Epithelial cadoherin specific antibody

Abstract

To provide epithelial cadherin-specific antibodies, and their use in the diagnosis and treatment of diseases such as cancer. [Solution] The above problem is solved by an antibody or antigen-binding fragment that specifically binds one or more O-mannosylated threonine residues of E-cadherin, wherein the one or more O-mannosylated threonine residues are located within amino acid positions 467 to 472 of the E-cadherin sequence.

Description

[Technical Field]

[0001] This invention relates to the fields of biology, medicine, and immunology. [Background technology]

[0002] Transmembrane proteins epithelial cadherins (E-cadherins: also known as CD324, cadherin-1, CAM120 / 80, and uvomorrulin) are members of the cadherin superfamily. In the art, E-cadherins are known as calcium-dependent intercellular adhesion glycoproteins with a molecular weight of approximately 120 kDa, consisting of five extracellular cadherin (EC) repeats (EC1-EC5), a transmembrane domain, and a highly conserved cytoplasmic tail. E-cadherins are an important type of intercellular adhesion protein that firmly holds epithelial cells together. E-cadherin downregulation can reduce the strength of cell adhesion within tissues, potentially resulting in increased cell motility and epithelial-mesenchymal transition (EMT). Loss of E-cadherin function or expression is associated with cancer progression and metastasis. [Overview of the project] [Problems that the invention aims to solve]

[0003] This invention relates to epithelial cadherin-specific antibodies and their use in the diagnosis and treatment of diseases such as cancer. [Means for solving the problem]

[0004] In a first embodiment, the present invention provides an E-cadherin-specific antibody and its antigen-binding fragment comprising the structural and functional features specified herein.

[0005] In various embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds one or more O-mannosylated threonine residues of an E-cadherin, the one or more O-mannosylated threonine residues located within amino acid positions 467-472 of the E-cadherin sequence, as shown in Figure 1A. In preferred embodiments, the binding of the antibody or antigen-binding fragment to the E-cadherin is influenced by the presence of an O-mannosylated threonine residue at position 467, O-mannosylated threonine residue at position 468, O-mannosylated threonine residue at position 470, O-mannosylated threonine residue at position 472, glutamic acid residue at position 463, serine residue at position 465, serine residue at position 469, and / or valine residue at position 477 of the E-cadherin sequence, as shown in Figure 1A, particularly the presence of an O-mannosylated threonine residue at position 467 and / or an O-mannosylated threonine residue at position 468 and / or an O-mannosylated threonine residue at position 470 of the E-cadherin sequence, as shown in Figure 1A. In some embodiments, the serine residues at positions 465 and / or 469 are O-mannosylated. In a preferred embodiment, the antibody or antigen-binding fragment binds O-mannosylated shortened 70kDa E-cadherin better than O-mannosylated full-length E-cadherin. In a preferred embodiment, the antibody or antigen-binding fragment binds O-mannosylated shortened 70kDa E-cadherin at least twice as well, more preferably at least three times as well, more preferably at least four times as well, and more preferably at least five times as well as O-mannosylated full-length E-cadherin.

[0006] In various embodiments, the present invention provides an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, wherein the antibody or antigen-binding fragment is: a. Heavy chain variable region CDR1 containing the amino acid sequence GFX1FSX2AW (where X1 is T or I, and X2 is N or Y); Alternatively, the heavy chain variable region CDR1 containing an amino acid sequence different from the GFX1FSX2AW sequence by one, two, or three conservative substitutions; b. Heavy chain variable region CDR2 containing the amino acid sequence IKSKIDG X1T X2 (where X1 is G or E and X2 is T or I); Alternatively, the heavy chain variable region CDR2 contains an amino acid sequence different from the IKSKIDG X1T X2 sequence by one, two, or three conservative substitutions; c. Heavy chain variable region CDR3 containing the amino acid sequence TPGVGX1NX2PYYFDR (where X1 is A or T and X2 is D or N); Alternatively, the heavy chain variable region CDR3 contains an amino acid sequence different from the TPGVGX1NX2PYYFDR sequence by one, two, or three conservative substitutions; d. Light chain variable region CDR1 containing the amino acid sequence QSVLCRSNNKNC; Alternatively, the light chain variable region CDR1 containing an amino acid sequence different from the QSVLCRSNNKNC sequence by one, two, or three conservative substitutions; e. Light chain variable region CDR2 containing the amino acid sequence WAX1 (X1 is either S or C); Alternatively, a light chain variable region CDR2 containing an amino acid sequence different from the WAX1 sequence by one, two, or three conservative substitutions; f. Light chain variable region CDR3 containing the amino acid sequence QQYSNTPQT; Alternatively, the light chain variable region CDR3 may contain an amino acid sequence different from the QQYSNTPQT sequence by one, two, or three conservative substitutions. One or more of them, and possibly each of them.

[0007] In a particular embodiment, the antibody or antigen-binding fragment includes a heavy chain variable region containing a sequence having at least 80% sequence identity with a VH sequence selected from the group consisting of SEQ ID NOs: 1 to 17, as shown in Table 1; and / or a light chain variable region containing a sequence having at least 80% sequence identity with a VL sequence selected from the group consisting of SEQ ID NOs: 18 to 22. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region.

[0008] In various embodiments, the antibody or antigen-binding fragment according to the present invention is a full-length antibody.

[0009] In various embodiments, the antibody or antigen-binding fragment according to the present invention is a human antibody or its antigen-binding fragment.

[0010] In various embodiments, the antibody or antigen-binding fragment according to the present invention is an IgA isotype. In various embodiments, the antibody or antigen-binding fragment according to the present invention is an IgM isotype. In various embodiments, the antibody or antigen-binding fragment according to the present invention is an IgD isotype. In a particular embodiment, the antibody or antigen-binding fragment is human IgA, IgM, or IgD.

[0011] In various embodiments, the antibody or antigen-binding fragment according to the invention is of the IgG isotype. In certain embodiments, the antibody or antigen-binding fragment is IgG1, IgG2, IgG3 or IgG4, preferably IgG1. In certain embodiments, the antibody or antigen-binding fragment is human IgG1, IgG2, IgG3 or IgG4, preferably human IgG1.

[0012] In various embodiments, the antibody or antigen-binding fragment according to the invention is defucosylated.

[0013] Certain embodiments provide an antibody or antigen-binding fragment thereof that competes with an antibody selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN and AT1636-IYEN for binding to O-mannosylated E-cadherin, preferably O-mannosylated truncated 70 kDa E-cadherin.

[0014] In certain embodiments, the antibody or antigen-binding fragment according to the invention has the following characteristics: - binds to the extracellular 3 (EC3) domain of O-mannosylated E-cadherin; - binds O-mannosylated truncated 70 kDa E-cadherin better than, preferably at least 2-fold better than, more preferably at least 3-fold better than, more preferably at least 4-fold better than, more preferably at least 5-fold better than O-mannosylated full-length E-cadherin; and - binds tumor cells co-expressing E-cadherin and O-mannosyltransferase, preferably TMTC3 It has one or more of these, preferably each of them.

[0015] In some embodiments, the antibody or antigen-binding fragment has the following characteristics: - Combines colon cancer subtypes CMS1, CMS2, CMS3, and CMS4; - It binds the colon cancer cell line SW948 better than healthy thymic medullary epithelial cells, dendritic cells, or Langerhans cells. It further includes at least one of the following.

[0016] In certain embodiments, the antibody or antigen-binding fragment according to the present invention is bound to another compound. In certain embodiments, the other compound is a therapeutic compound. In certain embodiments, the other compound is a compound selected from the group consisting of immunomodulatory compounds, T cell-binding compounds, natural killer cell (NK cell)-binding compounds, natural killer T cell (NKT cell)-binding compounds, gamma-delta T cell-binding compounds, CD3-specific binding compounds, TGFβ-specific binding compounds, cytokines, secondary antibodies or their antigen-binding moieties, detectable labels, drugs, chemotherapeutic agents, cytotoxic agents, toxic moieties, hormones, enzymes, and radioactive compounds. In some embodiments, the immunomodulatory compound is not the Fc tail of the antibody according to the present invention. In some embodiments, the immunomodulatory compound is a non-natural immunomodulatory compound.

[0017] An antibody or antigen-binding fragment according to the present invention, directly or indirectly bound to a therapeutic compound, is also referred to herein as an antibody-drug conjugate (ADC).

[0018] This invention also: - Antibodies or antigen-binding fragments according to the present invention; and - Immunomodulatory compounds The present invention provides a bispecific or multispecific binding compound, preferably a bispecific or multispecific antibody or its antigen-binding fragment, that can bind O-mannosylated E-cadherin.

[0019] In some embodiments, the immunomodulatory compound is not the Fc tail of the antibody according to the present invention. In some embodiments, the immunomodulatory compound is a non-natural immunomodulatory compound.

[0020] This invention also: - Antibodies or antigen-binding fragments according to the present invention; and - T cell binding compounds or natural killer cell (NK cell) binding compounds or natural killer T cell (NKT cell) binding compounds or gamma-delta T cell binding compounds The present invention provides a bispecific or multispecific binding compound, preferably a bispecific or multispecific antibody or its antigen-binding fragment, that can bind O-mannosylated E-cadherin.

[0021] This invention also: - Antibodies or antigen-binding fragments according to the present invention; and - CD3 specific binding compound The present invention provides a bispecific or multispecific binding compound, preferably a bispecific or multispecific antibody or its antigen-binding fragment, that can bind O-mannosylated E-cadherin.

[0022] This invention also: - Antibodies or antigen-binding fragments according to the present invention; and - KLRG1 specific binding compound The present invention provides a bispecific or multispecific binding compound, preferably a bispecific or multispecific antibody or its antigen-binding fragment, that can bind O-mannosylated E-cadherin.

[0023] This invention also: - Antibodies or antigen-binding fragments according to the present invention; and - CD103 specific binding compound The present invention provides a bispecific or multispecific binding compound, preferably a bispecific or multispecific antibody or its antigen-binding fragment, that can bind O-mannosylated E-cadherin.

[0024] This invention also: - Antibodies or antigen-binding fragments according to the present invention; and - TGFβ specific binding compound The present invention provides a bispecific or multispecific binding compound, preferably a bispecific or multispecific antibody or its antigen-binding fragment, that can bind O-mannosylated E-cadherin.

[0025] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - A Fab fragment of another antibody, preferably specific to T cells, NK cells, NKT cells, or gamma-delta T cells. Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0026] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and • One Fab fragment of another antibody that is CD3 specific Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0027] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - One Fab fragment of another antibody that is specific to KLRG1 or CD103 Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0028] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - One Fab fragment of another antibody that is specific to TGFβ Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0029] Certain embodiments provide chimeric antigen receptor (CAR) T cells capable of binding O-mannosylated E-cadherin, wherein the CAR T cells contain the heavy chain CDR1, CDR2, and CDR3 sequences of the antibody according to the present invention. Preferably, the CAR T cells also contain the light chain CDR1, CDR2, and CDR3 sequences of the antibody according to the present invention. Preferably, the CDR1-3 sequences are present on the surface of the CAR T cells in a single-chain format.

[0030] The present invention also provides nucleic acids having the structural and functional features specified herein. In various embodiments, the present invention provides isolated, synthesized, or recombinant nucleic acids that encode an antibody or antigen-binding fragment according to the present invention, or that encode at least the heavy chain variable region and / or light chain variable region of an antibody or antigen-binding fragment according to the present invention.

[0031] In a particular embodiment, the present invention provides a nucleic acid comprising a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 23 to 39, as shown in Table 1, and / or a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 40 to 44. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation is located outside the CDR region.

[0032] In certain embodiments, the nucleic acid according to the present invention includes DNA or RNA.

[0033] In certain embodiments, the nucleic acid according to the present invention includes cDNA, peptide nucleic acid (PNA), loc nucleic acid (LNA), or DNA / RNA helix.

[0034] In a particular embodiment, the nucleic acid according to the present invention is a codon optimized for expression in non-human host cells.

[0035] In a particular embodiment, the nucleic acid according to the present invention is a codon optimized for expression in HEK293T cells or CHO cells.

[0036] The present invention further provides a vector comprising nucleic acids according to the present invention. In some embodiments, the vector is a CAR T cell vector comprising nucleic acid sequences encoding an antigen recognition domain and a T cell activation domain. In some embodiments, the CAR T cell vector further comprises a nucleic acid sequence encoding a transmembrane domain.

[0037] The present invention further provides isolated or recombinant host cells or non-human animals comprising antibodies, antigen-binding fragments, nucleic acids, vectors, ADCs or CAR T cells according to the present invention. In certain embodiments, the host cells are mammalian cells, bacterial cells, plant cells, HEK293 T cells or CHO cells.

[0038] The present invention also provides compositions comprising antibodies, antigen-binding fragments, nucleic acid molecules, vectors, ADCs, CAR T cells, or host cells according to the present invention. In various embodiments, the compositions are pharmaceutical compositions comprising pharmaceutically acceptable carriers, diluents, or excipients.

[0039] The present invention also provides a kit of parts comprising antibodies, antigen-binding fragments, nucleic acid molecules, vectors, ADCs, CAR T cells, or host cells according to the present invention.

[0040] In a particular embodiment, the composition or kit of parts according to the present invention further comprises at least one other therapeutic agent.

[0041] The present invention also provides a method for producing an antibody or antigen-binding fragment according to the present invention, the method comprising culturing a host cell containing a nucleic acid or vector according to the present invention to enable the host cell to translate the nucleic acid or vector, thereby producing the antibody or antigen-binding fragment according to the present invention. The method preferably further comprises recovering the antibody or antigen-binding fragment from the host cell and / or from the culture medium. In some embodiments, the host cell is provided with a vector comprising both a nucleic acid sequence encoding the heavy chain of the antibody and a nucleic acid sequence encoding the light chain of the antibody. In some embodiments, the host cell is provided with at least two different vectors, one vector comprising a nucleic acid sequence encoding the heavy chain of the antibody and a second vector comprising a nucleic acid sequence encoding the light chain of the antibody.

[0042] Furthermore, antibodies or antigen-binding fragments are also provided when obtained by the method according to the present invention.

[0043] The present invention also provides antibodies or antigen-binding fragments or bispecific antibodies or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use as pharmaceuticals, prophylactic agents or diagnostic agents.

[0044] Also provided are antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use in methods for treating or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells. In some preferred embodiments, the disorder is E-cadherin-positive and TMTC3-positive cancer. In some embodiments, the cancer also includes tumor cells expressing TGFβ.

[0045] Various embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use according to the present invention, thereby being used in combination with other therapeutic agents useful for treating and / or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0046] The present invention also provides the use of antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for the manufacture of pharmaceuticals.

[0047] The present invention also provides the use of antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for the manufacture of pharmaceuticals for treating or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase. In certain embodiments, the cells are tumor cells. In certain embodiments, the O-mannosyltransferase is TMTC3. The present invention also provides the use of antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for the preparation of pharmaceuticals for treating or preventing E-cadherin-positive and TMTC3-positive cancers. In certain embodiments, the E-cadherin-positive and TMTC3-positive cancers are epithelial cancers. In some embodiments, the E-cadherin-positive and TMTC3-positive cancers are selected from the group consisting of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, colorectal cancer, colon cancer, stomach cancer, gastric cancer, gastroesophageal junction cancer, breast cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer, endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, kidney cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer, and peritoneal cancer.

[0048] In some embodiments, the E-cadherin-positive and TMTC3-positive cancers are selected from the group consisting of colorectal cancer, colon cancer, colon cancer subtype CMS1, colon cancer subtype CMS2, colon cancer subtype CMS3, colon cancer subtype CMS4, laryngeal cancer, head and neck cancer, breast cancer, pancreatic cancer, esophageal cancer, bladder cancer, lung cancer, stomach cancer, urinary tract cancer, prostate cancer, and ovarian cancer.

[0049] The present invention also provides a method for treating and / or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably tumor cells, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment according to the present invention, and / or a bispecific or multispecific antibody or ADC or CAR T cell according to the present invention, and / or a nucleic acid according to the present invention, and / or a vector or cell according to the present invention, and / or a composition or kit of parts according to the present invention, to an individual in need. The present invention further provides a method for treating and / or preventing at least partially E-cadherin-positive and TMTC3-positive cancers, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment according to the present invention, and / or a bispecific or multispecific antibody or ADC or CAR T cell according to the present invention, and / or a nucleic acid according to the present invention, and / or a vector or cell according to the present invention, and / or a composition or kit of parts according to the present invention, to an individual in need. The composition is preferably a pharmaceutical composition according to the present invention.

[0050] The present invention also provides the use of antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells according to the present invention to determine whether a sample contains cells, preferably tumor cells, that contain O-mannosylated E-cadherin.

[0051] Furthermore, a method is provided for determining whether cells, preferably tumor cells, containing O-mannosylated E-cadherin are present in a sample, the method being: - Contacting the sample with an antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell according to the present invention, and - To enable the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell to bind to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-containing tumor cells, if present, and - Determine whether the cells are bound to the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell, and thereby determine whether or not O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-containing tumor cells, are present in the sample. Includes.

[0052] Furthermore, a method is provided for determining whether cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells, are present in a sample, the method being: - Contacting the sample with an antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell according to the present invention, and - To enable the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell to bind to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells, if present, and - Determine whether cells are bound to the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cells, and thereby determine whether cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells, are present in the sample. Includes.

[0053] The present invention also provides a method for determining whether a human or non-human individual has cancer that is positive for O-mannosylated E-cadherin, the method being: - Contacting the tumor cells of the said individual with the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cells according to the present invention, - To enable the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell to bind to O-mannosylated E-cadherin-containing tumor cells if present, and - To determine whether tumor cells are bound to the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell, thereby determining whether the individual has cancer that is positive for O-mannosylated E-cadherin. Includes.

[0054] In some embodiments, the method is an ex vivo method. In other embodiments, the method is performed in vivo. [Brief explanation of the drawing]

[0055] [Figure 1A] (A) The full-length E-cadherin amino acid sequence (Uniprot Q9UII7), including the numbering used throughout the text, is shown. [Figure 1B] (B) Different domains of E-cadherin are shown. Adopted from Berx et al. Genomics 1995. Red indicates the predicted binding epitope for AT1636. [Figure 1C] (C) Further, a truncated 70kDa protein including transmembrane (italic) and intercellular domains, indicated as p70 in the examples, is shown. [Figure 2](A) SDS-PAGE analysis of immunoprecipitated samples with AT1002 and AT1636 antibodies. Arrows indicate protein "bands" analyzed by mass spectrometry after immunoprecipitation with AT1636. JKT = Jurkat-negative control T cell line, DLD1 is a colon cancer cell line, M = molecular weight marker, IP = immunoprecipitation, and AT1002 is the negative control antibody. (B) Western blot showing immunoprecipitation of full-length E-cadherin with EP700Y rabbit antibody (Abcam) and C-tail intracellular targeted antibody (BD Bioscience), as well as immunoprecipitation of p70 protein from DLD1 cells with AT1636. EP700Y; binds to the proximal EC5 domain of the extracellular membrane; C-tail intra, specific to the C-terminal intracellular domain of E-cadherin (BD Biosciences), and mouse anti-E-cadherin, as well as AT1636, used for detection, react with epitopes preferentially exposed to the p70E-cadherin type. [Figure 3] A graphical overview representing the full-length and shortened p70E-cadherin. The black lollipops indicate the reported O-mannose glycosylation sites (Larsen PNAS (2017) and Vester-Christensen, PNAS (2013)), the white and dark gray lollipops illustrate the predicted O-mannose glycosylation sites, and the light gray lollipops illustrate the sites we identified by mass spectrometry of AT1636 immunoprecipitated p70E-cadherin, which may be mannosylated. Amino acid residues shown in bold are important for AT1636 binding as determined by alanine scanning. The capitalized residues 472 and 474 are known to be O-mannosylated, and 470 is predicted to be mannosylated as described by Larsen et al 2017 and Vester-Christensen et al 2013. In the full-length E-cadherin (above), the antibody-binding region and β-catenin-binding region of SC10.17 (anti-CD324 monoclonal antibody and its use, U.S. Patent No. 9534058) and EP700Y are shown. [Figure 4]Flow cytometry analysis of AT1636 binding to DLD1 cells pretreated with different inhibitors. Histograms (solid lines, open histograms) of AT1636 at 5 μg / ml and control antibodies AT1002 and EP700Y are shown for DLD1 cells pretreated for 48 hours with Mann (mannosyltransferase inhibitor: 4-oxo-2-thioxo-3-thiazolidinyl acetate (Sigma)) or CMK (furin-containing convertase inhibitor: decanoyl-RVKR-CMK (Tocris)) inhibitors. Filled histograms indicate binding to untreated cells. [Figure 5] (A) Selection and isolation of subclones (red boxes) with increased binding to recombinant E-cadherin protein compared to the mean binding of the parental E-cadherin clone, 7G02. Cells were stained with recombinant E-cadherin protein and IgG(H+L)-Alexa647 and anti-mouse Fc-PE antibody. Single-cell cloning of gate cells was performed using a cell sorter (FACS ARIA, BD). (B) Selection of subclones with increased E-cadherin antigen binding compared to the parental 7G02 clone. AT1636GFP low-parental cells were mixed with GFP high-subclonal cells. This mixture of cells was stained for E-cadherin binding and BCR expression. The intensity of antigen binding related to BCR expression in subclones (blue) compared to parental 7G cells (orange) is shown. [Figure 6A] (A) Binding curves of AT1636 and the AT1636 high-affinity mutant to human CRC cell line DLD1, thoracic epithelial cell line MCF10a, and mouse CRC cell line CMT93, as detected by flow cytometry, are shown (central fluorescence intensity (MFI) of Alexa647 dye conjugated to goat anti-human secondary antibody (Invitrogen) is shown). EP700Y and SC10.17 antibodies do not cross-react with mouse E-cadherin. [Figure 6B] (B) The binding ratios of AT1002 to control antibodies AT1636 and AT1636-YN and -IYN variants, as detected by flow cytometry, are shown for cutaneous epithelial cell line A431, lung A546, and mouse CMT93 cell line. [Figure 7A] (A) SPR binding of AT1636, AT1636-NY, and -IYN variants to soluble p70E-cadherin. 5.0 μg / ml antibody was injected into spots immobilized with 2.0 μg / ml p70E-cadherin. EP700Y rabbit anti-human E-cadherin, specific to the EC5 domain, was used as a control. Binding was detected using IBIS multiplex SPR imaging. [Figure 7B] (B) ELISA assay to determine the binding of AT1636 and AT1636-IYN mutants to recombinant, immobilized full-length E-cadherin (left panel), p70 E-cadherin (center panel), and E-cadherin D3 domain containing the M470A substitution (which inhibits mannosylation of this residue) (right panel). SC10.17 antibody is used as a control antibody that binds to the full-length (EC1 domain) but not to the p70 and D3 domains. AT1002 is an influenza-specific negative human control antibody. [Figure 7C] (C) ELISA assay using a broad range of concentrations of AT1636 and its variants to bind recombinant, immobilized full-length E-cadherin (left panel), p70E-cadherin (center panel), and E-cadherin D3 domains containing the M470A substitution (which inhibits mannosylation of this residue) (right panel). AT1002 is an influenza-specific negative human control antibody. [Figure 8A] (A) Western blot showing the input and flow-through (FT) after AT1636 immunoprecipitation and specific elution of p70 from the AT1636 immunoprecipitation using high levels of mannopyranoside. [Figure 8B] (B) ELISA showing AT1636 binding to full-length E-cadherin derived from HEK cells (Sino Biological) (left panel) and the absence of AT1636 binding to E. coli-derived E-cadherin (Lsbio) (left panel). E. coli-produced E-cadherin is recognized by the EP700Y antibody. [Figure 9]Alanine substitution of the E-cadherin p70 truncated EC3 domain (D3) reveals several amino acids essential for AT1636 binding. AT1636 binding to recombinant small D3-FLAG mouse Fc fusion proteins was studied by ELISA during anti-mouse Fc capture. All proteins were expressed at similar levels in the culture supernatant, D3 wild-type binding was set to 1, and all were normalized to anti-FLAG detection. [Figure 10] The computational analysis shown is the combined mRNA expression of TMTC3 and E-cadherin in several tumor-specific cell lines; the number of tumor cell lines included for each tissue type is in the center of the circle. Light gray indicates that the proportion of tumor cell lines with high (≧7x) expression for both E-cadherin and TMTC3, and are therefore predicted to be recognized by AT1636. The cutoff value of 7 was selected based on flow cytometry analysis showing that such cell lines are likely to be conjugated by the AT1636 antibody, see Table 3. Tissues that are typically negative for both TMTC3 and E-cadherin are hematopoietic or lymphoid tissue, bone, and soft tissue. (Data obtained from Broad Institute: https: / / portals.broadinstitute.org / ccle, J. Barretina, Nature (2012)). [Figure 11] Flow cytometry analysis suggests increased binding of AT1636 to SK-MEL-5 cells transduced using a construct containing full-length E-cadherin. SK-MEL-5 is normally negative for E-cadherin but expresses TMTC3. AT1636 (solid line) does not bind to SK-MEL-5 (left), but binds when E-cadherin is overexpressed (center). EP700Y (right) is shown here bound to SK-MEL-5. The light gray curve is the background staining of the isotype control. [Figure 12]shRNA-induced TMTC3 knockdown results in reduced AT1636 binding, as determined by flow cytometry. In addition to a control scrambled shRNA vector, TMTC3-targeted shRNA probes were developed and tested. TMTC3 expression is potently reduced, as determined by qPCR (left). TMTC3 knockdown resulted in a >3-fold reduction in AT1636 binding (right panel, solid line). [Figure 13A] (A) A graphic representation of the structure of a monovalent T cell derivative (mTCE) consisting of AT1636 or AT1636-IYN fused to anti-CD3 UCHT1. [Figure 13B] (B) The compounds were tested in a 2D cell culture model. Luciferase and GFP-positive CRC cell lines DLD1, HT29, and HCT116 were cultured overnight at 5000 c / w (96 w) and then incubated with unstimulated total PBMCs as effector cells for 2 days. Cytotoxicity was established by measuring luciferase expression over 48 hours. [Figure 13C] (C) Graphic representation of the monovalent knob-in-hole (KiH) bispecificity format structure for both CD3εscFv derived from AT1636, AT1636-IYN, or AT1002 and UCHT1 antibodies. [Figure 13D] (D) Compounds were tested in a 2D cell culture model. Luciferase and GFP-positive CRC cell lines DLD1 and HT29 and melanoma cell line A375 were cultured overnight at 5000 c / w (96 w). Then, KiH bispecific monovalent AT1636, AT1636-IYN, and AT1002 were incubated with UCHT1 scFv CD3ε and subsequently cultured for 2 days with unstimulated total PBMCs as effector cells. Cytotoxicity was assessed by measuring luciferase activity at the end of the 48-hour incubation period. [Figure 14]Stable overexpression of p70E-cadherin and full-length E-cadherin in cell lines that normally express E-cadherin (DLD1, HCT116, and HT29) and cells that are normally negative for E-cadherin (SK-MEL-5). The left column shows cells transduced with an empty vector. With overexpression of p70E-cadherin, all cells exhibit a deadhesion morphological phenotype (suggesting an EMT phenotype). [Figure 15] Flow cytometry analysis of AT1636 binding to DLD1 cells cultured for extended periods with TGFβ compared to cells cultured in the absence of TGFβ. [Figure 16A] Reduced cell proliferation and cell number after the addition of TGFβ and AT1636-IYN. (A) A431 cell cultures in or without TGFβ and AT1636 and AT1636-IYN mutants. The top row panel shows A431 cells cultured for 5 days on tissue culture-treated plastic, and the bottom row panel shows A431 cells cultured on fibronectin-coated plastic using 10× magnification. The left panel shows cells in culture medium, the middle panel shows cells in the presence of TGFβ, and the right panel shows cells cultured with TGFβ and AT1636-IYN. Reduced (viable) cells and cells with lower adhesion were observed in wells cultured in the presence of TGFβ and AT1636-IYN. No differences were observed between cells cultured on plastic or fibronectin-coated plates, and no effect was observed (not shown) for AT1636-wt or AT1002 control antibodies. [Figure 16B] (B) Detailed representative overview using 20× magnification of A431 cells cultured in fibronectin-coated wells after 7 days of culture in the presence of TGFβ (left panel) and TGFβ and AT1636-IYN (right panel). In the right panel, rounder, drier single cells and cells with lower adhesion are observed. [Figure 17]Time-series analysis of the internalization of AT1636 and its variants in DLD1 cells detected by fluorescence microscopy (Incucyte) using pH-sensitive Zenon pHrodo iFL dye. All antibodies except the negative control AT1002 were internalized. [Figure 18] The cell surface coverage of full-length and CFSE-labeled CD103+ T cells incubated on plates conjugated with AT1636, its variants, and the CD103-specific antibody (MCA708) is shown. [Modes for carrying out the invention]

[0056] E-cadherin E-cadherins are present in humans and are encoded by the CDH1 gene, also known as CD324. The amino acid sequences of currently known human E-cadherins are shown in Figure 1A. E-cadherins are 120 kDa transmembrane glycoproteins that localize to adherent junctions in epithelial cells. E-cadherins are members of a large family of cadherins that can be classified into several subtypes: type I classical cadherins such as E-cadherin (CDH1), N-cadherin (CDH2), and P-cadherin (CDH3); type II classical cadherins such as VE-cadherin (CDH5) and OB-cadherin (CDH11); desmosome cadherins; seven-transmembrane cadherins; cadherins of the FAT and dachsous (DCHS) groups; and protocadherins (PCDH). E-cadherins are transmembrane proteins having three components: (1) an extracellular cadherin domain (EC) responsible for isomorphic cadherin-cadherin interactions, (2) a single-pass or seven-pass transmembrane domain, and (3) a cytoplasmic domain that acts as a connector between the cell surface, associated cytoplasmic catenin proteins, and the cytoskeleton. Cadherins are involved in the growth of organisms (embryonic development), wound healing, and tumor invasion and metastasis.

[0057] In addition to being a calcium-dependent adhesion molecule, E-cadherin is also a crucial regulator of epithelial junction formation. Its association with catenin requires cell-cell adhesion and polarization of epithelial cells / epithelial sheets between the outer and apical membranes. Tyrosine phosphorylation can disrupt these complexes, leading to alterations in cell adhesion properties. E-cadherin expression is frequently downregulated in highly invasive and poorly differentiated carcinomas. Increased expression of E-cadherin in these cells reduces invasiveness. Therefore, loss of E-cadherin expression or function appears to be a critical step in the progression of tumorigenesis. Furthermore, cadherin plays a vital role in epithelial-mesenchymal transition (EMT), the reversible process of cell invasion and migration. EMT is a highly diverse process that can be regulated by many external signals (inflammation, stress, hypoxia, immune responses, etc.). In particular, it is generally accepted that potent regulation of EMT-inducing transcription factors (Snail, E47, Twist, and Zeb families) is based on EMT, and that the binding of, for example, TGFβ, Wnt, integrins, and growth factors to cells results in the downregulation of E-cadherins, ZO-1, and desmoplakins, as well as the upregulation of vimentin, fibronectin, and N-cadherins. The cells then undergo a process that resembles a more "stem cell-like" phenotype. More recently, this model has been modified as it has been observed that cells can also "show" EMT without the downregulation or upregulation of known EMT markers. These have been named partial EMT, hybrid EMT / MET, and quasi-EMT, and most of the novel models propose systems in which cells can regulate protein expression (e.g., protein internalization, high / low protein turnover) or cells together (clusters) in different modes of activity / invasiveness. E-cadherins play a dominant role in these processes, and in this respect, E-cadherin O-mannosylation is considered an additional tool for regulating adhesion and morphological changes while tumor cells interact with the surrounding matrix.

[0058] Anti-E-cadherin antibody and its antigen-binding fragment The present invention provides an antibody capable of specifically binding to O-mannosylated E-cadherin and its antigen-binding fragment. In some embodiments, the antibody is isolated. In other embodiments, the antibody is synthetic or recombinant. Interestingly, the present invention provides an antibody and its antigen-binding fragment containing VH and VL sequences, based on the VH and VL sequences of a human antibody derived from a human individual suffering from metastatic stage IV colon cancer but in complete remission for many years after chemotherapy. In contrast, many currently known therapeutic antibodies are typically obtained by immunizing non-human animals such as mice, rats, camels, rabbits, or goats, and then possibly by undergoing a humanization process. Such humanized antibodies still carry the risk of adverse side effects due to the recipient's immune response to the non-human sequence. In addition, many prior art therapeutic antibodies or fragments are derived from artificial phage display libraries in which immunoglobulin heavy and light chains are randomly paired. In contrast, the present invention provides an antibody and antigen-binding fragment having naturally paired heavy and light chains, based on the sequence of an antibody evolving in vivo in a human patient in complete remission.

[0059] Because E-cadherin is widely expressed in many epithelial tissues, prior to the present invention, E-cadherin had not been considered as a choice antigen for therapeutic use in the art, particularly in light of the fact that E-cadherin is frequently downregulated in tumor cells to promote EMT. However, the present invention provides an antibody and its antigen-binding fragment that can specifically bind a truncated form of E-cadherin having a molecular weight of about 70 kDa, which is frequently upregulated in tumor cells.

[0060] As used herein, the term “antibody” encompasses a proteinaceous molecule and any antigen-binding fragment thereof. The proteinaceous molecule is preferably an immunoglobulin protein, meaning that they belong to the immunoglobulin class of proteins. In some embodiments, the antibody or its antigen-binding fragment comprises one or more domains that bind an epitope on an antigen, and such domains preferably originate from or share sequence homology with a variable domain of the antibody.

[0061] Complementarity-determining regions (CDRs) are hypervariable regions located in the heavy chain variable domain and the light chain variable domain. In full-length antibodies, CDRs 1-3 of the heavy chain and CDRs 1-3 of the connected light chain together form an antigen-binding site.

[0062] The fragment crystallizable (Fc) region of natural antibodies consists of two heavy chain CH2 and CH3 domains.

[0063] Typically, the antigen-binding fragment of an antibody can bind the same antigen as the antibody, although the degree of binding is not necessarily the same. In some embodiments, the antigen-binding fragment includes at least the heavy chain CDR3 region of the antibody. In some embodiments, the antigen-binding fragment includes at least the heavy chain CDR3 region and the light chain CDR3 region of the antibody. In some embodiments, the heavy chain and light chain CDR3 regions are paired with each other.

[0064] In various embodiments, the antigen-binding fragment of the antibody includes at least the heavy chain CDR1, CDR2, and CDR3 regions of the antibody. In various embodiments, the antigen-binding fragment of the antibody includes at least the VH domain. In various embodiments, the antigen-binding fragment of the antibody includes at least the heavy chain CDR1, CDR2, and CDR3 regions and the light chain CDR1, CDR2, and CDR3 regions of the antibody. In various embodiments, the antigen-binding fragment of the antibody includes at least the VL domain. In various embodiments, the antigen-binding fragment of the antibody includes at least the VH and VL domains.

[0065] Non-limiting examples of antibody or antigen-binding fragments according to the present invention include full-length antibodies, DuoBody® (a bispecific antibody containing two different IgG1 domains), single-domain antibodies or nanobodies (containing a single VH or VL domain), single-chain variable fragments (scFv; typically containing VH and VL domains linked by a short linker peptide), Fv fragments (containing VH and VL domains, typically without a linker), unibody®, Fd fragments (containing a VH domain and a CH1 domain), diabody (containing two VH domains and two VL domains, where VH is linked to VL by such a short linker and cannot pair with each other but can pair with VL and VH on another chain, thereby creating two antigen-binding sites), Fab fragments (containing a heavy chain constant domain CH1, a light chain constant domain CL, and heavy chain variable domains VH and VL), and F(ab')2 fragments (containing two Fab fragments linked by disulfide crosslinking).

[0066] In various embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH is paired with the VL.

[0067] Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the subject being treated (e.g., human antibodies for human subjects). In various embodiments, the antibodies of the present invention are full-length antibodies, preferably IgG or IgM or IgA full-length antibodies. As used herein, an IgG full-length antibody is a bivalent molecule comprising two gamma-class heavy chains and two light chains. As is well known to those skilled in the art, the heavy chain of an antibody is the larger of the two types of chains that make up an immunoglobulin molecule. The natural heavy chain typically includes a constant domain CH, which contains constant regions CH1, CH2, and CH3, as well as a variable domain (VH) involved in antigen binding. The light chain of an antibody is the smaller of the two types of chains that make up an immunoglobulin. The natural light chain typically includes a constant domain (CL) and a variable domain (VL). The light chain variable domain is often, though not always, associated with the variable domain of the heavy chain involved in antigen binding.

[0068] Full-length IgD antibodies are divalent molecules containing two heavy chains and two light chains of the delta class.

[0069] In the case of IgM, a full-length antibody is a decavalent or dodecavalent molecule containing five or six bound immunoglobulins, each having two antigen-binding sites formed by a heavy chain and a light chain of immunoglobulin monomers.

[0070] In the case of IgA, full-length antibodies can be monomers or dimers.

[0071] In some embodiments, the antibody or antigen-binding fragment according to the present invention is a human antibody or its antigen-binding fragment. The presence of a human amino acid sequence reduces the chance of adverse side effects during therapeutic use in human patients, as opposed to mouse or humanized antibodies, while the sequence of a non-human CDR or variable or constant region is associated with the risk of anti-mouse immune response in human recipients.

[0072] In various embodiments, the antibody or antigen-binding fragment according to the present invention is an IgG isotype, preferably IgG1. This is beneficial for medical applications in humans, for example, because IgG1 antibodies typically have a favorable half-life when administered in vivo to human organisms. Furthermore, the Fc tail of IgG1 enables effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). In some embodiments, the antibody or antigen-binding fragment according to the present invention is human IgG, preferably human IgG1.

[0073] In some embodiments, the antibody or antigen-binding fragment according to the present invention is of the IgG2 isotype. In some embodiments, the antibody or antigen-binding fragment according to the present invention is of the IgG3 isotype. In some embodiments, the antibody or antigen-binding fragment according to the present invention is of the IgG4 isotype.

[0074] In some embodiments, the antibody or antigen-binding fragment according to the present invention is an IgM isotype. In some embodiments, the antibody or antigen-binding fragment according to the present invention is an IgA isotype. In some embodiments, the antibody or antigen-binding fragment according to the present invention is an IgD isotype.

[0075] In various embodiments, the antibody or its antigen-binding fragment contains one or more mutations. Such mutations include, for example, amino acid substitutions, insertions, or deletions. As used herein, a full-length antibody that has one or more, preferably up to 20, amino acid residues deleted without essentially altering the binding characteristics of the resulting antibody is still considered a full-length antibody.

[0076] In some embodiments, the antibody or antigen-binding fragment according to the present invention has a modified Fc tail. In some embodiments, the Fc tail is modified by one or more amino acid substitutions and / or changes in glycosylation. In some embodiments, the Fc tail is modified to reduce ADCC activity. In some embodiments, the Fc tail is modified to enhance ADCC activity. In some embodiments, the antibody or antigen-binding fragment according to the present invention is defucosylated, thereby enhancing ADCC activity.

[0077] The terms “can bind,” “specifically,” “can specifically bind,” “specifically bindable,” and “bind” are interchangeable herein and refer to the interaction between an antibody, or its antigen-binding fragment, and its target (also called its antigen). This means that the antibody or antigen-binding fragment binds to the antigen preferentially to other antigens or amino acid sequences. Thus, while the antibody or antigen-binding fragment may bind nonspecifically to other antigens or amino acid sequences, the binding affinity of the antibody or antigen-binding fragment to its antigen is significantly higher than the nonspecific binding affinity of the antibody or antigen-binding fragment to other antigens or amino acid sequences.

[0078] Typically, the antibody or antigen-binding fragment of the present invention, modified in some way, retains at least 50% of its binding activity (compared to the parent antibody). Preferably, the antibody or antigen-binding fragment of the present invention retains at least 60%, 70%, 80%, 90%, 95%, or 100% of its binding activity compared to the parent antibody.

[0079] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises conserved or non-conserved amino acid substitutions that substantially do not alter its biological activity (the resulting variants are referred to herein as “conserved variants” or “functional variants,” respectively). In some embodiments, such conserved or functional variants retain at least 80%, 90%, 95%, or 100% of their binding activity compared to the parent antibody.

[0080] As used herein, a conservative substitution is one in which an amino acid residue is replaced by another residue that generally has similar properties (size, hydrophobicity, etc.), and therefore the overall functionality of the antibody is not inherently affected. Typically, substitutions of amino acid residues within the same class, as shown in Table 2, are considered conservative amino acid substitutions.

[0081] An antibody or antigen-binding fragment according to the present invention that can bind to O-mannosylated E-cadherin may also be specific to another compound if the O-mannosylated E-cadherin epitope to which the antibody or antigen-binding fragment binds is also coincidentally present in the other compound. In such cases, the antibody or antigen-binding fragment referred to herein as specific to O-mannosylated E-cadherin is also specific to such other compound containing the same type of O-mannosyl epitope. Such other O-mannosylated epitopes may be produced in vivo by another O-mannosyltransferase other than the O-mannosyltransferase that produces O-mannosylated E-cadherin in vivo. Therefore, the terms “binding” or “specific” do not exclude the binding of the antibody or antigen-binding fragment of the present invention to another protein or protein(s) containing the same type of O-mannosylated epitope.

[0082] "Binding affinity" refers to the sum of the non-covalent interactions between a single binding site of an antibody or antigen-binding fragment and its binding partner (e.g., antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of the binding pair (e.g., antibody and antigen). Affinity is generally expressed using the equilibrium dissociation constant (K). D It can be expressed as, k a k d This is calculated as a ratio to [a certain value], see, for example, Chen, Y., et al., (1999) J. Mol Biol 293:865-881. Affinity can be measured by common methods known in the art, such as surface plasmon resonance (SPR) assays using instruments like BiaCore (GE Healthcare), Octet (Fortebio), or IBIS-iSPR instruments from IBIS Technologies BV (Hengelo, Netherlands), or liquid-phase assays such as Kinexa.

[0083] As used herein, the terms “nucleic acid” and “nucleic acid molecule” are used interchangeably. In some embodiments, the nucleic acid or nucleic acid molecule according to the present invention comprises a chain of nucleotides, more preferably DNA, cDNA, or RNA. In some embodiments, the nucleic acid or nucleic acid molecule according to the present invention comprises non-natural nucleotides that exhibit the same function as natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks, such as DNA / RNA helices, peptide nucleic acids (PNAs), and / or lock nucleic acids (LNAs).

[0084] The percentage of amino acid or nucleic acid sequence identity, or the term "% sequence identity," is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that are identical to residues in a reference sequence after two sequences have been aligned and gaps introduced as necessary, and the maximum percentage identity has been achieved. Methods and computer programs for alignment are well known in the art, such as "Align2."

[0085] As used herein, the singular term "a" encompasses the term "one or more."

[0086] Typical E-cadherin-specific antibodies The present invention provides antibodies and their antigen-binding fragments that are specific to O-mannosylated E-cadherins and have specific structural and functional characteristics, as well as their therapeutic use for the treatment or prevention of disease. A non-limiting example of such disease is O-mannosylated E-cadherin-containing cancer.

[0087] As used herein, the term “O-mannosylated E-cadherin” refers to an E-cadherin protein comprising at least one threonine or serine residue having O-bonded mannose, meaning that mannose is bonded to the oxygen atom of threonine or serine. In some embodiments, the E-cadherin protein comprises at least one single O-mannosylated threonine or serine residue. The term “single O-mannosylated threonine residue” refers to a threonine residue comprising O-bonded mannose without a bond to O-bonded mannose of another sugar moiety. The term “single O-mannosylated serine residue” refers to a serine residue comprising O-bonded mannose without a bond to O-bonded mannose of another sugar moiety.

[0088] Various embodiments provide an antibody or antigen-binding fragment according to the present invention that is specific to O-mannosylated E-cadherin, the binding of the antibody or antigen-binding fragment to the E-cadherin depends on the presence of an O-mannosylated threonine residue at position 467, O-mannosylated threonine residue at position 468, O-mannosylated threonine residue at position 470, O-mannosylated threonine residue at position 472, a glutamic acid residue at position 463, a serine residue at position 465, a serine residue at position 469, and / or a valine residue at position 477 of the E-cadherin sequence, as shown in Figure 1A.

[0089] Some embodiments provide an antibody specific to O-mannosylated E-cadherin and an antigen-binding fragment thereof, wherein the binding of the antibody or antigen-binding fragment to the E-cadherin depends on the presence of one or more O-mannosylated threonine residues within the E-cadherin amino acid region 467-472, as shown in Figure 1A. In some embodiments, the antibody or antigen-binding fragment depends on the presence of an O-mannosylated threonine residue at position 467 and / or at position 468 and / or at position 470 and / or at position 472 of the E-cadherin sequence, as shown in Figure 1A. In some embodiments, the antibody or antigen-binding fragment depends on the presence of an O-mannosylated threonine residue at position 468 and an O-mannosylated threonine residue at position 470 of the E-cadherin sequence, as shown in Figure 1A. In some embodiments, the antibody or antigen-binding fragment depends on the presence of an O-mannosylated threonine residue at position 467, an O-mannosylated threonine residue at position 468, and an O-mannosylated threonine residue at position 470 of the E-cadherin sequence, as shown in Figure 1A. In some embodiments, the antibody or antigen-binding fragment depends on the presence of an O-mannosylated threonine residue at position 467, an O-mannosylated threonine residue at position 468, an O-mannosylated threonine residue at position 470, and an O-mannosylated threonine residue at position 472 of the E-cadherin sequence, as shown in Figure 1A. In some embodiments, the binding of the antibody or antigen-binding fragment to the E-cadherin further depends on the presence of a glutamic acid residue at position 463 and / or a serine residue at position 465 and / or a serine residue at position 469 and / or a valine residue at position 477 of the E-cadherin sequence, as shown in Figure 1A. In some embodiments, the serine residues at positions 465 and / or 469 are O-mannosylated.

[0090] As used herein, the binding of an antibody or antigen-binding fragment is "dependent" on a particular amino acid residue if the substitution of the amino acid residue with an alanine residue reduces the binding of the antibody or antigen-binding fragment to its antigen by at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, and more preferably at least 95%.

[0091] Some embodiments provide an antibody or antigen-binding fragment according to the present invention that is specific to O-mannosylated E-cadherins and specifically binds to an O-mannosylated threonine residue at position 467 and / or an O-mannosylated threonine residue at position 468 and / or an O-mannosylated threonine residue at position 470 and / or an O-mannosylated threonine residue at position 472 and / or a glutamic acid residue at position 463 and / or a serine residue at position 465 and / or a serine residue at position 469 and / or a valine residue at position 477 of an E-cadherin sequence, as shown in Figure 1A. In some embodiments, the serine residues at positions 465 and / or 469 are O-mannosylated.

[0092] Some embodiments of the present invention provide an antibody or antigen-binding fragment that is specific to O-mannosylated E-cadherin and specifically binds to one or more O-mannosylated threonine residues of E-cadherin, wherein the one or more O-mannosylated threonine residues are located within amino acid positions 467-472 of the E-cadherin sequence, as shown in Figure 1A.

[0093] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds one or more O-mannosylated threonine residues selected from the group consisting of an O-mannosylated threonine residue at position 467, an O-mannosylated threonine residue at position 468, an O-mannosylated threonine residue at position 470, and an O-mannosylated threonine residue at position 472 of an E-cadherin sequence, as shown in Figure 1A. Some embodiments provide an antibody and its antigen-binding fragment that specifically binds an O-mannosylated threonine residue at position 467 of an E-cadherin sequence, as shown in Figure 1A. Some embodiments provide an antibody and its antigen-binding fragment that specifically binds an O-mannosylated threonine residue at position 468 of an E-cadherin sequence, as shown in Figure 1A. Some embodiments provide an antibody and its antigen-binding fragment that specifically binds an O-mannosylated threonine residue at position 470 of an E-cadherin sequence, as shown in Figure 1A. Some embodiments provide antibodies and antigen-binding fragments that specifically bind an O-mannosylated threonine residue at position 472 of an E-cadherin sequence, as shown in Figure 1A.

[0094] Some embodiments provide antibodies and antigen-binding fragments that specifically bind O-mannosylated threonine residues at position 468 and 470 of an E-cadherin sequence, as shown in Figure 1A.

[0095] Some embodiments provide antibodies and antigen-binding fragments that specifically bind to O-mannosylated threonine residues at positions 467, 468, and 470 of an E-cadherin sequence, as shown in Figure 1A.

[0096] Some embodiments provide antibodies and antigen-binding fragments that specifically bind to O-mannosylated threonine residues at positions 467, 468, 470, and 472 of an E-cadherin sequence, as shown in Figure 1A.

[0097] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing T468 and T470 of the E-cadherin sequence, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, both threonine residues are O-mannosylated.

[0098] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing T467, T468, and T470 of the E-cadherin sequence, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, two of the threonine residues are O-mannosylated. In some embodiments, all three threonine residues are O-mannosylated.

[0099] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing T467, T468, T470, and T472 of the E-cadherin sequence, as shown in Figure 1A, wherein the threonine residues are O-mannosylated. In some embodiments, two of the threonine residues are O-mannosylated. In some embodiments, three of the threonine residues are O-mannosylated. In some embodiments, all four threonine residues are O-mannosylated.

[0100] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing T468, S469, and T470 of the E-cadherin sequence, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, both threonine residues are O-mannosylated. In some embodiments, the serine residue is O-mannosylated.

[0101] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing T467, T468, S469, and T470 of the E-cadherin sequence, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, the serine residues are O-mannosylated.

[0102] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing the E-cadherin sequence S465, T467, T468, S469 and T470, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0103] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing the E-cadherin sequence S465, T467, T468, S469, T470, and T472, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, at least three of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0104] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing E463, S465, T467, T468, S469, T470, and T472 of the E-cadherin sequence, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, at least three of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0105] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to a region of E-cadherin containing E463, S465, T467, T468, S469, T470, T472 and V477 of the E-cadherin sequence, as shown in Figure 1A, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, at least three of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0106] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to an E-cadherin epitope containing the sequence TST, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, both threonine residues are O-mannosylated. In some embodiments, the serine residue is O-mannosylated.

[0107] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to an E-cadherin epitope containing the sequence TTST, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, the serine residue is O-mannosylated.

[0108] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to an E-cadherin epitope containing the sequence STTST, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0109] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to an E-cadherin epitope comprising the sequence STTSTT, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, at least three of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0110] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to an E-cadherin epitope containing the sequence ESTTSTT, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, at least three of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0111] Some embodiments provide an antibody and its antigen-binding fragment that specifically binds to an E-cadherin epitope containing the sequence ESTTSTTV, wherein at least one of the threonine residues is O-mannosylated. In some embodiments, at least two of the threonine residues are O-mannosylated. In some embodiments, at least three of the threonine residues are O-mannosylated. In some embodiments, all of the threonine residues are O-mannosylated. In some embodiments, at least one serine residue is O-mannosylated. In some embodiments, both serine residues are O-mannosylated.

[0112] E-cadherin is known in the art as a product of the CDH1 gene and has a molecular weight of approximately 120 kDa in humans. However, this invention offers the surprising insight that an O-mannosylated shortened E-cadherin form also exists in nature. This shortened form, with a molecular weight of approximately 70 kDa, lacks the extracellular domains EC1 and EC2 of the full-length E-cadherin. Extracellular domains 5,4 and part of extracellular domain EC3 are still present in the shortened 70 kDa form. We offer the insight that this shortened 70 kDa form of E-cadherin is present on the surface of many types of epithelial cells and is frequently upregulated on tumor cells. Without being bound by theory, it is thought that the upregulation of the 70 kDa form of E-cadherin contributes to tumor growth, as the shortened 70 kDa form stimulates epithelial-mesenchymal transition (EMT), as shown in the examples, thereby increasing tumor cell migration and metastasis. Furthermore, since the shortened 70kDa E-cadherin has a lower ability to bind to immune cells compared to the full-length 120kDa E-cadherin, it is thought that upregulation of the 70kDa E-cadherin contributes to the tumor's immune evasion mechanism. According to the present invention, overexpression of the O-mannosylated 70kDa E-cadherin can promote evasion from immune cell recognition via CD3, KLRG1, or CD103, and promotes EMT without complete downregulation of E-cadherin. Therefore, upregulation of the shortened 70kDa E-cadherin on tumor cells may reduce the interaction between these tumor cells and immune cells, thereby helping tumors evade the immune response.

[0113] In some embodiments, the present invention provides an antibody and its antigen-binding fragment that binds the aforementioned O-mannosylated shortened 70kDa E-cadherin better than a well-known O-mannosylated full-length E-cadherin of about 120kDa. Preferred embodiments provide an antibody and its antigen-binding fragment that binds the O-mannosylated shortened 70kDa E-cadherin at least twice, more preferably at least three times, more preferably at least four times, and more preferably at least five times better than O-mannosylated full-length E-cadherin. This feature allows for improved tumor specificity and reduced adverse side effects caused by healthy tissue binding when the shortened 70kDa E-cadherin type is significantly upregulated on tumor cells. As used herein, the term “full-length E-cadherin” refers to a known CDH1 gene product having a molecular weight of about 120kDa in humans, as shown, for example, in Figure 1A. The term “abbreviated 70kDa E-cadherin” or “70kDa E-cadherin type” refers to a smaller E-cadherin type with a molecular weight of 60kDa to 80kDa, typically around 70kDa, and which occurs spontaneously on the surface of epithelial cells. As shown in Figure 1C, this spontaneously occurring abbreviated E-cadherin type with a molecular weight of 60kDa to 80kDa lacks the extracellular domains EC1 and EC2 of the full-length E-cadherin. Extracellular domains 5,4 and part of extracellular domain EC3 are still present in this abbreviated 70kDa type. The term "O-mannosylated shortened 70kDa E-cadherin" refers to the aforementioned shortened 70kDa E-cadherin protein, which contains at least one O-mannosylated threonine or serine residue, preferably an O-mannosylated threonine residue at at least position 467 and / or 468 and / or 470, as shown in Figure 1A.

[0114] In a particular embodiment, the anti-E-cadherin antibody or antigen-binding fragment of the present invention is: - Heavy chain variable region CDR3 containing the amino acid sequence TPGVGX1NX2PYYFDR (X1 is A or T, X2 is D or N); and - Light chain variable region CDR3 containing amino acid sequence QQYSNTPQT Includes.

[0115] A particular embodiment provides an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, wherein the antibody or antigen-binding fragment is: a. Heavy chain variable region CDR1 containing the amino acid sequence GFX1FSX2AW (where X1 is T or I, and X2 is N or Y); b. Heavy chain variable region CDR2 containing the amino acid sequence IKSKIDG X1T X2 (where X1 is G or E and X2 is T or I); c. Heavy chain variable region CDR3 containing the amino acid sequence TPGVGX1NX2PYYFDR (where X1 is A or T and X2 is D or N); d. Light chain variable region CDR1 containing the amino acid sequence QSVLCRSNNKNC; e. Light chain variable region CDR2 containing the amino acid sequence WAX1 (X1 is either S or C); f. Light chain variable region CDR3 containing the amino acid sequence QQYSNTPQT; Alternatively, the light chain variable region CDR3 may contain an amino acid sequence different from the QQYSNTPQT sequence by one, two, or three conservative substitutions. One or more of them, and possibly each of them.

[0116] In some cases, a conservative amino acid substitution is applied to at least one of the CDR sequences described above. In some embodiments, the conservative substitution includes the substitution of one or more amino acid residues of an amino acid class, as shown in Table 2, with another amino acid residue of the same amino acid class. Non-limiting examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue, and the substitution of one polar residue with another polar residue, such as arginine with lysine, glutamic acid with aspartic acid, or glutamine with asparagine. Preferably, after the conservative amino acid substitution, the preferred E-cadherin binding characteristics of the parent antibody are maintained or even improved. Preferably, such a mutant CDR sequence differs from the parent sequence by three or fewer, preferably two or fewer, and preferably one or fewer amino acids.

[0117] Therefore, some embodiments are: - Heavy chain variable region CDR3 containing the amino acid sequence TPGVGX1NX2PYYFDR (where X1 is A or T and X2 is D or N); or heavy chain variable region CDR3 containing an amino acid sequence different from the TPGVGX1NX2PYYFDR sequence by one, two or three conservative substitutions; and - Light chain variable region CDR3 containing the amino acid sequence QQYSNTPQT or light chain variable region CDR3 containing an amino acid sequence different from the QQYSNTPQT sequence by one, two, or three conservative substitutions. The present invention provides an anti-E-cadherin antibody or antigen-binding fragment comprising the present invention.

[0118] Furthermore, an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin is provided, wherein the antibody or antigen-binding fragment is: a. Heavy chain variable region CDR1 containing the amino acid sequence GFX1FSX2AW (where X1 is T or I, and X2 is N or Y); Alternatively, the heavy chain variable region CDR1 containing an amino acid sequence different from the GFX1FSX2AW sequence by one, two, or three conservative substitutions; b. Heavy chain variable region CDR2 containing the amino acid sequence IKSKIDGX1TX2 (where X1 is G or E and X2 is T or I); Alternatively, a heavy chain variable region CDR2 containing an amino acid sequence different from the IKSKIDGX1TX2 sequence by one, two, or three conservative substitutions; c. Heavy chain variable region CDR3 containing the amino acid sequence TPGVGX1NX2PYYFDR (where X1 is A or T and X2 is D or N); Alternatively, the heavy chain variable region CDR3 contains an amino acid sequence different from the TPGVGX1NX2PYYFDR sequence by one, two, or three conservative substitutions; d. Light chain variable region CDR1 containing the amino acid sequence QSVLCRSNNKNC; Alternatively, the light chain variable region CDR1 containing an amino acid sequence different from the QSVLCRSNNKNC sequence by one, two, or three conservative substitutions; e. Light chain variable region CDR2 containing the amino acid sequence WAX1 (X1 is either S or C); Alternatively, a light chain variable region CDR2 containing an amino acid sequence different from the WAX1 sequence by one, two, or three conservative substitutions; f. Light chain variable region CDR3 containing the amino acid sequence QQYSNTPQT; Alternatively, the light chain variable region CDR3 may contain an amino acid sequence different from the QQYSNTPQT sequence by one, two, or three conservative substitutions. One or more of them, and possibly each of them.

[0119] Table 1 provides sequences of preferred antibodies according to the present invention. These preferred antibodies are referred to herein as antibodies AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN. These antibodies conjugate O-mannosylated E-cadherins, particularly novel, approximately 70 kDa truncated E-cadherin types as described above herein. The heavy and light chain CDR sequences of these preferred antibodies are GFX1FSX2AW, IKSKIDGX1TX2, TPGVGX1NX2PYYFDR, QSVLCRSNNKNC, WAX1, and QQYSNTPQT, as described above in a) to f).

[0120] As used herein, the terms "AT1636", "E-C06", "D-H04", "D-A02", "D-E09", "E-A04", "E-B09", "C-A05", "C-A03", "C-B02", "C-D04-A", "C-D04-B", "F-C08", "D-G03", "D-F10", "C-E08", "D-B06", "D-G05", "D-H08", "C-H01", "D-C12 "D-C11", "E-C10", "AT1636-I", "AT1636-Y", "AT1636-E", "AT1636-N", "AT1636-YN", "AT1636-IYN", and "AT1636-IYEN" encompass all antibodies and antigen-binding fragments having at least the heavy chain and light chain CDR1-3 regions, preferably the heavy chain and light chain variable regions, of these antibodies as shown in Figure 1.

[0121] Based on the antibodies shown in Table 1, it is possible to conjugate O-mannosylated E-cadherin to produce an antibody or antigen-binding fragment containing at least one CDR sequence of the antibody shown in Table 1. Therefore, an antibody or antigen-binding fragment containing at least one CDR sequence of the antibody as shown in Table 1 is provided. The CDR sequence is preferably a CDR3 sequence of the antibody as shown in Table 1. In some embodiments, an antibody or antigen-binding fragment containing a heavy-chain CDR3 sequence and a light-chain CDR3 sequence of the antibody as shown in Table 1 is provided. Accordingly, some embodiments provide an antibody or antigen-binding fragment comprising heavy and light chain CDR3 sequences of an antibody selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN.

[0122] Some embodiments provide antibodies or antigen-binding fragments that conjugate O-mannosylated E-cadherin and include heavy chain CDR1-3 sequences of one or more antibodies shown in Table 1.

[0123] In some embodiments, an antibody or antigen-binding fragment is provided that contains the same antibody heavy chain CDR1, CDR2, and CDR3 sequences shown in Table 1. Therefore, according to this embodiment, the heavy chain CDR1, CDR2, and CDR3 sequences of the antibody AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, or AT1636-IYEN are present together in a single antibody or antigen-binding fragment. Such antibody or antigen-binding fragments can functionally pair with multiple different heavy chains, further comprising a general light chain as defined herein, which is a light chain that thereby maintains the antigen specificity of the heavy chain. This approach is based on the well-known fact that heavy chains are often the primary drivers of affinity and specificity. While pairing a general light chain with a given heavy chain typically provides a preferred structure, such general light chains do not significantly contribute to antigen specificity.

[0124] In some embodiments, the antibody or antigen-binding fragment according to the present invention comprises all three heavy chain CDRs and all three light chain CDRs of the same antibody shown in Table 1. Therefore, antibodies or antigen-binding fragments containing heavy and light chain CDR1-3 sequences of antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN are further provided.

[0125] The heavy chain variable region (VH) and light chain variable region (VL) sequences of antibodies AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN are also shown in Table 1. Based on these VH and / or VL sequences, it is further possible to conjugate O-mannosylated E-cadherins to produce antibodies or antigen-binding fragments containing the heavy chain variable region (VH) and / or light chain variable region (VL) of the antibodies shown in Table 1, or sequences having at least 80% sequence identity with them. Typically, 80% to 99% deformation of the VH and VL sequences is acceptable, but antigen-binding is maintained, especially when the CDR region remains unchanged. Therefore, antibodies and antigen-binding fragments containing VH or VL sequences having at least 80% sequence identity with the VH or VL sequences shown in Table 1 are also provided herein.

[0126] Therefore, antibodies or antigen-binding fragments thereof are provided, comprising the heavy chain variable region (VH) of an antibody shown in Table 1, or a sequence having at least 80% sequence identity thereto. Also provided are antibodies or antigen-binding fragments thereof comprising the light chain variable region (VL) of an antibody shown in Table 1, or a sequence having at least 80% sequence identity thereto. Some embodiments provide antibodies or antigen-binding fragments thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) of an antibody shown in Table 1, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide an antibody or antigen-binding fragment comprising the heavy chain variable region (VH) and / or light chain variable region (VL) of an antibody shown in Table 1, or sequences having at least 80% sequence identity thereto, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR3 sequence and light chain CDR3 sequence of the antibody as shown in Table 1. Preferably, the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequence and light chain CDR1-3 sequence of the antibody as shown in Table 1.

[0127] For example, in some embodiments, one or more framework residues of the VH or VL sequences shown in Table 1 are modified to reduce immunogenicity and / or to enhance the binding efficacy or stability of the resulting antibody or antigen-binding fragment. The framework sequence is optimized, for example, by mutating the nucleic acid molecule encoding such a framework sequence after testing the characteristics of the preferably resulting antibody—or its antigen-binding fragment. In this way, it is possible to obtain improved binding compounds.

[0128] In some embodiments, one or more framework residues are mutated back to the germline sequence from which the antibody AT1636 originates in order to reduce immunogenicity. Methods for comparing the framework region of a given antibody with the germline sequence from which the antibody originates are well known in the art.

[0129] In some embodiments, one or more framework residues of the VH or VL sequences shown in Table 1 are modified to remove one or more T cell epitopes, thereby reducing the potential immunogenicity of the resulting antibody or antigen-binding fragment. This is called deimmunization. Methods for deimmunizing the framework region of a given antibody or antigen-binding fragment are also well known in the art, for example, as described in De Groot et al, 2005.

[0130] In some embodiments, up to 10 amino acid residues of the framework residue of the VH or VL sequence shown in Table 1 are modified compared to the VH or VL sequence shown in Table 1. In some embodiments, up to 8 amino acid residues of the framework residue of the VH or VL sequence shown in Table 1 are modified. In some embodiments, up to 5 amino acid residues of the framework residue of the VH or VL sequence shown in Table 1 are modified. In some embodiments, up to 3 or 2 amino acid residues of the framework residue of the VH or VL sequence shown in Table 1 are modified. In some embodiments, one amino acid residue of the framework residue of the VH and VL sequences shown in Table 1 is modified.

[0131] Some embodiments include: - A heavy chain variable region containing a sequence having at least 80% sequence identity with a VH sequence selected from the group consisting of sequence numbers 1 to 17; and / or - Light chain variable region containing a sequence having at least 80% sequence identity with a VL sequence selected from the group consisting of sequence numbers 18-22. The present invention provides an antibody or antigen-binding fragment that can be conjugated to an O-mannosylated E-cadherin containing the present compound.

[0132] A preferred antibody according to the present invention is antibody AT1636. This antibody is preferred because it can bind O-mannosylated E-cadherins expressed on tumor cells, particularly the newly discovered approximately 70 kDa shortened E-cadherin form as described above herein. A particular advantage of AT1636 is that it binds this shortened 70 kDa E-cadherin form better than the full-length approximately 120 kDa E-cadherin. This feature of AT1636 typically enables improved tumor specificity when O-mannosylated shortened 70 kDa E-cadherins are upregulated on tumor cells. A further advantage of AT1636's preference for the shortened 70 kDa E-cadherin form is that full-length E-cadherins are widely expressed. Therefore, without the preference for the shortened 70kDa E-cadherin type, widely expressed full-length E-cadherin may function as a sink and / or may lead to undesirable effects. Furthermore, the expression level of full-length E-cadherin is very high, and therefore often cannot distinguish between healthy epithelial cells and tumor epithelial cells, while the preference for the shortened 70kDa E-cadherin type allows for higher tumor specificity. In addition, E-cadherin has an important barrier function, and therefore it is preferable to avoid significant interference by the healthy function of E-cadherin.

[0133] Furthermore, AT1636 is derived from a human individual with metastatic stage IV colon cancer who has been in complete remission for many years after chemotherapy, suggesting therapeutic efficacy. Interestingly, AT1636 is an IgG3 isotype. The presence of a human amino acid sequence reduces the chance of adverse side effects during therapeutic use in human patients.

[0134] In addition, AT1636 was selected for its ability to bind to O-mannosylated E-cadherin expressing colon cancer subtypes CMS1, CMS2, CMS3, and CMS4. AT1636 binds to tumor cells, particularly epithelial tumor cells, more specifically, O-mannosylated E-cadherin expressing cancer cells such as O-mannosylated E-cadherin expressing colon cancer cells, breast cancer cells, pancreatic cancer cells, bladder cancer cells, endometrial cancer cells, lung cancer cells, and esophageal cancer cells, as shown in the examples. Therefore, the antibody AT1636 is particularly suitable for the treatment and / or diagnosis of disorders associated with the presence of O-mannosylated E-cadherin expressing cells, especially cancer cells expressing the newly discovered shortened E-cadherin type of approximately 70 kDa.

[0135] The antibodies E-C10, D-C12, and D-C11 shown in Table 1 have the same heavy and light chain CDR1-C11 sequences as AT1636 and therefore possess the same binding specificity. These antibodies are also preferred antibodies according to the present invention because they can bind, among other things, O-mannosylated E-cadherins expressed on cells, particularly the newly discovered approximately 70 kDa truncated E-cadherin type as described herein, more specifically, one or more O-mannosylated threonine residues located within amino acid positions 467-472 of the E-cadherin sequence as shown in Figure 1A, and are therefore very suitable for the treatment and / or diagnosis of disorders associated with the presence of such O-mannosylated E-cadherin expressing cells, particularly cancer cells. The presence of human amino acid sequences reduces the opportunity for adverse side effects during therapeutic use in human patients.

[0136] The heavy chain CDR1-3 sequences of antibodies AT1636, E-C10, D-C12, and D-C11, as shown in Table 1, are GFTFSNAW, IKSKIDGGTT, and TPGVGANDPYYFDR. The light chain CDR1-3 sequences of these antibodies AT1636, E-C10, D-C12, and D-C11 are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide antibodies or antigen-binding fragments capable of conjugating O-mannosylated E-cadherins, comprising a heavy chain CDR1 containing the sequence GFTFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGGTT, a heavy chain CDR3 containing the sequence TPGVGANDPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0137] The VH sequence of antibody AT1636 is shown in Table 1 as SEQ ID NO: 1. The VL sequence of antibody AT1636 is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating an O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody AT1636 as shown in Table 1.

[0138] The VH sequence of antibody E-C10 is shown in Table 1 as SEQ ID NO: 1. The VL sequence of antibody E-C10 is shown in Table 1 as SEQ ID NO: 22. Therefore, some embodiments provide an antibody or antigen-binding fragment that can be conjugated to an O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 22, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 22, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody E-C10 as shown in Table 1.

[0139] The VH sequence of antibody D-C12 is shown in Table 1 as SEQ ID NO: 13. The VL sequence of antibody D-C12 is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 13 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 13 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody D-C12 as shown in Table 1.

[0140] The VH sequence of antibody D-C11 is shown in Table 1 as SEQ ID NO: 14. The VL sequence of antibody D-C11 is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment that can be conjugated to an O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 14 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 14 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody D-C11 as shown in Table 1.

[0141] Further provided are antibodies or antigen-binding fragments that compete with antibodies AT1636 or E-C10 or D-C12 or D-C11 for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0142] Further provided are antibodies or antigen-binding fragments that compete with antibodies AT1636 or E-C10 or D-C12 or D-C11 for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0143] Further provided are antibodies or antigen-binding fragments thereof that compete with antibodies AT1636 or E-C10 or D-C12 or D-C11 for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0144] The antibodies E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, AT1636-I, AT1636-Y, AT1636-E, and AT1636-N, shown in Table 1, are also preferred antibodies according to the present invention. These antibodies can also bind O-mannosylated E-cadherins expressed on cells, particularly the newly discovered approximately 70 kDa truncated E-cadherin type, more specifically, one or more O-mannosylated threonine residues located within amino acid positions 467-472 of the E-cadherin sequence as shown in Figure 1A, and are therefore highly suitable for the treatment and / or diagnosis of disorders associated with the presence of such O-mannosylated E-cadherin expressing cells, especially cancer cells. The presence of human amino acid sequences in these antibodies reduces the opportunity for adverse side effects during therapeutic use in human patients.

[0145] The heavy chain CDR1-3 sequences of antibodies E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, and AT1636-I, shown in Table 1, are GFIFSNAW, IKSKIDGGTT, and TPGVGANDPYYFDR. The light chain CDR1-3 sequences of these antibodies E-C06, DH04, D-A02, D-E09, E-A04, E-B09, and AT1636-I are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFIFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGGTT, a heavy chain CDR3 containing the sequence TPGVGANDPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0146] The VH sequences of antibodies E-C06 and D-H04 are shown in Table 1 as SEQ ID NO: 2. The VL sequences of antibodies E-C06 and D-H04 are shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide antibodies or antigen-binding fragments that can be conjugated to O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 2 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 2 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody E-C06 or D-H04 as shown in Table 1.

[0147] The VH sequences of antibodies D-A02, D-E09, E-A04, E-B09, and AT1636-I are shown in Table 1 as SEQ ID NO: 3. The VL sequences of antibodies D-A02, D-E09, E-A04, E-B09, and AT1636-I are shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide antibodies or antigen-binding fragments that can be conjugated to O-mannosylated E-cadherins, comprising a VH sequence such as that shown in SEQ ID NO: 3 and a VL sequence such as that shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region.Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 3 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibodies D-A02, D-E09, E-A04, E-B09, or AT1636-I as shown in Table 1.

[0148] Further provided are antibodies or antigen-binding fragments that compete with antibodies E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, or AT1636-I for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0149] Further provided are antibodies or antigen-binding fragments that compete with antibodies E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, or AT1636-I for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0150] Further provided are antibodies or antigen-binding fragments that compete with antibodies E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, or AT1636-I for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0151] The heavy chain CDR1-3 sequences of antibody C-A05, as shown in Table 1, are GFIFSNAW, IKSKIDGETT, and TPGVGANDPYYFDR. The light chain CDR1-3 sequences of antibody C-A05 are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFIFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGETT, a heavy chain CDR3 containing the sequence TPGVGANDPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0152] The VH sequence of antibody C-A05 is shown in Table 1 as SEQ ID NO: 4. The VL sequence of antibody C-A05 is shown in Table 1 as SEQ ID NO: 19. Therefore, some embodiments provide an antibody or antigen-binding fragment that can be conjugated to an O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 4 and a VL sequence as shown in SEQ ID NO: 19, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 4 and a VL sequence as shown in SEQ ID NO: 19, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody C-A05 as shown in Table 1.

[0153] Further provided are antibodies or antigen-binding fragments that compete with antibody C-A05 for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0154] Further provided are antibodies or antigen-binding fragments that compete with antibody C-A05 for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0155] Further provided are antibodies or antigen-binding fragments that compete with antibody C-A05 for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0156] The heavy chain CDR1-3 sequences of antibodies C-A03, C-B02, and AT1636-E, shown in Table 1, are GFTFSNAW, IKSKIDGETT, and TPGVGANDPYYFDR. The light chain CDR1-3 sequences of these antibodies C-A03, C-B02, and AT1636-E are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide antibodies or antigen-binding fragments capable of conjugating O-mannosylated E-cadherins, comprising a heavy chain CDR1 containing the sequence GFTFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGETT, a heavy chain CDR3 containing the sequence TPGVGANDPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0157] The VH sequences of antibodies C-A03, C-B02, and AT1636-E are shown in Table 1 as Sequence ID No. 5. The VL sequences of antibodies C-A03, C-B02, and AT1636-E are shown in Table 1 as Sequence ID No. 18. Therefore, some embodiments provide antibodies or antigen-binding fragments that can be conjugated to O-mannosylated E-cadherins, comprising a VH sequence such as that shown in Sequence ID No. 5 and a VL sequence such as that shown in Sequence ID No. 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating an O-mannosylated E-cadherin containing a VH sequence, such as that shown in SEQ ID NO: 5, and a VL sequence, such as that shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment contains heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of an antibody C-A03, C-B02, or AT1636E, as shown in Table 1.

[0158] Further provided are antibodies or antigen-binding fragments that compete with antibodies C-A03, C-B02, or AT1636-E for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0159] Further provided are antibodies or antigen-binding fragments that compete with antibodies C-A03, C-B02, or AT1636-E for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0160] Further provided are antibodies or antigen-binding fragments that compete with antibodies C-A03, C-B02, or AT1636-E for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0161] The heavy chain CDR1-3 sequences of antibody C-D04-A, shown in Table 1, are GFTFSNAW, IKSKIDGETT, and TPGVGANNPYYFDR. The light chain CDR1-3 sequences of antibody C-D04-A are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFTFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGETT, a heavy chain CDR3 containing the sequence TPGVGANNPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0162] The VH sequence of antibody C-D04-A is shown in Table 1 as SEQ ID NO: 6. The VL sequence of antibody C-D04-A is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 6 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 6 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody C-D04-A as shown in Table 1.

[0163] Further provided are antibodies or antigen-binding fragments that compete with antibody C-D04-A for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0164] Further provided are antibodies or antigen-binding fragments that compete with antibody C-D04-A for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0165] Further provided are antibodies or antigen-binding fragments that compete with antibody C-D04-A for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0166] The heavy chain CDR1-3 sequences of antibody C-D04-B, as shown in Table 1, are GFTFSNAW, IKSKIDGETT, and TPGVGANNPYYFDR. The light chain CDR1-3 sequences of antibody C-D04-B are QSVLCRSNNKNC, WAC, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFTFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGETT, a heavy chain CDR3 containing the sequence TPGVGANNPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAC, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0167] The VH sequence of antibody C-D04-B is shown in Table 1 as SEQ ID NO: 6. The VL sequence of antibody C-D04-B is shown in Table 1 as SEQ ID NO: 20. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 6 and a VL sequence as shown in SEQ ID NO: 20, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 6 and a VL sequence as shown in SEQ ID NO: 20, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody C-D04-B as shown in Table 1.

[0168] Further provided are antibodies or antigen-binding fragments that compete with antibody C-D04-B for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0169] Further provided are antibodies or antigen-binding fragments that compete with antibody C-D04-B for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0170] Further provided are antibodies or antigen-binding fragments that compete with antibody C-D04-B for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0171] The heavy chain CDR1-3 sequences of antibodies F-C08, D-G03, and AT1636-N, shown in Table 1, are GFTFSNAW, IKSKIDGGTT, and TPGVGANNPYYFDR. The light chain CDR1-3 sequences of these antibodies F-C08, D-G03, and AT1636-N are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFTFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGGTT, a heavy chain CDR3 containing the sequence TPGVGANNPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0172] The VH sequence of antibody F-C08 is shown in Table 1 as SEQ ID NO: 7. The VL sequence of antibody F-C08 is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment that can be conjugated to an O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 7 and a VL sequence as shown in SEQ ID NO: 18, or a sequence having at least 80% sequence identity with them. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 7 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody F-C08 as shown in Table 1.

[0173] The VH sequences of antibodies D-G03 and AT1636-N are shown in Table 1 as SEQ ID NO: 8. The VL sequences of antibodies D-G03 and AT1636-N are shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide antibodies or antigen-binding fragments that can be conjugated to O-mannosylated E-cadherins, comprising a VH sequence such as that shown in SEQ ID NO: 8 and a VL sequence such as that shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating an O-mannosylated E-cadherin containing a VH sequence, such as that shown in SEQ ID NO: 8, and a VL sequence, such as that shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment contains the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of an antibody D-G03 or AT1636-N, as shown in Table 1.

[0174] Further provided are antibodies or antigen-binding fragments that compete with antibodies F-C08, D-G03, or AT1636-N for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0175] Further provided are antibodies or antigen-binding fragments that compete with antibodies F-C08, D-G03, or AT1636-N for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0176] Further provided are antibodies or antigen-binding fragments that compete with antibodies F-C08, D-G03, or AT1636N for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0177] As shown in Table 1, the heavy chain CDR1-3 sequences of antibody D-F10 are GFTFSNAW, IKSKIDGGTT, and TPGVGTNNPYYFDR. The light chain CDR1-3 sequences of antibody C-A05 are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFTFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGGTT, a heavy chain CDR3 containing the sequence TPGVGTNNPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0178] The VH sequence of antibody D-F10 is shown in Table 1 as SEQ ID NO: 9. The VL sequence of antibody D-F10 is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 9 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 9 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody D-F10 as shown in Table 1.

[0179] Further provided are antibodies or antigen-binding fragments that compete with antibody D-F10 for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0180] Further provided are antibodies or antigen-binding fragments that compete with antibody D-F10 for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0181] Further provided are antibodies or antigen-binding fragments that compete with antibody D-F10 for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0182] The heavy chain CDR1-3 sequences of antibodies C-E08, D-B06, D-G05, AT1636-Y, and D-H08, shown in Table 1, are GFTFSYAW, IKSKIDGGTT, and TPGVGANDPYYFDR. The light chain CDR1-3 sequences of these antibodies C-E08, D-B06, D-G05, and D-H08 are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide antibodies or antigen-binding fragments capable of conjugating O-mannosylated E-cadherins, comprising a heavy chain CDR1 containing the sequence GFTFSYAW, a heavy chain CDR2 containing the sequence IKSKIDGGTT, a heavy chain CDR3 containing the sequence TPGVGANDPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0183] The VH sequences of antibodies C-E08, D-B06, and AT1636-Y are shown in Table 1 as SEQ ID NO: 10. The VL sequences of antibodies C-E08, D-B06, and AT1636-Y are shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide antibodies or antigen-binding fragments that can be conjugated to O-mannosylated E-cadherins, comprising a VH sequence such as that shown in SEQ ID NO: 10 and a VL sequence such as that shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating an O-mannosylated E-cadherin containing a VH sequence, such as that shown in SEQ ID NO: 10, and a VL sequence, such as that shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment contains heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of an antibody C-E08, D-B06, or AT1636-Y, as shown in Table 1.

[0184] The VH sequence of antibody D-G05 is shown in Table 1 as SEQ ID NO: 10. The VL sequence of antibody D-G05 is shown in Table 1 as SEQ ID NO: 21. Therefore, some embodiments provide an antibody or antigen-binding fragment that can be conjugated to an O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 10 and a VL sequence as shown in SEQ ID NO: 21, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 10 and a VL sequence as shown in SEQ ID NO: 21, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody D-G05 as shown in Table 1.

[0185] The VH sequence of antibody D-H08 is shown in Table 1 as SEQ ID NO: 11. The VL sequence of antibody D-H08 is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 11 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 11 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody D-H08 as shown in Table 1.

[0186] Further provided are antibodies or antigen-binding fragments that compete with antibodies C-E08, D-B06, D-G05, D-H08, or AT1636-Y for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0187] Further provided are antibodies or antigen-binding fragments that compete with antibodies C-E08, D-B06, D-G05, D-H08, or AT1636-Y for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0188] Further provided are antibodies or antigen-binding fragments that compete with antibodies C-E08, D-B06, D-G05, D-H08, or AT1636-Y for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0189] The heavy chain CDR1-3 sequences of antibody C-H01, as shown in Table 1, are GFTFSNAW, IKSKIDGGTI, and TPGVGANDPYYFDR. The light chain CDR1-3 sequences of antibody C-H01 are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFTFSNAW, a heavy chain CDR2 containing the sequence IKSKIDGGTI, a heavy chain CDR3 containing the sequence TPGVGANDPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0190] The VH sequence of antibody C-H01 is shown in Table 1 as SEQ ID NO: 12. The VL sequence of antibody C-H01 is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 12 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 12 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of antibody C-H01 as shown in Table 1.

[0191] Further provided are antibodies or antigen-binding fragments that compete with antibody C-H01 for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0192] Further provided are antibodies or antigen-binding fragments that compete with antibody C-H01 for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0193] Further provided are antibodies or antigen-binding fragments that compete with antibody C-H01 for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0194] Another preferred antibody according to the present invention is the antibody AT1636-YN. This antibody is preferred because it can bind O-mannosylated E-cadherins expressed on tumor cells, particularly the newly discovered shortened E-cadherin form of approximately 70 kDa. A particular advantage of AT1636-YN is the fact that it binds this shortened 70 kDa E-cadherin form better than the full-length E-cadherin of approximately 120 kDa. As described above herein, this feature typically enables improved tumor specificity when O-mannosylated shortened 70 kDa E-cadherins are upregulated on tumor cells. A further advantage of AT1636-YN's preference for the shortened 70 kDa E-cadherin form is that full-length E-cadherins are widely expressed. Therefore, without the preference for the shortened 70kDa E-cadherin type, widely expressed full-length E-cadherin may function as a sink and / or may lead to undesirable effects. Furthermore, the expression level of full-length E-cadherin is very high, and therefore often cannot distinguish between healthy epithelial cells and tumor epithelial cells, while the preference for the shortened 70kDa E-cadherin type allows for higher tumor specificity. In addition, E-cadherin has an important barrier function, and therefore it is preferable to avoid significant interference by the healthy function of E-cadherin.

[0195] In addition, AT1636-YN can bind to O-mannosylated E-cadherin expressing colon cancer subtypes CMS1, CMS2, CMS3, and CMS4. AT1636-YN binds to tumor cells, particularly epithelial tumor cells, more specifically, O-mannosylated E-cadherin expressing cancer cells such as colon cancer cells, breast cancer cells, pancreatic cancer cells, bladder cancer cells, endometrial cancer cells, lung cancer cells, and esophageal cancer cells. Therefore, the antibody AT1636-YN is particularly suitable for the treatment and / or diagnosis of disorders associated with the presence of O-mannosylated E-cadherin expressing cells, especially cancer cells expressing the newly discovered shortened E-cadherin type of approximately 70 kDa. The presence of a human amino acid sequence reduces the chance of adverse side effects during therapeutic use in human patients.

[0196] Furthermore, antibody AT1636-YN binds to epidermoid carcinoma cell line A431, lung cancer cell line A549, and mouse tumor cell line CMT93 better than antibody AT1636 (see Figure 6B).

[0197] The heavy chain CDR1-3 sequences of the antibody AT1636-YN, as shown in Table 1, are GFTFSYAW, IKSKIDGGTT, and TPGVGANNPYYFDR. The light chain CDR1-3 sequences of this antibody AT1636-YN are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFTFSYAW, a heavy chain CDR2 containing the sequence IKSKIDGGTT, a heavy chain CDR3 containing the sequence TPGVGANNPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0198] The VH sequence of antibody AT1636-YN is shown in Table 1 as SEQ ID NO: 15. The VL sequence of antibody AT1636-YN is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 15 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 15 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of the antibody AT1636-YN as shown in Table 1.

[0199] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-YN for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0200] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-YN for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0201] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-YN for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0202] Another preferred antibody according to the present invention is the antibody AT1636-IYN. This antibody is preferred because it can bind O-mannosylated E-cadherins expressed on tumor cells, particularly the newly discovered approximately 70 kDa shortened E-cadherin form. A particular advantage of AT1636-IYN is the fact that it binds this shortened 70 kDa E-cadherin form better than the full-length approximately 120 kDa E-cadherin. As described above in this specification, this feature typically enables improved tumor specificity when O-mannosylated shortened 70 kDa E-cadherins are upregulated on tumor cells. A further advantage of AT1636-IYN's preference for the shortened 70 kDa E-cadherin form is that full-length E-cadherins are widely expressed. Therefore, without the preference for the shortened 70kDa E-cadherin type, widely expressed full-length E-cadherin may function as a sink and / or may lead to undesirable effects. Furthermore, the expression level of full-length E-cadherin is very high, and therefore often cannot distinguish between healthy epithelial cells and tumor epithelial cells, while the preference for the shortened 70kDa E-cadherin type allows for higher tumor specificity. In addition, E-cadherin has an important barrier function, and therefore it is preferable to avoid significant interference by the healthy function of E-cadherin.

[0203] In addition, AT1636-IYN can bind to O-mannosylated E-cadherin expressing colon cancer subtypes CMS1, CMS2, CMS3, and CMS4. AT1636-IYN binds to tumor cells, particularly epithelial tumor cells, more specifically, O-mannosylated E-cadherin expressing cancer cells such as colon cancer cells, breast cancer cells, pancreatic cancer cells, bladder cancer cells, endometrial cancer cells, lung cancer cells, and esophageal cancer cells. Therefore, the antibody AT1636-IYN is particularly suitable for the treatment and / or diagnosis of disorders associated with the presence of O-mannosylated E-cadherin expressing cells, especially cancer cells expressing the newly discovered shortened E-cadherin type of approximately 70 kDa. The presence of human amino acid sequences reduces the likelihood of adverse side effects during therapeutic use in human patients.

[0204] Furthermore, antibody AT1636-IYN binds better to colon cell line DLD1, mammary epithelial cell line MCF10a, epidermal carcinoid cell line A431, lung cancer cell line A549, and mouse tumor cell line CMT93 than antibody AT1636 (see Figures 6A and 6B).

[0205] The heavy chain CDR1-3 sequences of the antibody AT1636-IYN, as shown in Table 1, are GFIFSYAW, IKSKIDGGTT, and TPGVGANNPYYFDR. The light chain CDR1-3 sequences of this antibody AT1636-IYN are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFIFSYAW, a heavy chain CDR2 containing the sequence IKSKIDGGTT, a heavy chain CDR3 containing the sequence TPGVGANNPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0206] The VH sequence of antibody AT1636-IYN is shown in Table 1 as SEQ ID NO: 16. The VL sequence of antibody AT1636-IYN is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 16 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 16 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of the antibody AT1636-IYN as shown in Table 1.

[0207] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-IYN for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0208] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-IYN for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0209] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-IYN for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0210] Another preferred antibody according to the present invention is the antibody AT1636-IYEN. This antibody is preferred because it can bind O-mannosylated E-cadherins expressed on tumor cells, particularly the newly discovered approximately 70 kDa shortened E-cadherin form. A particular advantage of AT1636-IYEN is the fact that it binds this shortened 70 kDa E-cadherin form better than the full-length approximately 120 kDa E-cadherin. As described above in this specification, this feature typically enables improved tumor specificity when O-mannosylated shortened 70 kDa E-cadherins are upregulated on tumor cells. A further advantage of AT1636-IYEN's preference for the shortened 70 kDa E-cadherin form is that full-length E-cadherins are widely expressed. Therefore, without the preference for the shortened 70kDa E-cadherin type, widely expressed full-length E-cadherin may function as a sink and / or may lead to undesirable effects. Furthermore, the expression level of full-length E-cadherin is very high, and therefore often cannot distinguish between healthy epithelial cells and tumor epithelial cells, while the preference for the shortened 70kDa E-cadherin type allows for higher tumor specificity. In addition, E-cadherin has an important barrier function, and therefore it is preferable to avoid significant interference by the healthy function of E-cadherin.

[0211] In addition, AT1636-IYEN can bind to O-mannosylated E-cadherin expressing colon cancer subtypes CMS1, CMS2, CMS3, and CMS4. AT1636-IYEN binds to tumor cells, particularly epithelial tumor cells, more specifically, O-mannosylated E-cadherin expressing cancer cells such as colon cancer cells, breast cancer cells, pancreatic cancer cells, bladder cancer cells, endometrial cancer cells, lung cancer cells, and esophageal cancer cells. Therefore, the antibody AT1636-IYEN is particularly suitable for the treatment and / or diagnosis of disorders associated with the presence of O-mannosylated E-cadherin expressing cells, especially cancer cells expressing the newly discovered shortened E-cadherin type of approximately 70 kDa. The presence of human amino acid sequences reduces the likelihood of adverse side effects during therapeutic use in human patients.

[0212] Furthermore, the antibody AT1636-IYEN binds to the colon cell line DLD1, the mammary epithelial cell line MCF10a, and the mouse tumor cell line CMT93 more effectively than the antibody AT1636 (see Figure 6A).

[0213] The heavy chain CDR1-3 sequences of the antibody AT1636-IYEN, as shown in Table 1, are GFIFSYAW, IKSKIDGETT, and TPGVGANNPYYFDR. The light chain CDR1-3 sequences of this antibody AT1636-IYEN are QSVLCRSNNKNC, WAS, and QQYSNTPQT. Therefore, some embodiments provide an antibody or antigen-binding fragment capable of conjugating O-mannosylated E-cadherin, comprising a heavy chain CDR1 containing the sequence GFIFSYAW, a heavy chain CDR2 containing the sequence IKSKIDGETT, a heavy chain CDR3 containing the sequence TPGVGANNPYYFDR, a light chain CDR1 containing the sequence QSVLCRSNNKNC, a light chain CDR2 containing the sequence WAS, and a light chain CDR3 containing the sequence QQYSNTPQT.

[0214] The VH sequence of antibody AT1636-IYEN is shown in Table 1 as SEQ ID NO: 17. The VL sequence of antibody AT1636-IYEN is shown in Table 1 as SEQ ID NO: 18. Therefore, some embodiments provide an antibody or antigen-binding fragment that can be conjugated to an O-mannosylated E-cadherin, comprising a VH sequence as shown in SEQ ID NO: 17 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region. Therefore, some embodiments provide antibodies or antigen-binding fragments that can conjugate O-mannosylated E-cadherins, comprising a VH sequence as shown in SEQ ID NO: 17 and a VL sequence as shown in SEQ ID NO: 18, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them, wherein the antibody or antigen-binding fragment comprises the heavy chain CDR1-3 sequences and light chain CDR1-3 sequences of the antibody AT1636-IYEN as shown in Table 1.

[0215] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-IYEN for binding to O-mannosylated E-cadherins, preferably to one or more O-mannosylated threonine residues located within amino acid positions 467-472 of an E-cadherin sequence as shown in Figure 1A.

[0216] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-IYEN for binding to O-mannosylated E-cadherin-containing cells, preferably O-mannosylated E-cadherin-positive tumor cells. The cells preferably express O-mannosylated E-cadherin on their surface.

[0217] Further provided are antibodies or antigen-binding fragments that compete with the antibody AT1636-IYEN for binding to cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0218] In some embodiments, the heavy and light chain CDR1-3 sequences of the antibody described above consist of the listed heavy and light chain CDR1-3 sequences.

[0219] In some embodiments, the heavy and light chain CDR1-3 sequences of antibodies AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, or AT1636-IYEN are combined with framework sequences of different antibodies. Human framework sequences are preferred. Sequences of human framework regions are available from publicly available DNA databases. In some preferred embodiments, human germline sequences are used for framework regions in antibody and antigen-binding fragments according to the present invention. Since these germline sequences typically do not contain somatic high-frequency mutations that could trigger an immunogenic response, the use of human germline sequences minimizes the risk of immunogenicity of the antibody.

[0220] In some embodiments, the antibody or antigen-binding fragment according to the present invention is a human antibody or its antigen-binding fragment. The presence of a human amino acid sequence reduces the chance of adverse side effects during therapeutic use in human patients compared to non-human antibodies.

[0221] Some embodiments provide antibodies according to the present invention that are full-length antibodies. Full-length antibodies are advantageous due to their preferred half-lives. The antibodies of the present invention are preferably IgG isotypes. In particular, IgG1 is preferred based on its long circulating half-life in humans. Furthermore, IgG1 antibodies are readily produced commercially, and their Fc tails enable effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). To prevent immunogenicity in humans, it is preferable that the antibodies according to the present invention are human antibodies or their antigen-binding fragments.

[0222] As described above in this specification, antibody AT1636 is an IgG3 isotype. Because IgG3 isotype antibodies are difficult to develop commercially due to their tendency to aggregate, some embodiments provide IgG1 isotype antibodies containing the heavy chain CDR1-3 and light chain CDR1-3 sequences of antibody AT1636. Some embodiments provide IgG1 antibodies containing the heavy chain CDR1-3 and light chain CDR1-3 sequences of antibodies selected from the group consisting of antibodies AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN.

[0223] Some embodiments provide an IgG1 antibody comprising the VH and VL sequences of an antibody selected from the group consisting of antibody AT1636, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or sequences having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with them. Preferably, the sequence changes in the VH and / or VL regions are located outside the CDR region.

[0224] The full-length IgG antibodies according to the present invention encompass antibodies in which mutations exist that provide desired characteristics. Such mutations must not be deletions of substantial portions of any antibody region. However, as described above in this specification, antibodies in which one or more amino acid residues are deleted without essentially altering the binding characteristics of the resulting antibody are included in the term “full-length antibody.” For example, an IgG antibody may have 1 to 20 amino acid residues in its constant region, insertions, deletions, or combinations thereof. For example, glycosylation can be reduced, as described below in this specification, and ADCC or CDC activity can be altered.

[0225] In some embodiments, the antibody or antigen-binding fragment according to the present invention has the following characteristics: - Binds to the extracellular (EC)3 domain of O-mannosylated E-cadherin; - The O-mannosylated shortened 70kDa E-cadherin is attached more effectively than the O-mannosylated full-length E-cadherin, preferably at least twice as effectively, more preferably at least three times as effectively, more preferably at least four times as effectively, and more preferably at least five times as effectively; - Binds tumor cells that co-express E-cadherin and O-mannosyltransferase, preferably TMTC3. It has one or more of these, preferably each of them.

[0226] In some embodiments, the antibody or antigen-binding fragment has the following characteristics: - Combines colon cancer subtypes CMS1, CMS2, CMS3, and CMS4; - It binds the colon cancer cell line SW948 better than healthy thymic medullary epithelial cells, dendritic cells, or Langerhans cells. It further includes at least one of the following.

[0227] Some preferred embodiments provide antibody and antigen-binding fragments according to the present invention having each of the features listed above. Such antibody and antigen-binding fragments have broad antitumor applicability in light of their ability to bind to different cancer types and different subtypes of colon cancer. Furthermore, in light of the preference for shortened 70kDa E-cadherin over full-length E-cadherin as detailed above herein, such antibody and antigen-binding fragments are suitable for improving tumor specificity when the shortened 70kDa E-cadherin type is significantly upregulated on tumor cells.

[0228] As shown in the examples, antibodies are provided that specifically bind one or more O-mannosylated threonine and / or serine residues of E-cadherin, the one or more O-mannosylated threonine and / or serine residues located within amino acid positions 467-472 of the E-cadherin sequence, as shown in Figure 1A. It is now known that it is possible to obtain or generate further antibodies competing for the same epitope of O-mannosylated E-cadherin. This can be done, for example, by immunizing non-human animals with an O-mannosylated E-cadherin peptide containing the aforementioned amino acid residues 467-472 of the E-cadherin sequence as shown in Figure 1A, or an immunogenic compound containing such a peptide, or a nucleic acid molecule encoding such a peptide, preferably followed by one or more booster doses. Alternatively, non-human animals can be immunized with TMTC3 and E-cadherin-expressing cells to express O-mannosylated E-cadherin on the cell surface. Furthermore, non-human animals can also be immunized using so-called DNA immunization techniques with nucleic acids such as cDNA that express both TMTC3 and E-cadherin.

[0229] Subsequently, antibodies and / or B cells specific to the epitope or peptide can be recovered from the non-human animal. In some embodiments, the obtained antibodies are humanized to optimize them for human treatment. In some embodiments, for binding to the peptide or O-mannosylated E-cadherin or its 70 kDa truncated form, AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B The obtained antibodies or B cells are tested for competition with antibodies selected from the group consisting of 06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or their antigen-binding fragments.

[0230] Animal immunization protocols, including appropriate administration procedures and adjuvants, procedures for obtaining and purifying antibodies and / or immune cells from such immunized animals, competitive experiments, and procedures for humanizing non-human antibodies, are well known in the art. See, for example, Hanly et al., 1995.

[0231] Alternatively, or further, cells co-expressing the peptide or TMCT3-E-cadherin are used to screen the phage display library to identify and / or isolate O-mannosylated E-cadherin-specific immunoglobulins, typically Fab fragments. The resulting antibodies, B cells, or Fab fragments are typically identified for binding to the peptide or O-mannosylated E-cadherin or its 70kDa truncated form, such as AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04A, C-D04-B, F-C08, D-G03, D The antibody or its antigen-binding fragment is selected from the group consisting of antibodies comprising -F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, and competes with the antigen-binding fragment. In some embodiments, a competitive assay is performed.

[0232] Also provided herein are nucleic acid molecules and vectors encoding at least one CDR sequence of an antibody or antigen-binding fragment according to the present invention. Thus, some embodiments provide an isolated, synthetic or recombinant nucleic acid, or vector, encoding at least one CDR sequence of an antibody or antigen-binding fragment according to the present invention. In some embodiments, at least the heavy chain CDR3 sequence and the light chain CDR3 sequence of an antibody or antigen-binding fragment according to the present invention are encoded. Thus, there is further provided an isolated, synthetic or recombinant nucleic acid, or vector, encoding at least the heavy chain CDR3 sequence and the light chain CDR3 sequence of an antibody or antigen-binding fragment according to the present invention. Preferably, at least the heavy chain CDR1-3 sequences and the light chain CDR1-3 sequences of an antibody or antigen-binding fragment according to the present invention are encoded. Thus, there is further provided an isolated, synthetic or recombinant nucleic acid, or vector, encoding at least the heavy chain CDR1-3 sequences and the light chain CDR1-3 sequences of an antibody or antigen-binding fragment according to the present invention. Preferably, the CDR sequence is the CDR sequence of an antibody as shown in Table 1.

[0233] Certain embodiments provide isolated, synthesized, or recombinant nucleic acids encoding at least the heavy chain variable region and / or light chain variable region of an antibody or antigen-binding fragment according to the present invention. In some embodiments, the nucleic acid encodes both the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment according to the present invention. Such nucleic acids are particularly suitable for the production of the antibody or antigen-binding fragment of the present invention in producing cells. In some embodiments, the nucleic acid comprises a nucleic acid sequence that is a codon optimized for certain producing cells, such as Escherichia coli, Chinese hamster ovary (CHO), NSO (mouse myeloma), or T293 cells, enabling the efficient production of the antibody or antigen-binding fragment of the present invention in these producing cells. Antibody production can be carried out by any recombinant antibody production system; it should be noted that the four producing cell systems described above are just a few examples of the many systems available to date. As used herein, the term “codon” means a set of three nucleotides that encode a particular amino acid residue. The term “optimized codon” means that one or more codons from the original, preferably human, nucleic acid sequence have been replaced with one or more codons preferred by certain producing cells. These substitution codons preferably encode the same amino acid residue as the original codon being substituted. Alternatively, one or more substitution codons encode different amino acid residues. This is not required, but preferably results in a conservative amino acid substitution. In the constant region and framework region, one or more amino acid substitutions are generally acceptable. In the CDR region, it is preferable to use codons that encode the same amino acid residue as the original codon being substituted, so that the resulting product has the same CDR amino acid sequence as the original antibody.

[0234] Preferred VH and VL amino acid and nucleotide sequences of antibodies according to the present invention are listed in Table 1. Since many amino acid residues are encoded by more than one different nucleic acid codon, different codons can be used for certain amino acid residues to optimize the use of codons for certain producing cells, for example, as described above. Furthermore, some nucleic acid sequence modifications resulting in different amino acid residues are also typically acceptable, particularly outside the sequence encoding the CDR. Thus, certain embodiments provide isolated, synthesized, or recombinant nucleic acids encoding at least the heavy chain variable region and / or light chain variable region of antibodies shown in Table 1. Some embodiments provide isolated, synthesized, or recombinant nucleic acids encoding heavy chain variable region amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 17, or amino acid sequences having at least 80% sequence identity with them. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably 100%. Preferably, the variation of the sequence in the VH region is located outside the CDR region. Some embodiments provide isolated, synthesized, or recombinant nucleic acids that encode light chain variable region amino acid sequences selected from the group consisting of SEQ ID NOs. 18-22, or amino acid sequences having at least 80% sequence identity with them.Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100%. Preferably, the modification of the sequence of the VL region is located outside the CDR region. Some embodiments provide an isolated, synthetic or recombinant nucleic acid encoding a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17 and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, or an amino acid sequence having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100%. Preferably, the modification of the sequence of the VH and / or VL region is located outside the CDR region.

[0235] Some embodiments provide isolated, synthesized, or recombinant nucleic acids having at least 80% sequence identity with VH or VL sequences as shown in Table 1. Preferred VH nucleic acid sequences of antibodies according to the present invention are listed in Table 1 as SEQ ID NOs: 23-39. Preferred VL nucleic acid sequences of antibodies according to the present invention are listed in Table 1 as SEQ ID NOs: 40-44. Therefore, nucleic acids are further provided that include a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 23-39, and / or a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 40-44. Preferably, the nucleic acid molecule according to the present invention includes a variable heavy chain coding sequence and a variable light chain coding sequence of the same antibody as shown in Table 1. Therefore, nucleic acids are also provided that include a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 23-39, and a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 40-44. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region.

[0236] In some embodiments, nucleic acid molecules encoding antibodies or antigen-binding fragments according to the present invention are provided. Further, nucleic acid molecules encoding antibodies selected from the group consisting of antibodies AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN are provided. In some embodiments, the nucleic acid is a codon optimized for expression in non-human host cells.

[0237] Vectors containing nucleic acid molecules according to the present invention are further provided. As used herein, “vectors containing nucleic acid molecules according to the present invention” are also referred to as “vectors according to the present invention.”

[0238] Methods for constructing vectors comprising one or more nucleic acid molecules according to the present invention are well known in the art. Not limited examples of suitable vectors and production platforms include retroviral and lentiviral vectors, bacterial or yeast plasmids, SV40 vectors, baculovirus vectors, phage DNA vectors, pUC vectors, plasmid vectors such as pBR322, vectors manufactured by Lonza such as the pCon Plus vector, production systems manufactured by Lentuler BioPharma such as the TurboCell® expression platform, and Fujifilm Diosynth expression platforms such as the Apollo® mammalian expression platform.

[0239] In some embodiments, the vector according to the present invention includes nucleic acid sequences encoding the VH and VL sequences of an antibody as shown in Table 1. The VH nucleic acid sequences of these antibodies are listed in Table 1 as SEQ ID NOs. 23-39, and the VL nucleic acid sequences of these antibodies are listed in Table 1 as SEQ ID NOs. 40-44. Therefore, vectors are further provided that include a nucleic acid sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 23-39, and / or a nucleic acid sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 40-44. Preferably, the vector according to the present invention includes a variable heavy chain coding sequence and a variable light chain coding sequence of an antibody as shown in Table 1. Therefore, vectors are also provided that include a nucleic acid sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 23-39, and a nucleic acid sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 40-44. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region of the antibody.

[0240] Some embodiments include: - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 23 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 23 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 44, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 24 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them. - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 25 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 26 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 41, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 27 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 28 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 28 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 42, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 29 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in SEQ ID NO: 30 and VL-coded nucleic acid sequences such as those shown in SEQ ID NO: 40, or sequences having at least 80% sequence identity with them; or - VH-coded nucleic acid sequences such as those shown in Sequence ID No. 31 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coding nucleic acid sequences such as those shown in Sequence ID No. 32 and VL-coding nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coding nucleic acid sequences such as those shown in SEQ ID NO: 32 and VL-coding nucleic acid sequences such as those shown in SEQ ID NO: 43, or sequences having at least 80% sequence identity with them; or VH-coded nucleic acid sequences such as those shown in Sequence ID No. 33 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coded nucleic acid sequences such as those shown in Sequence ID No. 34 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coding nucleic acid sequences such as those shown in Sequence ID No. 35 and VL-coding nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coded nucleic acid sequences such as those shown in Sequence ID No. 36 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coded nucleic acid sequences such as those shown in Sequence ID No. 37 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coded nucleic acid sequences such as those shown in Sequence ID No. 38 and VL-coded nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them; or VH-coding nucleic acid sequences such as those shown in Sequence ID No. 39 and VL-coding nucleic acid sequences such as those shown in Sequence ID No. 40, or sequences having at least 80% sequence identity with them. Provides a vector containing the following:

[0241] Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region.

[0242] In some embodiments, the vector according to the present invention is a CAR T cell vector comprising nucleic acid sequences encoding an antigen recognition domain and a T cell activation domain. In some embodiments, the antigen recognition domain comprises at least the heavy chain CDR1-3 sequences of the antibody according to the present invention. In some embodiments, the antigen recognition domain comprises at least the light chain CDR1-3 sequences of the antibody according to the present invention. In some embodiments, the antigen recognition domain comprises the heavy chain CDR1-3 sequences and the light chain CDR1-3 sequences of the antibody according to the present invention. In some embodiments, the antigen recognition domain comprises the VH sequence of the antibody according to the present invention, or a sequence having at least 80% sequence identity thereto. In some embodiments, the antigen recognition domain comprises the VL sequence of the antibody according to the present invention, or a sequence having at least 80% sequence identity thereto. In some embodiments, the antigen recognition domain comprises the VH and VL sequences of the antibody according to the present invention, or sequences having at least 80% sequence identity thereto. Preferably, the sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 100%. Preferably, the sequence variation in the VH and / or VL regions is located outside the CDR region.

[0243] In some embodiments, the antigen recognition domain is located in a single-chain format. In some embodiments, the CAR T cell vector further comprises a nucleic acid sequence encoding a transmembrane domain.

[0244] The vectors according to the present invention are useful, for example, for the in vitro production of the antibodies or antigen-binding fragments or CAR T cells of the present invention. This is done, for example, by introducing such nucleic acid molecules or vectors into cells, so that the nucleic acid translation mechanism of the cells produces the encoded antibodies or antigen-binding fragments or CAR T cells. In some embodiments, at least one nucleic acid molecule or vector according to the present invention is expressed in so-called producing cells, such as Escherichia coli, CHO, NSO, or T293 cells, some of which are suitable for commercial antibody production. In such cases, as described above herein, it is preferable to use nucleic acid molecules in which the original human sequence is a codon optimized for the producing cell, as provided herein. The proliferation of the producing cell results in a producing cell line that can consequently produce the antibodies or antigen-binding fragments according to the present invention. Preferably, the producing cell line is suitable for producing antibodies for use in humans. Therefore, the producing cell line preferably does not contain pathogens such as pathogenic microorganisms. In some embodiments, antibodies consisting of human sequences are produced by such a producing cell line.

[0245] In some embodiments, the CAR T cell vector according to the present invention is introduced into T cells to produce CAR T cells.

[0246] Therefore, isolated or recombinant host cells comprising at least one antibody, or antigen-binding fragment, or nucleic acid molecule, or vector according to the present invention are further provided. Such cells are preferably antibody-producing cells capable of large-scale antibody production. In some embodiments, the cells are mammalian cells, T cells, bacterial cells, plant cells, HEK293 T cells, CHO cells, production systems manufactured by Lonza such as the pCon Plus vector production system, production systems manufactured by Lentuler BioPharma such as the TurboCell® expression platform, or Fujifilm Diosynth expression platforms such as the Apollo® mammalian expression platform.

[0247] A method for producing an antibody or antigen-binding fragment according to the present invention is further provided, the method comprising culturing a host cell comprising a nucleic acid or vector according to the present invention, enabling the host cell to translate the nucleic acid or vector, thereby producing the antibody or antigen-binding fragment according to the present invention. The method according to the present invention preferably further comprises the step of recovering the antibody or antigen-binding fragment from the host cell and / or from the culture medium. In some embodiments, the antibody or antigen-binding fragment is an antibody as shown in Table 1, preferably an antibody selected from the group consisting of AT1636, AT1636I, AT1636Y, AT1636E, AT1636N, AT1636-YN, AT1636-IYN and AT1636-IYEN, and antigen-binding fragments thereof. In some preferred embodiments, the antibody or antigen-binding fragment is an antibody selected from the group consisting of AT1636-YN, AT1636-IYN and AT1636-IYEN, and antigen-binding fragments thereof.

[0248] Antibodies or antigen-binding fragments when obtained by the method according to the present invention are also provided herein. The binding compounds obtained according to the present invention are suitable for use in human therapy or diagnosis, for example, optionally after additional purification, isolation or processing steps.

[0249] In some embodiments, at least one nucleic acid molecule or vector according to the present invention is introduced into a non-human animal, for example for in vivo antibody production. Therefore, an isolated or recombinant non-human animal comprising an antibody, antigen-binding fragment, nucleic acid molecule or vector according to the present invention is further provided. Methods for producing transgenic non-human animals are known in the art.

[0250] Additional antibody modifications Further provided are antibodies according to the present invention in which one or more amino acid residues in the constant region are modified. In some embodiments, one or more amino acids in the Fc region are modified to reduce glycosylation. N-glycosylation is a commonly recognized post-translational modification of antibodies and is known to occur in glycosylation motifs containing the consensus sequence NXS or NXT (where N represents asparagine, X represents an amino acid residue, S represents serine, and T represents threonine). Fc glycosylation affects the structural characteristics of the Fc portion of the antibody, thereby affecting effector function and pharmacokinetics. Since Fc glycosylation may result in a shortened half-life and / or improved immunogenicity, glycosylation may be undesirable for therapeutic antibodies. In some embodiments, one or more amino acids in the Fc glycosylation region are modified compared to the original parent antibody to reduce or avoid glycosylation. For example, at least one of the N, S, and T residues in the glycosylation motif described above is modified. In some embodiments, the asparagine residue (N47) at position 47 of the CH2 domain is modified. In some embodiments, the threonine residue (T95) at position 95 of the CH2 domain is modified.

[0251] Alternatively, or furthermore, one or more glycosylation sites in the variable framework region of the antibody according to the present invention are modified to reduce or avoid glycosylation.

[0252] The constant domain of an antibody is responsible for various antibody characteristics such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cell-mediated cytotoxicity (CDC). The Fc region mediates antibody function by binding to different receptors on immune effector cells such as macrophages, natural killer cells, B cells, and neutrophils. Some of these receptors, such as CD16A (FcγRIIIA) and CD32A (FcγRIIA), activate immune effector cells to construct a response to antigens. Other receptors, such as CD32B, inhibit the activation of immune cells. In some embodiments, antibodies according to the present invention are designed to enhance ADCC activity. One technique for enhancing the ADCC activity of an antibody is defucosylation. Therefore, defucosylated antibodies or antigen-binding fragments according to the present invention are further provided.

[0253] Any known means in the art for obtaining defucosylated antibodies can be applied. Defucosylated antibodies can be obtained by using, for example, a cell line producing cells with reduced fucosylation ability, such as the Lec13 CHO mutant (Patnaik & Stanley, 2006). It is also possible to knock out the FUT8 gene encoding alpha-1,6-fucosyltransferase in cell lines such as CHO (Potelligent® technology) (Yamane-Ohnuki et al, 2004).

[0254] Alternatively, an antibody-producing cell line can be used that overexpresses N-acetylglucosaminyltransferase III (GnT III), resulting in a non-fucosylated antibody (GlycoMAb™ technology).

[0255] Alternatively, or even further, ADCC enhancement can be achieved using several other strategies, including glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche), and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), all of which aim to improve Fc binding to low-affinity activated FcγRIIIa and / or reduce binding to low-affinity inhibitory FcγRIIb. Chemoenzymatic modification has also been used for modifying Fc-binding N-glycans.

[0256] Apart from fucose, other sugar moieties are known to play a role in ADCC activity. In some embodiments, the antibody or antigen-binding fragment according to the present invention is supergalactosylated to enhance ADCC activity.

[0257] In some embodiments, at least one amino acid in the FcγR binding site within the Fc domain of the antibody of the present invention is modified to manipulate the Fc / FcR interaction. In some embodiments, amino acid mutations S298A, E333A, and K334A are introduced into the Fc domain of the antibody of the present invention. These mutations have been reported to enhance ADCC activity (Shields et al, 2001). In some embodiments, the ADCC activity of the antibody of the present invention is enhanced by introducing amino acid mutations S239D and I332E, sometimes in combination with amino acid mutation A330L (Lazar et al, 2006). In some embodiments, the ADCC activity of the antibody of the present invention is enhanced by introducing amino acid mutations L235V, F243L, R292P, Y300L, and P396L (Stavenhagen et al, 2007). Therefore, group: - S298A, E333A, K334A; - S239D, I332E; - S239D, I332E, A330L; and - L235V, F243L, R292P, Y300L, P396L Further provided are antibodies or antigen-binding fragments according to the present invention, comprising amino acid mutations selected from the above.

[0258] Several in vitro methods exist to determine the efficacy of antibodies in inducing ADCC. These include chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Typically, labeled target cell lines expressing a specific surface exposure antigen are incubated with antibodies specific to that antigen. After washing, effector cells expressing the Fc receptor CD16 are typically co-incubated with antibody-labeled target cells. Subsequently, target cell lysates are typically measured by the release of intracellular labels, for example, by a scintillation counter or spectrophotometric method. Alternatively, luciferase-based cytotoxicity assays can be used, incubating target cells expressing firefly luciferase with antibodies, such as bispecific or multispecific antibodies. After washing, effector cells are added and co-incubated. Subsequently, target cell death is typically measured by lysing the remaining target cells and measuring luciferin emission by spectrophotometric method.

[0259] In some embodiments, antibodies according to the present invention are designed to enhance CDC activity. One method for enhancing CDC is the introduction of amino acid mutations K326W and / or E333S into the Fc domain (Idusogie et al, 2001). In some embodiments, amino acid mutations S267E, H268F, and S324T are introduced into the Fc domain of antibodies according to the present invention to enhance CDC activity. Since these mutations have been reported to reduce ADCC activity, amino acid substitutions G236A and I332E are also preferably introduced to repair ADCC activity (Moore et al, 2010).

[0260] In some embodiments, the amino acid mutation E345R is introduced into the Fc domain of the antibody of the present invention to enhance CDC activity. In some embodiments, the amino acid mutations E345K and / or E430G are introduced into the Fc domain of the antibody of the present invention to enhance CDC and ADCC activity (De Jong et al, 2016).

[0261] Therefore, group: - K326W; - E333S; - K326W, E333S; - E345R; - E345K; - E430G; - E345K, E430G; - S267E, H268F, S324T; and - S267E, H268F, S324T, G236A, I332E Further provided are antibodies or antigen-binding fragments according to the present invention, comprising one or more amino acid mutations selected from the above.

[0262] While immune effector functions such as ADCC and CDC are beneficial in many therapeutic applications, reducing them is beneficial in other applications. Such applications include, for example, therapeutic approaches where the mechanism of action lies particularly in the Fab arm or other parts fused to the Fc region. In such cases, reduced Fc / FcR and / or Fc / C1q interactions may be beneficial in reducing tissue damage caused by immune effector functions. Therefore, reduced immune effector functions may be preferable when the use of antibodies according to the present invention does not require ADCC or CDC. The effector functions of antibodies according to the present invention are reduced, for example, by using IgG2 or IgG4 formats with reduced effector functions compared to IgG1. In some embodiments, the effector functions of antibodies according to the present invention are reduced by introducing an L235E mutation into the Fc region or by introducing one or more other mutations within amino acid positions 234-237. In some embodiments, the IgG1 antibody of the present invention is provided with amino acid substitutions L234A and L235A (LALA mutations) to reduce effector function (Lund et al, 1992). In some embodiments, the IgG1 antibody of the present invention is provided with amino acid substitutions L234A, L235A and P329G (LALA-PG mutation) to reduce effector function. In some embodiments, the IgG4 antibody of the present invention is provided with amino acid substitutions S228P and L235E (SPLE mutation). The introduction of amino acid substitution P329G is also beneficial in reducing effector function.

[0263] Therefore, group: - L235E; - L234A, L235A; - L234A, L235A, P329G; - S228P, L235E; and - S228P, L235E, P329G Further provided are antibodies or antigen-binding fragments according to the present invention, comprising one or more amino acid mutations selected from the above.

[0264] Bispecific or multispecific binding compounds Another aspect of the present invention provides an antibody or antigen-binding fragment according to the present invention that is bound to another compound. In some embodiments, the antibody or antigen-binding fragment according to the present invention is bound to another therapeutic moiety, such as a drug, chemotherapeutic agent, toxic moiety, cytotoxic agent, or radioactive compound, in order to form a so-called “antibody-drug conjugate” (ADC).

[0265] Some embodiments provide an ADC, which comprises an antibody or antigen-binding fragment and a unit of a cell growth inhibitor or cytotoxic agent according to the present invention. The drug unit may disrupt, for example, DNA strands (e.g., duocalmycin, calicheamicin, pyrrolobenzodiazepine [PBD], and SN-38 [active metabolite of irinotecan]) or microtubules (e.g., mytansin and auristatin), or induce cell death using topoisomerase or RNA polymerase inhibition (Chau et al, 2019). In some embodiments, the ADC includes a chemical linker unit between the unit of the cell growth inhibitor or cytotoxic agent and the antibody unit (Tsuchikama, 2018). In some embodiments, the linker cleaves under intracellular conditions, and the cleavage of the linker releases the drug unit from the antibody or antigen-binding fragment under intracellular conditions. In some embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. In some embodiments, the linker is cleaved by a cleavage agent present in the intracellular environment (e.g., within lysosomes, endosomes, or pituitaries). Non-limiting examples of cleavable linkers include disulfide-containing linkers that can be cleaved via disulfide exchange, acid-unstable linkers that can be cleaved at acidic pH, and linkers that can be cleaved by hydrolases, esterases, peptidases, and glucuronidases.

[0266] In some embodiments, the antibody or antigen-binding fragment is conjugated to a nucleic acid that can be a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In some embodiments, the antibody or antigen-binding fragment is conjugated to an aptamer or ribozyme instead of auristatin or a functional peptide analog or derivative thereof.

[0267] In some embodiments, the antibody-drug conjugate according to the invention comprises one or more radiolabeled amino acids, which are useful for both diagnostic and therapeutic purposes. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, for example, Junghans et al. 1996, U.S. Patent No. 4,681,581, U.S. Patent No. 4,735,210, U.S. Patent No. 5,101,827, U.S. Patent No. 5,102,990 (US RE35,50G), U.S. Patent No. 5,648,471 and U.S. Patent No. 5,697,902). In some embodiments, the antibody or antigen-binding fragment according to the invention is conjugated to a radioisotope or a radioisotope-containing chelate.

[0268] The antibodies and antigen-binding fragments thereof disclosed herein also 99 Tc, 90 Y, 111 In, 32 P, 14 C, 125 I, 3 H, 13 1I, 11 C, 15 0, 13 N, 18 F, 35 S, 51 Cr, 51To, 226Ra, 6oCo, 59Fe, 51Se, 152Eu, 67CU, 2nCi, 211At, 212Pb, 47Sc, 109Pd, 234Th, and 4oK, 151Gd, 55 Mn, 52 Tr, and 56It may be conjugated with labels such as Fe.

[0269] In some embodiments, the portion bound to the antibody or antigen-binding fragment according to the present invention is an immunomodulatory compound. Preferred examples of such immunomodulatory compounds are T cell-binding compounds, NK cell-binding compounds, NKT cell-binding compounds, or gamma-delta T cell-binding compounds. In some preferred embodiments, the T cell-binding compound is a CD3-specific binding compound, a KLRG1-specific binding compound, or a CD103-specific binding compound. When bound to the antibody or antigen-binding fragment according to the present invention, such a T cell-binding compound directs T cells to cells such as cancer cells expressing E-cadherin and O-mannosyltransferase, thereby inducing or enhancing a cytotoxic T cell response against the (cancer) cells.

[0270] Similarly, NK cell-binding compounds, NKT cell-binding compounds, or gamma-delta T cell-binding compounds are suitable for attracting NK cells, NKT cells, or gamma-delta T cells, respectively, to cells expressing E-cadherin and O-mannosyltransferase, thereby inducing cytotoxicity or other immunomediated activity.

[0271] In some preferred embodiments, the T cell binding compound is a CD3-specific binding compound. In some preferred embodiments, the T cell binding compound is a KLRG1-specific binding compound. In some preferred embodiments, the T cell binding compound is a CD103-specific binding compound.

[0272] In some embodiments, the antibody or antigen-binding fragment according to the present invention binds to a TGFβ-specific binding compound. This is particularly useful for directing the antibody or antigen-binding fragment according to the present invention to cells, preferably disease-specific cells such as tumor cells containing O-mannosylated E-cadherin and TGFβ. As shown in the examples, the antibody or antigen-binding fragment according to the present invention can inhibit tumor cell proliferation and / or increase tumor cell death, particularly well when the tumor expresses both O-mannosylated E-cadherin and TGFβ.

[0273] An overview of bispecific antibodies and antibody constructs in oncology is given in Suurs et al, 2019.

[0274] Therefore, some embodiments provide bispecific or multispecific binding compounds comprising an antibody or antigen-binding fragment and an immunomodulatory molecule according to the present invention.

[0275] Some embodiments provide bispecific or multispecific binding compounds comprising an antibody or antigen-binding fragment according to the present invention and a compound selected from the group consisting of T cell-binding compounds, NK cell-binding compounds, NKT cell-binding compounds, and gamma-delta T cell-binding compounds.

[0276] Some embodiments provide bispecific or multispecific conjugation compounds comprising an antibody or antigen-binding fragment and a CD3-specific conjugation compound according to the present invention.

[0277] Some embodiments provide bispecific or multispecific conjugation compounds comprising an antibody or antigen-binding fragment and a CD103-specific conjugation compound according to the present invention.

[0278] Some embodiments provide bispecific or multispecific binding compounds comprising an antibody or antigen-binding fragment and a KLRG1 specificity-binding compound according to the present invention.

[0279] Some embodiments provide bispecific or multispecific binding compounds comprising an antibody or antigen-binding fragment and a TGFβ-specific binding compound according to the present invention.

[0280] Some embodiments provide antibody or antigen-binding fragments according to the present invention that are bound to another tumor-binding compound. Such bispecific or multispecific compounds allow for increased or more specific binding to tumor cells, for example, if two or more bound compounds are specific to different epitopes on tumor cells. Therefore, such bispecific or multispecific compounds are very suitable for therapeutic or diagnostic applications.

[0281] In some embodiments, the antibody or antigen-binding fragment according to the present invention is bound to a label. This allows for the detection of E-cadherin-containing cells, such as E-cadherin-positive cancer cells, using such a label-binding compound. In some embodiments, the antibody or antigen-binding fragment according to the present invention is bound to a hormone or enzyme. This allows for the targeting of such a hormone or enzyme to E-cadherin-containing (cancer) cells. Other embodiments provide the antibody or antigen-binding fragment according to the present invention, which is bound to a secondary antibody or its antigen-binding fragment.

[0282] Accordingly, some embodiments provide antibodies or antigen-binding fragments according to the present invention, which are bound to another compound, preferably an immunomodulatory compound, a T cell-binding compound, an NK cell-binding compound, an NKT cell-binding compound, and a gamma-delta T cell-binding compound, a CD3-specific binding compound, a TGFβ-specific binding compound, a cytokine, a secondary antibody or its antigen-binding fragment, a detectable label, a drug, a chemotherapeutic agent, a cytotoxic agent, a toxic moiety, a hormone, an enzyme, and a radioactive compound.

[0283] In some embodiments, the secondary antibody or its antigen-binding fragment is also specific to O-mannosylated E-cadherin. Therefore, the present invention provides bispecific or multispecific binding compounds comprising an antibody or antigen-binding fragment and a secondary antibody or antigen-binding fragment that is also specific to O-mannosylated E-cadherin. The resulting binding compounds are monospecific to E-cadherin, and each Fab arm typically binds to its own E-cadherin epitope. In some embodiments, the epitopes recognized by the Fab fragments are different from each other. In other embodiments, the epitopes are the same. Fab arms may bind epitopes with different affinities. Alternatively, Fab arms may bind their epitopes with essentially the same affinity, and the K of the Fab arms D However, this means that they differ from each other by 30% or less, preferably 20% or less, or 10% or less.

[0284] In some embodiments, the secondary antibody or its antigen-binding fragment is also an antibody or antigen-binding fragment according to the present invention. Thus, a bispecific or multispecific conjugated compound comprising at least two antibodies or antigen-binding fragments according to the present invention is provided. In some embodiments, the at least two antibodies or antigen-binding fragments according to the present invention are bound to each other. In some embodiments, the bispecific or multispecific conjugated compound comprises at least two AT1636 antibodies or their antigen-binding moieties. In some embodiments, the bispecific or multispecific conjugated compound comprises at least two AT1636-I antibodies or their antigen-binding moieties. In some embodiments, the bispecific or multispecific conjugated compound comprises at least two AT1636-E antibodies or their antigen-binding moieties. In some embodiments, the bispecific or multispecific conjugated compound comprises at least two AT1636-N antibodies or their antigen-binding moieties. In some embodiments, the bispecific or multispecific conjugated compound comprises at least two AT1636-Y antibodies or their antigen-binding moieties. In some embodiments, the bispecific or multispecific conjugate compound comprises at least two AT1636-YN antibodies or their antigen-binding moieties. In some embodiments, the bispecific or multispecific conjugate compound comprises at least two AT1636-IYN antibodies or their antigen-binding moieties. In some embodiments, the bispecific or multispecific conjugate compound comprises at least two AT1636-IYEN antibodies or their antigen-binding moieties.

[0285] Some embodiments provide a conjugation compound capable of conjugating O-mannosylated E-cadherin, the compound comprising an antibody or antigen-binding fragment and a therapeutic or radioactive compound or toxic moiety according to the present invention.

[0286] In some embodiments, the antibody or antigen-binding fragment according to the present invention is bound to another E-cadherin-specific binding compound, such as a currently known anti-E-cadherin antibody or its antigen-binding fragment, in order to produce a bispecific or multispecific compound. In some embodiments, the heavy chain of the antibody or antigen-binding fragment according to the present invention is paired with the heavy chain of another E-cadherin-specific antibody in order to produce a bispecific antibody or its antigen-binding fragment. The bispecific or multispecific compounds according to the present invention enable, for example, increased binding to E-cadherin-containing cells. Therefore, such bispecific or multispecific compounds are very suitable for therapeutic or diagnostic applications. The bispecific or multispecific compounds according to the present invention can also be used in assays, where different E-cadherin-containing cells are bound to the same bispecific or multispecific binding compound.

[0287] Some embodiments provide a bispecific antibody, or its antigen-binding fragment, comprising one Fab fragment of an antibody according to the present invention and one Fab fragment of another antibody. In some embodiments, such a bispecific antibody comprises one Fab fragment of an antibody according to the present invention and one Fab fragment of another antibody which is preferably specific to T cells, NK cells, NKT cells, or gamma-delta T cells, for example, a Fab fragment which is specific to CD3, KLRG1, or CD103.

[0288] Therefore, some embodiments are: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - A Fab fragment of another antibody, preferably specific to T cells, NK cells, NKT cells, or gamma-delta T cells. The present invention provides a bispecific antibody or its antigen-binding fragment that can conjugate O-mannosylated E-cadherin.

[0289] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - One Fab fragment of another antibody that is CD3 specific Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0290] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - One Fab fragment of another antibody that is specific to KLRG1 Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0291] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - One Fab fragment of another antibody that is specific to CD103 Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0292] Also: - One Fab fragment of an antibody or antigen-binding fragment according to the present invention; and - One Fab fragment of another antibody that is specific to TGFβ Also provided are bispecific antibodies or antigen-binding fragments thereof that can conjugate O-mannosylated E-cadherins, including the antibody containing the antibody.

[0293] The antibody or antigen-binding fragment according to the present invention may bind to another moiety, such as a drug or immunomodulatory compound or label, via a linker such as an acid-unstable hydrazone linker, or via a peptide linker such as citrulline-valine, or via a thioether linkage, or by a saltase-catalyzed amide group transfer as described in detail in International Publication No. 2010 / 087994.

[0294] Saltase-catalyzed amide transfer is involved in the design of a saltase recognition site (LPETGG) to the heavy chain of an antibody, preferably to the C-terminal portion of the heavy chain, and to the portion bound to the antibody. The antibody and its portion further typically include tags for purification purposes, such as a GGGGS sequence and an HIS tag. Subsequently, a saltase-mediated amide transfer is performed, followed by click chemistry coupling. In saltase-catalyzed amide transfer, "click chemistry coupling" typically involves the chemical coupling of, for example, an alkyne-containing reagent with an azide-containing reagent added by saltase, for example, through the addition of glycine to the saltase motif on the heavy chain of the antibody and to the saltase motif on the portion bound to the antibody (such as a protein, peptide, or antibody). Thus, in one embodiment, the present invention provides an antibody according to the present invention in which the saltase recognition site (LPETGG) is designed to the heavy chain of the antibody, preferably to the C-terminal portion of the heavy chain, and the antibody further preferably includes purification tags such as a GGGGS sequence and an HIS tag.

[0295] In some embodiments, the antibody or antigen-binding fragment according to the present invention is bound to another portion via a thioether bond. In such cases, one or more cysteines are preferably incorporated into the antibody or antigen-binding fragment according to the present invention. Cysteines contain a thiol group, and therefore, the incorporation of one or more cysteines into the antibody or antigen-binding fragment according to the present invention, or the substitution of one or more amino acids with one or more cysteines, allows for binding to another portion of the antibody or antigen-binding fragment. The one or more cysteines are preferably introduced at a position that does not significantly affect the folding of the antibody or antigen-binding fragment and does not significantly alter the antigen-binding or effector function. Therefore, the present invention also relates to AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT163 The present invention provides an antibody or antigen-binding fragment comprising a heavy chain sequence of an antibody selected from the group consisting of 6-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, wherein at least one amino acid (other than cysteine) of the antibody is replaced by cysteine.

[0296] The present invention further provides chimeric antigen receptor (CAR) T cells comprising the heavy chain CDR1, CDR2, and CDR3 sequences of the antibody according to the present invention. In some embodiments, the CAR T cells further comprise the light chain CDR1, CDR2, and CDR3 sequences of the antibody according to the present invention. Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are engineered receptor proteins that can confer the ability of cells to bind to novel, specific targets. CARs combine both antigen-binding and cell-activating functions into a single receptor. Typically, CARs have a modular design comprising an antigen-binding domain that transmits an activation signal and one or more directly or indirectly bound intracellular domains. Depending on the number of co-stimulatory domains, CARs can be classified as first-generation (CD3z only), second-generation (one co-stimulatory domain + CD3z), or third-generation CARs (more than one co-stimulatory domain + CD3z). The introduction of CAR genes into T cells successfully redirects T cells with additional antigen specificity, providing the necessary signals to promote full T cell activation. Alternatively, CAR genes can also be introduced into other immune cells such as NK, NKT, or gamma-delta-T cells (Rafiq et al. 2019). The antigen-binding features of a CAR are preferably defined by an extracellular scFv. The scFv format is generally two variable domains linked by a flexible peptide sequence that is either oriented VH-linker-VL or VL-linker-VH. Other formats known in the art include tandem CARs, loop tandem CARs, and CARs that bind to common adapter molecules (Guedan et al. Mol Ther 2019).

[0297] The intracellular signaling domain of CARs typically includes an activation domain and one or more co-stimulatory domains. In the art, the majority of CARs activate CAR T cells via a CD3ζ-derived immunoreceptor tyrosine-based activation motif. The most widely studied co-stimulatory domains are derived from co-stimulatory molecules from the CD28 family (including CD28 and ICOS) or the tumor necrosis factor receptor (TNFR) family of genes (including 4-1BB(CD137), OX40, and CD27). Alternative domains include those derived from MYD88 or the killer cell immunoglobulin-like receptor 2DS2 (KIR2DS2; also known as DAP12, combined with co-expression of the TYRO protein tyrosine kinase-binding protein). Alternatively, the binding domain used for CAR-T cells can fusion to the extracellular N-terminus of any of the five other TCR subunits, resulting in the incorporation of each TCR fusion construct (TRuCs) into the TCR complex (Bauerle et al, 2019).

[0298] Strategies used in the art to genetically recombine cells to express CARs include virus-based and non-virus-based genetic engineering tools such as gamma retroviruses and lentiviral vectors. Other methods include, for example, transposon system-like sleeping beauty (SB) and piggyBac, mRNA, non-integrated lentiviruses, endonuclease enzymes (Guedan et al. 2019), and DNA nanocarriers for in-situ cell programming.

[0299] The CAR T cells according to the present invention bind O-mannosylated E-cadherin, preferably its 70 kDA truncated form, and are therefore highly suitable for use in immunotherapy against O-mannosylated E-cadherin-positive cancer cells. Therefore, some embodiments provide chimeric antigen receptor (CAR) T cells capable of binding O-mannosylated E-cadherin, wherein the CAR T cells contain the heavy chain CDR1, CDR2, and CDR3 sequences of the antibody according to the present invention. In some embodiments, the CAR T cells contain the heavy chain CDR1, CDR2, and CDR3 sequences of the antibody as shown in Table 1. In some embodiments, the CAR T cells further contain the CDR1, CDR2, and CDR3 sequences of the antibody as shown in Table 1. In some embodiments, the CAR The T cells contain heavy chain CDR1, CDR2, and CDR3 sequences, as well as light chain CDR1, CDR2, and CDR3 sequences, of antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN.

[0300] Some embodiments provide isolated or recombinant host cells or non-human animals containing bispecific or multispecific antibodies or CAR T cells according to the present invention.

[0301] Therapeutic use of anti-E-cadherin antibodies The antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention are suitable for use against cells expressing O-mannosylated E-cadherin. Furthermore, methods are provided for treating subjects, including human subjects requiring treatment with the antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention. Also provided are nucleic acid molecules or vectors, or cells containing nucleic acids according to the present invention, for use as pharmaceuticals and / or prophylactic agents. When one or more nucleic acid molecules (or vectors containing them) according to the present invention are administered, the nucleic acid molecules(s) are translated in situ into antibodies or antigen-binding fragments according to the present invention. The resulting antibodies or antigen-binding fragments according to the present invention subsequently interfere with or prevent damage associated with O-mannosylated E-cadherin-expressing cells, such as E-cadherin-positive and TMTC3-positive tumors. Similarly, the introduction of cells according to the present invention into patients requiring it results in the in vivo generation of therapeutic or prophylactic anti-O-mannosylated E-cadherin antibodies or antigen-binding fragments according to the present invention.

[0302] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use as pharmaceuticals or prophylactic agents. In some embodiments, the pharmaceutical or prophylactic agent is for disorders associated with cells expressing E-cadherin. In certain embodiments, the cells also express O-mannosyltransferase, enabling the binding of antibodies and antigen-binding fragments that are specific to the O-mannosylation of E-cadherin and O-mannosylated E-cadherin. Thus, some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or prophylacticizing disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably tumor cells.

[0303] In certain embodiments, the O-mannosyltransferase is TMTC3, which is well known for its E-cadherin O-mannosylation activity. Therefore, the present invention provides further antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use in methods for treating or preventing disorders associated with cells expressing E-cadherin and TMTC3, preferably tumor cells.

[0304] In some embodiments, the disorder associated with tumor cells expressing E-cadherin and O-mannosyltransferase is epithelial carcinoma. In some embodiments, the impairment associated with tumor cells expressing E-cadherin and O-mannosyltransferase is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, colorectal carcinoma, colon cancer, stomach cancer, gastric cancer, gastroesophageal junction cancer, breast cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer, endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, renal cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer, and peritoneal cancer. In some embodiments, the impairment associated with tumor cells expressing E-cadherin and O-mannosyltransferase is selected from the group consisting of colorectal cancer, colon cancer, colon cancer subtype CMS1, colon cancer subtype CMS2, colon cancer subtype CMS3, colon cancer subtype CMS4, laryngeal cancer, head and neck cancer, breast cancer, pancreatic cancer, esophageal cancer, bladder cancer, lung cancer, stomach cancer, urinary tract cancer, prostate cancer, and ovary cancer.

[0305] As used herein, tumor cells expressing E-cadherin are also referred to as “E-cadherin-expressing tumor cells” or “E-cadherin-positive tumor cells.” Tumor cells expressing both E-cadherin and TMTC3 are also referred herein to as “E-cadherin-expressing and TMTC3-expressing tumor cells” or “E-cadherin and TMTC3-expressing tumor cells” or “E-cadherin-positive and TMTC3-positive tumor cells” or “E-cadherin and TMTC3-positive tumor cells.” Cancers containing tumor cells expressing E-cadherin and TMTC3 are referred herein to as “E-cadherin-positive and TMTC3-positive cancers.”

[0306] The "subject" may be a human or an animal. In some embodiments, the subject is a mammalian individual such as a human, cat, dog, rabbit, mouse, rat, cow, goat, horse, pig, monkey, ape, or gorilla. In certain embodiments, the subject is a human individual.

[0307] As used herein, the term “disorders associated with cells expressing E-cadherin and O-mannosyltransferase” means any disease involving the presence of disease-specific cells expressing E-cadherin and O-mannosyltransferase. In some embodiments, such cells are disease-causing factors, as is frequently the case with tumor cells expressing E-cadherin and O-mannosyltransferase. In some embodiments, the presence of such cells causes adverse symptoms, such as inflammation and / or pain.

[0308] The term “treating or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase” may mean interfering with the onset or progression of said disorders and / or alleviating symptoms resulting from said disorders. For example, the term “treating or preventing disorders associated with tumor cells expressing E-cadherin and O-mannosyltransferase” may include preventing, interfering with, and / or slowing the growth of said tumor cells and / or alleviating symptoms resulting from the presence of said tumor cells in a patient.

[0309] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive cancers. The advantage of the O-mannosylated E-cadherin-specific antibodies and antigen-binding fragments according to the present invention is their specificity to (tumor) cells that express both E-cadherin and TMTC3 but bind to E-cadherin-positive cells that do not express TMTC3 to a fairly low degree. This allows for a reduction in adverse side effects, and therefore higher doses may be tolerated.

[0310] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive epithelial cancers.

[0311] Some embodiments include adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, colorectal cancer, colon cancer, stomach cancer, gastric cancer. cancer), esophagogastric junction cancer, breast cancer, pancreatic cancer, esophageal cancer, esophagogastric junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer The present invention provides antibodies or antigen-binding fragments or bispecific antibodies or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive cancers selected from the group consisting of cancer, endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, renal cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer and peritoneal cancer.

[0312] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive colorectal cancer.

[0313] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive colon cancer.

[0314] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive colorectal cancer subtype CMS1.

[0315] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or prophylacticizing E-cadherin-positive and TMTC3-positive colorectal cancer subtype CMS2.

[0316] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive colorectal cancer subtype CMS3.

[0317] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive colorectal cancer subtype CMS4.

[0318] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive pharyngeal cancer.

[0319] Some embodiments provide antibodies or antigen-binding fragments or bispecific antibodies or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive head and neck cancers.

[0320] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive breast cancer.

[0321] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive pancreatic cancer.

[0322] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive esophageal cancer.

[0323] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive bladder cancer.

[0324] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive lung cancer.

[0325] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive stomach cancer.

[0326] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive urinary tract cancers.

[0327] Some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive prostate cancer or ovary cancer.

[0328] In some embodiments, the antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention are used against E-cadherin-positive and TMTC3-positive cancers, including tumor cells expressing transforming growth factor β (TGFβ), preferably TGFβ1. As used herein, cancers including E-cadherin-expressing tumor cells, TMTC3-expressing tumor cells, and TGFβ-expressing tumor cells are referred to as "E-cadherin-positive, TMTC3-positive, and TGFβ-positive cancers." As shown in the examples, the antibodies or functional fragments according to the present invention bind particularly well to tumor cells in the presence of TGFβ. Combinations of the antibodies or antigen-binding fragments according to the present invention with TGFβ are particularly suitable for inhibiting tumor cell proliferation and / or increasing tumor cell death. Therefore, antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention are further provided for use in methods for treating or preventing E-cadherin-positive, TMTC3-positive, and TGFβ-positive cancers. An advantage of improved tumor cell proliferation inhibition in the presence of TGFβ is the possibility of using lower doses.

[0329] Preferred antibodies for use in any of the listed methods are antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, as well as their antigen-binding fragments having the same binding specificity.

[0330] Some embodiments provide the use of antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for the manufacture of pharmaceuticals.

[0331] Some embodiments provide the use of antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for the manufacture of pharmaceuticals for treating or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase. In certain embodiments, the cells are tumor cells. In certain embodiments, the O-mannosyltransferase is TMTC3. Some embodiments provide the use of antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for the preparation of pharmaceuticals for treating or preventing E-cadherin-positive and TMTC3-positive cancers. In some embodiments, the E-cadherin-positive and TMTC3-positive cancers are epithelial cancers. In some embodiments, the E-cadherin-positive and TMTC3-positive cancers are selected from the group consisting of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, colorectal cancer, colon cancer, stomach cancer, gastric cancer, gastroesophageal junction cancer, breast cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer, endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, kidney cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer, and peritoneal cancer. In some embodiments, the E-cadherin-positive and TMTC3-positive cancers are selected from the group consisting of colorectal cancer, colon cancer, colon cancer subtype CMS1, colon cancer subtype CMS2, colon cancer subtype CMS3, colon cancer subtype CMS4, laryngeal cancer, head and neck cancer, breast cancer, pancreatic cancer, esophageal cancer, bladder cancer, lung cancer, stomach cancer, urinary tract cancer, prostate cancer, and ovarian cancer.

[0332] Further embodiments provide compositions comprising antibodies or antigen-binding fragments according to the present invention. Some embodiments provide compositions comprising bispecific antibodies, multispecific antibodies, ADCs or CAR T cells according to the present invention. Compositions comprising nucleic acid molecules according to the present invention, as well as compositions comprising vectors or cells according to the present invention, are also provided. In some embodiments, the antibody is selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, CB02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN. In some embodiments, the composition according to the present invention comprises the antibody according to the present invention and another E-cadherin-specific antibody. The other E-cadherin-specific antibodies described above preferably bind to different E-cadherin epitopes compared to the antibodies according to the present invention. Such combinations of different E-cadherin-specific antibodies are particularly suitable for binding to and / or interfering with E-cadherin-positive cells, such as E-cadherin and TMTC3-positive tumor cells.

[0333] In some embodiments, the compositions according to the present invention are pharmaceutical compositions. Such pharmaceutical compositions also preferably include a pharmaceutically acceptable carrier, diluent and / or excipient. Non-limiting examples of suitable carriers include, for example, keyhole limpet hemocyanin (KLH), serum albumin (e.g., BSA or RSA), and ovalbumin. In some specific embodiments, the suitable carrier includes, for example, a solution such as physiological saline. The pharmaceutical compositions according to the present invention are preferably suitable for human use.

[0334] The present invention further provides a method for treating and / or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably but not limited to tumor cells, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment according to the present invention, and / or a bispecific or multispecific antibody or ADC or CAR T cell according to the present invention, and / or a nucleic acid according to the present invention, and / or a vector or cell according to the present invention, and / or a composition or kit of parts according to the present invention, to an individual in need. The present invention further provides a method for treating and / or preventing at least partially E-cadherin-positive and TMTC3-positive cancers, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment according to the present invention, and / or a bispecific or multispecific antibody or ADC or CAR T cell according to the present invention, and / or a nucleic acid according to the present invention, and / or a vector or cell according to the present invention, and / or a composition or kit of parts according to the present invention, to an individual in need. The composition is preferably a pharmaceutical composition according to the present invention. The antibody or antigen-binding fragment or nucleic acid molecule or vector or ADC or CAR T cell or pharmaceutical composition according to the present invention is preferably administered by one or more injections. In some embodiments, the antibody or antigen-binding fragment or nucleic acid molecule or vector or ADC or CAR T cell or pharmaceutical composition according to the present invention is administered intravenously. Alternatively, other routes of administration known in the art may be used. Non-limiting examples of doses of the binding compounds according to the present invention range from 0.1 mg to 10 mg per kg of body weight.

[0335] Some embodiments provide antibodies or antigen-binding fragments or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention, which can be combined with other therapeutic agents, preferably anticancer agents and / or immunomodulatory compounds. For example, antibodies or antigen-binding fragments according to the present invention can be combined with other agents useful in treating and / or preventing disorders associated with cells expressing O-mannosyltransferases such as E-cadherin and TMTC3, preferably tumor cells. Therefore, the present invention provides antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells for use in methods for treating or preventing disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells, thereby combining the said antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention with other therapeutic agents useful for treating and / or preventing said disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells.

[0336] Further, antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention are provided for use in methods for treating or preventing E-cadherin-positive and TMTC3-positive cancers, thereby allowing the antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention to be combined with other therapeutic agents for treating and / or preventing the cancers.

[0337] In some embodiments, the other therapeutic agent is a chemotherapeutic agent.

[0338] In some embodiments, the other therapeutic agent is a cell proliferation inhibitor or a cytotoxic agent.

[0339] In some embodiments, the other therapeutic agent is a therapeutic nucleic acid. In some embodiments, the nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRAN molecule), or an immunostimulant nucleic acid (e.g., an immunostimulant CpG motif-containing DNA molecule). In some embodiments, the nucleic acid is an aptamer or a ribozyme.

[0340] In some embodiments, the other therapeutic agent includes a radiolabeled amino acid.

[0341] In some embodiments, the other therapeutic agent comprises a radioisotope or a radioisotope-containing chelate.

[0342] The impairment associated with tumor cells expressing E-cadherin and O-mannosyltransferase is preferably E-cadherin-positive and TMTC3-positive cancer. In some embodiments, the impairment associated with tumor cells expressing E-cadherin and O-mannosyltransferase is epithelial carcinoma. In some embodiments, the impairment associated with tumor cells expressing E-cadherin and O-mannosyltransferase is a cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, colorectal carcinoma, colon cancer, stomach cancer, gastric cancer, gastroesophageal junction cancer, breast cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer, endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, renal cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer, and peritoneal cancer. In some specific embodiments, the impairment associated with tumor cells expressing E-cadherin and O-mannosyltransferase is a cancer selected from the group consisting of colorectal cancer, colon cancer, colon cancer subtype CMS1, colon cancer subtype CMS2, colon cancer subtype CMS3, colon cancer subtype CMS4, laryngeal cancer, head and neck cancer, breast cancer, pancreatic cancer, esophageal cancer, bladder cancer, lung cancer, stomach cancer, urinary tract cancer, prostate cancer, and ovary cancer.

[0343] Compositions and kits of parts comprising combinations of antibodies or antigen-binding fragments or ADCs or CAR T cells or nucleic acids or vectors or host cells and another therapeutic agent according to the present invention are also provided herein. Some embodiments provide kits of parts or compositions comprising antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acid molecules or vectors or host cells according to the present invention and another therapeutic agent for the treatment or prevention of disorders associated with cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells. In some embodiments, the composition is a pharmaceutical composition. The disorder is preferably E-cadherin-positive and TMTC3-positive cancer.

[0344] In some embodiments, the composition is a pharmaceutical composition. The disorder is preferably E-cadherin-positive and TMTC3-positive cancer.

[0345] A kit of parts according to the present invention may comprise one or more containers filled with compositions comprising antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAT T cells or nucleic acid molecules or vectors or host cells and other therapeutic agents. The kit of parts or the one or more containers may further comprise one or more pharmaceutically acceptable carriers, diluents or excipients. In connection with such a kit of parts or container(s), various materials such as instructions for use or notices in the form prescribed by government agencies regulating the manufacture, use or sale of pharmaceuticals may be included, and the notices reflect approval by the agent for manufacture, use or sale. In some embodiments, a kit of parts according to the present invention comprises instructions for use.

[0346] Some embodiments provide methods for treating or preventing disorders in human or non-human organisms associated with cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, preferably tumor cells, the methods comprising administering to the organism a therapeutically effective amount of an antibody or antigen-binding fragment or a bispecific or multispecific antibody or ADC or CAR T cell or nucleic acid or vector or host cell or composition or kit of parts, in combination with a further therapeutic agent or therapeutic procedure. The further therapeutic agent is preferably one of the agents described herein.

[0347] Further applications of E-cadherin-specific antibodies The antibodies, antigen-binding fragments, ADCs, and CAR T cells according to the present invention are also particularly useful for detecting O-mannosylated E-cadherin expressing cells. For example, if an individual, preferably a human, is suspected of having a disorder related to O-mannosylated E-cadherin expressing cells, a sample from the individual can be tested for the presence of O-mannosylated E-cadherin expressing cells (also referred herein as O-mannosylated E-cadherin positive cells) using the antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention. In some embodiments, the sample is mixed with the antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention that specifically bind O-mannosylated E-cadherin positive cells if such cells are present in the sample. O-mannosylated E-cadherin-positive cells, such as O-mannosylated E-cadherin-positive tumor cells, which bind to antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention, can be isolated from a sample and / or detected using any method known in the art, for example, isolation using magnetic beads, streptavidin-coated beads, or isolation through the use of a secondary antibody immobilized on a column. Alternatively, or further, the antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention are labeled so that they can be detected. Such antibodies or antigen-binding fragments or ADCs or CAR T cells are labeled, for example, fluorescently, enzymatically, or radioactively, using fluorophores such as rare earth chelates, fluorescein or its derivatives, rhodamine or its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescein, 152E u, dansyl, umbelliferone, luciferin, luminal labeling, isoluminal labeling, aromatic acridinium ester labeling, imidazole labeling, acridinium (acridimium) salt labeling, oxalate ester labeling, aequorin labeling, 2,3-dihydrophthalazinedione, biotin / avidin, spin labeling, or stable free radicals.In some embodiments, the antibody or antigen-binding fragment or ADC or CAR T cell according to the present invention is detected using a labeled secondary antibody directed against the antibody or antigen-binding fragment or ADC or CAR T cell.

[0348] As provided herein, screening assays can be performed using methods known in the art, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), Western blot assays, and immunohistochemical staining assays.

[0349] Labeled antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention are incubated with a cell-containing sample of an individual, such as a blood sample or tissue sample, and then unbound compounds are washed away. Subsequently, it is determined whether the labeled antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention bind to O-mannosylated E-cadherin-positive cells. In some embodiments, unlabeled antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention are brought into contact with a cell-containing sample. After incubation, one or more washing steps are performed, preferably to remove unbound compounds. Subsequently, the antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention bind to O-mannosylated E-cadherin-positive cells, for example, using a marker such as a fluorescent compound, or a detection antibody conjugated to horseradish peroxidase or alkaline phosphatase. After a further washing step, it is preferable to determine whether the detection antibody is bound, for example, by measuring luminescence or by adding a substrate of horseradish peroxidase or alkaline phosphatase. These detection techniques are well known in the art.

[0350] When the antibody or antigen-binding fragment or ADC or CAR T cells according to the present invention appear to bind to components of a patient's sample, this suggests the presence of O-mannosylated E-cadherin-positive cells. Thus, disease-specific cells, such as O-mannosylated E-cadherin-positive tumor cells, can be detected. Furthermore, the presence of disease-specific O-mannosylated E-cadherin-positive cells, such as O-mannosylated E-cadherin-positive tumor cells, suggests that treatment with the antibody or antigen-binding fragment or ADC or CAR T cells according to the present invention has a beneficial effect. Therefore, some embodiments provide the use of the antibody or antigen-binding fragment or bispecific or multispecific antibody or ADC or CAR T cells according to the present invention to determine whether a sample contains cells expressing O-mannosylated E-cadherin. In some embodiments, the antibody or antigen-binding fragment or bispecific or multispecific antibody or ADC or CAR T cells according to the present invention are used to determine whether a sample contains tumor cells expressing O-mannosylated E-cadherin.

[0351] Furthermore, a method is provided for determining whether cells expressing O-mannosylated E-cadherin, preferably tumor cells, are present in a sample, the method being: - Contacting the sample with an antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell according to the present invention, and - To enable the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell to bind to cells that express O-mannosylated E-cadherin, preferably tumor cells, if present, and - Determine whether the cells are bound to the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell, and thereby determine whether or not cells expressing O-mannosylated E-cadherin, preferably tumor cells, are present in the sample. Includes.

[0352] Antibodies AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D 04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT16 36-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, as well as their antigen-binding fragments, are particularly suitable for detecting O-mannosylated E-cadherin expressing cells, such as O-mannosylated E-cadherin positive tumor cells. Therefore, the use of antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or their antigen-binding fragments, is further provided for determining whether a sample contains O-mannosylated E-cadherin-containing cells.Some embodiments include, for example, O-mannosylated E-cadherin expressing epithelial cancer cells, or adenocarcinomas, squamous cell carcinomas, adenosquamous cell carcinomas, undifferentiated carcinomas, large cell carcinomas, small cell carcinomas, colorectal cancers, colon cancers, stomach cancers, gastric cancers. cancer), esophagogastric junction cancer, breast cancer, pancreatic cancer, esophageal cancer, esophagogastric junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer A selection from the group consisting of cancer, endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, renal cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer, and peritoneal cancer, preferably colorectal cancer cells, colon cancer cells, colon cancer subtype CMS1 cells, colon cancer subtype CMS2 cells, colon cancer subtype CMS3 cells, colon cancer subtype CMS4 cells, pharyngeal cancer cells, head and neck cancer, breast cancer cells, pancreatic cancer cells, esophageal cancer cells, bladder cancer cells, lung cancer cells, stomach cancer cells, urinary tract cancer cells, prostate cancer cells, and ovarian cancer cells. To determine whether a sample contains tumor cells containing O-mannosylated E-cadherin, such as cancer cells, selected from a group of cancer cells, use AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F The present invention provides the use of antibodies selected from the group consisting of 10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or antigen-binding fragments thereof.

[0353] Furthermore, a method is provided for determining whether cells containing O-mannosylated E-cadherin, preferably tumor cells, are present in a sample, the method being: - Contacting the sample with an antibody selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or an antigen-binding fragment thereof, and - To enable the antibody or antigen-binding fragment to bind to cells containing O-mannosylated E-cadherin, preferably tumor cells, if present, and - To determine whether or not cells are bound to the antibody or antigen-binding fragment, thereby determining whether or not cells containing O-mannosylated E-cadherin, preferably tumor cells, are present in the sample. Includes.

[0354] Some embodiments provide a method according to the present invention, wherein the sample includes a blood sample, a bone marrow sample, or a biopsy. In some embodiments, the biopsy is from the intestine and is preferably tested for gastrointestinal cancer, colorectal cancer, colon cancer, esophageal cancer, or stomach cancer. In some embodiments, the biopsy is from pancreatic tissue or lung tissue or breast tissue or pharyngeal tissue or squamous epithelial tissue or liver tissue or ovarian tissue or prostate tissue or urinary tract tissue or bladder tissue or brain tissue. In some embodiments, the sample is a blood sample and is useful, for example, for testing for the presence of multiple myeloma and / or metastasis of any of the solid tumors described above.

[0355] The test results of the method according to the present invention are useful for typing samples. For example, if a sample of an individual appears to contain malignant O-mannosylated E-cadherin-positive cells, the sample is typed as containing disease-associated cells. Such typing can then be used to diagnose disorders associated with O-mannosylated E-cadherin-expressing cells. Therefore, some embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use as diagnostic agents. Further embodiments provide antibodies or antigen-binding fragments or bispecific or multispecific antibodies or ADCs or CAR T cells or nucleic acids or vectors or host cells according to the present invention for use in the diagnosis of disorders associated with cells containing O-mannosylated E-cadherin, preferably tumor cells. The disorder is preferably an epithelial cancer, preferably an adenocarcinoma, a squamous cell carcinoma, an adenosquamous cell carcinoma, an undifferentiated carcinoma, a large cell carcinoma, a small cell carcinoma, a colorectal cancer, a colon cancer, a stomach cancer, a gastric cancer. cancer), esophagogastric junction cancer, breast cancer, pancreatic cancer, esophageal cancer, esophagogastric junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer A cancer is selected from the group consisting of endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, renal cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer, and peritoneal cancer, more preferably from the group consisting of colorectal cancer, colon cancer, colon cancer subtype CMS1, colon cancer subtype CMS2, colon cancer subtype CMS3, colon cancer subtype CMS4, laryngeal cancer, head and neck cancer, breast cancer, pancreatic cancer, esophageal cancer, bladder cancer, lung cancer, stomach cancer, urinary tract cancer, prostate cancer, and ovarian cancer.

[0356] In some preferred embodiments, antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or their antigen-binding fragments, are used for the detection and diagnosis described above. Therefore, antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or their antigen-binding fragments, are also provided for use in the diagnosis of disorders associated with O-mannosylated E-cadherin-containing cells.Some embodiments include epithelial carcinoma, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, colorectal cancer, colon cancer, stomach cancer, gastric cancer. cancer), esophagogastric junction cancer, breast cancer, pancreatic cancer, esophageal cancer, esophagogastric junction cancer, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, urinary tract cancer, prostate cancer, brain tumor, thyroid cancer, laryngeal cancer, carcinoid cancer, liver cancer, hepatocellular carcinoma, head and neck cancer, ovary cancer, cervical cancer, ovarian cancer More preferably selected from the group consisting of cancer, endometrial cancer, carcinoma in situ, clear cell carcinoma, melanoma, multiple myeloma, renal cancer, renal cell carcinoma, renal transitional cell carcinoma, fallopian tube cancer and peritoneal cancer, colorectal cancer, colon cancer, colon cancer subtype CMS1, colon cancer subtype CMS2, colon cancer subtype CMS3, colon cancer subtype CMS4, laryngeal cancer, head and neck cancer, breast cancer, pancreatic cancer, esophageal cancer, bladder cancer, lung cancer, stomach cancer, urinary tract cancer, prostate cancer and ovarian cancer. The present invention provides antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or antigen-binding fragments thereof, for use in the diagnosis of E-cadherin-positive and TMTC3-positive cancer cells, selected from the group consisting of cancer.

[0357] Furthermore, a method is provided for determining whether a human or non-human individual has cancer involving O-mannosylated E-cadherin, and this method is: - Contacting the cells of the said individual with an antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell according to the present invention, - To enable the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell to bind to tumor cells containing O-mannosylated E-cadherin, if present, and - To determine whether tumor cells are bound to the aforementioned antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell, thereby determining whether the individual has cancer containing O-mannosylated E-cadherin. This includes. In some embodiments, the method is an ex vivo method. In other embodiments, the method is an in vivo imaging method.

[0358] Appropriate imaging techniques include SPECT imaging (single-photon emission tomography) and PET imaging (positron emission tomography). Appropriate labeling is, for example, iodine-123 in combination with SPECT imaging. 123 1) and Technetium-99m (9m 9 Tc), or, for example, combined with PET imaging 11 C, 13 N, 15 0 or 18 Contains F, or indium-111 (see, for example, Gordon et al., (2005) International Rev. Neurobiol. 67:385-440).

[0359] Non-limiting cases of O-mannosylated E-cadherin-positive cancer are listed above. Preferably, antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or their antigen-binding fragments, are used in the method. Therefore, some embodiments provide a method for determining whether a human or non-human individual has cancer that expresses O-mannosylated E-cadherin, the method being: - Contacting the cells of the individual with an antibody selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or an antigen-binding fragment thereof. - To enable the binding of tumor cells containing O-mannosylated E-cadherin, if present, to the antibody or antigen-binding fragment, and - To determine whether tumor cells are bound to the antibody or antigen-binding fragment, and thereby determine whether the individual has cancer containing O-mannosylated E-cadherin. Includes.

[0360] In some embodiments, it is determined whether an individual has cancer expressing E-cadherin and O-mannosyltransferase, preferably TMTC3. As described above herein, the presence of cancer containing O-mannosylated E-cadherin suggests that treatment using antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention has beneficial effects. Therefore: - Contacting a sample from the said individual with an antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell according to the present invention, and - To enable the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cells to bind to tumor cells expressing E-cadherin and O-mannosyltransferase, preferably TMTC3, if present, and - To determine whether tumor cells are bound to the antibody or antigen-binding fragment or bispecific antibody or multispecific antibody or ADC or CAR T cell, thereby determining whether the individual has cancer expressing E-cadherin and O-mannosyltransferase, preferably TMTC3. A method is also provided for determining whether an individual has cancer expressing E-cadherin and O-mannosyltransferase, preferably TMTC3. Preferably, the individual is human.

[0361] Another aspect of the present invention provides a method for determining whether treatment of a cancer patient using an antibody or antigen-binding fragment or ADC or CAR T cells according to the present invention has the potential for improved prospective outcomes of the treatment compared to the mean population of cancer patients, the method comprising determining whether a sample of the cancer patient contains O-mannosylated E-cadherin-positive tumor cells. If this is true, then antibodies according to the present invention, such as antibodies selected from the group consisting of AT1636, E-C06, D-H04, D-A02, D-E09, E-A04, E-B09, C-A05, C-A03, C-B02, C-D04-A, C-D04-B, F-C08, D-G03, D-F10, C-E08, D-B06, D-G05, D-H08, C-H01, D-C12, D-C11, E-C10, AT1636-I, AT1636-Y, AT1636-E, AT1636-N, AT1636-YN, AT1636-IYN, and AT1636-IYEN, or their antigen-binding fragments, are particularly suitable for combating such cancers. Therefore, if it is known that an individual's cancer cells contain O-mannosylated E-cadherin on their surface, the likelihood of treatment success increases. Thus, a screening method is further provided which includes determining whether an individual's disease-specific cells, preferably tumor cells, contain O-mannosylated E-cadherin on their surface. In some embodiments, it is determined whether the disease-specific cells express E-cadherin and O-mannosyltransferase, preferably TMTC3. In some embodiments, it is further determined whether the disease-specific cells express TGFβ. If disease-specific cells, such as cancer cells, express E-cadherin and O-mannosyltransferase, preferably TMTC3, and TGFβ, the likelihood of treatment with the antibody or antigen-binding fragment or ADC or CAR T cells according to the present invention is further increased.

[0362] Since the presence of O-mannosylated E-cadherin is typically a result of the expression of E-cadherin and O-mannosyltransferases such as TMTC3, some embodiments provide a screening method that includes determining whether disease-specific cells of an individual, preferably tumor cells, express E-cadherin and O-mannosyltransferase, particularly TMTC3. Some embodiments provide a screening method that includes determining whether disease-specific cells of an individual, preferably tumor cells, express E-cadherin and O-mannosyltransferase, particularly TMTC3, and TGFβ.

[0363] In some embodiments, such a method according to the present invention is: - A step of contacting a disease-specific cell-containing sample from an individual with a binding compound specific to O-mannosylated E-cadherin, preferably an antibody or antigen-binding fragment; - A step that enables the binding compound to bind to disease-specific cells of the sample, and - A step to determine whether the binding compound is bound to disease-specific cells in the sample. The binding of the conjugated compound to disease-specific cells of the sample suggests that the patient has a significant potential for positive outcomes from treatment using antibodies or antigen-binding fragments or ADCs or CAR T cells according to the present invention.

[0364] In some embodiments, the disease-specific cells are tumor cells.

[0365] In some embodiments, it is further determined whether the disease-specific cells express TGFβ.

[0366] While this application may describe features as part of the same embodiment or as part of a separate embodiment, the scope of the invention also includes embodiments that include all or any combination of some of the features described herein. [Examples]

[0367] The present invention is further illustrated in the following embodiments. These embodiments do not limit the scope of the present invention, but merely serve to clarify it.

[0368] [Table 1-1] [Table 1-2]

[0369] [Table 2-1] [Table 2-2] [Table 2-3]

[0370] [Table 3]

[0371] [Table 4-1] [Table 4-2] [Table 4-3]

[0372] [Table 5]

[0373] References Aiello, NM, Maddipati, R, Norgard, RJ, Balli, D, Li, J, Yuan, S, Yamazoe, T, et al. EMT Subtype Influences Epithelial Plasticity and Mode of Cell Migration. Developmental Cell 2018; 45: 681-695.e4. Atwell, S, Ridgway, JB, Wells, JA, Carter, P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. Journal of Molecular Biology 1997; 270: 26-35. Baeuerle, P.A. et al. Nature Communications (2019)10: 2087 Barretina, J, Caponigro, G, Stransky, N, Venkatesan, K, Margolin, AA, Kim, S, Wilson, CJ, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012; 483: 603-607. Bartels MF, et al. (2016) Protein O-mannosylation in the murine brain: Occurrence of mono-O-mannosyl glycans and identification of new substrates. PLoS One 11:e0166119. Bartels, L, de Jong, G, Gillissen, MA, Yasuda, E, Kattler, V, Bru, C, Fatmawati, C, et al. A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia. Cancer Research 2019; 79: 3372-3382. Bartels, L. et al. A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia. Methods 2019, accepted for publication Carvalho S, et al. (2016) O-mannosylation and N-glycosylation: Two coordinated mechanisms regulating the tumour suppressor functions of E-cadherin in cancer. Oncotarget 7:65231-65246. Chau C. et al. Lancet 2019: 394: 793-804 Chen, Y et al. (1999) J. Mol. Biol. 293:865-881 Gauthier L. et al. (2019) Cell 177, 1701-1713 De Groot, A.S. et al. (2005) Dev. Biol. 122: 171-194 Guedan, S. et al. (2019) Molecular Therapy: Methods & Clinical Development. Vol 12: 145-156 Hanly et al. (1995) ILAR Journal. 37(3): 93-118 Idusogie, EE et al. (2001) J. Immunol. 166: 2571–2575 According to Junghans et al. , in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) Kwakkenbos, MJ et al. Genetic manipulation of B cells for the isolation of rare therapeutic antibodies from the human repertoire. Methods 2013; 1-6 Larsen , ISB , Narimatsu , Y , Joshi , HJ , Siukstaite , L , Harrison , OJ , Brasch , J , Goodman , KM , et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. Proc Natl Acad Sci USA 2017; 114: 11163–11168. Lazar, GA et al. ( 2006 ) Proc . Natl. Acad. Sci. U.S.A. 103: 4005–4010 Lommel M, et al. (2013) Protein O-mannosylation is crucial for E-cadherin-mediated cell adhesion. Proc Natl Acad Sci USA 110:21024-21029. Lund, J et al (1992) Mol. Immunol. 29: 53-59 Merchant, AM, Zhu, Z, Yuan, JQ, Goddard, A, Adams, CW, Presta, LG, Carter, P. An efficient route to human bispecific IgG. Nature Biotechnology, Published online: 01 August 2008; | doi:10.1038 / nbt0808-886 1998; 16: 677-681. Moore, GL et al. (2010) MAbs 2(2): 181-189 Padmanaban, V, Krol, I, Suhail, Y, Szczerba, BM, Aceto, N, Bader, JS, Ewald, AJ. E-cadherin is required for metastasis in multiplemodels of breast cancer. Nature 2019; 1-31. Patnaik & Stanley (2006) Methods Enzymol. 416: 159-182 Racape, M, Duong Van Huyen, J-P, Danger, R, Giral, M, Bleicher, F, Foucher, Y, Pallier, A, et al. The Involvement of SMILE / TMTC3 in Endoplasmic Reticulum Stress Response. PLoS ONE 2011; 6: e19321. Rafiq et al. 17 december 2019, nature reviews, clinical oncology Shields, RL et al. (2001) J. Biol Chem 276: 6591-6604 Stavenhagen, JB et al. (2007) Cancer Res. 67: 8882-8890 Sunryd, JC, Cheon, B, Graham, JB, Giorda, KM, Fissore, RA, Hebert, DN. TMTC1 and TMTC2 are novel endoplasmic reticulum tetratricopeptide repeat-containing adapter proteins involved in calcium homeostasis. Journal of Biological Chemistry 2014; 289: 16085-16099. Suurs F.V. et al (2019) Pharmacology & Therapeutics 201: 103-119 Tauriello, DVF, Palomo-Ponce, S, Stork, D, Berenguer-Llergo, A, Badia-Ramentol, J, Iglesias, M, Sevillano, M, et al. TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis. Nature 2018; 554: 538-543 Tsuchikama K. et al. (2018). Protein Cell 9(1):33-46 Vester-Christensen, MB, Halim, A, Joshi, HJ, Steentoft, C, Bennett, EP, Levery, SB, Vakhrushev, SY, et al. Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins. Proc Natl Acad Sci USA 2013; 110: 21018-21023. Yamane-Ohnuki, N et al. (2004) Biotechnol. Bioeng 87:614-622 US 4,681,581 US 4,735,210 US 5,101,827 US 5,102,990 (US RE35,50G) US 5,648,471 US 5,697,902 US9534058 (B2) WO 2010 / 087994 WO 2013 / 081463 WO 2015 / 093949

[0374] Examples Example 1: Discovery of AT1636 antibody Patient and healthy human materials The research protocol was approved by the Medical Ethics Committee of the Academic Medical Center, Amsterdam, Netherlands. All participants signed informed consent. Whole peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood after Ficoll gradient centrifugation and frozen until use.

[0375] Generation of colon cancer-specific clone AT1636 Naive and memory IgG B cells were isolated from patients with Lynch syndrome who were carriers of a pathogenic gene variant in the MSH6 gene, diagnosed with stage IV colorectal cancer (CRC) and liver metastases, and who had successfully received treatment with Avastin, capecitabine, and oxaliplatin. B cells were isolated from peripheral blood obtained from these patients 9 years after the last treatment. Naive and memory IgG B cells were immortalized by retroviral transduction of the Bcl6 and Bcl-xL genes and the reporter gene GFP. The immortalized B cells were then seeded at concentrations of 5, 10, or 20 cells per well (hereinafter referred to as microculture) and grown in IL-21 and CD40L. Subsequently, the supernatant of the grown B cell microcultures was screened for specific antibodies that bind to the colon cell lines COLO-205, CACO-2, and DLD1 cells (ATCC). Anti-human IgG-PE (Southern Biotech) was used as a secondary antibody, and the bound antibodies were detected by flow cytometry (BD).

[0376] An unrelated control antibody (AT1002) that specifically binds to the influenza HA antigen (described in International Publication No. 2013 / 081463) was included as a negative control in the experiment.

[0377] Microcultures showing specific binding of the supernatant to colon cell lines were selected, seeded at a concentration of 1 cell / well, and clonal cultures were obtained. After proliferation, the supernatant of the clonal cultures was tested for the presence of antibodies that specifically bind to colon cell lines using flow cytometry as described above. One of the obtained colon-specific B cell clones, named 7G02, produced IgG3 antibodies bound to two of the three colon cell lines.

[0378] Cloning of the colon cancer-specific antibody AT1636 To identify antibodies produced by 7G02, total mRNA was isolated using the TriPure / chloroform method (Roche) according to the manufacturer's instructions. Next, cDNA was generated using reverse transcriptase (SuperScript III, Invitrogen) and a random hexamer (Promega). The IgG variable domains of the heavy and light chains were amplified by PCR (FastStart Taq DNA polymerase, Roche) according to the manufacturer's procedure, applying a leader-specific primer combining CH1 (heavy chain) and Ckappa (light chain) specific primers. The amplicons were used for Sangerdideoxy fluorescence sequencing (BDT, Invitrogen) using congener primers similar to those used for amplification. At least five clones were sequenced to exclude reverse transcriptase or DNA polymerase-induced mutations.

[0379] Next, synthetic codon-optimized DNA fragments (GeneArt) encoding the complete heavy and light chain regions of 7G02 were subcloned into a Double Gene pXC-based expression vector (Lonza). The constructs were checked for integrity by DNA sequencing. From this, the human IgG1 / kappa recombinant antibody for 7G02 was designated as AT1636.

[0380] Next, a pXC dual gene vector was stably transfected into CHO-GS cells to generate a stable pool (GS Xceed platform, Lonza). The stable pool was expanded and used for 7 days of shaking flask, fed batch cell culture for IgG production. The cell-removed supernatant containing recombinant AT1636 antibody was collected and purified using protein A chromatography with an AKTA purification system (General Electric Life Sciences). The antibody was eluted using a buffer of 0.1 M citrate, 150 mM NaCl, pH 3.5, followed by neutralization with 1 M Tris-HCl, pH 9.0, and then re-buffered in TBS-TS by size exclusion chromatography. The concentration was established using a NanoDrop spectrophotometer (OD280, Thermo Fisher). The monomer content of the purified antibody was confirmed to be >90% using size exclusion chromatography. The integrity of the purified protein was established by SDS-PAGE.

[0381] Flow cytometry coupling characteristics of AT1636 Recombinant AT1636 antibody was tested for binding to a panel of cell lines and primary cell material using flow cytometry. Essentially, cells were incubated with antibody solution for 30–60 minutes at 4°C, followed by two washes with 150 μl of PBS 1% BSA. Antibody binding was detected with anti-human IgG-RPE (Southern Biotech) or Alexa Fluor647-conjugated polyclonal BCR antibody (Invitrogen) and analyzed using FACSCanto II or LSRFortessa (Becton Dickinson & Co.). AT1636 shows binding to epithelial cell lines that co-express E-cadherin and TMCT3 (according to the Cancer Cell Line Encyclopedia (https: / / portals.broadinstitute.org / ccle)) (see Table 3).

[0382] Example 2: Target identification of AT1636 Immunoprecipitation To identify the target of the AT1636 antibody, immunoprecipitation (IP) of the target was performed using cells from the colon cancer cell line DLD1 (ATCC CCL-221) and the T cell line Jurkat (negative control). Cells were lysed using a lysis buffer supplemented with protease and phosphatase inhibitors (Roche) (0.5% Triton X114 (Sigma), 0.5% DOC; 0.1% SDS, 150 mM NaCl, 10 mM Tris-HCl pH 7.4, 1.5 mM MgCl2). After lysis, the insoluble fraction was removed by centrifugation. Subsequently, the lysate was pre-cleaned with an unrelated antibody (RSV antibody palivizumab) conjugated to Protein G Dynabeads (Invitrogen) and streptavidin beads (Invitrogen) to remove nonspecific binding proteins. Subsequently, the pre-cleaned lysates were incubated with 50 μg of Protein G Dynabeads-conjugated AT1636 antibody or bead-conjugated influenza-specific antibody AT1002 as a negative control for 3 hours at 4°C. The antibody-incubated beads were washed three times with lysis buffer, and the conjugated proteins were eluted from the beads using 1× SDS-PAGE sample buffer (Bio-Rad) + 0.1 M DTT. The samples were separated on an SDS-PAGE gel. 85% of the IP samples were run on preparative SDS-PAGE, and the immunoprecipitated proteins were visualized by Imperial protein staining (Pierce). Specific immunoprecipitated proteins of 70 kDa and 85 kDa were excised between AT1636 and AT1002 (negative control) immunoprecipitates from DLD1 vs. Jurkat T cells (as a negative control) (see Figure 2a). The bands were subjected to mass spectrometry. Proteins were subjected to reduction with dithiothreitol, alkylation with iodoacetamide, and in-gel trypsin digestion using a Proteiner DP digestion robot (Bruker Daltonics, Bremen, Germany). Trypsin peptides were analyzed by online C18 nanoHPLC MS / MS using a system consisting of an Easy nLC 1000 gradient HPLC system (Thermo, Bremen, Germany) and a LUMOS mass spectrometer (Thermo).Subsequently, the protein was identified by searching the human Uniprot database for mass spectrometry data using the Mascot algorithm (Mascot v2.2.04, Matrix Science). An MS tolerance of 10 ppm and an MS / MS tolerance of 0.02 Da were used. Trypsin was designated as the enzyme of choice, allowing for the absence of up to two cleavage sites. Carbamide methylcysteine ​​was selected as the fixed modification, and methionine oxidation and N-terminal acetylation were selected as variable modifications. The results from the database search were analyzed and visualized using scaffolding (www.proteomesoftware.com). E-cadherin was found to be O-mannosylated. To identify O-mannosylation, modifications of serine and threonine by hexoses were selected as variable modifications. Semitrypsin was used as enzyme specificity to identify the non-trypsin N-terminus.

[0383] Mass spectrometry revealed that the immunoprecipitated protein with AT1636 was a truncated 70 kDa E-cadherin (CDH1, referred to herein as the truncated p70 form) with 24% sequence coverage of E-cadherin in the excised 70 kDa band, while beta-catenin was found in the 85 kDa band (76% protein coverage). The peptide corresponding to the outermost C-terminal domain of E-cadherin was not detected, suggesting a truncated protein (see cartoon of full-length and truncated E-cadherin in Figure 3). Further N-terminal acetylation experiments revealed that the N-terminal residue was glutamic acid 463 (numbered by Uniport P12830 entry).

[0384] Western blot The specific binding of AT1636 to p70E-cadherin was confirmed by Western blotting. AT1636 reactivity was compared with that of commercially available EP700Y (Abcam, rabbit antibody) and a mouse antibody specific to the cytoplasmic domain of E-cadherin (clone 36 / E, BD Biosciences). EP700Y has been shown to bind to the EC5 domain of human E-cadherin and therefore binds both full-length and p70E-cadherin, as is also true for intracellular C-tail antibodies. E-cadherin antibody immunoprecipitation was performed from DLD1 cells using equal volumes of lysate (10 mg) and antibody (2.5 μg). Intake (40 μg) and IP samples (all) were electrophoresed by SDS-PAGE and transferred to PVDF membranes (Bio-Rad) for immunoblotting. When using an antibody that binds the intracellular domain of E-cadherin and the EP700Y antibody, both full-length (120kDa) E-cadherin and 70kDa protein were immunoprecipitated, whereas with AT1636, mainly 70kDa protein was immunoprecipitated (Figure 2b). Therefore, AT1636 preferentially binds p70 to full-length E-cadherin, as indicated by the enrichment of p70. In signal concentration quantification, we observed a 7-fold enrichment of p70 compared to full-length E-cadherin in AT1636 IP compared to EP700Y IP.

[0385] In Figure 3, the figure summarizes the shortening observed in p70, with most of the EC1, EC2, and EC3 domains of the full-length E-cadherin removed, leaving the EC4 and EC5 domains in addition to the short peptide of the D3 domain. Some antibody binding regions and β-catenin interaction domains are also shown.

[0386] Proteolytic cleavage of the AT1636 target To investigate whether E-cadherin is proteolytically cleaved to generate p70, we inhibited furin and related convertases using a furin / convertase inhibitor added to DLD1 cells (decanoyl-RVKR-CMK(Tocris)). Cells were cultured for 48 hours in the absence or presence of the indicated concentrations of the inhibitor and refreshed once. Cells were harvested and subjected to flow cytometry using the indicated antibody (Figure 4). Incubation of DLD1 cells with CMK was reduced but did not completely abolish the binding of AT1636 to DLD1 cells (Figure 4), suggesting that p70 cleavage is partially mediated by furin and other related convertases.

[0387] In addition to the unique cleavage of E-cadherins within the EC3 domain, it is known that E-cadherins can be O-mannosylated (ISBLarsen, PNAS (2017), MBVester-Christensen, PNAS (2013), M. Lommel, PNAS (2013), and S. Carvalho S, Oncotarget (2016)). At least two O-mannosylated threonines (Thr residues 472 and 474), possibly one at position 470, are adjacent to the cleavage site and possible binding domains of AT1636. To study the dependence of p70 O-mannosylation on AT1636 binding, CMK experiments and similar experiments were performed using a mannosyltransferase inhibitor (Mann, oxo-2-thioxo-3-thiazolidinyl acetate, Sigma). The two right-hand columns in Figure 4 show a decrease in AT1636 binding to DLD1 cells treated with Mann, suggesting that p70O-mannosylation within the AT1636 binding region is required for binding.

[0388] Example 3: Generation of high-affinity AT1636 mutant Production of recombinant soluble p70E-cadherin protein Full-length E-cadherin cDNA and p70 E-cadherin cDNA (Figure 1), corresponding to a portion of EC3 up to the N-terminus at position 463, excluding both the EC5 and EC4 domains and both the intracellular and transmembrane (TM) domains, were obtained from GeneArt. Subsequently, proteins containing only the C tag were produced by cloning into pCMV3, pcDNA3, and pXC19 vectors containing a FLAG tag on a mouse F-tail with saltase and HIS tags. The vectors were transiently transfected into Expi293 or CHO cells, and recombinant proteins were purified with a C-tag affinity matrix or Protein A Sepharose. Eluted proteins were rebuffered in TBS-TS by size exclusion chromatography. Concentrations were established using a NanoDrop spectrophotometer (OD280, Thermo Fisher). Monomer content of the purified antibodies was confirmed to be >90% using size exclusion chromatography. Integrity of the purified proteins was established by SDS-PAGE.

[0389] Generation of GFP-highly expressing 7G02 B cells Following the Bcl6 and Bcl-xL genes, the retrovirus used to transduce 7G02 B cells also contained the GFP gene as a reporter for successful B cell transduction. 7G02 B cells were subjected to a second retroviral transduction using retroviruses containing the Bcl6, Bcl-xL, and GFP genes. This resulted in 7G02 B cells with higher GFP expression than the original 7G02 B cells. The high-GFP-expressing 7G02 B cells were subjected to cell sorting using FACSAria III (BD Bioscience) to generate a homogeneous population of 7G02 B cells stably expressing high levels of GFP. The sequences of the heavy and light chain variable domains of the 7G02-GFP-high-cell antibody were determined by isolating total RNA using TriPure / chloroform (Roche / Merck) according to the manufacturer's protocol. cDNA was then generated using reverse transcriptase (Invitrogen). cDNA encoding the variable domains of the antibody heavy and light chains was amplified by PCR using VH and VL primers and subjected to DNA sequencing. The sequences of the variable domains of the antibody heavy and light chains in 7G02-GFP-high B cells were identical to those of 7G02-GFP-low B cells.

[0390] Isolation of 7G02 B cell clones with increased target binding. Using soluble E-cadherin protein, subclones with increased antigen binding compared to the original 7G02 B cell clone were selected using the AIMProve method, as described by Kwakkenbos et al. (MJ Kwakkenbos, Methods (2013)). In short, 7G02 B cell GFP-high clones were expanded, and the proliferated cells were incubated with recombinant soluble E-cadherin mouse Fc fusion protein (see above). Subsequently, the cells were washed and co-incubated with Alexa Fluor647-conjugated polyclonal antibody (Invitrogen) that specifically binds the heavy and light chains of the B cell receptor (BCR) to assess BCR expression levels, and the bound E-cadherin protein was visualized using R-phycoerythrin-labeled polyclonal anti-mouse Fc antibody (Jackson ImmunoResearch). Cells were analyzed by flow cytometry, and cells showing higher recombinant E-cadherin protein binding in relation to BCR expression compared to the mean 7G02 B cell population were classi...

Claims

[Claim 1] An antibody or antigen-binding fragment that specifically binds one or more O-mannosylated threonine residues of an E-cadherin, wherein the one or more O-mannosylated threonine residues are located within amino acid positions 467 to 472 of the E-cadherin sequence as shown in Figure 1A.

Images