Antibodies against LYPD3

Abstract

The present invention relates to an anti-LYPD3 antibody that binds to LYPD3 in an O-glycosylation-dependent manner. Thereby, the antibody is specific to tumor-associated LYPD3. The present invention further provides pharmaceutical compositions containing said anti-LYPD3 antibodies and their use in treating cancer. The present inventors have developed anti-LYPD3 antibodies with enhanced tumor specificity. These antibodies bind to tumor-associated LYPD3 in an O-glycosylation-dependent manner and recognize the O-glycan structures present on LYPD3 in cancer cells.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of antibodies. In particular, anti-LYPD3 antibodies that exhibit strong antigen binding in a glycosylation-dependent manner are provided. In certain embodiments, the present invention relates to anti-LYPD3 antibodies for therapeutic and diagnostic use. [Background technology]

[0002] Background of the Invention Today, antibodies are widely used drugs in medicine and research. In medicine, they find 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 infections. In these therapies, antibodies can have therapeutic activity in 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] In the treatment of cancer, the important feature of therapeutic antibody is its specificity to tumor tissue.This means that antibody should target epitopes that are found exclusively or mainly in cancer cells, but are found to a lesser extent in cells of normal tissue.Therefore, the therapeutic activity of antibody, such as inducing immune response to target cells or destroying cells by cytotoxic payload, can specifically act at tumor site.The respective activity in normal tissue caused by antibody binding to non-tumor cells can cause serious side effects.Therefore, increasing the specificity to tumor cells reduces the risk of adverse effects and improves the safety of the expected treatment.

[0004] A potential cancer antigen is LYPD3 (C4.4A). This protein is a glycosylphosphatidylinositol (GPI)-anchored, highly glycosylated cell surface protein that has been shown to be upregulated in migrating keratinocytes during wound healing. It was first described as a metastasis-associated cell surface protein in rat pancreatic tumor cells and has since been implicated in carcinogenesis in several different cancers. In cancer, LYPD3 has been suggested to be specifically involved in tumor cell invasion through interactions with the extracellular matrix.

[0005] LYPD3 is strongly overexpressed in non-small cell lung cancer (NSCLC), with preferential expression in the squamous cell carcinoma (SCC) subtype compared with the other two most common NSCLC subtypes, adenocarcinoma (AC) and large cell carcinoma (LCC). Lung cancer is the most frequently diagnosed cancer, with an estimated 1.8 million new cases in 2012. NSCLC accounts for 85% of all lung cancers, while SCC is the second most common histological subtype, occurring in 30% of NSCLC cases and responsible for approximately 400,000 deaths annually worldwide.

[0006] LYPD3 overexpression has also been detected in SCC of the head and neck (HNSCC), including the esophageal SCC (ESCC) subtype. At the transcriptional level, approximately 50% of primary lung cancers and 75% of lung cancer metastases express LYPD3 mRNA, but expression is not detected in normal lung tissue. Furthermore, LYPD3 is expressed in colorectal and breast cancers.

[0007] LYPD3 overexpression has been shown to correlate with an aggressive phenotype and poor prognosis in NSCLC, colorectal cancer, and ESCC, but is associated with a favorable prognosis in breast cancer, making LYPD3 an attractive target for antitumor immunotherapy.

[0008] However, under normal physiological conditions, LYPD3 is expressed in skin keratinocytes, esophageal endothelial cells, and placental cells, which may lead to unwanted side effects in cancer therapy using antibodies that cannot distinguish between cancer-associated LYPD3 and LYPD3 on normal tissues.

[0009] Therefore, there is a need in the art to provide tumor-specific anti-LYPD3 antibodies. Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention The present inventors have developed anti-LYPD3 antibodies with enhanced tumor specificity. These antibodies bind to tumor-associated LYPD3 in an O-glycosylation-dependent manner and recognize the O-glycan structures present on LYPD3 in cancer cells. O-glycosylation in cancer cells contains abundant short-chain structures, particularly monosaccharides, disaccharides, and trisaccharides, such as Thomsen-Friedenreich antigen (TF; Galβ1-3GalNAcα1-), sialylated Thomsen-Friedenreich antigen (sTF), Thomsen nouvelle antigen (Tn; GalNAcα1-), and sialylated Thomsen nouvelle antigen (Siaα2-6GalNAcα1-). In contrast, O-glycosylation in normal cells encompasses much longer oligosaccharide chains. The developed antibodies specifically bind to LYPD3 bearing short cancer cell-derived O-glycosylation, and therefore distinguish between tumor-associated LYPD3 (i.e., LYPD3 present on cancer cells) and LYPD3 on cells of normal tissues. Thus, the antibodies described in the present invention preferentially bind to LYPD3 on tumor cells, thus having excellent cancer specificity, reduced binding to normal tissues, and reduced risk of adverse effects in cancer immunotherapy.

[0011] Mature LYPD3 consists of two Ly-6 (leukocyte antigen 6) / uPAR / α-neurotoxin domains (LU domains) and is extensively modified by post-translational glycosylation. It contains five N-glycosylation sites located in or close to the second LU domain and approximately 15 O-linked carbohydrates clustered in a C-terminal Ser / Thr / Pro-rich region. The antibodies described in the present invention recognize LYPD3 in an O-glycosylation-dependent manner by binding to amino acids 234-303 of the human LYPD3 sequence (SEQ ID NO: 137), particularly the C-terminal STP-rich region at amino acids 247-297. This region contains the O-glycosylation sites of LYPD3.

[0012] In view of the above, in a first aspect, the present invention relates to an antibody capable of specifically binding to human LYPD3 glycosylated at an epitope comprising an oligosaccharide structure selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1- attached to a serine or threonine residue of LYPD3.

[0013] In a second aspect, the present invention relates to an anti-LYPD3 antibody comprising: (i) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO:4, CDR-L2 having the amino acid sequence of SEQ ID NO:5, and CDR-L3 having the amino acid sequence of SEQ ID NO:6; or (ii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; or (iii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 17, CDR-H2 having the amino acid sequence of SEQ ID NO: 18, and CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 20, CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or (iv) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO:28, CDR-L2 having the amino acid sequence of SEQ ID NO:29, and CDR-L3 having the amino acid sequence of SEQ ID NO:30; or (v) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; or (vi) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 41, CDR-H2 having the amino acid sequence of SEQ ID NO: 42, and CDR-H3 having the amino acid sequence of SEQ ID NO: 43; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 44, CDR-L2 having the amino acid sequence of SEQ ID NO: 45, and CDR-L3 having the amino acid sequence of SEQ ID NO: 46; or (vii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 49, CDR-H2 having the amino acid sequence of SEQ ID NO: 50, and CDR-H3 having the amino acid sequence of SEQ ID NO: 51; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 52, CDR-L2 having the amino acid sequence of SEQ ID NO: 53, and CDR-L3 having the amino acid sequence of SEQ ID NO: 54; or (viii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 58, and CDR-H3 having the amino acid sequence of SEQ ID NO: 59; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 60, CDR-L2 having the amino acid sequence of SEQ ID NO: 61, and CDR-L3 having the amino acid sequence of SEQ ID NO: 62; or (ix) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 65, CDR-H2 having the amino acid sequence of SEQ ID NO: 66, and CDR-H3 having the amino acid sequence of SEQ ID NO: 67; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 68, CDR-L2 having the amino acid sequence of SEQ ID NO: 69, and CDR-L3 having the amino acid sequence of SEQ ID NO: 70; or (x) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 73, CDR-H2 having the amino acid sequence of SEQ ID NO: 74, and CDR-H3 having the amino acid sequence of SEQ ID NO: 75; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 76, CDR-L2 having the amino acid sequence of SEQ ID NO: 77, and CDR-L3 having the amino acid sequence of SEQ ID NO: 78; or (xi) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 81, CDR-H2 having the amino acid sequence of SEQ ID NO: 82, and CDR-H3 having the amino acid sequence of SEQ ID NO: 83; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 84, CDR-L2 having the amino acid sequence of SEQ ID NO: 85, and CDR-L3 having the amino acid sequence of SEQ ID NO: 86; or (xii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 89, CDR-H2 having the amino acid sequence of SEQ ID NO: 90, and CDR-H3 having the amino acid sequence of SEQ ID NO: 91; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 92, CDR-L2 having the amino acid sequence of SEQ ID NO: 93, and CDR-L3 having the amino acid sequence of SEQ ID NO: 94; or (xiii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 97, CDR-H2 having the amino acid sequence of SEQ ID NO: 98, and CDR-H3 having the amino acid sequence of SEQ ID NO: 99; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 100, CDR-L2 having the amino acid sequence of SEQ ID NO: 101, and CDR-L3 having the amino acid sequence of SEQ ID NO: 102; or (xiv) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 105, CDR-H2 having the amino acid sequence of SEQ ID NO: 106, and CDR-H3 having the amino acid sequence of SEQ ID NO: 107; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 108, CDR-L2 having the amino acid sequence of SEQ ID NO: 109, and CDR-L3 having the amino acid sequence of SEQ ID NO: 110; or (xv) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 113, CDR-H2 having the amino acid sequence of SEQ ID NO: 114, and CDR-H3 having the amino acid sequence of SEQ ID NO: 115; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 116, CDR-L2 having the amino acid sequence of SEQ ID NO: 117, and CDR-L3 having the amino acid sequence of SEQ ID NO: 118; or (xvi) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 121, CDR-H2 having the amino acid sequence of SEQ ID NO: 122, and CDR-H3 having the amino acid sequence of SEQ ID NO: 123; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 124, CDR-L2 having the amino acid sequence of SEQ ID NO: 125, and CDR-L3 having the amino acid sequence of SEQ ID NO: 126; or (xvii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 129, CDR-H2 having the amino acid sequence of SEQ ID NO: 130, and CDR-H3 having the amino acid sequence of SEQ ID NO: 131; and A light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 132, CDR-L2 having the amino acid sequence of SEQ ID NO: 133, and CDR-L3 having the amino acid sequence of SEQ ID NO: 134.

[0014] In a third aspect, the present invention provides a nucleic acid encoding an antibody according to the first or second aspect of the invention. Additionally, in a fourth aspect, there is provided 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 fifth aspect, a host cell comprising a nucleic acid or expression cassette or vector according to the invention.

[0015] In a sixth aspect, the present invention provides a conjugate comprising an antibody according to the first or second aspect of the invention conjugated to a further agent.

[0016] In a seventh aspect, the present invention relates to a composition comprising an antibody according to the first or second aspect of the invention, a nucleic acid according to the third aspect of the invention, an expression cassette or vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention or a conjugate according to the sixth aspect of the invention.

[0017] According to an eighth 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.

[0018] 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 indicating preferred embodiments of the present application are given by way of example only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

[0019] definition As used herein, the following expressions are intended to have the meanings normally and preferably indicated below, except to the extent that the context in which they are used indicates otherwise.

[0020] As 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 listed elements does not contain additional elements, as well as embodiments in which the subject matter "comprising" the specifically listed elements can and / or actually contains additional elements. Similarly, the term "having" should be understood as the term "comprising," and also includes and specifically refers to the terms "essentially consisting of" and "consisting of." The term "essentially consisting of," when possible, particularly refers to embodiments in which the subject matter contains 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less of additional elements in addition to the specifically listed elements from which the subject matter essentially consists.

[0021] 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 contains 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 regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each variable region contains three CDRs and four FRs. The amino acid residues of the CDRs are determined, inter alia, based on the IMGT system and CDR localization.

[0022] The variable regions of the heavy and light chains contain binding domains that interact with antigens. The heavy chain constant region can be of any type, such as a gamma, delta, alpha, mu, or epsilon heavy chain. Preferably, the antibody heavy chain is a gamma chain. Furthermore, the light chain constant region can be of any type, 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 light chains, respectively, whose constant region amino acid sequences are derived from naturally occurring heavy chain or light chain constant region amino acid sequences, particularly human heavy chain 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 the kappa light chain is at least 95%, particularly at least 98%, identical to the amino acid sequence of the constant domain of one of the allotypes of human kappa antibody light chains. The constant region 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 antibody, a human antibody, or a chimeric antibody.

[0023] The antigen-binding portion of an antibody generally refers to the full length of the antibody or one or more fragments thereof that retain the ability to specifically bind to an antigen.It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full length antibody.Examples of antibody binding fragments include: Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; F(ab)2 fragments, which are bivalent fragments that are linked by disulfide bridges at the hinge region and contain two Fab fragments that each bind to the same antigen; Fd fragments, which are composed of VH and CH1 domains; Fv fragments, which are composed of the VL and VH domains of a single arm of an antibody; and dAb fragments, which are composed of the VH domain.

[0024] The "Fab portion" of an antibody specifically refers to a portion of the antibody that contains the heavy and light chain variable regions (VH and VL) and the first domains of 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 to only the VH, VL, CH1, and CL regions present in the antibody. Preferably, the "Fab portion" refers to a portion of an antibody that corresponds to a fragment obtained by digesting a natural antibody with papain that contains the antigen-binding activity of the antibody. 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.

[0025] The "Fc portion" of an antibody refers specifically to the portion of the antibody containing heavy chain constant regions 2, 3, and, where applicable, 4 (CH2, CH3, and CH4). In particular, the Fc portion contains two of each of these regions. If an antibody does not contain all of these regions, the term "Fc portion" refers only to the CH2, CH3, and CH4 regions present in the antibody. Preferably, the Fc portion contains at least the CH2 region of the antibody. Preferably, the "Fc portion" refers to the portion of an antibody corresponding to a fragment obtained by digesting a native antibody with papain that does not contain the antigen-binding activity of the antibody. In particular, the Fc portion of an antibody is capable of binding to an Fc receptor and thus contains, for example, an Fc receptor binding site or Fc receptor binding ability.

[0026] According to the present invention, the term "chimeric antibody" refers in particular to an antibody in which the constant region is derived from a human antibody or a human antibody consensus sequence, and at least one, and preferably both, variable regions are derived from a non-human antibody, e.g., a rodent antibody such as a murine antibody.

[0027] According to the present invention, the term "humanized antibody" particularly refers to a non-human antibody comprising human constant and variable regions, the amino acid sequence of which has been modified to reduce the immunogenicity of the antibody 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 human-derived framework regions. If necessary, some residues in the human framework regions can be backmutated toward those of the parent non-human antibody, for example, 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 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, during CDR grafting, human framework regions are usually selected based on their homology to non-human framework regions, whereas in superhumanization, the criterion for selecting human framework regions is the similarity of CDRs.In human string content optimization, the difference between the non-human antibody sequence and the human germline sequence is scored, and then the antibody is mutated to minimize the score.In addition, humanized antibodies can also be obtained by an empirical method, using a large library of human framework regions or human antibodies to generate multiple antibody humanization candidates, and then determining the most promising candidate through a screening method.Also, using the above-mentioned rational approach, several humanized antibody candidates can be generated and then screened, for example, for their antigen binding.

[0028] 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.

[0029] As used herein, the term "antibody" refers to a population of antibodies of the same type in certain embodiments. In particular, all antibodies in the antibody population exhibit the characteristics used to define the antibody. In certain embodiments, all antibodies in the antibody population have the same amino acid sequence. Reference to a particular type of antibody, such as an anti-LYPD3 antibody, specifically refers to a population of antibodies of this type.

[0030] The term "antibody" as used herein includes full-length antibodies as well as fragments and derivatives of said antibodies. In particular, a "fragment or derivative" of an antibody is a protein or glycoprotein derived from said antibody and capable of binding to the same antigen, in particular the same epitope as the antibody. Thus, the fragment or derivative of an antibody herein generally refers to a functional fragment or derivative. In a particularly preferred embodiment, the fragment or derivative of an antibody 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) a Fab fragment, which is a monovalent fragment consisting of the variable regions and the first constant domain of each heavy and light chain; (ii) an F(ab)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which is a heavy chain variable region and the first constant domain, CH1; (iv) an Fv fragment, which is a single-arm Fv fragment consisting of the heavy and light chain variable regions of an antibody; (v) an scFv fragment, which is a Fv fragment consisting of a single polypeptide chain; (vi) an (Fv)2 fragment consisting of two covalently linked Fv fragments; (vii) a heavy chain variable domain; and (viii) a multibody, which is a multibody consisting of a heavy chain variable region and a light chain variable region covalently linked to each other such that the association between the heavy chain variable region and the light chain variable region is intermolecular, not intramolecular. Antibody derivatives particularly include antibodies that bind to the same antigen as the parent antibody but have a different amino acid sequence from the parent antibody from which they are derived. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.

[0031] A target amino acid sequence is "derived from" or "corresponding to" a reference amino acid sequence if it 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 of the heavy chain variable region (FRH1) 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, particularly 100% identical, to the corresponding portion of the reference amino acid sequence over its entire length. According to the present invention, the "homology" or "identity" of an amino acid sequence or nucleotide sequence is preferably determined over the entire length of the reference sequence or over the entire length of the corresponding portion of the reference sequence to which the homology or identity is defined. Antibodies derived from a parent antibody that are defined by one or more amino acid sequences, such as specific CDR sequences or specific variable region sequences, are particularly antibodies that have amino acid sequences, such as CDR sequences or variable region sequences, that are at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homologous or identical, particularly identical, to the respective amino acid sequence of the parent antibody. In certain embodiments, an antibody derived from a parent antibody (i.e., a derivative of the parent antibody) comprises the same CDR sequences as the parent antibody, but differs in the sequence of the remainder of the variable region.

[0032] The term "antibody" as used herein also refers to multivalent and multispecific antibodies, i.e., antibody constructs having more than two binding sites, each binding to the same epitope, as well as antibody constructs having one or more binding sites binding to a first epitope and one or more binding sites binding to a second epitope, and optionally further binding sites binding to further epitopes.

[0033] "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 its binding to another target. The agent binds more strongly to a target, such as an epitope, with a lower dissociation constant (K) than to the second target. d ) binds to a first target more strongly than to a second target. Preferably, the dissociation constant for a target to which the agent specifically binds is 10-fold, 30-fold, more than 100-fold, or more than 500-fold lower than the dissociation constant for a target to which the agent does not specifically bind. Furthermore, the term "specific binding" particularly refers to binding to a target with 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 specific for a particular antigen are particularly preferred as they exhibit a binding affinity between binding partners having a molecular weight 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-LYPD3 antibody" refers to an antibody that specifically binds to LYPD3, preferably with an 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 As used herein, the term "binding" refers specifically to specific binding.

[0034] The term "epitope" as used herein refers to amino acid residues and glycan structures on an antigen of an antibody that are directly contacted by or in direct proximity to amino acids of the antibody, particularly amino acids of the CDRs of the antibody, and that influence binding of the antibody to its antigen.

[0035] The term "LYPD3" according to the present invention particularly refers to the human LYPD3 protein, in particular the mature human LYPD3 protein. LYPD3 as used herein particularly refers to the human LYPD3 protein described in UniProt entry O95274. LYPD3 particularly comprises, and particularly consists of, the amino acid sequence of positions 31 to 326 of SEQ ID NO: 137, or an amino acid sequence that is at least 90%, particularly at least 95%, identical to positions 31 to 326 of SEQ ID NO: 137 over its entire length. In particular, LYPD3 may be post-translationally modified and may have O-glycosylation, N-glycosylation, and / or a glycosylphosphatidylinositol (GPI) anchor. For example, LYPD3 can have O-glycosylation at one or more positions corresponding to Ser223, Ser232, Thr247, Thr248, Ser251, Thr252, Thr253, Ser254, Thr256, Thr257, Ser258, Thr259, Ser260, Thr266, Ser267, Thr268, Thr269, Thr276, Ser277, Thr279, Ser289, Thr297, Ser307 and Ser309 of SEQ ID NO: 137.

[0036] The term "GalNAcα1-", also known as "Tn", "Tn antigen" or "Thomsen nouvelle antigen", refers to a monosaccharide structure consisting of an N-acetylgalactosaminyl residue attached via an α-glycosidic bond to a supporting structure, particularly a serine or threonine residue of a protein or peptide.

[0037] The term "sialylated GalNAcα1-," also known as "sTn," "sialylated Tn antigen," or "sialylated Thomsen nouvelle antigen," refers to a disaccharide structure consisting of an N-acetylgalactosaminyl residue attached via an α-glycosidic bond to a serine or threonine residue of a supporting structure, particularly a protein or peptide. To this structure, a sialic acid residue is attached via an α2-6 bond to the N-acetylgalactosaminyl residue, resulting in the disaccharide structure Siaα2-6GalNAcα1-.

[0038] The term "Galβ1-3GalNAcα1-," also known as "TF," "TF antigen," "T antigen," or "Thomsen Friedenreich antigen," refers to a disaccharide structure consisting of a galactosyl residue attached via a β1-3 bond to an N-acetylgalactosaminyl residue attached via an α1-glycosidic bond to a serine or threonine residue of a supporting structure, especially a protein or peptide.

[0039] The term "sialylated Galβ1-3GalNAcα1-," also referred to as "sTF," "sTF antigen," "sialylated TF antigen," or "sialylated Thomsen-Friedenreich antigen," refers to a trisaccharide or tetrasaccharide structure consisting of a galactosyl residue attached via a β1-3 bond to an N-acetylgalactosaminyl residue attached via an α1-glycosidic bond to a serine or threonine residue of a supporting structure, particularly a protein or peptide, to which a sialic acid residue is attached via an α2-3 bond to the galactosyl residue and / or a sialic acid residue is attached via an α2-6 bond to the N-acetylgalactosaminyl residue, resulting in the trisaccharide structures Siaα2-3Galβ1-3GalNAcα1- and Galβ1-3(Siaα2-6)GalNAcα1- or the tetrasaccharide structure Siaα2-6(Siaα2-3Galβ1-3)GalNAcα1-.

[0040] In the above structures, Gal represents a galactose residue, and GalNAc represents an N-acetylgalactosamine residue. "β1-3", "α2-3" and "α2-6" refer to the linkage between two adjacent monosaccharide residues, specifically between the carbon atom C1 or C2, respectively, of the left monosaccharide and the carbon atom C3 or C6, respectively, of the right monosaccharide, which may be at the α or β position (as shown in the scheme below for glucose): [ka]

[0041] The term "GalNAcα1-" indicates that the GalNAc residue at the reducing end of the oligosaccharide is linked to the supporting structure via its carbon atom C1 in the α configuration.

[0042] The term "sialic acid" particularly refers to any N- or O-substituted derivative of neuraminic acid. It can refer to both 5-N-acetylneuraminic acid (NeuNAc) and 5-N-glycolylneuraminic acid (NeuGc), but preferably refers only to 5-N-acetylneuraminic acid.

[0043] The terms "glycan," "glycan structure," "carbohydrate," "glycan," and "glycan structure" are generally used interchangeably herein.

[0044] 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 via 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 attached to atoms of the compounds. When a conjugate is composed of three or more compounds, the compounds can be linked, for example, in a chain, with one compound attached to the next, or with several compounds each attached to a central compound.

[0045] The term "nucleic acid" includes single-stranded nucleic acids, double-stranded nucleic acids, and ribonucleic acids and deoxyribonucleic acids. It may contain naturally occurring as well as synthetic nucleotides, and may be modified naturally or synthetically, for example, by methylation, 5'- and / or 3'-capping.

[0046] The term "expression cassette" specifically refers to a nucleic acid construct capable of enabling and regulating the expression of a coding nucleic acid sequence introduced therein. Expression cassettes may contain promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. While the exact structure of an expression cassette may vary depending on the species or cell type, it generally contains 5'-untranscribed sequences and 5'- and 3'-untranslated sequences involved in initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, and CAAT sequences. More specifically, the 5'-untranscribed expression control sequences include a promoter region containing a promoter sequence for transcriptional control of an operably linked nucleic acid. Expression cassettes may also contain enhancer sequences or upstream activator sequences.

[0047] 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., initiate transcription in response to an inducing agent, or "constitutive," meaning that transcription is not controlled by the inducing agent. A gene under the control of an inducible promoter is not expressed, or is expressed only weakly, in the absence of the inducing agent. In the presence of the inducing agent, the gene is switched on, or transcription levels increase. This is generally mediated by the binding of specific transcription factors.

[0048] As used herein, the term "vector" is used in its most general sense and includes any intermediate vehicle for nucleic acids that allows said nucleic acids to be introduced into, for example, prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, bacteriophages, or viral genomes. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, that can replicate independently of chromosomal DNA.

[0049] According to the present invention, the term "host cell" relates to any cell that can be transformed or transfected with an exogenous nucleic acid. The term "host cell" according to the present invention includes prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., mammalian cells, in particular human or hamster cells, yeast cells and insect cells). Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats or primates, are particularly preferred. Cells can be derived from a number of tissue types and include primary cells and cell lines. The nucleic acid can be present in the host cell in the form of a single copy or two or more copies and, in one embodiment, is expressed in the host cell.

[0050] 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, etc., or a rodent, such as a mouse or rat. In a particularly preferred embodiment, the patient is a human.

[0051] The term "cancer" according to the present invention includes, in particular, leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, bladder cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer, and metastases thereof. The term cancer according to the present invention also includes cancer metastasis. The term cancer further refers to and / or includes cancer stem cells, in particular cancer stem cells of the specific types of cancer listed above.

[0052] "Tumor" means a group of cells or tissues formed by miscontrolled cell growth. Tumors may show partial or complete lack of structural organization and functional coordination with normal tissue, and usually form a distinct mass of tissue that can be either benign or malignant.

[0053] "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, entry into the systemic circulation, and establishment for growth in normal tissues elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells are usually similar to those of the original tumor. This means, for example, that if breast cancer metastasizes to the lungs, the secondary tumor is made up of abnormal breast cells rather than abnormal lung cells. In that case, the lung tumor is called metastatic breast cancer, not lung cancer.

[0054] The term "pharmaceutical composition" refers specifically to a composition suitable for administration 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, preservative, and tonicity adjuster. DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description of the Invention The present invention is based on the development of anti-LYPD3 antibodies that specifically bind to tumor-associated LYPD3. These antibodies were generated by using LYPD3 or LYPD3 fragments bearing O-glycan structures produced by cancer cells and selecting for antibodies that bind to LYPD3 in an O-glycosylation-dependent manner. The O-glycosylation produced by normal cells differs significantly from that produced by cancer cells. For example, while normal cells produce large O-glycan structures, LYPD3 on tumor cells primarily bears mono-, di-, and trisaccharides, such as GalNAcα1-(Tn), sialylated GalNAcα1-(sTn), Galβ1-3GalNAcα1-(TF), and sialylated Galβ1-3GalNAcα1-(sTF). The antibodies described in the present invention recognize and bind to these small O-glycan structures of tumor-associated LYPD3. Thus, these antibodies bind to LYPD3 on tumor cells with higher affinity than to LYPD3 in normal tissues and can distinguish between tumor-associated and non-tumor-associated LYPD3.

[0056] 1. Anti-LYPD3 antibody In light of these findings, in a first aspect, the present invention provides an antibody capable of specifically binding to glycosylated human LYPD3 at an epitope comprising an oligosaccharide structure selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1- attached to a serine or threonine residue of LYPD3. Preferably, the antibody is capable of specifically binding to glycosylated human LYPD3 at an epitope comprising one or more GalNAcα1- and / or Galβ1-3GalNAcα1- oligosaccharide structures attached to a serine and / or threonine residue of LYPD3.

[0057] An antibody capable of specifically binding to glycosylated human LYPD3 binds to glycosylated human LYPD3 with higher binding affinity than non-glycosylated human LYPD3. In certain embodiments, an antibody capable of specifically binding to glycosylated human LYPD3 binds to glycosylated human LYPD3 with higher binding affinity than an unrelated protein bearing the same glycan structure. As used herein, the term "higher binding affinity" particularly refers to a difference in dissociation constant of at least 10-fold, particularly at least 25-fold, and particularly at least 100-fold.

[0058] In certain embodiments, the antibody is capable of binding to human LYPD3 at an epitope comprising two or more oligosaccharide structures, e.g., two, three, or four oligosaccharide structures, attached to serine and / or threonine residues of LYPD3. One or more of these additional oligosaccharide structures may also be selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1-. In certain embodiments, the antibody is capable of binding to human LYPD3 at an epitope comprising two oligosaccharide structures, each attached to a serine or threonine residue of LYPD3, each selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1-. In particular, the antibody is capable of binding to human LYPD3 at an epitope comprising three oligosaccharide structures, each selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1-, and each attached to a serine or threonine residue in LYPD3.

[0059] The one or more oligosaccharide structures are particularly present in the C-terminal Ser / Thr / Pro-rich domain of LYPD3. In a particular embodiment, at least one, and in particular all, of the oligosaccharide structures are attached to a serine or threonine residue within positions 234 to 303 of SEQ ID NO: 137. In particular, at least one, and in particular all, of the oligosaccharide structures are attached to a serine or threonine residue within positions 247 to 297 of SEQ ID NO: 137.

[0060] The antibody is capable of binding to human LYPD3 in a combined peptide and carbohydrate epitope. For example, the epitope comprises one or more amino acids from positions 234 to 303 of SEQ ID NO: 137. In particular, the epitope comprises one or more amino acids from positions 247 to 297 of SEQ ID NO: 137. In particular embodiments, at least 25%, particularly at least 50%, and particularly at least 75% of the amino acids of the epitope are located between positions 234 to 303 of SEQ ID NO: 137, particularly between positions 247 to 297 of SEQ ID NO: 137. Preferably, all amino acids of the epitope are located between positions 234 to 303 of SEQ ID NO: 137.

[0061] In this regard, the term "capable of binding to human LYPD3 at a combined peptide and carbohydrate epitope" means that the antibody directly interacts with the amino acids and one or more glycan structures of LYPD3, or directly interacts only with the amino acids of LYPD3, or directly interacts only with the carbohydrate chains of LYPD3. In all cases, the binding affinity of the antibody to LYPD3 is higher when oligosaccharide structures selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1- are attached to the amino acids of LYPD3 compared to non-glycosylated LYPD3. In embodiments where the antibody directly interacts only with the amino acids of LYPD3 but not with the carbohydrate chains, the higher affinity for O-glycosylated LYPD3 is due to a conformational change in the polypeptide chain induced by the oligosaccharides attached to LYPD3.

[0062] In certain embodiments, the antibody specifically binds to tumor-associated LYPD3. The antibody binds with higher binding affinity to glycosylated human LYPD3, particularly compared to oligosaccharide structures selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1- attached to carrier molecules unrelated to LYPD3. For example, the carrier molecule can be polyacrylamide (PAA) or random peptides. In certain embodiments, the antibody is capable of binding to tumor-associated LYPD3 with higher binding affinity than LYPD3 expressed by cells of normal tissues.

[0063] In certain embodiments, the antibodies can bind to human LYPD3 glycosylated with one or more of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1-. Thus, the antibodies bind to LYPD3 when glycosylated with GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, sialylated Galβ1-3GalNAcα1-, or any mixture of two or all of these glycan structures. In these embodiments, LYPD3 can also carry other glycan structures, so long as at least one of the above glycan structures is also present. In particular, these antibodies specifically bind to human LYPD3 glycosylated with one or more of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1-.

[0064] In certain embodiments, the antibodies are capable of binding to human LYPD3 glycosylated with either or both GalNAcα1- and Galβ1-3GalNAcα1-. Thus, the antibodies bind to LYPD3 when glycosylated with GalNAcα1-, Galβ1-3GalNAcα1-, or any mixture of these two glycan structures. In these embodiments, LYPD3 may also carry other glycan structures, so long as at least one of the above glycan structures is also present. In particular, these antibodies specifically bind to human LYPD3 glycosylated with either or both GalNAcα1- and Galβ1-3GalNAcα1-.

[0065] In certain embodiments, the antibodies are capable of binding to human LYPD3 glycosylated with either or both Galβ1-3GalNAcα1- and sialylated Galβ1-3GalNAcα1-. Thus, the antibodies bind to LYPD3 when glycosylated with Galβ1-3GalNAcα1-, sialylated Galβ1-3GalNAcα1-, or any mixture of these two glycan structures. In these embodiments, LYPD3 may also carry other glycan structures, so long as at least one of the above glycan structures is also present. In particular, these antibodies specifically bind to human LYPD3 glycosylated with either or both Galβ1-3GalNAcα1- and sialylated Galβ1-3GalNAcα1-.

[0066] In certain embodiments, the antibodies are capable of binding to human LYPD3 glycosylated with Galβ1-3GalNAcα1-. Thus, the antibodies bind to LYPD3 when it is glycosylated with Galβ1-3GalNAcα1-. In these embodiments, LYPD3 can also carry other glycan structures, provided that Galβ1-3GalNAcα1- is also present. In particular, these antibodies specifically bind to human LYPD3 glycosylated with Galβ1-3GalNAcα1-.

[0067] In a second aspect, the present invention provides an anti-LYPD3 antibody comprising: (i) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO:4, CDR-L2 having the amino acid sequence of SEQ ID NO:5, and CDR-L3 having the amino acid sequence of SEQ ID NO:6; or (ii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; or (iii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 17, CDR-H2 having the amino acid sequence of SEQ ID NO: 18, and CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 20, CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or (iv) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO:28, CDR-L2 having the amino acid sequence of SEQ ID NO:29, and CDR-L3 having the amino acid sequence of SEQ ID NO:30; or (v) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; or (vi) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 41, CDR-H2 having the amino acid sequence of SEQ ID NO: 42, and CDR-H3 having the amino acid sequence of SEQ ID NO: 43; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 44, CDR-L2 having the amino acid sequence of SEQ ID NO: 45, and CDR-L3 having the amino acid sequence of SEQ ID NO: 46; or (vii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 49, CDR-H2 having the amino acid sequence of SEQ ID NO: 50, and CDR-H3 having the amino acid sequence of SEQ ID NO: 51; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 52, CDR-L2 having the amino acid sequence of SEQ ID NO: 53, and CDR-L3 having the amino acid sequence of SEQ ID NO: 54; or (viii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 58, and CDR-H3 having the amino acid sequence of SEQ ID NO: 59; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 60, CDR-L2 having the amino acid sequence of SEQ ID NO: 61, and CDR-L3 having the amino acid sequence of SEQ ID NO: 62; or (ix) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 65, CDR-H2 having the amino acid sequence of SEQ ID NO: 66, and CDR-H3 having the amino acid sequence of SEQ ID NO: 67; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 68, CDR-L2 having the amino acid sequence of SEQ ID NO: 69, and CDR-L3 having the amino acid sequence of SEQ ID NO: 70; or (x) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 73, CDR-H2 having the amino acid sequence of SEQ ID NO: 74, and CDR-H3 having the amino acid sequence of SEQ ID NO: 75; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 76, CDR-L2 having the amino acid sequence of SEQ ID NO: 77, and CDR-L3 having the amino acid sequence of SEQ ID NO: 78; or (xi) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 81, CDR-H2 having the amino acid sequence of SEQ ID NO: 82, and CDR-H3 having the amino acid sequence of SEQ ID NO: 83; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 84, CDR-L2 having the amino acid sequence of SEQ ID NO: 85, and CDR-L3 having the amino acid sequence of SEQ ID NO: 86; or (xii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 89, CDR-H2 having the amino acid sequence of SEQ ID NO: 90, and CDR-H3 having the amino acid sequence of SEQ ID NO: 91; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 92, CDR-L2 having the amino acid sequence of SEQ ID NO: 93, and CDR-L3 having the amino acid sequence of SEQ ID NO: 94; or (xiii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 97, CDR-H2 having the amino acid sequence of SEQ ID NO: 98, and CDR-H3 having the amino acid sequence of SEQ ID NO: 99; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 100, CDR-L2 having the amino acid sequence of SEQ ID NO: 101, and CDR-L3 having the amino acid sequence of SEQ ID NO: 102; or (xiv) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 105, CDR-H2 having the amino acid sequence of SEQ ID NO: 106, and CDR-H3 having the amino acid sequence of SEQ ID NO: 107; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 108, CDR-L2 having the amino acid sequence of SEQ ID NO: 109, and CDR-L3 having the amino acid sequence of SEQ ID NO: 110; or (xv) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 113, CDR-H2 having the amino acid sequence of SEQ ID NO: 114, and CDR-H3 having the amino acid sequence of SEQ ID NO: 115; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 116, CDR-L2 having the amino acid sequence of SEQ ID NO: 117, and CDR-L3 having the amino acid sequence of SEQ ID NO: 118; or (xvi) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 121, CDR-H2 having the amino acid sequence of SEQ ID NO: 122, and CDR-H3 having the amino acid sequence of SEQ ID NO: 123; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 124, CDR-L2 having the amino acid sequence of SEQ ID NO: 125, and CDR-L3 having the amino acid sequence of SEQ ID NO: 126; or (xvii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 129, CDR-H2 having the amino acid sequence of SEQ ID NO: 130, and CDR-H3 having the amino acid sequence of SEQ ID NO: 131; and A light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 132, CDR-L2 having the amino acid sequence of SEQ ID NO: 133, and CDR-L3 having the amino acid sequence of SEQ ID NO: 134.

[0068] In certain embodiments, the anti-LYPD3 antibody comprises the heavy chain variable region and light chain variable region described in any one or more of items (i) to (viii) above. In preferred embodiments, the anti-LYPD3 antibody comprises the heavy chain variable region and light chain variable region described in item (ii) above. In preferred embodiments, the anti-LYPD3 antibody comprises the heavy chain variable region and light chain variable region described in item (v) above.

[0069] In certain embodiments, the anti-LYPD3 antibody may have one, two, or three amino acid substitutions, particularly one or two, and particularly one amino acid substitution, in a total of six CDR sequences. In these embodiments, the anti-LYPD3 antibody retains the antigen specificity of the antibody without said amino acid substitutions. As used herein, "amino acid substitution" also includes amino acid additions and amino acid deletions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0070] The antibody according to the second aspect of the invention in particular exhibits one or more binding activities as defined for the antibody according to the first aspect, in particular the antibody according to the second aspect is an antibody according to the first aspect.

[0071] In certain embodiments, an antibody according to the invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 7, comprising complementarity determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region having an amino acid sequence at least 60% identical to the amino acid sequence of SEQ ID NO:8 over its entire length, and comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO:4, CDR-L2 having the amino acid sequence of SEQ ID NO:5, and CDR-L3 having the amino acid sequence of SEQ ID NO:6; (ii) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 15, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 16, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; (iii) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 23, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 17, CDR-H2 having the amino acid sequence of SEQ ID NO: 18, and CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 24, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 20, CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; (iv) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 31, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 32, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 28, CDR-L2 having the amino acid sequence of SEQ ID NO: 29, and CDR-L3 having the amino acid sequence of SEQ ID NO: 30; (v) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 39, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 40, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; (vi) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 47, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 41, CDR-H2 having the amino acid sequence of SEQ ID NO: 42, and CDR-H3 having the amino acid sequence of SEQ ID NO: 43; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 48, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 44, CDR-L2 having the amino acid sequence of SEQ ID NO: 45, and CDR-L3 having the amino acid sequence of SEQ ID NO: 46; (vii) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 55, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 49, CDR-H2 having the amino acid sequence of SEQ ID NO: 50, and CDR-H3 having the amino acid sequence of SEQ ID NO: 51; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO:56, comprising CDR-L1 having the amino acid sequence of SEQ ID NO:52, CDR-L2 having the amino acid sequence of SEQ ID NO:53, and CDR-L3 having the amino acid sequence of SEQ ID NO:54; and (viii) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 63, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 58, and CDR-H3 having the amino acid sequence of SEQ ID NO: 59; and A light chain variable region having an amino acid sequence at least 60% identical to the amino acid sequence of SEQ ID NO: 64 over its entire length, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 60, CDR-L2 having the amino acid sequence of SEQ ID NO: 61, and CDR-L3 having the amino acid sequence of SEQ ID NO: 62.

[0072] In preferred embodiments, the anti-LYPD3 antibody comprises the heavy and light chain variable regions described in item (ii) above. In preferred embodiments, the anti-LYPD3 antibody comprises the heavy and light chain variable regions described in item (v) above. In the above embodiments, the sequence identity may be particularly at least 70%, preferably at least 80%, more preferably at least 90%. The above embodiments are particularly humanized versions of the respective antibodies, in which the amino acid sequence changes are substitutions for amino acid residues of the relevant human antibody sequence.

[0073] In certain embodiments, the anti-LYPD3 antibody may further have 1, 2, or 3 amino acid substitutions, particularly 1 or 2, and particularly 1 amino acid substitution, in all six CDR sequences. In these embodiments, the anti-LYPD3 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0074] In a further embodiment, an antibody according to the invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (ix) a heavy chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 71 over its entire length, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 65, CDR-H2 having the amino acid sequence of SEQ ID NO: 66, and CDR-H3 having the amino acid sequence of SEQ ID NO: 67; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 72, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 68, CDR-L2 having the amino acid sequence of SEQ ID NO: 69, and CDR-L3 having the amino acid sequence of SEQ ID NO: 70; (x) a heavy chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 79 over its entire length, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 73, CDR-H2 having the amino acid sequence of SEQ ID NO: 74, and CDR-H3 having the amino acid sequence of SEQ ID NO: 75; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 80, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 76, CDR-L2 having the amino acid sequence of SEQ ID NO: 77, and CDR-L3 having the amino acid sequence of SEQ ID NO: 78; (xi) a heavy chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 87 over its entire length, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 81, CDR-H2 having the amino acid sequence of SEQ ID NO: 82, and CDR-H3 having the amino acid sequence of SEQ ID NO: 83; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 88, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 84, CDR-L2 having the amino acid sequence of SEQ ID NO: 85, and CDR-L3 having the amino acid sequence of SEQ ID NO: 86; (xii) a heavy chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 95, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 89, CDR-H2 having the amino acid sequence of SEQ ID NO: 90, and CDR-H3 having the amino acid sequence of SEQ ID NO: 91; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 96, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 92, CDR-L2 having the amino acid sequence of SEQ ID NO: 93, and CDR-L3 having the amino acid sequence of SEQ ID NO: 94; (xiii) a heavy chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 103 over its entire length, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 97, CDR-H2 having the amino acid sequence of SEQ ID NO: 98, and CDR-H3 having the amino acid sequence of SEQ ID NO: 99; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 104, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 100, CDR-L2 having the amino acid sequence of SEQ ID NO: 101, and CDR-L3 having the amino acid sequence of SEQ ID NO: 102; (xiv) a heavy chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 111, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 105, CDR-H2 having the amino acid sequence of SEQ ID NO: 106, and CDR-H3 having the amino acid sequence of SEQ ID NO: 107; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 112, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 108, CDR-L2 having the amino acid sequence of SEQ ID NO: 109, and CDR-L3 having the amino acid sequence of SEQ ID NO: 110; (xv) a heavy chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 119, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 113, CDR-H2 having the amino acid sequence of SEQ ID NO: 114, and CDR-H3 having the amino acid sequence of SEQ ID NO: 115; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 120, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 116, CDR-L2 having the amino acid sequence of SEQ ID NO: 117, and CDR-L3 having the amino acid sequence of SEQ ID NO: 118; (xvi) a heavy chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 127, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 121, CDR-H2 having the amino acid sequence of SEQ ID NO: 122, and CDR-H3 having the amino acid sequence of SEQ ID NO: 123; and a light chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 128, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 124, CDR-L2 having the amino acid sequence of SEQ ID NO: 125, and CDR-L3 having the amino acid sequence of SEQ ID NO: 126; and (xvii) a heavy chain variable region having an amino acid sequence at least 90% identical over its entire length to the amino acid sequence of SEQ ID NO: 135, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 129, CDR-H2 having the amino acid sequence of SEQ ID NO: 130, and CDR-H3 having the amino acid sequence of SEQ ID NO: 131; and A light chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 136 over its entire length, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 132, CDR-L2 having the amino acid sequence of SEQ ID NO: 133, and CDR-L3 having the amino acid sequence of SEQ ID NO: 134.

[0075] In the above embodiments, the sequence identity may particularly be at least 95%. In certain embodiments, the anti-LYPD3 antibody may further have one, two, or three amino acid substitutions, particularly one or two, and particularly one amino acid substitution, in all six CDR sequences. In these embodiments, the anti-LYPD3 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0076] In particular, the antibodies according to the present invention comprise a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40; (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56; and (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64.

[0077] In a preferred embodiment, the anti-LYPD3 antibody comprises the heavy chain variable region and light chain variable region described in item (ii) above. In a preferred embodiment, the anti-LYPD3 antibody comprises the heavy chain variable region and light chain variable region described in item (v) above. In a specific embodiment, the antibody is a humanized version of any one of the antibodies described in items (i) to (viii) above. The humanized version has at least 60%, particularly at least 70%, preferably at least 80%, and more preferably at least 90% amino acid sequence identity with the respective antibody, particularly over the entire length of the original sequence. In a specific embodiment, the antibody according to the present invention is a humanized antibody derived from an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, or derived from an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

[0078] In particular, the antibody according to the present invention is a humanized antibody comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 139, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 143; (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 145; (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 146; (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 138, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 145; and (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 145.

[0079] The antibodies according to the invention may also comprise a heavy chain variable region and a light chain variable region selected from the group consisting of: (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 80; (xi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88; (xii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96; (xiii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 103, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 104; (xiv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 111, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 112; (xv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 119, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 120; (xvi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 127, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 128; and (xvii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 136.

[0080] In embodiments in which the heavy and / or light chain variable regions comprise an amino acid sequence having a certain identity to the amino acid sequence of SEQ ID NO: 7, 8, 15, 16, 23, 24, 31, 32, 39, 40, 47, 48, 55, 56, 63, 64, 71, 72, 79, 80, 87, 88, 95, 96, 103, 104, 111, 112, 119, 120, 127, 128, 135, or 136, sequence deviations from said amino acid sequence are located particularly in the framework regions, but not in the CDRs. Thus, in these embodiments, the heavy and light chain variable regions comprise the respective CDR sequences as defined herein.

[0081] In certain embodiments, the antibody is capable of binding to human LYPD3 glycosylated with one or more of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1-, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising complementarity determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6; (ii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 49, CDR-H2 having the amino acid sequence of SEQ ID NO: 50, and CDR-H3 having the amino acid sequence of SEQ ID NO: 51; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 52, CDR-L2 having the amino acid sequence of SEQ ID NO: 53, and CDR-L3 having the amino acid sequence of SEQ ID NO: 54; (iii) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 7, comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO:8, and comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO:4, CDR-L2 having the amino acid sequence of SEQ ID NO:5, and CDR-L3 having the amino acid sequence of SEQ ID NO:6; (iv) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 55, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 49, CDR-H2 having the amino acid sequence of SEQ ID NO: 50, and CDR-H3 having the amino acid sequence of SEQ ID NO: 51; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 56, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 52, CDR-L2 having the amino acid sequence of SEQ ID NO: 53, and CDR-L3 having the amino acid sequence of SEQ ID NO: 54; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; and (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56.

[0082] In certain embodiments, the antibody is capable of binding to human LYPD3 glycosylated with either or both GalNAcα1- and Galβ1-3GalNAcα1-, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; (ii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 41, CDR-H2 having the amino acid sequence of SEQ ID NO: 42, and CDR-H3 having the amino acid sequence of SEQ ID NO: 43; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 44, CDR-L2 having the amino acid sequence of SEQ ID NO: 45, and CDR-L3 having the amino acid sequence of SEQ ID NO: 46; (iii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 58, and CDR-H3 having the amino acid sequence of SEQ ID NO: 59; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 60, CDR-L2 having the amino acid sequence of SEQ ID NO: 61, and CDR-L3 having the amino acid sequence of SEQ ID NO: 62; (iv) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 39, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 40, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; (v) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 47, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 41, CDR-H2 having the amino acid sequence of SEQ ID NO: 42, and CDR-H3 having the amino acid sequence of SEQ ID NO: 43; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 48, and comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 44, CDR-L2 having the amino acid sequence of SEQ ID NO: 45, and CDR-L3 having the amino acid sequence of SEQ ID NO: 46; (vi) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 63, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 58, and CDR-H3 having the amino acid sequence of SEQ ID NO: 59; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 64, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 60, CDR-L2 having the amino acid sequence of SEQ ID NO: 61, and CDR-L3 having the amino acid sequence of SEQ ID NO: 62; (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40; (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 139, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (xi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 144; (xi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 143; (xii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 145; (xiii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 146; (xiv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 138, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 145; (xv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 145; (xvi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; and (xvii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64.

[0083] In a preferred embodiment, the antibody is capable of binding to human LYPD3 glycosylated with either or both GalNAcα1- and Galβ1-3GalNAcα1-, and comprises a heavy chain variable region and a light chain variable region described in any one of items (i), (iv), (vii), and (viii) to (xv) above.

[0084] In certain embodiments, the antibody is capable of binding to human LYPD3 glycosylated with either or both Galβ1-3GalNAcα1- and sialylated Galβ1-3GalNAcα1-, and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 28, CDR-L2 having the amino acid sequence of SEQ ID NO: 29, and CDR-L3 having the amino acid sequence of SEQ ID NO: 30; (ii) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 31, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 32, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 28, CDR-L2 having the amino acid sequence of SEQ ID NO: 3295, and CDR-L3 having the amino acid sequence of SEQ ID NO: 30; and (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32.

[0085] In certain embodiments, the antibody is capable of binding to Galβ1-3GalNAcα1-glycosylated human LYPD3 and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising complementarity determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; (ii) a heavy chain variable region comprising complementarity-determining regions (CDRs) CDR-H1 having the amino acid sequence of SEQ ID NO: 17, CDR-H2 having the amino acid sequence of SEQ ID NO: 18, and CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising complementarity determining regions (CDRs) CDR-L1 having the amino acid sequence of SEQ ID NO: 20, CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; (iii) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 15, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 16, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; (iv) a heavy chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 23, comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 17, CDR-H2 having the amino acid sequence of SEQ ID NO: 18, and CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a light chain variable region having an amino acid sequence at least 60% identical over its entire length to the amino acid sequence of SEQ ID NO: 24, comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 20, CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; and (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24.

[0086] In a preferred embodiment, the antibody is capable of binding to Galβ1-3GalNAcα1-glycosylated human LYPD3 and comprises a heavy chain variable region and a light chain variable region described in any one of items (i), (iii) and (v) above.

[0087] In the above embodiments, the sequence identity may in particular be at least 70%, preferably at least 80%, more preferably at least 90%. In the above embodiments referring to sequence identity, the antibodies are in particular humanized versions of the respective antibodies in which the amino acid sequence changes are substitutions for amino acid residues of the relevant human antibody sequence.

[0088] In certain embodiments, the anti-LYPD3 antibody may further have 1, 2, or 3 amino acid substitutions, particularly 1 or 2, and particularly 1 amino acid substitution, in all six CDR sequences. In these embodiments, the anti-LYPD3 antibody retains the antigen specificity of the antibody without said amino acid substitutions.

[0089] In certain embodiments, the antibody comprises an Fc region. The antibody may be, in particular, a whole antibody. In particular, the antibody may comprise two heavy chains and two light chains. The antibody may be of any isotype, in particular an IgG type antibody, particularly IgG1, IgG2, or IgG4. In certain embodiments, the antibody is an IgG1 type antibody. The antibody may in particular bind to one or more human Fc receptors, in particular a human Fcγ receptor such as Fcγ receptor IIIa. In certain embodiments, the anti-LYPD3 antibody is a chimeric, humanized, or human antibody.

[0090] In further embodiments, the anti-LYPD3 antibody is an antibody fragment. In particular, the fragment is selected from the group consisting of: (i) a Fab fragment; (ii) a F(ab)2 fragment; (iii) a Fd fragment; (iv) a Fv fragment; (v) a scFv fragment; and (vi) a (Fv)2 fragment. In certain embodiments, the anti-LYPD3 antibody does not include an Fc region.

[0091] In certain embodiments, the anti-LYPD3 antibody is glycosylated, particularly N-glycosylated. In particular, the antibody has a glycosylation site in the second constant domain (CH2) of the heavy chain. Antibodies typically have two heavy chains with identical amino acid sequences. Therefore, the antibody preferably has at least two glycosylation sites, one in each of its two CH2 domains. This glycosylation site is particularly located 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 homologous regions of other antibody isotypes. The actual location of this conserved glycosylation site may vary within the amino acid sequence of the antibody due to optional additional amino acids or other sequence modifications that may be present in the variable region.

[0092] In a preferred embodiment, the anti-LYPD3 antibody does not contain N-glycolylneuraminic acid (NeuGc) or detectable amounts of NeuGc. Furthermore, the antibody preferably does not contain Galili epitopes (Galα1,3-Gal structures) or detectable amounts of Galili epitopes. In particular, the relative amount of glycans bearing NeuGc and / or Galα1,3-Gal structures is less than 0.1% or even less than 0.02% of the total amount of glycans attached to the Fc portion of antibodies in the antibody population.

[0093] In other embodiments, the anti-LYPD3 antibody is aglycosylated in its CH2 domain. In these embodiments, the antibody CH2 domain can 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 Fcγ receptor binding and therefore reduced effector function. In further embodiments, the anti-LYPD3 antibody can 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.

[0094] The present invention further provides anti-LYPD3 antibodies that compete for LYPD3 binding with the antibodies described herein, particularly the antibodies described in the second aspect of the invention. In certain embodiments, the competing anti-LYPD3 antibody competes with an antibody comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40; (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56; and (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64.

[0095] Assays for determining the competitive binding of two antibodies are well known in the art.For example, ELISA can be used, in which LYPD3 is immobilized, a first antibody is labeled, and an excess of a second antibody is added to the immobilized LYPD3.If the label can be detected in the sample containing immobilized LYPD3 after washing, competitive binding is not observed.In a preferred embodiment, a control experiment is carried out in which the second antibody is labeled and the first antibody is added in excess.Alternatively, one antibody can be immobilized, LYPD3 can be labeled, and the other antibody can be added in excess.

[0096] 2. Generation of Anti-LYPD3 Antibodies The anti-LYPD3 antibody is preferably produced recombinantly in a host cell. Thus, the antibody is particularly a monoclonal antibody. The host cell used to produce the antibody can be any host cell that can be used to produce antibodies. 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.

[0097] In certain embodiments, the anti-LYPD3 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 the anti-LYPD3 antibody and suitable production procedures are described in WO 2008 / 028686. In a specific embodiment, the anti-LYPD3 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 by Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin (DE), at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstrasse 7B, 38124 Braunschweig (DE) under the 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 high sialylation, high bisecting GlycNAc, high galactosylation, and high 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 very low. Other suitable cell lines include K562, a human myeloid leukemia cell line present in the American Type Culture Collection (ATCC CCL-243), CHO cells, and cell lines derived therefrom. In a specific embodiment, the anti-LYPD3 antibody is expressed in CHO cells, particularly CHO dhfr cells. - It is produced recombinantly in cells.

[0098] 3. Anti-LYPD3 Antibody Conjugates In certain embodiments, the anti-LYPD3 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 antibody can be conjugated to one or more additional agents. When two or more additional agents are present in the conjugate, these additional agents can be the same or different, and particularly all the same. The conjugation of the additional agent to the antibody can be achieved using any method known in the art. The additional agent can be covalently or non-covalently attached to the antibody, particularly by fusion or chemical coupling. In certain embodiments, the additional agent is covalently attached to the antibody, particularly via a linker moiety. The linker moiety can be any chemical entity suitable for binding the additional agent to the antibody.

[0099] The additional agent is preferably useful for 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 one of a different species and / or of a different specificity than the anti-LYPD3 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.

[0100] In certain embodiments, the additional agent is a polypeptide or protein. This polypeptide or protein may be fused to a polypeptide chain of the anti-LYPD3 antibody, in particular. In certain embodiments, the additional agent is a polypeptide or protein fused to the C-terminus of the antibody light chain of the anti-LYPD3 antibody. In embodiments where the anti-LYPD3 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 anti-LYPD3 antibody. In embodiments where the 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 antibody does not comprise one or more light chains and one or more heavy chains, for example, when the 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 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.

[0101] 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 activation targets such as CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM), and CD258 (LIGHT), and inhibitory targets such as CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA, and phosphatidylserine, as well as their respective ligands, such as PDL1. In a further embodiment, the additional polypeptide or protein agent is an anti-cancer antibody directed against a tumor-associated antigen. Exemplary suitable tumor targets and anti-cancer antibodies that can be used as fusion partners are described below in connection with combination therapy.

[0102] 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.

[0103] A conjugate comprising an anti-LYPD3 antibody conjugated to an additional agent is, in particular, a chimeric antigen receptor (CAR). Such a conjugate is also referred to herein as an anti-LYPD3 CAR.

[0104] In these embodiments, the additional agent is an antigen receptor, particularly a T cell receptor or T cell co-receptor, or a part and / or chimera thereof. In particular, an anti-LYPD3 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 antibody and the transmembrane domain.

[0105] In these embodiments, the anti-LYPD3 antibody is particularly 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, for example, on 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 include the cytoplasmic domain of the zeta chain of the T cell receptor. Furthermore, the intracellular T cell signaling domain may include 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).

[0106] An exemplary CAR may comprise, from N- to C-terminus, (i) an anti-LYPD3 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.

[0107] Alternatively, an anti-LYPD3 antibody, particularly in a single-chain format such as an scFv, can be N-terminally fused to a CD3 chain of the T cell receptor complex, particularly the CD3ε chain, to form a chimeric antigen receptor. Alternatively, an anti-LYPD3 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 on T cells or NK cells.

[0108] In certain embodiments, the additional agent is a cytotoxic or chemotherapeutic agent, particularly a cytotoxin. Specific examples of chemotherapeutic agents that can be conjugated as the additional agent include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes such as taxol, topoisomerase inhibitors such as irinotecan and topotecan, antineoplastic agents such as doxorubicin, or microtubule inhibitors such as auristatins and maytansine / maytansinoids.

[0109] Chemotherapeutic agents include, inter alia, V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizing agents, auristatins, dolastatins, maytansines, maytansinoids, amatoxins, methionine aminopeptidases, inhibitors of the nuclear export of protein 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 attached to the anti-LYPD3 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.

[0110] 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 attached to the anti-LYPD3 antibody. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is an auristatin, particularly monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), or auristatin T. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 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 attached to the anti-LYPD3 antibody is a DNA alkylating agent, particularly an indolinobenzodiazepine or an oxazolidinobenzodiazepine. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is a DNA damaging agent, particularly calicheamicin. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 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 aspects, the chemotherapeutic agent attached to the anti-LYPD3 antibody is an inhibitor of microtubule formation, particularly tubulysin, ansamitocin, podophyllotoxin, or vinblastine. In some aspects, the chemotherapeutic agent attached to the anti-LYPD3 antibody is a microtubule stabilizer, particularly paclitaxel or epothilone. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is an actin stabilizer, particularly phallotoxin.In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is a topoisomerase II inhibitor, particularly teniposide, XK469, razoxane, amsacrine, idarubicin, or mevalon. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is a platinum compound, particularly cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is a ribosome inhibitor, particularly ricin, saporin, abrin, diphtheria toxin, or exotoxin A. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is an RNA polymerase II inhibitor, particularly an amatoxin, such as amanitin. In some embodiments, the chemotherapeutic agent attached to the anti-LYPD3 antibody is a bacterial toxin, particularly anthrax toxin. Suitable antibody drug conjugates are also described in European Patent No. 16151774.3 and Luxembourg Patent No. 92659, which are expressly incorporated herein by reference.

[0111] Further suitable toxins that can be conjugated to anti-LYPD3 antibodies are described below with respect to combination therapy.

[0112] 4. Nucleic acid encoding anti-LYPD3 antibody In a further aspect, the present invention provides a nucleic acid encoding an anti-LYPD3 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 adapted to 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 such as cDNA or single-stranded RNA such as 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 antibody.

[0113] When the anti-LYPD3 antibody is composed of two or more different amino acid chains, such as a light chain and a heavy chain, the nucleic acid may be, for example, 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 create separate amino acid chains. Alternatively, the nucleic acid may 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 may be a single nucleic acid molecule containing one coding region encoding the heavy chain and one coding region separated by a linker peptide containing a self-cleaving peptide such as a 2A peptide and / or a protease recognition site such as a furin recognition site. In addition to the coding region encoding the antibody, the nucleic acid may also contain additional nucleic acid sequences or other modifications that, for example, may encode other proteins, affect the transcription and / or translation of one or more coding regions, affect the stability or other physical or chemical properties of the nucleic acid, or may have no function at all.

[0114] In certain embodiments, the nucleic acid is a viral vector that can be used to infect human cells. These viral vectors are suitable for human therapy, for example, by directing viral infection and / or replication to diseased cells, such as tumor cells, or, in embodiments where the anti-LYPD3 antibody is in the form of a chimeric antigen receptor, for modifying T cells to obtain CAR T cells.

[0115] 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. Furthermore, the expression cassette or vector may comprise additional 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 for expressing antibodies, and vectors comprising the respective expression cassettes, are well known in the art and therefore do not require further description here.

[0116] 5.Host cells The present invention further provides a host cell comprising a nucleic acid described in the present invention or an expression cassette or vector described in the present invention. The host cell may be any host cell. It may be an isolated cell or a cell contained in a tissue. Preferably, the host cell is a cultured cell, particularly a primary cell or a cell of an established cell line, preferably a tumor-derived cell. Suitable host cells are particularly eukaryotic host cells, particularly mammalian host cells. Exemplary host cells include yeast cells, such as Pichia pastoris cell lines, insect cells, such as SF9 and SF21 cell lines, plant cells, avian cells, such as the EB66 duck cell line, rodent cells, such as CHO, 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.

[0117] In a preferred embodiment of the present invention, the host cell is a CHO cell or a cell derived from a human myeloid leukemia cell. Preferably, it is selected from the following cells or cell lines: K562, KG1, MUTZ-3, CHO, or a cell or cell line derived therefrom. The host cell is preferably selected from the group consisting of CHO, NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, and a cell or cell line derived from any of the aforementioned host cells. These cell lines and their characteristics are described in detail in PCT Application WO 2008 / 028686. In a specific embodiment, the host cell is optimized for the expression of glycoproteins, particularly antibodies, with a specific glycosylation pattern. Preferably, the codon usage in the coding region and / or promoter of the nucleic acid described in the present invention, as well as in the additional elements of the expression cassette or vector, is adapted to the type of host cell used, and more preferably, is optimized. Preferably, the anti-LYPD3 antibody is produced by the above-mentioned host cell or cell line.

[0118] The present invention further provides a host cell carrying an anti-LYPD3 CAR. Such a host cell is also referred to herein as a CAR cell. The CAR cell particularly comprises a nucleic acid described in the present invention encoding an anti-LYPD3 CAR, or an expression cassette or vector described in the present invention. In certain embodiments, the CAR cell is engineered to express an anti-LYPD3 CAR, for example, by introducing a vector containing an expression cassette for the anti-LYPD3 CAR.

[0119] In certain embodiments, CAR cell is leukocyte, particularly lymphocyte such as T cell, NK cell and NKT cell, or monocyte such as macrophage.CAR cell is particularly primary leukocyte.In certain embodiments, CAR cell is selected from the group consisting of primary T cell, primary NK cell, primary NKT cell and primary macrophage, particularly primary T cell.

[0120] 6. Pharmaceutical Compositions and Therapeutic Uses In another aspect, the present invention provides a composition comprising an anti-LYPD3 antibody, a nucleic acid, an expression cassette or vector, a host cell, or a conjugate. The composition may also contain two or more of these components. In addition, the composition may contain one or more additional components selected from the group consisting of solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, the components of the composition are preferably all pharmaceutically acceptable. The composition may be a solid or fluid composition, particularly a (preferably aqueous) solution, emulsion, or suspension, or a lyophilized powder.

[0121] The anti-LYPD3 antibody or its conjugate or CAR cell is particularly useful in medicine, particularly for the treatment, diagnosis, prognosis, and / or monitoring of diseases, particularly the diseases described herein, such as cancer and infectious diseases, preferably cancer. Thus, in a further aspect, the present invention provides an anti-LYPD3 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, particularly diseases associated with LYPD3. Exemplary diseases include diseases associated with abnormal cell proliferation, such as cancer, adolescent idiopathic scoliosis, cholesteryl ester transfer protein (CETP) deficiency, fish eye disease, combined hyperlipidemia, citrullinemia, and familial hypercholesterolemia.

[0122] In a preferred embodiment, the disease is cancer. Preferably, the cancer is selected from the group consisting of head and neck cancer, colon cancer, colorectal cancer, hepatocellular carcinoma, skin cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, esophageal cancer, lung cancer, and genital cancer. The cancer may be, for example, head and neck squamous cell carcinoma, esophageal cancer, breast cancer, cervical cancer, skin cancer, or squamous cell carcinoma of the anal region, rectum, oral cavity, lip, bucca cavioris, nose, penis, vulva, epiglottis, tongue, or skin.

[0123] The cancer is LYPD3-positive, and particularly comprises cancer cells bearing LYPD3 on their cell surface. In certain embodiments, the anti-LYPD3 antibody is used in combination with another anti-cancer therapeutic agent. The additional therapeutic agent may be any known anti-cancer drug, particularly an antibody against a cancer antigen. Antibodies suitable for use in combination with anti-LYPD3 antibodies 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 (Rituxan, Mab anti-CTLA-4 antibodies, such as ipilimumab and tremelimumab, anti-PD1 / PD-L1 antibodies, such as pembrolizumab, nivolumab, cemiplimab, atezolizumab, durvalumab, and avelumab, antibodies against TNF and TNFR superfamily members, such as urelumab, MEDI6469, TRX518, and varilumab; 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. Further suitable antibodies for use in combination with anti-LYPD3 antibodies include bispecific antibodies, particularly immune cell-engaging bispecific antibodies, such as blinatumomab (targeting CD19 and CD3), odronextamab (targeting CD20 and CD3), Regn4018 (targeting MUC16 and CD3), Regn4336 (targeting PSMA and CD3), and Regn7075 (targeting EGFR and CD28).

[0124] Additional anti-cancer therapeutic agents that can be used in combination with the anti-LYPD3 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; thromatase inhibitors, 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), etc.; topoisomerase inhibitors, such as ibuprofen rinotecan, 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](ethanediato-O,O') and the like. 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 anti-LYPD3 antibodies are described above with respect to agents that can be conjugated to the antibodies.

[0125] Treatment with anti-LYPD3 antibodies can further be combined with immunostimulants, cytokines, chemokines, radiation therapy, therapeutic cells such as natural or engineered immune cells (CAR-T cells, TCR-T cells, CAR-NK cells, CAR-monocytes, CAR-NKT cells, etc.), vaccines such as protein, peptide or RNA vaccines, B-Raf inhibitors such as vemurafenib, dexamethasone, protease inhibitors such as bortezomib, and lenalidomide.

[0126] For use in treating cancers in which cells express LYPD3, the antibody can be coupled to an additional agent, such as those described above. The additional agent is preferably a cytotoxic agent, such as a radionuclide or cytotoxin. Exemplary cytotoxic agents are described above. Cytotoxic agents also include precursor compounds that only express cytotoxic activity upon activation, for example, by light irradiation or an enzymatic reaction in vivo. One or more of the anti-cancer therapeutic agents described above can also be used as additional agents to couple to the anti-LYPD3 antibody. Furthermore, the antibody can 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.

[0127] For use as a detection agent in the diagnosis, prognosis and / or monitoring of disease, the antibody is preferably coupled to a labeling agent capable of producing a detectable signal. In particular, the labeling agent may be a radionuclide, a fluorophore or an enzyme. [Brief explanation of the drawings]

[0128] [Figure 1]Figure 1 shows the biosynthetic pathways of the Tn and TF carbohydrate antigens and their structures, in which squares represent GalNAc, circles represent Gal, and diamonds represent sialic acid. [Figure 2] Figure 2 shows the carbohydrate-dependent target binding of anti-LYPD3 antibodies to A) LYPD3-ECD or B) the STP-rich C-terminal portion of ECD (LYPD3-STP-ECD). Binding to equimolar amounts of antigen (35 nM) was assessed by antigen ELISA using 5 μg / ml of a-LYPD3 mAb. To control for antigen coating on the ELISA plate, aLYPD3 pAb or aMBL Ab was used. Proteins were expressed and purified in transfected NM-F9 cells with the respective O-glycosylation patterns (F9, O-glyc.), and de-O-glycosylated proteins were generated by enzymatic digestion of these NM-F9 proteins (F9 de-O-glyc.). Binding to recombinantly expressed and purified LYPD3-STP-ECD from O-glycosylation-deficient CHO cells was also tested (aglyc.). A high OD signal indicates strong binding between the antigen and the antibody. [Figure 3] Figure 3 shows the binding of anti-LYPD3 clones to different glycosylated LYPD3 variants. Binding to an equimolar amount of antigen (35 nM) was assessed by antigen ELISA using the indicated concentrations of aLYPD3 mAb. aLYPD3 pAb or aMBL Ab was used as a coating control, and aTF and aTn antibodies were used as glycosylation controls. TF and Tn-bearing proteins were recombinantly expressed and purified from NM-F9 cells; de-O-glycosylated proteins were obtained by enzymatic digestion of NM-F9-derived proteins; and Tn proteins were obtained by degalactosylation of NM-F9-derived proteins as described in Example 1. A high OD signal indicates strong binding between the antigen and antibody. [Figure 4]Figure 4 shows the analysis of anti-LYPD3 clones tested for glycan binding by ELISA. Binding to PAA-conjugated glycans coated at 10 μg / ml was assessed by antigen ELISA using 5 μg / ml of aLYPD3 mAb; aTn, aTF, and a-sTn mAbs were used to control coating of the four indicated glycans onto ELISA plates. A high OD signal indicates strong binding of the antigen to the antibody. [Figure 5] Figure 5 shows the binding curve of anti-huLYPD3 antibody to NM-F9-derived LYPD3-STP in an ELISA. The anti-LYPD3 antibody was titrated on an ELISA plate coated with the LYPD3-STP-ECD target protein. A high OD signal indicates strong binding between the antibody and the antigen. [Figure 6] Figure 6 shows the carbohydrate-dependent antigen binding of anti-LYPD3 antibodies to LYPD3-ECD compared with aLYPD3 mAb, aTF, and aTn. Binding to equimolar amounts of antigen was assessed by antigen ELISA using a-LYPD3 mAb. Proteins were expressed and purified in transfected NM-F9 or NM-H9D8 cells with the respective O-glycosylation patterns (NM-F9: primarily TF; H9D8: primarily sTF and TF). Desialylated proteins were generated by enzymatic treatment with sialidase (H9D8, de-sialyl: primarily TF), and degalactosylated proteins were generated by treatment with galactosidase (NM-F9, TN-glycyl: primarily Tn). A high OD signal indicates strong binding between the antigen and antibody. [Figure 7]Figure 7 shows the binding of aLYPD3 clones to cellular O-glycosylated and de-O-glycosylated LYPD3. Cell lines with different LYPD3 expression (LYPD3-F9 = high O-glycosylation, high LYPD3; NM-F9 = high O-glycosylation, low LYPD3; LYPD3-HEK-O-glycosylation KO = no O-glycosylation, high LYPD3) were stained with 1 μg / ml of aLYPD3 clones, control aLYPD3 pAb and mAb, and aTF mAb. The signal-to-noise ratio of stained cells is shown: the MFI (median fluorescence intensity) of stained cells divided by the MFI of isotype-stained cells. [Figure 8] Figure 8 shows the titration of aLYPD3 clones compared to a control aLYPD3 mAb on LYPD3-F9 cells expressing high levels of O-glycosylated LYPD3. Cells were stained with different concentrations of aLYPD3 clones and aLYPD3 mAb control and detected with a fluorophore-conjugated anti-human IgG secondary reagent. The MFI of live cells is shown. [Figure 9] Figure 9 shows titration of aLYPD3 clones on LYPD3-F9 cells, which express high levels of O-glycosylated LYPD3. Cells were stained with different concentrations of aLYPD3 clones and detected with a fluorophore-conjugated anti-human IgG secondary reagent. The MFI of live cells is shown. [Figure 10] Figure 10 shows binding of aLYPD3 clones to normal human epithelial mammary cells (HMECs). HMECs from healthy donors were stained with 10 μg / ml of aLYPD3 clones and a fluorophore-conjugated anti-human IgG secondary reagent. LYPD3 and TFa expression was confirmed using control aLYPD3 pAb and mAb and aTF mAb. The signal-to-noise ratio of stained cells is shown: MFI (median fluorescence intensity) of stained cells divided by the MFI of isotype-stained cells. [Figure 11-1]Figure 11 shows the binding of the aLYPD3 clone to Caov-3 cells, ZR-75-1 cells with and without sialidase treatment, and MDA-MB-231 cells after sialidase treatment. (A) Caov-3 cells were stained with 10 μg / ml of the aLYPD3 clone and a fluorophore-conjugated anti-human IgG secondary reagent. (B) ZR-75-1 cells were treated with or without sialidase (5 mU / ml for 30 minutes), washed, and stained with 10 μg / ml of the aLYPD3 clone and a fluorophore-conjugated anti-human IgG secondary reagent. (C) MDA-MB-231 cells were treated with sialidase (5 mU / ml for 30 minutes), then stained with 10 μg / ml of the aLYPD3 clone and a fluorophore-conjugated anti-human IgG secondary reagent. Expression of LYPD3 and TFa was confirmed using control aLYPD3 pAb and mAb and aTF mAb. The signal-to-noise ratio of stained cells is shown: MFI (median fluorescence intensity) of stained cells divided by the MFI of isotype-stained cells. [Figure 11-2] Same as above. [Figure 12] Figure 12 shows the ability of aLYPD3 clone 17B3 to inhibit the proliferation of LYPD3-F9 cells. Cells were incubated with various concentrations of aLYPD3 antibody 17B3 and a fixed amount of Protein G-MMAE. Proliferation was measured after 4 days. An irrelevant human IgG1 control shows no inhibition. Proliferation is shown as a relative percentage to the antibody-free medium control based on luminescence signal. [Figure 13-1] FIG. 13 shows the binding of selected anti-LYPD3 clones to healthy esophageal tissue sections compared to the aLYPD3 pAb control antibody. [Figure 13-2] Same as above. [Figure 14]Figure 14 shows the binding of humanized aLYPD3 antibodies to cellular O-glycans and cellular O-glycosylation and de-O-glycosylation of LYPD3, as determined by flow cytometry. (A) Cell lines with different LYPD3 expression and O-glycosylation (LYPD3-ECD HEK O-glycosylation KO = no O-glycosylation, high LYPD3; mock F9 O-glycosylation = high O-glycosylation with TF, low LYPD3; LYPD3-ECD F9 = high O-glycosylation with TF, high LYPD3) were stained with 10 μg / ml of humanized aLYPD3 antibodies (21E9-219 to 21E9-228), parental antibody (parental 21E9), and control antibodies against LYPD3 (aLYPD3 pAb and aLYPD3 mAb). (B) The cell line NM-F9 expressing LYPD3 with TF O-glycosylation was stained with humanized antibodies at different concentrations (0.1 μg / ml and 10 μg / ml). The signal-to-noise ratio of stained cells is shown: MFI (median fluorescence intensity) of stained cells divided by the MFI of isotype-stained cells. [Figure 15] Figure 15 shows binding of humanized aLYPD3 antibody to O-glycosylated LYPD3-STP-ECD in F9 cells. Binding to equimolar amounts of antigen was assessed by antigen ELISA using the indicated concentrations of humanized aLYPD3 mAb and parental aLYPD3 mAb. Proteins were expressed in transfected NM-F9 cells, purified, and subjected to TF O-glycosylation. A high OD signal indicates strong binding of the antigen to the antibody. [Figure 16] Figure 16 shows the glycan-specific binding of the humanized aLYPD3 antibody. Binding to equimolar amounts of antigen was assessed by antigen ELISA using the indicated concentrations of aLYPD3 mAb. Proteins were expressed and purified in transfected NM-F9 cells with the respective O-glycosylation patterns, and were either untreated (F9 O-glyc.: primarily TF), degalactosylated (F9 O-glyc.de-gal.: primarily Tn), or deglycosylated (F9 de-O-glyc.: no O-glycosylation). A high OD signal indicates strong binding between the antigen and antibody. [Example]

[0129] Example 1: Antigen production For optimized selection of antibodies that specifically bind to human LYPD3 with tumor-associated glycosylation patterns, we generated different O-glycosylation mutants of human LYPD3. Because the C-terminal portion of the extracellular domain of huLYPD3 (aa 234-303) contains numerous Ser / Thr / Pro residues and therefore a high density of O-glycosylation sites, this aa sequence of huLYPD3 was selected as a target structure ("LYPD3-STP") for antibody generation, which is likely to generate glyco-dependent anti-LYPD3 antibodies using the antibody generation approach described in Example 2.

[0130] Different cell lines were transfected with DNA encoding the huLYPD3 extracellular domain (ECD; aa 31–303), specifically the C-terminal Ser / Thr / Pro-rich domain (step-ECD; aa 234–303) of huLYPD3, to generate cells expressing soluble LYPD3-ECD or LYPD3-STP-ECD. The soluble protein constructs were recombinantly expressed as fusion proteins with, for example, a tandem Strep-tag® (Twin-Strep-tag®, IBA, Germany), maltose-binding lectin (herein referred to as MBL), or a polyhistidine tag, allowing purification from cell supernatants by affinity chromatography and detection of the protein via the fusion moiety independent of LYPD3. Furthermore, different cell lines were transfected with DNA encoding the complete human LYPD3 propeptide sequence (aa 31–346) or the C-terminal Ser / Thr / Pro-rich domain of the huLYPD3 propeptide (aa 234–346) together with a C-terminal EGFP fusion to generate cells expressing membrane-bound LYPD3 or LYPD3-STP, respectively, on the cell surface.

[0131] Cancer cell line-derived NM-F9 and NM-H9D8 cells were used to express human LYPD3 and LYPD3-STP proteins with tumor-associated O-glycosylation patterns. NM-H9D8 cells provide LYPD3 glycosylation with high sialylation (primarily sTF and sTn), while sialylation-deficient NM-F9 cells primarily produce LYPD3 carrying TF and less Tn. O-glycosylation-deficient (O-glyc KO) CHO (CH-O-delete; GlycoDisplay Apps, Copenhagen, Denmark) or HEK cells (HEK GALE / GALEK2 KO, Kerafast, Inc., Boston, MA, USA) were used to generate LYPD3 mutants without O-glycosylation.

[0132] Tn-LYPD3-STP-ECD is either recombinantly expressed in HEK cells deficient in the production of core 1 O-glycan extensions (HEK293 cosmc KO, Glycodisplay) or generated by enzymatic digestion of NM-F9-derived LYPD3-STP-ECD with β-galactosidase (GalactEXO, Genovis) to remove galactose residues from the TF structure, resulting in GalNAc linked to serine or threonine, termed the Tn antigen.

[0133] Soluble LYPD3 protein lacking O-glycosylation was purified directly from the supernatant of transfected O-glycosylation-deficient CHO cells or generated from protein purified from NM-H9D8 or NM-F9 transfectants by enzymatic digestion. The latter was performed by applying either soluble sialidase, O-glycosidase and α-N-acetylgalactosaminidase, or immobilized β-galactosidase and GalNAcase enzyme to a microspin column to remove sialic acids (α2-3, α2-6, and α2-8), core 1 O-glycans, and GalNAc residues on the glycoprotein (SialExo, Oglyzor, GalNAcEXO, GalactEXO, all Genovis).

[0134] Binding of anti-huLYPD3 antibodies to purified soluble protein was assessed by ELISA assay as described in Example 3, while binding to differently glycosylated membrane-bound LYPD3 was analyzed using flow cytometry.

[0135] Example 2: Generation of anti-LYPD3 antibodies Monoclonal antibodies that specifically recognize the STP-rich domain of huLYPD3, which has a tumor-associated glycosylation pattern, were generated by phage display technology or by animal immunization. In both cases, huLYPD3 bearing the O-glycan structures produced by cancer cells in the protein and cellular forms described in Example 1 was an essential part of the antibody generation procedure. All antibodies were selected to bind to human LYPD3 in an O-glycosylation-dependent manner.

[0136] Using HybriFree technology, LYPD3-specific antibodies were isolated from splenocytes of chickens and rabbits immunized with either purified LYPD3-ECD or the STP-rich ECD portion (LYPD3-STP-ECD) derived from transfected NM-F9 cells and boosted with LYPD3-ECD. After the final immunization, splenocytes were isolated from animals that demonstrated antigen-specific antibody responses in serum or chicken egg yolk preparations, as assessed by flow cytometry (as described in Example 4) and / or ELISA assays (as described in Example 3). Splenic B cells with antigen specificity for O-glycosylated LYPD3-STP were first enriched by depleting cells that exhibited unwanted protein binding. This included binding to unglycosylated LYPD3-STP-ECD and / or recombinantly expressed protein fusion partners lacking the LYPD3 sequence, and / or binding to the same protein fusion partner, as well as unrelated glycoproteins containing similar glycan structures attached to heterologous protein sequences, similar to O-glycosylated LYPD3-STP-ECD. In this negative selection step, spleen cells were first incubated with off-target proteins immobilized on microtiter plates and / or present in the panning solution. Unbound cells were then transferred to plates coated with O-glycosylated LYPD3-STP-ECD or LYPD3-ECD protein purified from NM-F9 cells to capture splenic B cells with target specificity. Antibody variable domain cDNAs were amplified from the captured cells and cloned into plasmids carrying separate expression cassettes for the IgG heavy and light chains for the construction of a combinatorial human IgG1-encoding library in mammalian expression vectors. Plasmid DNA from the resulting antibody library pool was transfected into CHO cells for transient production of chimeric antibodies.Antibody minipool cell culture supernatants were tested for target-specific binding by ELISA using various on-target and off-target proteins. Furthermore, LYPD3-transfected HEK O-glycosylated KO cells and / or LYPD3-transfected F9 cells were tested by flow cytometry in comparison with untransfected NM-F9 cells. Proteins used for ELISA screening included O-glycosylated LYPD3-ECD and LYPD3-STP-ECD, as well as unglycosylated LYPD3-ECD and LYPD3-STP-ECD, both expressed and purified in NM-F9 cells. Single clones were generated from the antibody pool that showed specific binding to O-glycosylated LYPD3-STP. The VH and VL cDNAs of the antigen-specific single clones were sequenced, and antibodies with unique sequences were expressed in CHO cells. The generation of antigen-specific antibodies in the single clone supernatants was confirmed by ELISA and flow cytometry before antibody purification.

[0137] The following glycosylation-specific anti-huLYPD3 antibodies were obtained by immunization of chickens or rabbits: [Table A]

[0138] Fully human glycospecific anti-huLYPD3 antibodies were isolated from the Yumab ​​naive human antibody library (YUMAB GmbH, Braunschweig, Germany) by phage display technology. Briefly, bacteriophages with antigen specificity for O-glycosylated LYPD3-STP were enriched from the library through three rounds of antibody selection using a different strategy combining subsequent panning reactions against purified protein and cells. In each step, positive selection of scFv-generated phages was achieved by binding to O-glycosylated LYPD3-STP-ECD or LYPD3-ECD, both produced from NM-F9 cells and immobilized on microtiter plates. For competition, purified, recombinantly expressed protein fusion partners without the LYPD3 sequence and both LYPD3-ECD and LYPD3-STP-ECD without O-glycosylation were present in the panning solution to deplete antibodies recognizing these structures. In some strategies, phage binding to untransfected NM-F9 cells was depleted, followed by a second and / or third enrichment step by incubation with LYPD3-STP-transfected NM-F9 cells. In other strategies, binding to LYPD3-transfected NM-F9 cells or neuraminidase-treated Caov-3 cells (an ovarian adenocarcinoma cell line expressing high levels of LYPD3 and TFa glycans) was used for antibody selection after depletion of untransfected NM-F9-binding phage. Soluble scFv antibodies were generated from single clones from each selection strategy and screened by ELISA against LYPD3-ECD and LYPD3-STP-ECD proteins with and without O-glycosylation. scFv antibodies with glycosylated LYPD3 binding by ELISA were also tested by flow cytometry for specific binding to LYPD3- or LYPD3-STP-transfected NM-F9 cells, as well as to neuraminidase-treated Caov-3 cells.After sequencing the VH and VL DNA of clones that specifically recognized O-glycosylated LYPD3-STP in ELISA and LYPD3-positive cells, the unique antibody VH and VL sequences were cloned into a mouse IgG2a expression vector for transfection of HEK cells.

[0139] The following glycosylation-specific anti-huLYPD3 antibodies were obtained by isolation from a phage display library of human antibodies: [Table B]

[0140] All selected antibodies were purified from mammalian cell culture supernatants by protein A affinity chromatography. The purity and integrity of the mAbs were confirmed using SDS-PAGE and analytical SEC.

[0141] In some experiments, the VH and VL sequences of the anti-human LYPD3 antibody were also cloned into a mouse IgG1 expression vector for the purification of chimeric antibodies with a mouse IgG backbone. Both purified chimeric anti-LYPD3 expressed as human IgG1 or mouse IgG1 with the same VH and VL combinations exhibited similar binding properties to soluble and membrane-bound proteins, as confirmed by ELISA and flow cytometry assays.

[0142] Example 3: Antigen ELISA with on-target and off-target controls The antibodies were analyzed in an antigen ELISA assay for specific binding to LYPD3 bearing tumor-associated glycans.

[0143] Briefly, different glycosylated and non-glycosylated protein antigens or glycans conjugated to carbohydrate-polyacrylamide conjugates (carbohydrate PAA conjugates, GlycoNZ) were coated onto 96-well plates overnight, nonspecific binding was blocked, and test antibody samples were added. In some assays, an anti-huLYPD3 huIgG1 control monoclonal Ab purified from CHO cell supernatant after expression of antibody sequences derived from patent application WO 2011 / 070088 (SEQ ID NOs: 51 and 52 of WO 2011 / 070088) ("aLYPD3 mAb") was used. This control anti-huLYPD3 antibody binds to the extracellular domain of human LYPD3 in a glycosylation-independent manner. To control protein antigen coating on the ELISA plate, we used anti-huLYPD3 rabbit polyclonal antibody (R&D Systems, catalog no. AF5428) ("aLYPD3 pAb") or a-MBL antibody (Novusbio) ("aMBL") for constructs expressed as MBL fusion proteins. Antibodies recognizing specific glycan structures were used to detect glycosylation of coated proteins. The anti-TFa ("aTF") antibody clone HH8 was kindly provided by Professor Clausen, University of Copenhagen. The antibody specifically recognizing Tn ("aTn") was purchased from SBH Sciences or provided by Professor Clausen, University of Copenhagen (clone 5F4). A peroxidase-conjugated anti-IgG secondary antibody was then added, followed by a 3,3',5,5'-tetramethylbenzidine (TMB) substrate reaction. The antibody binding rate was determined by measuring the absorbance at 450 nm and at 620 nm or 630 nm as the reference wavelength using a multimode microplate reader (PerkinElmer EnSpire 2300 or Tecan Spark).

[0144] The carbohydrate-dependent binding of the generated a-huLYPD3 antibodies to the STP-rich domain of huLYPD3 was assessed by antigen ELISA (Figure 2). All antibodies showed significant binding to O-glycosylated LYPD3-STP-ECD and also to O-glycosylated LYPD3-ECD proteins containing the LYPD3-STP sequence, confirming that the epitopes recognized by these antibodies reside in the aa sequence R234 to H303 of huLYPD3. Absence of O-glycosylation on the tested LYPD3 proteins, achieved by enzymatic de-O-glycosylation or by recombinant expression and purification from O-glycosylation-deficient cells, abrogated the binding of the a-LYPD3 antibodies. This clearly demonstrates that the O-glycosylated LYPD3 epitope is required for antibody binding, thus confirming the carbohydrate-dependent nature of LYPD3 binding for all selected antibodies. However, the a-LYPD3 control mAb was able to recognize LYPD3-ECD independently of O-glycosylation and failed to bind to any of the LYPD3-STP-ECD mutants. Thus, this antibody binds to the LYPD3 epitope located at aa 31–233 of huLYP3 in a glycosylation-independent manner.

[0145] Furthermore, anti-huLYPD3 antibody clones appear to elicit diverse fine specificities regarding the type of LYPD3 O-glycosylation. This is evident from the varying degrees of recognition of LYPD3 protein expressed and purified from NM-H9D8, which provides high sialylation bearing primarily sTF and some TF, of NM-H9D8-expressed LYPD3 treated with sialidase, which bears primarily TF, and of degalactosylated LYPD3-STP-ECD, which bears primarily Tn-glycosylation (Tn glyc). The relative amounts of different O-glycosylated structures on LYPD3 were determined for LYPD3 expressed in different cell lines with and without enzyme treatment: [Table 1]

[0146] The antibodies analyzed showed strong and highly specific binding to O-glycosylated LYPD3 but did not show significant binding to unrelated O-glycosylated proteins (Figure 3) or pure carbohydrate antigens (Figure 4). Thus, the binding of anti-LYPD3 antibody clones also specifically depends on the LYPD3 protein backbone.

[0147] Anti-LYPD3 antibodies were compared for their binding affinity to glycosylated LYPD3-STP-ECD (expressed in NM-F9 cells). All anti-LYPD3 antibodies showed high affinity, dose-dependent binding to the O-glycosylated LYPD3-STP target structure, with EC values ​​of 4 × 10. -10 ~4×10 -11 The difference was between M (Fig. 5).

[0148] The anti-LYPD3 clones 17B3 and 21E9 were further compared for their binding affinities to TF-bearing LYPD3 (expressed in NM-F9 or NM-H9D8 cells and treated with sialidase), LYPD3 bearing mainly sTF (sialylated TF) and some TF (expressed in NM-H9D8 cells), and Tn-bearing LYPD3 (expressed in NM-F9 cells and treated with galactosidase). Both clones showed strong binding to TF-glycosylated LYPD3, whereas 21E9 additionally recognized Tn-glycosylated LYPD3 (Figure 6).

[0149] Example 4: Binding of O-glycosylated and de-O-glycosylated LYPD3 expressed in cell lines We tested the binding of aLYPD3 clones to cell lines with different glycosylation states of LYPD3. LYPD3-transfected NM-F9 cells (LYPD3-F9), which express LYPD3 and carry nonsialylated O-glycans, non-transfected NM-F9 cells (corresponding to WT cells), which express endogenous levels of LYPD3, and LYPD3-transfected HEK-O-glyc KO cells (LYPD3-HEK-O-glyc.KO), which express LYPD3 but lack O-glycans, were stained with aLYPD3 clones and detected via fluorophore-conjugated anti-human IgG secondary reagents. LYPD3 and TFa expression was confirmed using control aLYPD3 pAb and mAb and aTF mAb. An irrelevant hIgG1 control was included to determine background staining. DAPI was used to distinguish live from dead cells. Cells were analyzed using a Canto II (BD) flow cytometer.

[0150] The results showed that all aLYPD3 clones bound to O-glycosylated LYPD3 on LYPD3-F9 and NM-F9 cells, but not to non-O-glycosylated LYPD3 on LYPD3-HEK-O-glyc KO cells. Binding to NM-F9 cells was comparable to staining with aLYPD3 pAb and mAb (Figure 7).

[0151] Example 5: Titration of aLYPD3 clones on LYPD3-F9 cells To compare the binding affinity of different aLYPD3 clones, LYPD3-F9 cells expressing high levels of O-glyco.LYPD3 were stained with different concentrations of aLYPD3 clones, and bound antibodies were detected with a fluorophore-conjugated anti-human IgG secondary reagent. An irrelevant hIgG1 control was included to determine background staining, and DAPI was used to distinguish live from dead cells. Cells were analyzed using a Canto II (BD) flow cytometer.

[0152] All aLYPD3 clones showed dose-dependent binding to LYPD3-F9 cells (Figures 8 and 9). Binding of the aLYPD3 clones was in the same range as the aLYPD3 mAb control (Figure 8). Clones 16F9, 19G10, 21E9, 22A11, and 25A6 showed very similar binding affinities, slightly higher than those of clones 17B3, 19B4, and 26A4 (Figure 9).

[0153] Example 6: aLYPD3 clones do not bind to LYPD3 expressed on normal human epithelial mammary cells To demonstrate that the aLYPD3 clones do not bind to the LYPD3 glycoforms found primarily on normal human cells, human epithelial mammary cells (HMECs) from healthy donors were stained with the aLYPD3 clones and detected via a fluorophore-conjugated anti-human IgG secondary reagent. LYPD3 and TFa expression was confirmed using control aLYPD3 pAb and mAb and aTF mAb. An irrelevant hIgG1 control was included to determine background staining. DAPI was used to distinguish live from dead cells. Cells were analyzed using a Canto II (BD) flow cytometer.

[0154] Although HMECs expressed moderate levels of LYPD3 (LYPD3 positivity approximately 40%), they expressed very little TFa, as determined with control antibodies aLYPD3 pAb and mAb and aTF mAb. None of the aLYPD3 clones showed binding to LYPD3 expressed on HMECs (Fig. 10).

[0155] Example 7: Binding of aLYPD3 clones to tumor cell lines To examine the binding of aLYPD3 clones to tumor cell lines, we selected different cell lines with varying expression levels of LYPD3 and TFa: Caov-3 (an ovarian adenocarcinoma cell line, LYPD3+), ZR-75-1 (a breast cancer cell line, LYPD3+), and MDA-MB-231 (a breast adenocarcinoma cell line, LYPD3-). MDA-MB-231 and ZR-75-1 were treated with sialidase to remove sialic acid from the cell surface. The tumor cell lines were stained with the aLYPD3 clones, and binding was detected using a fluorophore-conjugated anti-human IgG secondary reagent. LYPD3 and TFa expression was confirmed using a control aLYPD3 pAb and aTF mAb. An irrelevant hIgG1 control was included to determine background staining. DAPI was used to distinguish live from dead cells. Cells were analyzed using a Canto II (BD) flow cytometer.

[0156] All aLYPD3 clones tested bound to Caov-3, a cell line that expresses high levels of LYPD3 and TFα. Binding was comparable to that of the aLYPD3 pAb (Figure 11A). Therefore, because the ZR-75-1 cell line expresses lower levels of TFα compared to Caov-3, we examined the binding of aLYPD3 clones to ZR-75-1 before and after sialidase treatment to further expose TFα molecules. All aLYPD3 clones bound to sialidase-treated ZR-75-1 (Figure 11B). Several aLYPD3 clones (16F9, 19G10, 25A6, and 26A4) also showed binding to untreated ZR-75-1. The aLYPD3 clones did not bind to MDA-MB-231 cells, which express high levels of TFα after sialidase treatment but do not express LYPD3 (Figure 11C).

[0157] Example 8: Inhibition of proliferation using protein G drug conjugated aLYPD3 antibody To investigate the potential of the aLYPD3 clone to deliver cytotoxic drugs to target cells, a protein G drug conjugate assay was performed. 5,000 LYPD3-F9 cells / well were seeded in flat-bottom 96-well plates in the presence of the indicated test antibody dilutions and a fixed concentration of protein G preloaded with the toxin MMAE (Protein G-MMAE, Levena Biopharma). After 4 days of incubation, cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions and analyzed on a TECAN Infinite F200 microplate reader (Tecan). Proliferation rates were calculated based on the luminescent signal relative to a medium control without antibody.

[0158] The results demonstrated that aLYPD3 clone 17B3 conjugated to protein G-MMAE inhibited the proliferation of LYPD3-F9 in a dose-dependent manner, indicating effective internalization of the aLYPD3 clone (Figure 12).

[0159] Example 9: Tissue binding of carbohydrate-specific anti-LYPD3 antibodies Formalin-fixed, paraffin-embedded tissue sections were stained with 10 μg / ml of aLYPD3 clones and detected using either an HRP-labeled polymer conjugated to an anti-mouse or anti-human secondary antibody. A commercially available polyclonal aLYPD3 antibody was included to detect LYPD3 protein levels in each tissue.

[0160] Staining of cancer tissue sections demonstrated binding of aLYPD3 clones to head and neck squamous cell carcinoma, esophageal cancer, breast cancer, cervical cancer, and skin cancer of different origins. Importantly, reactivity to healthy tissues was absent or reduced compared to the protein-specific control aLYPD3 pAb shown in Figure 13 and Table 2. In conclusion, aLYPD3 clones were found to react with several cancer tissue sections but not with their healthy counterparts. In particular, clones 17B3 and 21E9 were shown to strongly interact with SCC cancer tissue sections from the anal region / rectum, oral cavity, lip, bucca cavioris, nose, penis, vulva, epiglottis, tongue, and skin (data not shown). [Table 2] Binding of aLYPD3 clones to healthy tissues compared to pAb control antibodies is shown and classified according to staining intensity: + moderate to strong staining, + / - weak staining, - no staining observed.

[0161] In particular, the 17B3 and 21E9 clones were shown to interact strongly with SCC tissue sections from the anal region / rectum, oral cavity, lips, bucca cavioris, nose, penis, vulva, epiglottis, tongue, and skin.

[0162] Example 10: Humanization of the rabbit heavy and light chain variable regions of anti-huLYPD3 antibody 21E9 The nucleic acid sequences encoding the rabbit heavy and light chain variable regions of the monoclonal anti-huLYPD3 antibody 21E9 (SEQ ID NOs: 39 and 40) were ligated to the sequences of the human constant gamma 1 region (CH) and the human constant kappa region (CL), respectively.

[0163] Based on these chimeric clones, humanized antibodies were constructed. To this end, point mutations were introduced into the nucleic acid sequences of the rabbit VH and VL framework regions to generate the corresponding human framework regions. The target human framework regions were selected from a database compiled from NCBI GenBank entries (cleaned to remove incomplete and non-human sequences) to include both germline and mature antibody frameworks. In particular, the most relevant framework regions were selected from the library according to their overall sequence similarity and their CDR loop classification. Taking all the obtained data into consideration, a set of different humanized variable light and heavy chain sequences of the parent rabbit antibody was designed. Some variants contained backmutations relative to the rabbit sequence at key positions. Eight humanized variants of the light chain variable region were cloned into a kappa chain vector, and eight humanized variants of the heavy chain variable region were cloned into a gamma chain vector.

[0164] The resulting antibodies containing different combinations of heavy and light chains (a total of 64 antibody variants) were transiently expressed and screened for their expression and LYPD3 binding in ELISA according to Example 3. The following humanized antibody heavy and light chain variable regions were selected for further analysis: [Table 3]

[0165] Nine selected antibody combinations of humanized VH and VL sequences were produced as human IgG1 / κ by transient expression in CHO cells and purified by protein A chromatography. [Table 4]

[0166] Their binding specificity and affinity were analyzed and compared to the parent chimeric antibody in ELISA and flow cytometry assays as described in Examples 3 and 4. The data are summarized in Figures 14, 15, and 16. All humanized antibody variants showed the same binding affinity and specificity as the parent antibody 21E9 for LYPD3 bearing TF or Tn. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0167] Identification of deposited biological material The cell line DSM ACC 2606 was deposited by Nemod Biotherapeutics GmbH & Co. KG, Robert-Rossle-Str. 10, 13125 Berlin (DE) at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, 38124 Braunschweig (DE) on the date indicated in the table below. This biological material was transferred from Nemod Biotherapeutics GmbH & Co. KG to Glycotope GmbH, which therefore reserves the right to refer to this biological material.

[0168] The cell lines DSM ACC2806, DSM ACC2807 and DSM ACC2856 were deposited by Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin (DE) at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig (DE) on the dates indicated in the table below. [Table C]

Claims

1. An antibody that has the ability to specifically bind to glycosylated human LYPD3 in an epitope containing an oligosaccharide structure selected from the group consisting of GalNAcα1-, sialylated GalNAcα1-, Galβ1-3GalNAcα1-, and sialylated Galβ1-3GalNAcα1- that is attached to a serine or threonine residue of LYPD3.

2. The antibody according to claim 1, wherein the oligosaccharide structure is attached to a serine or threonine residue between positions 234 and 303 of SEQ ID NO:

137.

3. The antibody according to claim 1, wherein the epitope comprises one or more amino acids within positions 234 to 303 of SEQ ID NO: 137, and in particular, at least 50% of the amino acids of the epitope are located within positions 234 to 303 of SEQ ID NO:

137.

4. The antibody according to claim 1, which has the ability to specifically bind to human LYPD3 glycosylated with either GalNAcα1- or Galβ1-3GalNAcα1- or both.

5. The antibody according to claim 1, which has the ability to specifically bind to human LYPD3 glycosylated with Galβ1-3GalNAcα1-.

6. Antibodies that have the ability to bind to LYPD3 and include the following: (i) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and A light chain variable region comprising a complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6; or (ii) A heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and A light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; or (iii) A heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 17, CDR-H2 having the amino acid sequence of SEQ ID NO: 18, and CDR-H3 having the amino acid sequence of SEQ ID NO: 19, and A light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 20, CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and CDR-L3 having the amino acid sequence of SEQ ID NO: 22; or (iv) A heavy chain variable region including CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27, and A light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 28, CDR-L2 having the amino acid sequence of SEQ ID NO: 29, and CDR-L3 having the amino acid sequence of SEQ ID NO: 30; or (v) A heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35, and A light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; or (vi) A heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 41, CDR-H2 having the amino acid sequence of SEQ ID NO: 42, and CDR-H3 having the amino acid sequence of SEQ ID NO: 43, and A light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 44, CDR-L2 having the amino acid sequence of SEQ ID NO: 45, and CDR-L3 having the amino acid sequence of SEQ ID NO: 46; or (vii) A heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 49, CDR-H2 having the amino acid sequence of SEQ ID NO: 50, and CDR-H3 having the amino acid sequence of SEQ ID NO: 51, and A light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 52, CDR-L2 having the amino acid sequence of SEQ ID NO: 53, and CDR-L3 having the amino acid sequence of SEQ ID NO: 54; or (viiii) A heavy chain variable region including CDR-H1 having the amino acid sequence of SEQ ID NO: 57, CDR-H2 having the amino acid sequence of SEQ ID NO: 58, and CDR-H3 having the amino acid sequence of SEQ ID NO: 59, and A light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 60, CDR-L2 having the amino acid sequence of SEQ ID NO: 61, and CDR-L3 having the amino acid sequence of SEQ ID NO: 62; or (ix) A heavy chain variable region and a light chain variable region according to any one of the above items (i) to (viiii), comprising a total of one, two, or three amino acid substitutions in the six CDR sequences.

7. The antibody according to claim 6, (i) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 8; or (ii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 15, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 16; or (iii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 23, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 24; or (iv) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 31, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 32; or (v) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 39, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 40; or (vi) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 47, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 48; or (vii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 55, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 56; or (viiii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 63, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 64; or (ix) The heavy chain variable region and the light chain variable region each contain an amino acid sequence that is at least 60% identical to one of the heavy chain variable region sequences and light chain variable region sequences described in items (i) to (viiii) above, over their entire length, and the antibody is preferably a humanized version of the antibody described in any one of items (i) to (viiii) above.

8. The antibody according to claim 6, further comprising an Fc region.

9. The antibody according to claim 6, (i) A heavy chain variable region including the complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35, and Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; or (ii) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 39 and having at least 60% of the same amino acid sequence over its entire length, including CDR-H1 having the amino acid sequence of SEQ ID NO: 33, CDR-H2 having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 having the amino acid sequence of SEQ ID NO: 35, and A light chain variable region having the same amino acid sequence as the amino acid sequence of SEQ ID NO: 40 for at least 60% of its entire length, and including CDR-L1 having the amino acid sequence of SEQ ID NO: 36, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; Includes, The antibody is an antibody that has the ability to specifically bind to human LYPD3 glycosylated with either GalNAcα1- or Galβ1-3GalNAcα1-, or both.

10. The antibody according to claim 6, (i) A heavy chain variable region including the complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and A light chain variable region comprising the complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; or (ii) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and having at least 60% of the same amino acid sequence over its entire length, including CDR-H1 having the amino acid sequence of SEQ ID NO: 9, CDR-H2 having the amino acid sequence of SEQ ID NO: 10, and CDR-H3 having the amino acid sequence of SEQ ID NO: 11, and A light chain variable region having the same amino acid sequence as the amino acid sequence of SEQ ID NO: 16 for at least 60% of its entire length, and including CDR-L1 having the amino acid sequence of SEQ ID NO: 12, CDR-L2 having the amino acid sequence of SEQ ID NO: 13, and CDR-L3 having the amino acid sequence of SEQ ID NO: 14; Includes, The antibody described above is an antibody that has the ability to specifically bind to human LYPD3 glycosylated with Galβ1-3GalNAcα1-.

11. Humanized antibodies, (i) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 139, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 144; or (ii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 140, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 144; or (iii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 141, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 144; or (iv) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 142, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 144; or (v) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 142, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 143; or (vi) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 142, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 145; or (vii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 142, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 146; or (viiii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 138, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 145; or (ix) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 140, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

145. The antibody according to claim 6.

12. The antibody according to claim 6, wherein the antibody is of type IgG1, type IgG2, type IgG3, or type IgG4, and is particularly an antibody of type IgG1.

13. A conjugate comprising the antibody according to any one of claims 1 to 12, conjugated with a further drug.

14. The conjugate according to claim 13, wherein the further agent is a cytotoxic agent, a tumor-specific antibody, or an immune checkpoint blocker or activator antibody.

15. The conjugate according to claim 13, which is a chimeric antigen receptor.

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

17. A nucleic acid encoding the conjugate according to claim 13, wherein the further agent is a polypeptide or protein fused to the antibody.

18. An expression cassette or vector comprising the nucleic acid according to claim 16 and a promoter operably connected to the nucleic acid.

19. A host cell containing the nucleic acid described in claim 16.

20. An expression cassette or vector comprising the nucleic acid according to claim 17 and a promoter operably connected to the nucleic acid.

21. A host cell comprising the nucleic acid described in Claim 20.

22. A host cell which is a leukocyte containing a nucleic acid encoding the conjugate described in Claim 15.

23. A composition comprising the antibody according to any one of claims 1 to 12.

24. A composition comprising the conjugate described in Claim 13.

25. A composition comprising the host cells described in Claim 22.

26. The composition according to claim 23, which optionally further comprises one or more components selected from the group consisting of solvents, diluents, and excipients.

27. A composition for use in pharmaceuticals, comprising the antibody according to any one of claims 1 to 12.

28. A composition comprising the conjugate described in claim 13 for use in pharmaceuticals.

29. A composition comprising the host cells described in Claim 22 for use in pharmaceuticals.

30. The composition according to claim 27 for use in the treatment of cancer, adolescent idiopathic scoliosis, cholesteryl ester transfer protein (CETP) deficiency, fish eye disease, combined hyperlipidemia, citrullinemia, or familial hypercholesterolemia.

31. The composition according to claim 30, wherein the cancer is selected from the group consisting of head and neck cancer, colon cancer, colorectal cancer, hepatocellular carcinoma, skin cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, esophageal cancer, lung cancer, and cancers of the reproductive organs, particularly non-small cell lung cancer (NSCLC) and squamous cell carcinoma (SCC).

32. The composition according to claim 28 for use in the treatment of cancer, adolescent idiopathic scoliosis, cholesteryl ester transfer protein (CETP) deficiency, fish eye disease, combined hyperlipidemia, citrullinemia, or familial hypercholesterolemia.

33. The composition according to claim 32, wherein the cancer is selected from the group consisting of head and neck cancer, colon cancer, colorectal cancer, hepatocellular carcinoma, skin cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, esophageal cancer, lung cancer, and cancers of the reproductive organs, particularly non-small cell lung cancer (NSCLC) and squamous cell carcinoma (SCC).

34. The composition according to claim 29 for use in the treatment of cancer, adolescent idiopathic scoliosis, cholesteryl ester transfer protein (CETP) deficiency, fish eye disease, combined hyperlipidemia, citrullinemia, or familial hypercholesterolemia.

35. The composition according to claim 34, wherein the cancer is selected from the group consisting of head and neck cancer, colon cancer, colorectal cancer, hepatocellular carcinoma, skin cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, esophageal cancer, lung cancer, and cancers of the reproductive organs, particularly non-small cell lung cancer (NSCLC) and squamous cell carcinoma (SCC).

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