanized VH sequence. The following humanized antibodies were generated as described in Example 1:

[0129] Table 3

[0130]

[0131] As described in Example 2, the binding of these humanized antibody variants to Lewis Y and Lewis b was analyzed. All antibody variants showed strong, highly specific binding to Lewis Y. No significant binding to Lewis b was detected (see Example 2). Figure 6 ).

[0132] Example 4: Comparison of antigen binding and specificity of humanized variant AA9-3-10.1 with known anti-LeY antibodies

[0133] The binding specificity of the humanized antibody variant AA9-3-10.1 to different, closely related carbohydrate antigens (βD-galactose, Lewis b, Lewis X, 3'-O-su-Lewis X, lactose-N-tetrasaccharide (LNT), Neu5Acα2-5Galβ, and Lewis Y) was compared with the binding specificity to known antibodies h3S193 and BR96.

[0134] In short, the antigen (conjugated to polyacrylamide) was coated onto a 96-well MaxiSorp plate (Nunc Thermo Scientific) and left overnight. Non-specific binding was blocked, and test samples were added at different concentrations. Then, anti-human IgG (H+L) POD secondary antibody was added, followed by a TMB substrate reaction. The binding antibody was measured at 450 / 630 nm using an EnSpire 2300 multilabel reader (PerkinElmer) or at 450 / 620 nm using a Tecan SPARK plate reader.

[0135] The humanized antibody variant AA9-3-10.1 exhibited stronger binding to Lewis Y and significantly improved specificity. At low antibody concentrations, h3S193 bound to Lewis Y much less, while BR96 bound significantly to βD-galactose and Lewis X. Furthermore, at higher concentrations, both h3S193 and BR96 bound to Neu5Acα2-5Galβ (see...). Figure 7 and 8 ).

[0136] A novel approach to determining binding constants and affinities is fluorescence proximity sensing using single-stranded DNA (96mer) and ligand-conjugated complementary DNA on a DRX2 instrument (Dynamic Biosensor) chip. In this study, streptavidin was used as the ligand to capture biotinylated polyacrylamide-conjugated Lewis Y or Lewis b. Binding of the antigen to the humanized antibody variant AA9-3-10.1 or a competing anti-Lewis Y antibody resulted in fluorescence changes. On and off rates could be calculated during association and dissociation. AA9-3-10.1 and the competing anti-Lewis Y antibody were diluted to 300, 60, and 12 nM in PE140 buffer and applied to the chip to bind the antigen. For experiments using AA9-3-10.1 on Lewis b, 3000, 600, and 120 nM were used because very low signals were expected. Binding curves were evaluated by single-exponential global fitting (instrument software). Due to higher sensitivity, faster interactions could be monitored compared to surface plasmon resonance (SPR). This leads to binding kinetics that differ from SPR and are more similar to KinExA, the "gold standard" method for measuring in liquid systems.

[0137] The assay showed a strong and highly specific binding of AA9-3-10.1 to Lewis Y. The data confirmed the results of the ELISA assay.

[0138] Table 4

[0139]

[0140] nd: Uncertain

[0141] Example 5: Comparison of binding of humanized variant AA9-3-10.1 to tumor cells with known anti-LeY antibodies

[0142] The binding characteristics of humanized anti-Lewis Y antibody variants AA9-3-10.1, BR96, and 3S193 (all hIgG1) to human cancer cell lines Ls-174T, T-47D, H9D8, and Colo-205 were analyzed by flow cytometry. Unrelated hIgG1 was used as a negative control. Briefly, tumor cells were harvested and incubated in the dark at 4°C with serially diluted designated antibodies. Cells were then washed and incubated in the dark at 4°C with a goat anti-hIgG PE-conjugated secondary antibody. After an additional washing step, cells were stained with DAPI to distinguish between live and dead cells and analyzed by flow cytometry.

[0143] The humanized anti-Lewis Y antibody variant AA9-3-10.1 showed concentration-dependent binding to Lewis Y-positive cell lines Ls-174T and T-47D, but no binding to Lewis Y-negative cell lines H9D8 and Colo-205. In contrast, competing anti-Lewis Y antibodies BR96 and 3S193 showed stronger binding to all four tumor cell lines than the humanized anti-Lewis Y antibody variant AA9-3-10.1 (see [link to AA9-3-10.1]). Figure 9 The stronger binding of BR96 and 3S193 is likely due to cross-reactivity. For example, Colo-205 has been described as being negative for Lewis Y (Westwood et al., 2005) but positive for Lewis b (Noble et al., 2013).

[0144] Example 6: Comparison of binding of humanized variant AA9-3-10.1 to blood cells containing known anti-LeY antibodies

[0145] The binding of humanized anti-Lewis Y antibody variants AA9-3-10.1, BR96, and 3S193 (as mIgG1) to leukocytes was determined using flow cytometry. Therefore, whole blood from five healthy volunteers was used. In the first step, erythrocytes were lysed, and the remaining leukocytes were incubated with the designated antibody [10 μg / ml] at room temperature. Unrelated mIgG1 was used as a negative control. Cells were then washed and incubated with the secondary antibody mIgG AF647 conjugated at room temperature. Following the washing step, cells were stained with the anti-human CD45 PacificBlue conjugated antibody at room temperature. After an additional washing step, cells were analyzed by flow cytometry. Immune cell subsets, granulocytes, monocytes, and lymphocytes, were distinguished by their CD45 expression and granularity.

[0146] The humanized anti-Lewis Y antibody variant AA9-3-10.1 binds little or no to leukocyte subsets, while BR96 binds strongly to granulocytes, and 3S193 binds to granulocytes, lymphocytes, and monocytes (see [link to relevant documentation]). Figure 10 This demonstrates the superior specificity of the humanized anti-Lewis Y antibody variant against tumor cells and its very low cross-reactivity with normal tissue cells.

[0147] Example 7: Inhibition of proliferation of different tumor cell lines using a humanized anti-Lewis Y antibody variant AA9-3-10.1 conjugated with a toxin.

[0148] To demonstrate the efficacy of the humanized anti-Lewis Y antibody variant in killing tumor cells, proliferation inhibition assays were performed on different tumor cell lines. As a cytotoxin, MMAE is conjugated to protein G, which in turn binds to the antibody, forming an antibody-toxin conjugate.

[0149] Cell lines Ls-174T, T-47D, MCF-7 (CSC-rich), Ovcar-3, and HSC-4 were seeded at 5000 cells / well in 96-well plates and incubated for 4 days with specified concentrations of ProtG MMAE in the presence of humanized anti-Lewis Y antibody variant AA9-3-10.1 (as hIgG1) or an unrelated isotype control. The number of viable cells was determined using the commercial CellTiterGlo luminescent cell viability assay. The percentage of proliferation was determined relative to the culture medium-only control.

[0150] The toxin-conjugated humanized anti-Lewis Y antibody variant AA9-3-10.1 was able to inhibit the proliferation of various Lewis Y-expressing tumor cell lines, demonstrating effective antibody internalization (see [link to relevant documentation]). Figure 11 ).

[0151] Example 8: Immunohistochemical staining of humanized anti-Lewis Y antibody variant AA9-3-10.1 in different tumor tissues of different cancer types

[0152] Immunohistochemical analysis was performed to assess the binding of the humanized anti-Lewis Y antibody variant AA9-3-10.1 to various cancer indications. Briefly, tissue microarray slides from breast cancer (BRC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), head and neck cancer (HNC), small cell lung cancer (SCLC), and ovarian cancer (OvCa) were deparaffinized and rehydrated in descending alcohol sequence. After antigen recovery, endogenous peroxidase and nonspecific binding were blocked. Binding of the humanized anti-Lewis Y antibody variant AA9-3-10.1 as mIgG1 [6.5 μg / ml] was detected using the secondary antibody Envision Flex anti-mouse Ig-HRP and DAB+ staining solution. Finally, slides were counterstained with Mayer's hematoxylin, mounted, and evaluated under a microscope. The binding of the humanized anti-Lewis Y antibody variant AA9-3-10.1 was reported using an immune response score (IRS; range 0-12), which was calculated by multiplying staining intensity (range 0-3) by the percentage of stained cells (range 0-4).

[0153] The humanized anti-Lewis Y antibody variant AA9-3-10.1 provides high-proportion staining for tumor tissues of breast cancer (BRC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), head and neck cancer (HNC), small cell lung cancer (SCLC), and ovarian cancer (OVCa).

[0154] Table 5

[0155]

[0156] sequence list

[0157]

[0158]

[0159]

[0160] Identification of Preserved Biological Materials

[0161] Cell lines DSM ACC 2806, DSM ACC 2807, and DSM ACC 2856 are deposited at DSMZ (German Center for Microbial and Cell Culture Collection), Inhoffenstraβe 7B, 38124 Braunschweig (DE), Glycotope GmbH, Robert- -Str.10, 13125 Berlin (DE), dates are shown in the table below.

[0162] Cell line name Collection Number collector Preservation date NM-H9D8 DSM ACC 2806 Glycotope GmbH September 15, 2006 NM-H9D8-E6 DSM ACC 2807 Glycotope GmbH October 5, 2006 NM-H9D8-E6Q12 DSM ACC 2856 Glycotope GmbH August 8, 2007 sequence list <110> Glycotope GmbH <120> Humanized antibodies against Lewis Y <130> 63 136 K <150> EP 20 183 237.5 <151> 2020-06-30 <160> 32 <170> BiSSAP 1.3.6 <210> 1 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile Asp Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 2 <211> 115 <212> PRT <213> Synthetic Sequence <220> <223> Heavy Chain Variable Region <400> 2 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile Asp Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 3 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region <400> 3 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 4 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region <400> 4 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile Asp Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Met 35 40 45[[ID=X]] Gly Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 5 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region <400> 5 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 6 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region <400> 6 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile Asp Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 7 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 7 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 8 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 8 Lys Val Gln Leu Val Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Lys Gln Thr Pro Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 9 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 9 Lys Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 10 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <220> <221> VARIANT <222> 1 <223> Xaa is either Gln or Lys <220> <221> VARIANT <222> 9 <223> Xaa is either Ala or Pro. <220> <221> VARIANT <222> 12 <223> Xaa is Lys or Val <220> <221> VARIANT <222> 34 <223> Xaa is Ile or Met <220> <221> VARIANT <222> 38 <223> Xaa is either Arg or Lys <220> <221> VARIANT <222> 40 <223> Xaa is Thr or Ala <220> <221> VARIANT <222> 43 <223> Xaa is either Lys or Gln. <220> <221> VARIANT <222> 44 <223> Xaa is either Gly or Ser <220> <221> VARIANT <222> 55 <223> Xaa is either Gln or Asn <220> <221> VARIANT <222> 67 <223> Xaa is either Lys or Arg <220> <221> VARIANT <222> 68 <223> Xaa is either Ala or Val <220> <221> VARIANT <222> 70 <223> Xaa is either Met or Leu <220> <221> VARIANT <222> 72 <223> Xaa is Val or Thr <220> <221> VARIANT <222> 74 <223> Xaa is Lys or Thr <400> 10 Xaa Val Gln Leu Val Gln Ser Gly Xaa Glu Val Xaa Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Xaa Asp Trp Val Xaa Gln Xaa Pro Gly Xaa Xaa Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Xaa Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Xaa Xaa Thr Xaa Thr Xaa Asp Xaa Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 11 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <220> <221> VARIANT <222> 9 <223> Xaa is either Ala or Pro. <220> <221> VARIANT <222> 12 <223> Xaa is Lys or Val <220> <221> VARIANT <222> 40 <223> Xaa is Thr or Ala <220> <221> VARIANT <222> 43 <223> Xaa is either Lys or Gln. <220> <221> VARIANT <222> 67 <223> Xaa is either Lys or Arg <220> <221> VARIANT <222> 68 <223> Xaa is either Ala or Val <220> <221> VARIANT <222> 72 <223> Xaa is Val or Thr <220> <221> VARIANT <222> 74 <223> Xaa is either Lys or Thr <400> 11 Gln Val Gln Leu Val Gln Ser Gly Xaa Glu Val Xaa Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile Asp Trp Val Arg Gln Xaa Pro Gly Xaa Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Xaa Xaa Thr Met Thr Xaa Asp Xaa Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 12 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR‑H1 <400> 12 Asp Tyr Asn Ile Asp 1 5 <210> 13 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR‑H1 <400> 13 Asp Tyr Asn Met Asp 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR-H2 <400> 14 Tyr Ile Tyr Pro Tyr Gln Gly Tyr Ser Asp Tyr Asn Gln Lys Phe Lys 1 5 10 15 Ser <210> 15 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR-H2 <400> 15 Tyr Ile Tyr Pro Tyr Asn Gly Tyr Ser Asp Tyr Asn Gln Lys Phe Lys 1 5 10 15 Ser <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR-H3 <400> 16 Gln Leu Gly Pro Gly Thr Phe 1 5 <210> 17 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <400> 17 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Gly 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ala 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 18 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region <400> 18 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Thr Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Gly 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ala 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Ala Lys Leu Glu Ile Lys 100 105 110 <210> 19 <211> 112 <212> PRT <213> artificial sequence <220> <223> Chain link <400> 19 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Ser Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Lys Arg Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Arg 85 90 95 Thr His Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 20 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 20 Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Ser Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Lys Arg Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Lys Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Arg 85 90 95 Thr His Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 21 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <400> 21 Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Ser Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Leu Gln Leu Leu Ile Tyr Glu Val Ser Lys Arg Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Arg 85 90 95 Thr His Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> twenty two <211> 112 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <220> <221> VARIANT <222> 4 <223> Xaa is either Met or Leu <220> <221> VARIANT <222> 19 <223> Xaa is either Ala or Thr <220> <221> VARIANT <222> twenty four <223> Xaa is either Lys or Thr <220> <221> VARIANT <222> 30 <223> Xaa is Leu or Val <220> <221> VARIANT <222> 32 <223> Xaa is either Gly or Ser <220> <221> VARIANT <222> 35 <223> Xaa is either Lys or Asn. <220> <221> VARIANT <222> 36 <223> Xaa is Thr or Ser <220> <221> VARIANT <222> 39 <223> Xaa is either Asn or Asp. <220> <221> VARIANT <222> 41 <223> Xaa is either Leu or Tyr <220> <221> VARIANT <222> 49 <223> Xaa is Pro or Leu <220> <221> VARIANT <222> 50 <223> Xaa is either Lys or Gln. <220> <221> VARIANT <222> 55 <223> Xaa is Leu or Glu <220> <221> VARIANT <222> 58 <223> Xaa is either Asn or Lys <220> <221> VARIANT <222> 59 <223> Xaa is either Leu or Arg <220> <221> VARIANT <222> 60 <223> Xaa is either Glu or Asn <220> <221> VARIANT <222> 74 <223> Xaa is Thr or Lys <220> <221> VARIANT <222> 88 <223> Xaa is Val or Leu <220> <221> VARIANT <222> 94 <223> Xaa is Leu or Phe <220> <221> VARIANT <222> 96 <223> Xaa is Ala or Arg <220> <221> VARIANT <222> 99 <223> Xaa is Phe or Leu <220> <221> VARIANT <222> 105 <223> Xaa is Gln or Ala <220> <221> VARIANT <222> 107 <223> Xaa is Thr or Ala <400> 22 Asp Ile Val Xaa Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Xaa Ser Ile Ser Cys Xaa Ser Ser Gln Ser Leu Xaa His Xaa 20 25 30 Asn Gly Xaa Xaa Tyr Leu Xaa Trp Xaa Leu Gln Lys Pro Gly Gln Ser 35 40 45 Xaa Xaa Leu Leu Ile Tyr Xaa Val Ser Xaa Xaa Xaa Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Xaa Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Xaa Gly Val Tyr Tyr Cys Xaa Gln Xaa 85 90 95 Thr His Xaa Pro Leu Thr Phe Gly Xaa Gly Xaa Lys Leu Glu Ile Lys 100 105 110 <210> twenty three <211> 112 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <220> <221> VARIANT <222> 19 <223> Xaa is either Ala or Thr <220> <221> VARIANT <222> 88 <223> Xaa is Val or Leu <220> <221> VARIANT <222> 105 <223> Xaa is Gln or Ala <220> <221> VARIANT <222> 107 <223> Xaa is Thr or Ala <400> twenty three Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Xaa Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Gly 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Xaa Gly Val Tyr Tyr Cys Leu Gln Ala 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Xaa Gly Xaa Lys Leu Glu Ile Lys 100 105 110 <210> 24 <211> 16 <212> PRT <213> artificial sequence <220> <223> CDR‑L1 <400> 24 Lys Ser Ser Gln Ser Leu Leu His Gly Asn Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> artificial sequence <220> <223> CDR‑L1 <400> 25 Thr Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Ser Tyr Leu Asp 1 5 10 15 <210> 26 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 26 Leu Val Ser Asn Leu Glu Ser 1 5 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR-L2 <400> 27 Glu Val Ser Lys Arg Asn Ser 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR-L3 <400> 28 Leu Gln Ala Thr His Phe Pro Leu Thr 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR-L3 <400> 29 Phe Gln Arg Thr His Leu Pro Leu Thr 1 5 <210> 30 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 30 Lys Val Lys Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Lys Gln Thr His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Tyr Ser Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Gly Pro Gly Thr Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 31 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 31 Asp Ile Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Thr Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Gly 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ala 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Ala Lys Leu Glu Leu Lys 100 105 110 <210> 32 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region <400> 32 Asp Ile Val Leu Thr Gln Ser Pro Leu Phe Leu His Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Ser Tyr Leu Asp Trp His Leu Gln Lys Ser Asp Gln Ser 35 40 45 Leu Gln Leu Leu Ile Tyr Glu Val Ser Lys Arg Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Lys Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Arg 85 90 95 Thr His Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110

Claims

1. A humanized antibody capable of binding Lewis Y, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 or 10, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

22.

2. The antibody according to claim 1, wherein the heavy chain variable region comprises a complementarity-determining region CDR-H1 consisting of the amino acid sequence of SEQ ID NO:12, a CDR-H2 consisting of the amino acid sequence of SEQ ID NO:14, and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO:

16.

3. The antibody according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO:1 to 7 and SEQ ID NO:

9.

4. The antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

11.

5. The antibody according to claim 1, wherein the light chain variable region comprises a complementarity-determining region CDR-L1 consisting of the amino acid sequence of SEQ ID NO:24, a CDR-L2 consisting of the amino acid sequence of SEQ ID NO:26, and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO:

28.

6. The antibody according to claim 1, wherein the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 17 to 21.

7. The antibody according to claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

23.

8. The antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

23.

9. The antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

17.

10. The antibody according to claim 1, further comprising an Fc region.

11. The antibody according to claim 10, wherein it is an IgG1, IgG2, IgG3 or IgG4 antibody.

12. The antibody according to claim 1, which specifically binds to Lewis Y but not to Lewis b.

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

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

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

16. A conjugate comprising an antibody conjugated with any one of claims 1 to 12, wherein the additional reagent is a cytotoxic agent.

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

18. The composition according to claim 17, further comprising one or more components selected from solvents, diluents and excipients, as a pharmaceutical composition.

19. Use of the antibody according to any one of claims 1 to 12, the conjugate according to claim 16, or the composition according to claim 17 or 18 in the preparation of a medicament for treating cancer.

20. The use according to claim 19, wherein the cancer is selected from lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, gastric cancer, leukemia, lymphoma, multiple myeloma, head and neck cancer, pancreatic cancer, liver cancer, prostate cancer, and bladder cancer.

Images