Humanized antibody against Lewis Y

Abstract

The present invention relates to humanized anti-Lewis Y antibodies that specifically bind to 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 antigens. In particular, the present invention is directed to humanized anti-Lewis Y antibodies that are useful in the treatment of cancer. Thus, in a first aspect, the present invention is directed to a humanized antibody capable of binding to 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: 10 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and 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.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of antibodies. Humanized anti-Lewis Y antibodies are provided that exhibit excellent target specificity and affinity, particularly for glycan antigens. In certain embodiments, the present invention is directed to humanized anti-Lewis Y antibodies for therapeutic and diagnostic use. [Background technology]

[0002] Background of the Invention Nowadays, antibodies are widely used active substances in the fields of medicine and research.In medicine, they have applications in many different fields.For example, antibodies are used as therapeutic agents in the treatment and prevention of various diseases, such as cancer, cardiovascular disease, inflammatory disease, macular degeneration, transplant rejection, multiple sclerosis and viral infection.In these treatments, antibodies can have therapeutic activity by themselves, for example, by blocking receptors or messenger molecules, thereby inhibiting their disease-related functions, or by recruiting and activating components of the patient's immune system.

[0003] 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 to obtain hybridoma cells. A single hybridoma cell is then isolated by dilution cloning to generate cell clones that all produce the same monoclonal antibody.

[0004] However, in therapeutic applications, these monoclonal antibodies have the problem that 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 removes them from the circulation. Furthermore, systemic inflammatory effects can be caused. A solution to this problem is to replace certain constant regions of the monoclonal antibody with the corresponding regions of a human antibody. Replacing only the heavy and light chain constant regions results in a chimeric antibody, while additional replacement of the framework regions of the heavy and light chain variable regions results in a so-called humanized antibody.

[0005] Purified antibodies are used in many applications in research. They are most commonly used to identify and locate biomolecules, especially proteins. Biomolecules can be detected after they are isolated, 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 isolation procedures for specific biomaterials, especially proteins, where the antibody specifically separates the biomaterial of interest from a sample containing it.

[0006] In all these applications, tight binding and specific recognition of the antigen are crucial for the antibodies used, resulting in higher activity and lower cross-reactivity, and fewer adverse side effects, especially in therapeutic applications. However, when humanizing monoclonal antibodies, the affinity and specificity of the engineered antibody are often reduced.

[0007] An interesting and important group of antibodies are those directed against carbohydrate moieties. Histo-blood group glycans, in particular, are attractive targets for antibodies because they are often tumor-specific or tumor-associated antigens. For example, cells of several different epithelial cancers, including breast, bladder, colon, gastric, pancreatic, prostate, ovarian, and small cell lung cancer, express the carbohydrate structure Lewis Y [Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-], also known as LeY, which is more abundant than any other cell surface protein antigen. In each tumor, a high percentage of tumor cells, including even cancer stem cells, are LeY-positive. Therefore, these carbohydrate antigens are potential targets for tumor imaging and active or passive immunotherapy.

[0008] Antibodies against these carbohydrate structures are often associated with several problems. Undesirable side effects can occur, particularly due to the presence of identical or closely related carbohydrate structures found on normal tissues, such as the expression of H2 on erythrocytes or the presence of Lewis X on human myeloid cells, such as mature granulocytes. For example, several antibodies against LeY have failed in clinical trials because they cross-react with LeX and exhibited undesirable efficacy and / or safety profiles. In addition, cross-reactivity to other glycan epitopes is a common problem. For example, most existing antibodies that recognize Lewis Y exhibit cross-reactivity to other carbohydrate structures, such as Lewis b [Fucα1-4(Fucα1-2Galβ1-3)GlcNAcβ-], potentially impairing their therapeutic potential. Furthermore, carbohydrate antigens often generate IgM-type immune responses, which are considered to be a poorly suited antibody format for therapeutic use. Using recombinant antibody technology, class switching from IgM to IgG is possible. However, the low intrinsic affinity of carbohydrate-binding antibodies results in a significant loss of functional affinity.

[0009] 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 by chain shuffling (which contains the heavy chain of A70-A / A9 and the light chain of A70-C / C8). However, these are chimeric antibodies with murine variable regions that can cause the problems of non-human antibodies discussed above. Therefore, humanization of these antibodies would be beneficial. Unfortunately, humanized antibodies often have lower affinity and specificity for their target antigens than their non-human or chimeric counterparts. This can be achieved by altering the overall three-dimensional structure of the variable regions, particularly the conformation and orientation of the complementarity-determining regions (CDRs), through framework region replacement. This is particularly problematic for antibodies against carbohydrate antigens, which generally have low antigen affinity and specificity. For example, antibody A70-A / A9 exhibits cross-reactivity with Lewis b.

[0010] Therefore, there is a need in the art to provide humanized anti-Lewis Y antibodies with specifically high antigen-binding affinity and antigen specificity. Summary of the Invention [Means for solving the problem]

[0011] Summary of the Invention The present inventors have found humanized anti-Lewis Y antibodies that have antigen-binding affinities comparable to the parent chimeric antibodies from which they are derived. Furthermore, these humanized antibodies exhibit enhanced antigen specificity in that cross-reactivity with Lewis b of the parent chimeric antibody is prevented in the humanized version.

[0012] Thus, in a first aspect, the present invention is directed to a humanized antibody capable of binding to 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: 10 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and 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.

[0013] In a second aspect, the present invention provides a nucleic acid encoding an antibody according to the invention. Furthermore, in a third aspect, the present invention provides an expression cassette or vector comprising a nucleic acid according to the invention and a promoter operably linked to said nucleic acid, and in a fourth aspect, a host cell comprising a nucleic acid or expression cassette or vector according to the invention.

[0014] In a fifth aspect, the present invention provides a conjugate comprising an antibody according to the invention conjugated to a further agent.

[0015] In a sixth aspect, the present invention is directed to a composition comprising an antibody according to the invention, a nucleic acid according to the invention, an expression cassette or vector according to the invention, a host cell according to the invention, or a conjugate according to the invention.

[0016] According to a seventh aspect, the present invention provides an antibody, a nucleic acid, an expression cassette or vector, a host cell, a composition, or a conjugate according to the invention for use in medicine, in particular in the treatment of cancer.

[0017] Other objects, features, advantages, and aspects of the present invention will become apparent to those skilled in the art from the following description and the appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the present application, are given by way of example only. Various changes and modifications within the inventive spirit and scope of the present disclosure will become readily apparent to those skilled in the art upon reading the following.

[0018] definition As used herein, the following expressions are generally intended to have the meanings preferably set forth below, unless the context in which they are used indicates otherwise.

[0019] When used herein, the term "comprising" not only means literally, but also includes and specifically refers to the terms "essentially consisting of" and "consisting of." Thus, the term "comprising" refers to embodiments in which the subject matter "comprising" the specifically described elements does not include additional elements, as well as embodiments in which the subject matter "comprising" the specifically described elements can and / or does include additional elements. Similarly, the term "having" should be understood as the term "comprising," and is similarly understood to include and specifically refer to the terms "essentially consisting of" and "consisting of." The term "essentially consisting of" refers, where possible, to embodiments in which the subject matter, in addition to the specifically described elements that the subject matter essentially consists of, includes 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less of additional elements.

[0020] The term "antibody" specifically refers to a protein comprising at least two heavy chains and two light chains connected 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 comprises three, or in the case of IgM or IgE antibodies, four heavy chain constant domains (CH1, CH2, CH3, and CH4). The first constant domain, CH1, is adjacent to the variable region and may be connected to the second constant domain, CH2, by 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), which are separated by more conserved regions called framework regions (FRs). Each variable region contains three CDRs and four FRs. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The heavy chain constant region can be any type of constant region, such as a gamma, delta, alpha, mu, or epsilon heavy chain. Preferably, the antibody heavy chain is a gamma chain. Furthermore, the light chain constant region can also be any type of constant region, such as a kappa or lambda light chain. Preferably, the antibody light chain is a kappa chain. The terms "gamma (delta, alpha, mu, or epsilon) heavy chain" and "kappa (lambda) light chain" refer to antibody heavy chains or antibody light chains, respectively, having constant region amino acid sequences derived from naturally occurring heavy or light chain constant region amino acid sequences, particularly human heavy or light chain constant region amino acid sequences. In particular, the amino acid sequence of the constant domain of a gamma (particularly gamma 1) heavy chain is at least 95%, particularly at least 98%, identical to the amino acid sequence of the constant domain of a human gamma (particularly one of the human gamma 1 allotypes) antibody heavy chain. Furthermore, the amino acid sequence of the constant domain of a kappa 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 human kappa antibody light chain allotypes. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibody can be, for example, a humanized, human, or chimeric antibody.

[0021] Antigen-binding portion of antibody generally refers to the whole antibody or one or more fragments that retain the specific binding ability with antigen.It has been shown that the antigen-binding function of antibody can be performed by fragments of whole antibody.Examples of antibody binding fragments include Fab fragment, i.e., monovalent fragment consisting of VL, VH, CL and CH1 domain; F(ab)2 fragment, i.e., bivalent fragment that contains two Fab fragments that each bind to the same antigen and are linked by disulfide bridges at hinge region; Fd fragment consisting of VH and CH1 domain; Fv fragment consisting of VL and VH domain of single arm of antibody; and dAb fragment consisting of VH domain.

[0022] The "Fab portion" of an antibody refers to a portion of the antibody that contains, in particular, the first domains of the heavy and light chain variable regions (VH and VL) and the heavy and light chain constant regions (CH1 and CL). If an antibody does not contain all of these regions, the term "Fab portion" refers only to the portions of the VH, VL, CH1, and CL regions present in the antibody. Preferably, the "Fab portion" refers to that portion of an antibody that corresponds to a fragment containing the antigen-binding activity of the antibody, obtained by digesting a natural antibody with papain. In particular, the Fab portion of an antibody encompasses its antigen-binding site or antigen-binding ability. Preferably, the Fab portion comprises at least the VH, VL, CH1, and CL regions of the antibody. H Includes the area.

[0023] The "Fc portion" of an antibody refers in particular to the portion of the antibody comprising heavy chain constant regions 2, 3, and, if applicable, 4 (CH2, CH3, and CH4). In particular, the Fc portion comprises two of each of these regions. If an antibody does not comprise all of these regions, the term "Fc portion" refers only to the portions of the CH2, CH3, and CH4 regions present in the antibody. Preferably, the Fc portion comprises at least the CH2 region of the antibody. Preferably, the "Fc portion" refers to that portion of an antibody corresponding to a fragment not containing the antigen-binding activity of the antibody, obtained by digesting a native antibody with papain. In particular, the Fc portion of an antibody is capable of binding to an Fc receptor and thus comprises, for example, an Fc receptor binding site or Fc receptor binding ability.

[0024] In accordance with the present invention, the term "chimeric antibody" refers in particular to an antibody in which the constant regions are derived from a human antibody or human antibody consensus sequence, and at least one, and preferably both, variable regions are derived from a non-human antibody, such as a rodent antibody, e.g., a murine antibody.

[0025] According to the present invention, the term "humanized antibody" refers to a non-human antibody comprising human constant and variable regions whose amino acid sequences have been modified to reduce the immunogenicity of the antibody, particularly when administered to a human body. An exemplary method for constructing a humanized antibody is CDR grafting, in which the CDRs or specificity-determining residues (SDRs) of a non-human antibody are combined with framework regions of human origin. If necessary, some residues in the human framework regions may be backmutated relative to those of the parent non-human antibody, e.g., to increase or restore antigen-binding affinity. Other humanization methods include, for example, resurfacing, superhumanization, and human string content optimization. In the resurfacing method, only those residues in the non-human framework regions located on the surface of the antibody are replaced with residues present in the corresponding human antibody sequence at said positions. Superhumanization essentially corresponds to CDR grafting. However, while in CDR grafting, the human framework regions are usually selected based on their homology with the non-human framework regions, in superhumanization, the human framework regions are selected based on the similarity of the CDRs. In human string content optimization, the differences between a non-human antibody sequence and a human germline sequence are scored, and the antibody is then mutated to minimize the score. Furthermore, humanized antibodies can also be obtained by empirical methods in which multiple antibody humanization candidates are generated using human framework regions or large libraries of human antibodies, and the most promising candidates are then determined by screening methods. Similarly, several humanized antibody candidates can be generated by the above-described rational approach and then screened, for example, for their antigen binding.

[0026] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin.

[0027] The term "antibody," as used herein, in certain embodiments, refers to a population of antibodies of the same type. In particular, all antibodies in a population of antibodies exhibit the characteristics used to define the antibody. In certain embodiments, all antibodies in a population of antibodies have the same amino acid sequence. Reference to a particular type of antibody, such as an anti-Lewis Y antibody, specifically refers to a population of antibodies of that type.

[0028] The term "antibody", as used herein, also includes fragments and derivatives of said antibody. A "fragment or derivative" of an antibody is a protein or glycoprotein that is derived in particular from said antibody and is capable of binding to the same antigen, in particular the same epitope as the antibody. Thus, a fragment or derivative of an antibody herein generally refers to a functional fragment or derivative. In a particularly preferred embodiment, the antibody fragment or derivative comprises a heavy chain variable region. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody or its derivative. Examples of antibody fragments include (i) Fab fragments, i.e., monovalent fragments consisting of the variable regions of each heavy and light chain and the first constant domain; (ii) F(ab)2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the variable region of the heavy chain and the first constant domain, CH1; (iv) Fv fragments consisting of the heavy and light chain variable regions of a single arm of an antibody; (v) scFv fragments, i.e., Fv fragments consisting of a single polypeptide chain; (vi) (Fv)2 fragments consisting of two Fv fragments covalently linked together; (vii) heavy chain variable domains; and (viii) multibodies consisting of heavy and light chain variable regions covalently linked together such that association of the heavy and light chain variable regions can occur only intermolecularly, not intramolecularly. Antibody derivatives specifically include antibodies that bind to the same antigen as the parent antibody but have amino acid sequences different from those of the parent antibody from which they are derived. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.

[0029] A target amino acid sequence is "derived from" or "corresponding to" a reference amino acid sequence if the target amino acid sequence shares at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the corresponding portion of the reference amino acid sequence over its entire length. A "corresponding portion" means, for example, that framework region 1 (FRH1) of the heavy chain variable region of a target antibody corresponds to framework region 1 of the heavy chain variable region of a reference antibody. In certain embodiments, a target amino acid sequence "derived from" or "corresponding to" a 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 an amino acid sequence or nucleotide sequence is determined in accordance with the present invention, preferably over the entire length of the reference sequence or over the entire length of the corresponding portion of the reference sequence to which homology or identity is defined. Antibodies derived from a parent antibody defined by one or more amino acid sequences, e.g., particularly particular CDR sequences or particular variable region sequences, are antibodies having amino acid sequences, e.g., CDR sequences or variable region sequences, that are at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homologous or identical, particularly identical, to the respective amino acid sequences of the parent antibody. In certain embodiments, antibodies derived from (i.e., are derivatives of) a parent antibody contain the same CDR sequences as the parent antibody, but differ in the sequence of the remainder of the variable region.

[0030] The term "antibody", as used herein, also refers to multivalent and multispecific antibodies, i.e., antibody constructs having more than two binding sites that each bind to the same epitope, as well as antibody constructs having one or more binding sites that bind to a first epitope and one or more binding sites that bind to a second epitope, and optionally even further binding sites that bind to additional epitopes.

[0031] "Specific binding" preferably means that an agent, such as an antibody, binds more strongly to a target, such as an epitope, for which it is specific compared to binding to another target. An agent binds more strongly to a target, such as an epitope, with a dissociation constant (K) that is lower than the dissociation constant for a second target. d ), an agent binds more strongly to a first target compared to a second target. Preferably, the dissociation constant of a target to which an agent specifically binds is 100-fold lower, 200-fold lower, 500-fold lower, or 1000-fold lower than the dissociation constant of a target to which the agent does not specifically bind. Furthermore, the term "specifically binds" particularly refers to a dissociation constant of at least 10 5 M -1 , preferably at least 10 6 M -1 , more preferably at least 10 7 M -1 , e.g. at least 10 8 M -1 The affinity constant K a Antibodies that are specific for a particular antigen are those that have a binding affinity of at least 10 5 M -1 , preferably at least 10 6 M -1 , more preferably at least 10 7 M -1 K a For example, the term "anti-Lewis Y antibody" refers to an antibody that specifically binds to Lewis Y, and preferably has a binding affinity of at least 10 5 M -1 , preferably at least 10 6 M -1 , more preferably at least 10 7 M -1 K a It refers to an antibody that is capable of binding to Lewis Y with an affinity having

[0032] The term "A70-A / A9" as used herein specifically refers to a human / mouse chimeric antibody having the heavy and light chain variable region amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 31, respectively.

[0033] The term "Lewis Y" or "LeY" according to the present invention refers in particular to the carbohydrate structure (or oligosaccharide) Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-, which may in particular be attached to a support 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" each refer to the linkage of two adjacent monosaccharide residues, in particular the linkage between the carbon atom C1 of the left monosaccharide and the carbon atom C2, C4, or C3 of the right monosaccharide, where the linkage at the carbon atom C1 can be in the α- or β-position (as shown in the following scheme for glucose): [ka]

[0034] The term "GlcNAcβ-" indicates that the GlcNAc residue at the reducing end of the Lewis Y oligosaccharide is linked to the supporting structure in the β configuration. A schematic representation of the structure of Lewis Y and related blood group antigens is shown in Figure 1.

[0035] Lewis Y is a carbohydrate antigen of the human blood group system. It is expressed in many tumors and can be used as a tumor-associated or tumor-specific antigen for targeted cancer therapy.

[0036] The term "sialic acid" refers in particular to any N- or O-substituted derivative of neuraminic acid. It can refer to both 5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid, but preferably refers only to 5-N-acetylneuraminic acid.

[0037] The terms "glycan," "glycan structure," "carbohydrate," "glycan chain," and "carbohydrate structure" are generally used interchangeably herein.

[0038] In a "conjugate," two or more compounds are linked together. In certain embodiments, at least some of the properties of each compound are retained in the conjugate. Linkage can be achieved by covalent or non-covalent bonding. Preferably, the compounds of the conjugate are linked by covalent bonds. The different compounds of the conjugate can be directly linked to each other through one or more covalent bonds between atoms of the compounds. Alternatively, the compounds can be linked to each other through chemical moieties such as linker molecules, where the linker is covalently bonded to an atom of the compound. When a conjugate is composed of more than two compounds, the compounds can be linked, for example, in a chain conformation, where one compound is linked to the next compound, or there can be several compounds, each of which can be linked to one central compound.

[0039] The term "nucleic acid" includes single- and double-stranded nucleic acids and ribonucleic acids and deoxyribonucleic acids, which may contain naturally occurring as well as synthetic nucleotides and can be modified naturally or synthetically, for example, by methylation, 5'-capping and / or 3'-capping.

[0040] The term "expression cassette" refers specifically to a nucleic acid construct capable of enabling and regulating the expression of a coding nucleic acid sequence introduced therein. Expression cassettes may contain promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The exact structure of an expression cassette may vary depending on the species or cell type, but generally includes 5'-untranscribed and 5'- and 3'-untranslated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. More specifically, 5'-untranscribed expression control sequences include promoter regions containing promoter sequences for transcriptional control of operably linked nucleic acids. Expression cassettes may also include enhancer sequences or upstream activator sequences.

[0041] According to the present invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of a nucleic acid sequence to be expressed, which controls the expression of the sequence by providing a recognition and binding site for RNA polymerase. A "promoter" may contain additional recognition and binding sites for additional factors involved in regulating gene transcription. A promoter may control the transcription of prokaryotic or eukaryotic genes. Furthermore, a promoter may be "inducible," i.e., capable of initiating transcription in response to an inducing agent, or "constitutive," in which transcription is not controlled by an inducing agent. A gene under the control of an inducible promoter is not expressed, or is expressed only to a small extent, in the absence of an inducing agent. In the presence of an inducing agent, the gene is switched on, or transcription levels are increased, which is generally mediated by the binding of specific transcription factors.

[0042] The term "vector" is used herein in its most general sense and includes any intermediate vehicle for nucleic acid that allows said nucleic acid to be introduced into, for example, prokaryotic and / or eukaryotic cells and, if necessary, integrated into the genome. This type of vector is preferably replicated and / or expressed in the cell. Vectors include plasmids, phagemids, bacteriophages, or viral genomes. The term "plasmid," as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex that can replicate independently of chromosomal DNA.

[0043] According to the present invention, the term "host cell" relates to any cell that can be transformed or transfected with an exogenous nucleic acid. According to the present invention, the term "host cell" includes prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., mammalian cells, particularly human or hamster cells, yeast cells, and insect cells). Mammalian cells, such as human, mouse, hamster, pig, goat, or primate cells, are particularly preferred. Cells may be derived from multiple tissue types and may include primary cells and cell lines. The nucleic acid may be present in the host cell in the form of a single copy or in the form of two or more copies, and in one embodiment is expressed in the host cell.

[0044] The term "patient" according to the present invention means a human, a non-human primate, or another animal, in particular a mammal, such as a cow, horse, pig, sheep, goat, dog, cat, or rodent, such as a mouse or rat. In a particularly preferred embodiment, the patient is a human.

[0045] The term "cancer" according to the present invention particularly 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, as well as metastases thereof. 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 mentioned above.

[0046] "Tumor" means a group of cells or tissues formed by misregulated cell growth. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissue and usually form a palpable mass of tissue that can be either benign or malignant.

[0047] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process, usually involving the detachment of cancer cells from the primary tumor, their entry into the systemic circulation, and their establishment and growth in normal tissue 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 of the original tumor. This means, for example, that if breast cancer metastasizes to the lung, the secondary tumor will be made up of abnormal breast cells, not abnormal lung cells. Thus, a lung tumor would be called metastatic breast cancer, not lung cancer.

[0048] The term "pharmaceutical composition" refers to a composition suitable for administration, particularly to humans or animals, i.e., a composition containing pharmaceutically acceptable components. Preferably, a pharmaceutical composition comprises an active compound or a salt or prodrug thereof together with a carrier, diluent, or pharmaceutical excipient, such as a buffer, a preservative, and a tonicity adjuster. (Mode for Carrying Out the Invention)

[0049] Detailed Description of the Invention The present invention is based on the development of humanized anti-Lewis Y antibodies with antigen-binding affinities similar to those of the corresponding chimeric antibodies. The inventors were further able to demonstrate that the humanized antibodies unexpectedly have improved antigen specificity. In addition to its primary specificity for Lewis Y, the parent chimeric antibody exhibits significant binding to Lewis b. Such cross-reactivity is common for antibodies that bind to carbohydrate antigens, since these antigens can be quite small and very similar. For example, Lewis Y and Lewis b differ only in that the attachment points of the fucose arm and the fucose-galactose arm are switched at the central GlcNAc residue (see Figure 1). Nevertheless, the humanized anti-Lewis Y antibodies provided by the present invention surprisingly do not exhibit any cross-reactivity with other carbohydrate antigens, and in particular, no cross-reactivity with Lewis b.

[0050] In view of these findings, the present invention provides a humanized antibody capable of binding to 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: 10 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and 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.

[0051] Furthermore, a humanized antibody may exhibit antigen-binding properties similar to those of a reference antibody comprising 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 the human / mouse chimeric antibody A70-A / A9. In particular, a humanized antibody according to the present invention may specifically bind to the same antigen as the reference antibody, preferably with comparable affinity. That is, the humanized antibody preferably binds to the antigen with an affinity having a dissociation constant that is at most 1000-fold higher, more preferably at most 200-fold higher, at most 100-fold higher, at most 20-fold higher, or at most 10-fold higher than that of the reference antibody. Most preferably, the dissociation constant is approximately the same as that of the reference antibody, particularly no more than 2-fold higher. Furthermore, a humanized antibody preferably exhibits cross-reactivity with a reference antibody comprising 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, a humanized antibody, when present in a sufficiently high concentration, can block the binding of a reference antibody to Lewis Y. This can occur if a humanized antibody according to the invention already binds to Lewis Y, thereby preventing the binding of the reference antibody to Lewis Y.

[0052] In certain embodiments, the humanized antibody specifically binds to Lewis Y. In particular, the humanized antibody does not exhibit significant binding affinity for Lewis b and / or does not specifically bind to Lewis b. In certain embodiments, the dissociation constant of the humanized antibody for binding to Lewis Y is at least 10-fold lower than the dissociation constant of the humanized antibody for binding to Lewis b. In particular, the dissociation constant is at least 20-fold, at most 50-fold, or at most 100-fold lower for binding to Lewis Y compared to binding to Lewis b.

[0053] In certain embodiments, 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: 10. In particular, 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: 10.

[0054] In certain embodiments, the heavy chain variable region of the humanized antibody comprises complementarity determining regions CDR-H1 having the amino acid sequence of SEQ ID NO: 12 or 13, CDR-H2 having the amino acid sequence of SEQ ID NO: 14 or 15, and CDR-H3 having the amino acid sequence of SEQ ID NO: 16. In particular, 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 has three specific CDRs having the amino acid sequences of SEQ ID NOs: 12, 14, and 16, or SEQ ID NOs: 13, 15, and 16. Thus, any sequence deviations relative to SEQ ID NO: 10 are located in the framework regions but not in the CDRs.

[0055] In certain 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. In particular, 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. In particular, 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 certain embodiments, 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 NOs: 12, 14, and 16.

[0056] Specifically, the humanized antibody may comprise a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 9. In particular, the heavy chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6, particularly SEQ ID NO: 1 or 4, preferably SEQ ID NO: 1. In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical, particularly at least 93%, at least 95%, or particularly at least 98% identical to the amino acid sequence of SEQ ID NO: 1. In these embodiments, the heavy chain variable region preferably has three specific CDRs having the amino acid sequences of SEQ ID NOs: 12, 14, and 16.

[0057] In certain 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. In particular, 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.

[0058] In certain embodiments, the light chain variable region of the humanized antibody comprises complementarity determining regions CDR-L1 having the amino acid sequence of SEQ ID NO: 24 or 25, CDR-L2 having the amino acid sequence of SEQ ID NO: 26 or 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28 or 29. In particular, 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 has three specific CDRs having the amino acid sequences of SEQ ID NOs: 24, 26, and 28, or SEQ ID NOs: 25, 27, and 29. Thus, any sequence deviations from SEQ ID NO: 22 are located in the framework regions but not in the CDRs.

[0059] In certain 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. In particular, 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. In particular, 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 certain embodiments, 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 NOs: 24, 26, and 28.

[0060] Specifically, the humanized antibody may comprise a light chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 17 to 21. In particular, the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 17 or 18, particularly SEQ ID NO: 17. In certain embodiments, 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: 17, particularly an amino acid sequence that is at least 93%, at least 95%, or particularly at least 98% identical to the amino acid sequence of SEQ ID NO: 17.

[0061] In certain embodiments, the humanized antibody has a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and additionally having three specific CDRs having the amino acid sequences of SEQ ID NOs: 12, 14, and 16, or SEQ ID NOs: 13, 15, and 16; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 22, and additionally having three specific CDRs having the amino acid sequences of SEQ ID NOs: 24, 26, and 28, or SEQ ID NOs: 25, 27, and 29. In certain 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.

[0062] In certain embodiments, the humanized antibody has a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:11, and additionally having three specific CDRs having the amino acid sequences of SEQ ID NOs:12, 14, and 16; and a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:23, and additionally having three specific CDRs having the amino acid sequences of SEQ ID NOs:24, 26, and 28. In certain 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.

[0063] In certain embodiments, the humanized antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7 and 9, particularly 1-6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 or 18, particularly 17. In particular, the humanized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17. In particular, the humanized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17. In other embodiments, the humanized antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19.

[0064] In a preferred embodiment, the humanized antibody comprises an Fc region. The humanized antibody may be a whole antibody. The humanized antibody may be of any isotype, particularly an IgG antibody, particularly an IgG1, IgG2, or IgG4. In a specific embodiment, the humanized antibody is an IgG1 antibody. The humanized antibody may specifically bind to one or more human Fc receptors, particularly human Fcγ receptors, such as Fcγ receptor IIIa.

[0065] In further embodiments, the humanized anti-Lewis Y antibody is a fragment of an antibody. In particular, the fragment is selected from the group consisting of: (i) a Fab fragment; (ii) an F(ab)2 fragment; (iii) an Fd fragment; (iv) an Fv fragment; (v) an scFv fragment; and (vi) an (Fv)2 fragment. In certain embodiments, the humanized anti-Lewis Y antibody does not comprise an Fc region.

[0066] In certain embodiments, the humanized anti-Lewis Y antibody is glycosylated, particularly N-glycosylated. In particular, humanized antibodies have a glycosylation site in the second constant domain (CH2) of the heavy chain. Antibodies usually have two heavy chains with identical amino acid sequences. Therefore, humanized antibodies preferably have at least two glycosylation sites, one in each of their two CH2 domains. This glycosylation site is particularly at the amino acid position corresponding to amino acid position 297 of the heavy chain according to Kabat numbering and has the amino acid sequence motif Asn Xaa Ser / Thr, where Xaa can be any amino acid except proline. N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of 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 acid sequences that may be present in the variable regions or other sequence modifications.

[0067] In a preferred embodiment, the humanized anti-Lewis Y antibody does not contain N-glycolylneuraminic acid (NeuGc) or detectable amounts of NeuGc. Furthermore, the humanized antibody preferably does not contain Galili epitopes (Galα1,3-Gal structures) or detectable amounts of Galili epitopes. In particular, the relative amount of glycans having 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 portion of humanized antibodies in the antibody population.

[0068] In other embodiments, the humanized anti-Lewis Y antibody is not glycosylated in its CH2 domain. In these embodiments, the CH2 domain of the antibody may be mutated, for example, by substituting the asparagine residue at position 297 (or a corresponding position) of the heavy chain with any other amino acid, such as alanine or glutamine. Antibodies lacking glycosylation in the CH2 domain have reduced binding to Fcγ receptors and thus reduced 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.

[0069] The humanized anti-Lewis Y antibody is preferably produced recombinantly in a host cell. Thus, the humanized antibody is particularly a monoclonal antibody. The host cell used to produce the humanized antibody can be any host cell that can be used 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 EB66 duck cell lines, rodent cells, such as CHO, NS0, SP2 / 0, and YB2 / 0 cell lines, and human cells, such as HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz-3, and KG1 cell lines.

[0070] In certain embodiments, the humanized anti-Lewis Y antibody is recombinantly produced in a human cell line, particularly a human myeloid leukemia cell line. Preferred human cell lines that can be used to produce anti-Lewis Y antibodies as well as suitable production procedures are described in WO2008 / 028686A2. In certain embodiments, the humanized anti-Lewis Y antibody is obtained by expression in a human myeloid leukemia cell line selected from the group consisting of NM-H9D8, NM-H9D8-E6, and NM-H9D8-E6Q12. These cell lines have been deposited in accordance with the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstrasse 7B, 38124 Braunschweig (DE) by Glycotope GmbH, Robert-Roessle-Str. 10, 13125 Berlin (DE) under accession numbers DSM ACC2806 (NM-H9D8; deposited September 15, 2006), DSM ACC2807 (NM-H9D8-E6; deposited October 5, 2006), and DSM ACC2856 (NM-H9D8-E6Q12; deposited August 8, 2007). NM-H9D8 cells provide a glycosylation pattern with a high degree of sialylation, a high degree of bisecting GlycNAc, a high degree of galactosylation, and a high degree of fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells provide a glycosylation pattern similar to that of NM-H9D8 cells, except that the degree of fucosylation is much lower. Other suitable cell lines include K562, a human myeloid leukemia cell line present at the American Type Culture Collection (ATCC CCL-243), CHO cells, and cell lines derived therefrom. In a specific embodiment, the humanized anti-Lewis Y antibody is expressed in CHO cells, particularly CHO dhfr - It is produced recombinantly in cells.

[0071] In certain embodiments, the humanized anti-Lewis Y antibody is provided as a conjugate comprising the antibody conjugated to an additional agent, such as a detectable marker or a therapeutically active substance. The humanized antibody can be conjugated to one or more additional agents. When more than one additional agent is present in the conjugate, these additional agents can be the same or different, and particularly all identical. The conjugation of the additional agent to the humanized antibody can be achieved using any method known in the art. The additional agent can be covalently bound to the antibody, particularly by fusion or chemical coupling, or non-covalently. In certain embodiments, the additional agent is covalently bound to the humanized antibody, particularly via a linker moiety. The linker moiety can be any chemical entity suitable for binding the additional agent to the humanized antibody.

[0072] The additional agent is preferably useful in the treatment, diagnosis, prognosis, and / or monitoring of a disease, particularly cancer. For example, the additional agent may be selected from the group consisting of a radionuclide, a chemotherapeutic agent, an antibody, a bispecific antibody or antibody fragment, particularly an antibody or antibody fragment of a different species and / or of a different specificity than the humanized anti-Lewis Y antibody, an enzyme, an interacting domain, a detectable label, a toxin, a cytolytic component, an immunomodulator, an immune effector, a cytokine, a chemokine, an MHC class I or class II antigen, and a liposome.

[0073] In certain embodiments, the additional agent is a polypeptide or protein. This polypeptide or protein may be fused to a polypeptide chain, particularly a humanized antibody. In certain embodiments, the additional agent is a polypeptide or protein, 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 additional agent is a polypeptide or protein, fused to the C-terminus of each of the two antibody light chains. In further embodiments, the additional agent is a polypeptide or protein, 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 additional agent is a polypeptide or protein, fused to the C-terminus of each of the two antibody heavy chains. The additional agents may 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, in embodiments where the humanized antibody is an antibody fragment, the additional agent is a polypeptide or protein, fused to the C-terminus or N-terminus of the polypeptide chain of the humanized antibody. Suitable examples of such additional agents that are polypeptides or proteins may be selected from the group consisting of cytokines, chemokines, antibodies, antigen-binding fragments, enzymes, and interaction domains.

[0074] In certain embodiments, the additional polypeptide or protein agent is a checkpoint antibody that blocks and / or induces activation signals. Examples of the respective 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 inhibitory targets, and their respective ligands, such as PDL1. In further embodiments, the additional polypeptide or protein agent is an anti-cancer antibody against a tumor-associated antigen. Exemplary suitable tumor targets and anti-cancer antibodies that can be used as fusion partners are described below in relation to combination therapy.

[0075] In further embodiments, the additional agent that is a polypeptide or protein is an immunomodulatory compound, such as a chemokine, cytokine, or growth factor. Suitable cytokines in this regard include interferons, such as interferon-α, interferon-β, and interferon-γ, and interleukins, such as IL-15. Suitable growth factors include G-CSF and GM-CSF.

[0076] In certain embodiments, the additional agent, which is a polypeptide or protein, is an antigen receptor, particularly a T cell receptor, or a T cell co-receptor, or a part and / or chimera thereof. In particular, a humanized anti-Lewis Y antibody is fused to a transmembrane domain and an intracellular T cell signaling domain to form a chimeric antigen receptor (CAR). The intracellular domain is particularly derived from one or more T cell receptors or co-receptors. Optionally, the CAR further comprises a hinge region between the humanized antibody and the transmembrane domain.

[0077] In these embodiments, the humanized anti-Lewis Y antibody is a single-chain antibody fragment, particularly an scFv fragment, comprising a heavy chain variable region and a light chain variable region in one polypeptide chain. The hinge region may be based on, for example, the hinge region or membrane proximal region of a member of the immunoglobulin superfamily. Exemplary hinge regions include those derived from IgG, CD8, and CD28. The transmembrane domain may be a hydrophobic alpha helix spanning the cell membrane, such as that derived from CD28. The intracellular T cell signaling domain may particularly comprise the cytoplasmic domain of the zeta chain of the T cell receptor. In addition, the intracellular T cell signaling domain may comprise an additional domain of a T cell costimulatory protein. Exemplary additional domains include signaling domains derived from CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0078] An exemplary CAR may comprise, from N- to 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 zeta chain.

[0079] Alternatively, a humanized anti-Lewis Y antibody, particularly in a single-chain format such as an scFv, can be fused at its N-terminus to the CD3 chain of the T cell receptor complex, particularly the CD3ε chain, to form a chimeric antigen receptor, or a humanized anti-Lewis Y antibody, particularly in a single-chain format such as an scFv, can be fused to a binding domain capable of specifically binding to a naturally occurring or engineered receptor, particularly on T cells or NK cells.

[0080] In certain embodiments, the additional agent is a cytotoxic agent or chemotherapeutic agent, particularly a cytotoxin. Specific examples of chemotherapeutic agents that can be conjugated as additional agents include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxins, taxanes such as taxol, topoisomerase inhibitors such as irinotecan and topotecan, anti-neoplastic agents such as doxorubicin, or microtubule inhibitors such as auristatins and maytansine / maytansinoids.

[0081] The chemotherapeutic agent may in particular be selected from the group consisting of V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansines, maytansinoids, amatoxins, methionine aminopeptidases, inhibitors of protein nuclear transport CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of mitochondrial phosphoryl transfer reactions, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, inhibitors of microtubule formation, microtubule stabilizers, actin stabilizers, topoisomerase II inhibitors, platinum compounds, ribosome inhibitors, RNA polymerase II inhibitors, and bacterial toxins. In certain embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is selected from the group consisting of an auristatin, a maytansinoid, a topoisomerase I inhibitor, a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binder.

[0082] In some embodiments, the chemotherapeutic agent is maytansine or a maytansinoid. Specific examples of maytansinoids useful for conjugation include maytansinol, N 2’ -Deacetyl-N 2’-(3-mercapto-1-oxopropyl)-maytansine (DM1), N 2’ -Deacetyl-N 2’ -(4-mercapto-1-oxopentyl)-maytansine (DM3), and N 2’ -Deacetyl-N 2’and -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). In particular, DM1 or DM4 is conjugated to the anti-LeY antibody. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is an auristatin, particularly monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), or auristatin T. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a DNA minor groove binder, particularly a pyrrolobenzodiazepine (PBD), a pyrrolobenzodiazepine dimer (PBD dimer), a duocarmycin, a duocarmycin-hydroxybenzamide-azaindole (DUBA), a seco-duocarmycin-hydroxybenzamide-azaindole (seco-DUBA), or doxorubicin. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a DNA alkylating agent, particularly an indolinobenzodiazepine or an oxazolidinobenzodiazepine. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a DNA damaging agent, particularly calicheamicin. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a topoisomerase I inhibitor, particularly camptothecin and its derivatives, such as 7-ethyl-10-hydroxy-camptothecin (SN-38), (S)-9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan), (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (exatecan (DX-8951f)), and DXd. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a microtubule formation inhibitor, particularly tubulysin, ansamitocin, podophyllotoxin, or vinblastine. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a microtubule stabilizer, particularly paclitaxel or epothilone. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is an actin stabilizer, particularly phallotoxin.In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a topoisomerase II inhibitor, particularly teniposide, XK469, razoxane, amsacrine, idarubicin, or mevalon. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a platinum compound, particularly cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a ribosome inhibitor, particularly ricin, saporin, abrin, diphtheria toxin, or exotoxin A. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is an RNA polymerase II inhibitor, particularly an amatoxin, such as amanitin. In some embodiments, the chemotherapeutic agent conjugated to the anti-LeY antibody is a bacterial toxin, particularly anthrax toxin. Suitable antibody drug conjugates are also described in EP 16 151 774.3, and LU 92659, to which express reference is made herein.

[0083] Further suitable toxins that can be conjugated to humanized anti-Lewis Y antibodies are described below with respect to combination therapy.

[0084] In a further aspect, the present invention provides a nucleic acid encoding a humanized anti-Lewis Y antibody. The nucleic acid sequence of the nucleic acid can have any nucleotide sequence suitable for encoding an antibody. Preferably, however, the nucleic acid sequence is at least partially compatible with the specific codon usage of the host cell or organism in which the nucleic acid is expressed, particularly human codon usage. The nucleic acid can be double-stranded or single-stranded DNA or RNA, preferably double-stranded DNA, e.g., cDNA, or single-stranded RNA, e.g., mRNA. It can be one continuous nucleic acid molecule or can be composed of several nucleic acid molecules, each encoding a different portion of the humanized antibody.

[0085] When a humanized antibody is composed of more than one different amino acid chain, e.g., a light chain and a heavy chain, the nucleic acid can be a single nucleic acid molecule containing several coding regions each encoding one of the antibody amino acid chains, preferably separated by regulatory elements such as an IRES element to generate separate amino acid chains, or the nucleic acid can be composed of several nucleic acid molecules, each containing one or more coding regions each encoding one of the antibody amino acid chains. Alternatively, the nucleic acid can be a single nucleic acid molecule containing one coding region encoding the heavy and light chains separated by a linker peptide containing a self-cleaving peptide, e.g., a 2A peptide and / or a protease recognition site, such as a furin recognition site. In addition to the coding region encoding the humanized antibody, the nucleic acid can contain additional nucleic acid sequences or other modifications that, for example, may encode other proteins, affect the transcription and / or translation of the coding region(s), affect the stability or other physical or chemical properties of the nucleic acid, or may have no function at all.

[0086] In certain embodiments, the nucleic acid is a viral vector that can be used to infect human cells. These viral vectors can be suitable for human therapy, for example, by directing viral infection and / or replication to diseased cells, such as tumor cells, or by modifying T cells, in embodiments where the humanized antibody is in the form of a chimeric antigen receptor to obtain CAR T cells.

[0087] In a further aspect, the present invention provides an expression cassette or vector comprising a nucleic acid according to the present invention and a promoter operably linked to said nucleic acid.In addition, the expression cassette or vector may comprise further elements, in particular elements capable of influencing and / or regulating the transcription and / or translation of the nucleic acid, the amplification and / or replication of the expression cassette or vector, the integration of the expression cassette or vector into the genome of the host cell, and / or the copy number of the expression cassette or vector in the host cell.Suitable expression cassettes and vectors, including the respective expression cassettes for expressing antibodies, are well known in the art and therefore need not be described in detail herein.

[0088] The present invention further provides a host cell comprising a nucleic acid according to the present invention or an expression cassette or vector according to the present invention. The host cell can be any host cell. It can be an isolated cell or a cell contained in a tissue. Preferably, the host cell is a cultured cell, particularly a primary cell or a cell of an established cell line, preferably a tumor-derived cell. Preferably, the host cell is a bacterial cell, such as E. coli, a yeast cell, such as a Saccharomyces cell, particularly S. cerevisiae, an insect cell, such as Sf9 cell, or a mammalian cell, particularly a human cell, such as a tumor-derived human cell, a hamster cell, such as a CHO cell, or a primate cell. In a preferred embodiment of the present invention, the host cell is derived from a human myeloid leukemia cell. Preferably, the host cell is selected from the following cells or cell lines: K562, KG1, MUTZ-3, CHO, or a cell or cell line derived therefrom, or a mixture of cells or cell lines comprising at least one of the above cells. The host cell is preferably selected from the group consisting of NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, and cells or cell lines derived from any one of the aforementioned host cells, or a mixture of cells or cell lines comprising at least one of the aforementioned cells. These cell lines and their characteristics are described in detail in PCT application WO2008 / 028686A2. In a preferred embodiment, the host cell is optimized for the expression of glycoproteins, particularly antibodies with specific glycosylation patterns. Preferably, the codon usage in the coding region of the nucleic acid and / or promoter and additional elements of the expression cassette or vector according to the present invention is compatible with, and more preferably optimized for, the type of host cell used. Preferably, the humanized antibody is produced by the above-mentioned host cell or cell line.

[0089] In another aspect, the present invention provides a composition comprising a humanized antibody, a nucleic acid, an expression cassette or vector, a host cell, or a conjugate. The composition may also contain more than one of these components. Furthermore, the composition may contain one or more additional components selected from the group consisting of solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, all components of the composition are preferably pharmaceutically acceptable. The composition may be a solid or fluid composition, particularly preferably an aqueous solution, emulsion, or suspension, or a lyophilized powder.

[0090] Humanized anti-Lewis Y antibodies or conjugates thereof are particularly useful in medicine, particularly in the treatment, diagnosis, prognosis, and / or monitoring of diseases, particularly those diseases described herein, such as cancer and infectious diseases, preferably cancer. Thus, in a further aspect, the present invention provides a humanized antibody, nucleic acid, expression cassette or vector, host cell, conjugate, or composition for use in medicine. Preferably, the use in medicine is in 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 include particularly viral and bacterial infections, particularly infections caused by viruses or bacteria that have Lewis Y on their surface. Exemplary infectious diseases include infections caused by Helicobacter bacteria.

[0091] In a preferred embodiment, the disease is cancer, particularly epithelial cancer, especially advanced epithelial cancer. Preferably, the cancer is selected from the group consisting of lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, gastric 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.

[0092] In certain embodiments, the disease to be treated is a disease associated with abnormal cell growth, such as cancer. The cancer is Lewis Y positive, and in particular comprises cancer cells that have Lewis Y on their cell surface. In certain embodiments, the humanized anti-Lewis Y antibody is used in combination with another anti-cancer therapeutic agent. The additional therapeutic agent may be any known anti-cancer drug, and in particular an antibody against a cancer antigen. The humanized anti-Lewis Y antibody Suitable antibodies for combination with the Y antibody include anti-EGFR antibodies, such as cetuximab (Erbitux), tomzotuximab, panitumomab (Vectibix), and nimotuzumab (Theraloc), anti-HER2 antibodies, such as trastuzumab (Herceptin), timigituzumab, and pertuzumab; anti-VEGF antibodies, such as bevacizumab (Avastin) and vanuzizumab; anti-CD52 antibodies, such as alemtuzumab (Campath); anti-CD30 antibodies, such as brentuximab (Adcetris); anti-CD33 antibodies, such as gemtuzumab (Mylotarg); anti-CD20 antibodies, such as rituximab (Ritux). These include mabs (Rituxan, Mabthera), tositumomab (Bexxar), and ibritumomab (Zevalin); anti-CTLA4 antibodies such as ipilimumab and tremelimumab, anti-PD1 / PD-L1 antibodies such as pembrolizumab, nivolumab, atezolizumab, and avelumab, antibodies against TNF and TNFR superfamily members such as urelumab, MEDI6469, TRX518, and valilumab; CSF1R antibodies such as emactuzumab; anti-B7-H3 antibodies such as enoblitutuzumab; anti-LAG3 antibodies; anti-4-1BB antibodies; anti-ICOS antibodies; and anti-OX-40 antibodies.

[0093] Additional anti-cancer therapeutic agents that may be combined with the humanized anti-Lewis Y antibody and, optionally, one or more additional antibodies include taxanes, such as paclitaxel (Taxol), docetaxel (Taxotere), and SB-T-1214; cyclophosphamide; lapatinib; erlotinib; imatinib; pazopanib; capecitabine; cytarabine; vinorelbine; gemcitabine; anthracyclines, such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone; aromatase inhibitors. agents such as aminoglutethimide, testolactone (Teslac), anastrozole (Arimidex), letrozole (Femara), exemestane (Aromasin), vorozole (Rivizor), formestane (Lentaron), fadrozole (Afema), 4-hydroxyandrostenedione, 1,4,6-androstatriene-3,17-dione (ATD), and 4-androstene-3,6,17-trione (6-OXO); topoisomerase inhibitors such as irinoside tecan, topotecan, camptothecin, lamellarin D, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticine, aurintricarboxylic acid, and HU-331; platinum-based chemotherapeutic agents, such as cis-diamminedichloroplatinum(II) (cisplatin), cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), and [(1R,2R)-cyclohexane-1,2-diamine](ethanedioate-O,O' ) platinum(II) (oxaliplatin); antimetabolites, particularly antifolates such as methotrexate, pemetrexed, raltitrexed, and pralatrexate, pyrimidine analogs such as fluorouracil, gemcitabine, floxuridine, 5-fluorouracil and tegafur-uracil, and purine analogs; and inhibitors of the enzyme poly ADP-ribose polymerase (PARP inhibitors) such as olaparib, rucaparib, niraparib, and talazoparib. Further suitable toxins that can be used in combination with humanized anti-Lewis Y antibodies are described above with respect to agents that can be conjugated to humanized antibodies.

[0094] Treatment with humanized anti-Lewis Y antibodies may further be combined with immunostimulants, cytokines, chemokines, radiation therapy, vaccines such as protein, peptide, or RNA vaccines, B-Raf inhibitors such as vemurafenib, dexamethasone, protease inhibitors such as bortezomib, and lenalidomide.

[0095] For use in treating cancers in which cells express Lewis Y, the humanized antibody may be coupled to the aforementioned additional agents, which are preferably cytotoxic agents, such as radionuclides or cytotoxins. Exemplary cytotoxic agents are described above. Cytotoxic agents also include precursor compounds that only develop cytotoxic activity upon activation, for example, by irradiation with light or by an enzymatic reaction in the body. One or more of the anti-cancer therapeutic agents described above may also be used as additional agents for coupling with the humanized anti-Lewis Y antibody. Furthermore, the humanized antibody may be engineered to enhance its ability to activate a patient's immune response, particularly its ability to activate ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity). For example, this can be achieved by optimizing the amino acid sequence and / or glycosylation pattern of the antibody, particularly its constant region.

[0096] For use as a detection agent in the diagnosis, prognosis, and / or monitoring of disease, the humanized antibody is preferably coupled to a labeling agent capable of producing a detectable signal, which may in particular be a radionuclide, a fluorophore, or an enzyme. [Brief explanation of the drawings]

[0097] [Figure 1] Figure 1 shows a schematic diagram of the Lewis carbohydrate antigen family. Lewis antigens are a set of related glycans that have fucose linked alpha 1-3 (Lewis X, Y) or alpha 1-4 (Lewis A, B) to a GlcNAc monosaccharide.

[0098] [Figure 2-1] Figure 2 shows the results of an antigen ELISA assay. Different humanized anti-Lewis Y antibody variants were tested for their binding to Lewis Y, Lewis b, and Globo H. A high OD signal indicates strong binding of the antibody to the antigen. A: Binding of 25 ng / mL of the antibody variant to Lewis Y (LeY), Lewis b (Leb), and Globo H. B: Binding of 25 ng / mL and 12.5 ng / mL of the antibody variant to Lewis Y. C: Binding of 100 ng / mL, 50 ng / mL, and 25 ng / mL of the antibody variant to Lewis b. D: Binding of 100 ng / mL, 50 ng / mL, and 25 ng / mL of the antibody variant to Globo H. Control (ctrl.+): parental human / mouse chimeric antibody AA9. [Figure 2-2] Figure 2 shows the results of an antigen ELISA assay. Different humanized anti-Lewis Y antibody variants were tested for their binding to Lewis Y, Lewis b, and Globo H. A high OD signal indicates strong binding of the antibody to the antigen. A: Binding of 25 ng / mL of the antibody variant to Lewis Y (LeY), Lewis b (Leb), and Globo H. B: Binding of 25 ng / mL and 12.5 ng / mL of the antibody variant to Lewis Y. C: Binding of 100 ng / mL, 50 ng / mL, and 25 ng / mL of the antibody variant to Lewis b. D: Binding of 100 ng / mL, 50 ng / mL, and 25 ng / mL of the antibody variant to Globo H. Control (ctrl.+): parental human / mouse chimeric antibody AA9.

[0099] [Figure 3]Figure 3 shows the results of an antigen ELISA assay. Binding curves for different humanized anti-Lewis Y antibody variants to Lewis Y (A) and Lewis b (B) were determined. Control 1 (ctrl. + AA9): parent human / mouse chimeric antibody AA9. Control 2 (ctrl. + chain shuffling): chain-shuffled antibody containing the heavy chain of human / mouse chimeric antibody AA9 and the light chain of human / mouse chimeric antibody CC8.

[0100] [Figure 4] Figure 4 shows the results of an antigen ELISA assay. The binding curves of the humanized anti-Lewis Y antibody AA9-3-10.1 to Lewis Y and Lewis b were determined. Control (ctrl.): parental human / mouse chimeric antibody AA9.

[0101] [Figure 5] Figure 5 shows the results of an antigen ELISA assay. The binding of the humanized anti-Lewis Y antibody AA9-3-10.1 (humanized AA9) and the parental human / mouse chimeric antibody AA9 (chimeric AA9) to different carbohydrate antigens was tested at an antibody concentration of 50 ng / mL.

[0102] [Figure 6-1] Figure 6 shows the results of an antigen ELISA assay. Different sequence-optimized versions of the humanized anti-Lewis Y antibody AA9-3-10 were tested for binding to Lewis Y and Lewis b. A high OD signal indicates strong binding of the antibody to the antigen. A: Binding of 50 ng / mL of the antibody variant to Lewis Y (LeY) and Lewis b (Leb). B: Binding of 50 ng / mL and 25 ng / mL of the antibody variant to Lewis Y. C: Binding of 100 ng / mL and 50 ng / mL of the antibody variant to Lewis b. Control (pos.-ctrl.-Set2 128+129): parental human / mouse chimeric antibody AA9. [Figure 6-2]Figure 6 shows the results of an antigen ELISA assay. Different sequence-optimized versions of the humanized anti-Lewis Y antibody AA9-3-10 were tested for binding to Lewis Y and Lewis b. A high OD signal indicates strong binding of the antibody to the antigen. A: Binding of 50 ng / mL of the antibody variant to Lewis Y (LeY) and Lewis b (Leb). B: Binding of 50 ng / mL and 25 ng / mL of the antibody variant to Lewis Y. C: Binding of 100 ng / mL and 50 ng / mL of the antibody variant to Lewis b. Control (pos.-ctrl.-Set2 128+129): parental human / mouse chimeric antibody AA9.

[0103] [Figure 7] Figure 7 shows the results of an antigen ELISA assay. Different anti-Lewis Y antibodies were tested for their specificity 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.

[0104] [Figure 8] Figure 8 shows the results of an antigen ELISA assay. The binding curves of the anti-Lewis Y antibodies AA9-3-10.1, h3S193, and BR96 to Lewis Y were determined.

[0105] [Figure 9] Figure 9 shows the 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. An irrelevant hIgG1 was used as a negative control. Binding is reported as a percentage of antibody-positive cells of total viable cells.

[0106] [Figure 10]Figure 10 shows 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. An irrelevant hIgG1 was used as a reference. Binding to granulocytes, lymphocytes, and monocytes is reported as median fluorescence intensity (MFI).

[0107] [Figure 11] Figure 11 shows growth inhibition experiments of toxin-coupled AA9-3-10.1 (as hIgG1) and isotype control using different tumor cell lines. Proliferation is reported as percentage proliferation compared to medium control. [Example]

[0108] Example 1 Humanization of the murine heavy and light chain variable regions of anti-LeY antibody Nucleic acid sequences encoding the murine heavy and light chain variable regions of two monoclonal anti-LeY antibodies (AA9: SEQ ID NO: 30 and 31; CC8: SEQ ID NO: 32) were ligated to the genomic sequences of the human constant γ1 region (CH) and the human constant κ region (CL), respectively.

[0109] Based on these chimeric clones, humanized antibodies were constructed. To this end, point mutations were introduced into the nucleic acid sequences of the mouse framework regions of VH and VL to generate corresponding human framework regions. The target human framework regions were selected from a human germline antibody library. The most closely related framework regions were selected from the library based on their overall sequence similarity and their CDR loop classification. All the obtained data were taken into consideration to design a set of different variable sequences for the humanized variable light and variable heavy chains of both parent mouse antibodies. Some of the variants contained back mutations to the mouse sequences at critical positions. The humanized variants of the light chain variable region were cloned into a kappa chain vector, and the humanized variants of the heavy chain variable region were cloned into a gamma chain vector.

[0110] Antibodies containing different combinations of the resulting heavy and light chains were produced and screened for their expression and LeY binding profiles. The following humanized antibody heavy and light chain variable regions were selected for further analysis: [Table 1]

[0111] Example 2 Binding of humanized antibody variants to different carbohydrate antigens After expression of different constructs in NM-H9D8 cells, the titers of the humanized antibody variants were determined and their concentrations were adjusted. The humanized antibodies were then analyzed for binding to Lewis Y, Lewis b, and Globo H in an antigen ELISA. Briefly, antigens (coupled to polyacrylamide) were coated onto 96-well MaxiSorp plates (Nunc ThermoScientific) overnight. Nonspecific binding was blocked, and test samples were added at different concentrations. Subsequently, an anti-human IgG-Fc POD secondary antibody was added, followed by a TMB substrate reaction. Bound antibody was determined by measurement at 450 / 630 nm using an EnSpire2300 multilabel reader (PerkinElmer) or 450 / 620 nm using a Tecan SPARK plate reader.

[0112] All variants showed significant binding to Lewis Y, similar to that of the parent chimeric antibody. Furthermore, binding to Globo H was negative for all antibodies. Surprisingly, however, selected humanized antibody variants showed significantly reduced cross-reactivity to Lewis b (see Figures 2, 3, and 4).

[0113] The 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 for their fine specificity using an antigen ELISA with Lewis Y, Galα1,2-Gal, and β-N-acetyl-D-glucosamine-6-sulfate. The analyzed humanized antibodies showed strong and highly specific binding to Lewis Y but no significant binding to other carbohydrate antigens. Binding to the following carbohydrate antigens was tested at an antibody concentration of 50 ng / mL (see also Figure 5). [Table 2-1] [Table 2-2]

[0114] Example 3 Generation of further humanized variants based on antibody AA9-3-10 The humanized antibody variant AA9-3-10 (comprising 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. [Table 3]

[0115] The binding of these humanized antibody variants to Lewis Y and Lewis b was analyzed as described in Example 2. All antibody variants show strong and highly specific binding to Lewis Y. No significant binding to Lewis b could be detected (see Figure 6).

[0116] Example 4 Comparison of antigen binding and specificity of humanized variant AA9-3-10.1 with known anti-LeY antibodies 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, lacto-N-tetraose (LNT), Neu5Acα2-5Galβ, and Lewis Y) was compared with that of the known antibodies h3S193 and BR96.

[0117] Briefly, antigen (coupled to polyacrylamide gel) was coated onto a 96-well MaxiSorp plate (Nunc ThermoScientific) overnight. Nonspecific binding was blocked, and test samples were added at different concentrations. Anti-human IgG (H+L) POD secondary antibody was then added, followed by a TMB substrate reaction. Bound antibody was determined by measuring at 450 / 630 nm using an EnSpire 2300 multilabel reader (PerkinElmer) or at 450 / 620 nm using a Tecan SPARK plate reader.

[0118] The humanized antibody variant AA9-3-10.1 shows stronger binding to Lewis Y and significantly improved specificity. Binding of h3S193 to Lewis Y is much lower at low antibody concentrations, while BR96 shows significant binding to βD-galactose and Lewis X. Furthermore, both h3S193 and BR96 bind to Neu5Acα2-5Galβ at higher concentrations (see Figures 7 and 8).

[0119] A novel method for determining binding constants and affinities is fluorescence proximity sensing, using single-stranded DNA (96mer) spotted on a chip and complementary DNA coupled to a ligand in a DRX2 instrument (Dynamic Biosensors). In this study, streptavidin was used as the ligand to capture Lewis Y or Lewis b coupled to biotinylated polyacrylamide gel. Binding of the humanized antibody variant AA9-3-10.1 or a competing anti-Lewis Y antibody to the antigen resulted in a change in fluorescence. On and off rates during association and dissociation could be calculated. AA9-3-10.1 and a competing anti-Lewis Y antibody were diluted to 300, 60, and 12 nM in PE140 buffer and applied to the antigen bound to the chip. In experiments with AA9-3-10.1 for Lewis b, concentrations of 3000, 600, and 120 nM were used because a very low signal was expected. Binding curves were evaluated by a single-exponential global fit (instrument software). Higher sensitivity allows for monitoring more rapid interactions compared to surface plasmon resonance (SPR). This results in binding kinetics that are more comparable to the "gold standard" method KinExA, which differs from SPR but is measured in a liquid system.

[0120] The binding assay showed that AA9-3-10.1 bound strongly and highly specifically to Lewis Y. The data confirmed the results of the ELISA assay. [Table 4]

[0121] Example 5 Comparison of tumor cell binding between humanized variant AA9-3-10.1 and known anti-LeY antibodies The binding properties of humanized anti-Lewis Y antibody variants AA9-3-10.1, BR96, and 3S193 (all as hIgG1) to the human cancer cell lines Ls-174T, T-47D, H9D8, and Colo-205 were analyzed by flow cytometry. An irrelevant hIgG1 was used as a negative control. Briefly, tumor cells were harvested and incubated with serial dilutions of the indicated antibodies in the dark at 4°C. The cells were then washed and incubated with a secondary goat anti-hIgG PE-conjugated antibody in the dark at 4°C. After a further washing step, the cells were stained with DAPI to distinguish between viable and dead cells and analyzed via flow cytometry.

[0122] The humanized anti-Lewis Y antibody variant AA9-3-10.1 showed concentration-dependent binding to the Lewis Y-positive cell lines Ls-174T and T-47D and no binding to the Lewis Y-negative cell lines H9D8 and Colo-205, whereas the 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 Figure 9). The stronger binding of BR96 and 3S193 was most likely due to cross-reactivity. For example, Colo-205 has been described as negative for Lewis Y (Westwood et al., 2005) but positive for Lewis b (Noble et al., 2013).

[0123] Example 6 Comparison of blood cell binding of humanized variant AA9-3-10.1 with known anti-LeY antibodies The binding of the humanized anti-Lewis Y antibody variants AA9-3-10.1, BR96, and 3S193 (as mIgG1) to leukocytes was determined using flow cytometry. Whole blood from five healthy volunteers was used. In the first step, red blood cells were lysed, and the remaining leukocytes were incubated with the indicated antibodies at room temperature (10 μg / ml). An irrelevant mIgG1 was used as a negative control. Cells were then washed and incubated with a secondary anti-mIgG AF647-conjugated antibody at room temperature. After a washing step, cells were stained with an anti-human CD45 Pacific Blue-conjugated antibody at room temperature. After a further washing step, cells were analyzed via flow cytometry. Immune cell subpopulations, granulocytes, monocytes, and lymphocytes, were differentiated based on their CD45 expression and granularity.

[0124] The humanized anti-Lewis Y antibody variant AA9-3-10.1 shows no or very weak binding to leukocyte subsets, whereas BR96 binds strongly to granulocytes and 3S193 binds to granulocytes, lymphocytes, and monocytes (see Figure 10), demonstrating the excellent specificity of the humanized anti-Lewis Y antibody variants for tumor cells and their very low cross-reactivity with normal tissue cells.

[0125] Example 7 Growth inhibition of different tumor cell lines using the toxin-coupled humanized anti-Lewis Y antibody variant AA9-3-10.1 To demonstrate the efficacy of the humanized anti-Lewis Y antibody variants in killing tumor cells, growth inhibition assays were performed using different tumor cell lines. As a cytotoxin, MMAE was coupled to protein G, which binds to antibodies, thereby forming an antibody-toxin conjugate.

[0126] Cell lines Ls-174T, T-47D, MCF-7 (CSC-enriched), Ovcar-3, and HSC-4 were seeded at 5,000 cells / well in culture medium in 96-well flat-bottom plates and incubated for 4 days in the presence of the indicated concentrations of the humanized anti-Lewis Y antibody variant AA9-3-10.1 (as hIgG1) or an irrelevant isotype control, and ProtG-MMAE. Viable cell numbers were determined using a commercially available CellTiter-Glo luminescent cell viability assay. The percentage of proliferation was determined relative to a medium-only control.

[0127] The toxin-coupled humanized anti-Lewis Y antibody variant AA9-3-10.1 was able to inhibit the growth of various Lewis Y-expressing tumor cell lines, indicating effective internalization of the antibody (see Figure 11).

[0128] Example 8 Immunohistochemical staining of different tumor tissues from various cancer types with humanized anti-Lewis Y antibody variant AA9-3-10.1 The binding of the humanized anti-Lewis Y antibody variant AA9-3-10.1 to different cancer indications was analyzed by immunohistochemistry. Briefly, tissue microarray slides from breast cancer (BRC), non-small cell lung cancer (NSCLC), colon cancer (CRC), head and neck cancer (HNC), small cell lung cancer (SCLC), and ovarian cancer (OvCa) were deparaffinized and rewetted with a decreasing alcohol series. After antigen retrieval, endogenous peroxidase and nonspecific binding were blocked. Binding of the humanized anti-Lewis Y antibody variant AA9-3-10.1 mIgG1 (6.5 μg / ml) was detected with 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. Binding of the humanized anti-Lewis Y antibody variant AA9-3-10.1 is reported using an immunoreactivity score (IRS; range 0-12), calculated by multiplying the staining intensity (range 0-3) by the percentage of stained cells (range 0-4).

[0129] The humanized anti-Lewis Y antibody variant AA9-3-10.1 stains tumor tissues from breast cancer (BRC), non-small cell lung cancer (NSCLC), colon cancer (CRC), head and neck cancer (HNC), small cell lung cancer (SCLC), and ovarian cancer (OVCa) in a high percentage of cases. [Table 5] Sequence Listing [Table 6-1] [Table 6-2]

[0130] Identification of the deposited biological material The cell lines DSM ACC 2806, DSM ACC 2807, and DSM ACC 2856 were deposited by Glycotope GmbH, Robert-Roessle-Str. 10, 13125 Berlin (DE) with DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig (DE) on the dates indicated in the table below. [Table 7]

Claims

1. A humanized antibody capable of binding to 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: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

22.

2. The antibody of claim 1, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9.

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

11.

4. The antibody described in claim 3, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.

5. The antibody of any one of claims 1 to 4, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21.

6. The antibody of any one of claims 1 to 4, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

23.

7. The antibody described in claim 6, wherein the light chain variable region comprises an amino acid sequence selected from the group of SEQ ID NOs: 17 and 18.

8. The antibody of any one of claims 1 to 7, 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 of any one of claims 1 to 8, 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 of any one of claims 1 to 9, further comprising an Fc region.

11. The antibody of claim 10, which is an IgG1, IgG2, IgG3, or IgG4 type antibody.

12. The antibody according to any one of claims 1 to 11, which is capable of specifically binding to Lewis Y but not to Lewis b.

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

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

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

16. A conjugate comprising an antibody according to any one of claims 1 to 12 conjugated to a further agent.

17. 17. The conjugate of claim 16, wherein the additional agent is a cytotoxic agent, a tumor-specific antibody, or an immune checkpoint blocking or activating antibody.

18. 17. The conjugate of claim 16, which is a chimeric antigen receptor.

19. 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 any one of claims 16 to 18.

20. The composition of claim 19, which is a pharmaceutical composition further comprising one or more components selected from the group consisting of solvents, diluents, and excipients.

21. An antibody according to any one of claims 1 to 12, a conjugate according to any one of claims 16 to 18, or a composition according to claim 20 for use in medicine.

22. 22. The antibody, conjugate, or composition of claim 21 for use in treating cancer, an infectious disease, or an immunodeficiency disorder.

23. 23. The antibody, conjugate, or composition of claim 22, wherein the cancer is selected from the group consisting of 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