Anti-glycemic-MUC1 antibodies and their uses

Abstract

This disclosure relates to anti-glyco-MUC1 antibodies that specifically bind to cancer-specific glycosylated variants of MUC1, their antigen-binding fragments and associated fusion proteins and antibody-drug conjugates, as well as nucleic acids encoding such biomolecules. This disclosure also relates to the use of antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates, and nucleic acids in cancer therapy.

Description

[0001] 1. Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 691,887, filed June 29, 2018, and U.S. Provisional Application No. 62 / 802,865, filed February 8, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence List

[0004] This application contains a sequence list, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 26, 2019, and is named GOT-002WO_SL, with a size of 33,365 bytes. 3. Background Technology

[0006] Human mucin MUC1 is a polymorphic transmembrane glycoprotein expressed on the apical surface of monolayer epithelium and glandular epithelium (Taylor-Papadimitriou et al., 1999, Biochim. Biophys. Acta, 1455:301–313). MUC1 is highly overexpressed and aberrantly O-glycosylated in adenocarcinoma. The extracellular domain of the mucin contains a variable number of tandem repeats (TRs) of 20 amino acid residues (25–125), which have five potential O-glycosylation sites. O-glycans are incompletely processed in cancer cells, leading to the expression of the pan-cancer carbohydrate antigen Tn (GalNAcα1-O-Ser / Thr) (Springer, 1984, Science 224:1198–1206). Simple mucin-type O-glycans (Tn) are widely expressed in adenocarcinomas (including breast and ovarian cancers) but show limited distribution in normal adult tissues (Springer, 1984, Science 224:1198–1206). Expression of these O-glycans in cancer is associated with poor prognosis, and there is an increase in natural antibodies against these carbohydrate haptens in cancer patients (Miles, et al., 1995, Br. J. Cancer. 71:1074–1076; Soares et al., 1996, Pathol. Res. Pract. 192:1181–1186; Werther et al., 1996, Int. J. Cancer. 69:193–199). There is a need in the art for therapeutic approaches utilizing glyco-MUC1 epitopes overexpressed in cancer cells. 4. Overview of the Invention

[0008] This disclosure captures tumor specificity of glycopeptide variants by providing therapeutic and diagnostic agents based on antibodies and antigen-binding fragments that are selective for cancer-specific epitopes of glyco-MUC1.

[0009] This disclosure provides anti-glyco-MUC1 antibodies that bind to cancer-specific glycosylated variants of MUC1 and their antigen-binding fragments. This disclosure also provides fusion proteins and antibody-drug conjugates comprising anti-glyco-MUC1 antibodies and antigen-binding fragments, as well as nucleic acids encoding anti-glyco-MUC1 antibodies, antigen-binding fragments, and fusion proteins.

[0010] This disclosure also provides methods for using anti-glyco-MUC1 antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates, and nucleic acids in cancer therapy.

[0011] In some aspects, this disclosure provides bispecific and other multispecific anti-glyco-MUC1 antibodies and antigen-binding fragments that bind to cancer-specific glycosylated variants and second epitopes of MUC1. The second epitope may be on MUC1 itself, on another protein co-expressed with MUC1 on cancer cells, or on another protein presented on different cells, such as activated T cells. Furthermore, nucleic acids encoding such antibodies are disclosed, including nucleic acids containing codon-optimized coding regions and nucleic acids containing coding regions not codon-optimized for expression in specific host cells.

[0012] Anti-glyco-MUC1 antibodies and binding fragments can be in the form of fusion proteins containing fusion couplers. Fusion couplers can be used to provide a secondary function, such as signal transduction function of the signal transduction domain of a T cell signaling protein, a peptide regulator of T cell activation, or an enzymatic component of a labeling system. Exemplary T cell signaling proteins include 4-1BB, CO3C, and fusion peptides such as CD28-CD3-ζ and 4-1BB-CD3-ζ. 4-1BB or CD137 is a co-stimulatory receptor for T cells; CD3-ζ is a signal transduction component of a T cell antigen receptor. The portion providing the secondary function can be a regulator of T cell activation, such as IL-15, IL-15Ra, or an IL-15 / IL-15Ra fusion, or it can encode an enzymatic component of a labeling system for labeling or monitoring the extent and / or location of binding in vivo or in vitro. In some embodiments of this disclosure, constructs encoding these preventative and therapeutic biomolecules located in the context of T cells, such as autologous T cells, provide a powerful platform for recruiting adopted T cells to prevent or treat a variety of cancers.

[0013] In some respects, the anti-glyco-MUC1 antibodies and antigen-binding fragments of this disclosure comprise the heavy chain variable sequences and / or light chain variable sequences (or encoded by nucleotide sequences) shown in Tables 1A and 1B. For clarity, when the term "anti-glyco-MUC1 antibody" is used herein, unless the context otherwise requires, it is intended to include monospecific and multispecific (including bispecific) anti-glyco-MUC1 antibodies, antigen-binding fragments of monospecific and multispecific antibodies, and fusion proteins and conjugates containing antibodies and their antigen-binding fragments. Similarly, when the term "anti-glyco-MUC1 antibody or antigen-binding fragment" is used, unless the context otherwise requires, it is also intended to include monospecific and multispecific (including bispecific) anti-glyco-MUC1 antibodies and their antigen-binding fragments, and fusion proteins and conjugates containing such antibodies and antigen-binding fragments.

[0014] In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain and / or light chain CDR sequences (or encoded by nucleotide sequences) listed in Tables 1-3. The CDR sequences listed in Tables 1A and 1B include CDR sequences as defined by the IMGT (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77), Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and Chothia (Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948) protocols used to define CDR boundaries. The CDR sequences listed in Tables 1C, 1D, and 1E are common sequences derived from the CDR sequences listed in Tables 1A and 1B according to the definitions of IMGT, Kabat, and Chothia, respectively. The CDR sequences listed in Tables 2A and 2B are overlapping combination regions of the CDR sequences listed in Tables 1A and 1B, where the IMGT, Kabat, and Chothia sequences are shown in bold underlined text. The CDR sequences listed in Table 2C are overlapping combination regions of the common CDR sequences listed in Tables 1C, 1D, and 1E. The CDR sequences listed in Tables 3A and 3B are overlapping common regions of the CDR sequences shown in Tables 1A and 1B, respectively. The CDR sequences listed in Table 3C are overlapping common regions of the CDR sequences listed in Tables 1C, 1D, and 1E. The frame sequence of such anti-glycan-MUC1 antibody and antigen-binding fragment can be a natural mouse frame sequence of the VH and VL sequences listed in Table 1A or Table 1B, or it can be a non-natural (e.g., humanized or human) frame sequence.

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[0034] In some aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises a CDR containing the amino acid sequence of any combination of CDRs listed in embodiments 3 to 41. Therefore, in some embodiments, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises CDR-H1 containing the amino acid sequence of SEQ ID NO:93, CDR-H2 containing the amino acid sequence of SEQ ID NO:94, CDR-H3 containing the amino acid sequence of SEQ ID NO:95, CDR-L1 containing the amino acid sequence of SEQ ID NO:96, CDR-L2 containing the amino acid sequence of SEQ ID NO:97, and CDR-L3 containing the amino acid sequence of SEQ ID NO:98. In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, 9, 15, 25, 31, 37, 45, 51, 57, 63, 69, 75, 81, 87, or 93. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 4, 10, 16, 26, 32, 38, 46, 52, 58, 64, 70, 76, 82, 88, or 94. In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 5, 11, 17, 27, 33, 39, 47, 53, 59, 65, 71, 77, 83, 89, or 95. In some embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 6, 12, 18, 28, 34, 40, 48, 54, 60, 66, 72, 78, 84, 90, or 96. In some embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 7, 13, 19, 29, 35, 41, 49, 55, 61, 67, 73, 79, 85, 91, or 97. In some implementations, CDR-L3 contains the amino acid sequence of SEQ ID NO: 8, 14, 20, 30, 36, 42, 50, 56, 62, 68, 74, 80, 86 or 92.

[0035] In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 3-5 and the light chain CDR of SEQ ID NOS: 6-8. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 9-11 and the light chain CDR of SEQ ID NOS: 12-14. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 15-17 and the light chain CDR of SEQ ID NOS: 18-20. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 25-27 and the light chain CDR of SEQ ID NOS: 28-30. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 31-33 and the light chain CDR of SEQ ID NOS: 34-36. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 37-39 and the light chain CDR of SEQ ID NOS: 40-42. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 45-47 and the light chain CDR of SEQ ID NOS: 48-50. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 51-53 and the light chain CDR of SEQ ID NOS: 54-56. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 57-59 and the light chain CDR of SEQ ID NOS: 60-62. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 63-65 and the light chain CDR of SEQ ID NOS: 66-68. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 69-71 and the light chain CDR of SEQ ID NOS: 72-74. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 75-77 and the light chain CDR of SEQ ID NOS: 78-80. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No: 81-83 and the light chain CDR of SEQ ID NOS: 84-86.In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain CDR of SEQ ID No:87-89 and the light chain CDR of SEQ ID NOS:90-92.

[0036] The antibody and antigen binding fragment disclosed herein may be mouse, chimeric, humanized, or human.

[0037] In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure competes with antibody or antigen-binding fragments comprising heavy chain and light chain variable regions, respectively, SEQ ID NO: 1 and 2. In other respects, this disclosure provides an anti-MUC1 antibody or antigen-binding fragment having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1 and 2, respectively.

[0038] In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure competes with antibody or antigen-binding fragments comprising heavy and light chain variable regions, respectively, SEQ ID NO: 23 and 24. In other respects, this disclosure provides an anti-MUC1 antibody or antigen-binding fragment having heavy and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 23 and 24, respectively.

[0039] In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment disclosed herein is a single-chain variable fragment (scFv). An exemplary scFv comprises a heavy chain variable fragment located at the N-terminus of a light chain variable fragment. In some embodiments, the scFv heavy chain variable fragment and light chain variable fragment are covalently bound to an adapter sequence of 4-15 amino acids. The scFv may be in the form of a bispecific T-cell adaptor or within a chimeric antigen receptor (CAR).

[0040] The anti-glyco-MUC1 antibody and antigen-binding fragment can be in the form of a multimer of a single-chain variable fragment, a bispecific single-chain variable fragment, or a multimer of a bispecific single-chain variable fragment. In some embodiments, the multimer of the single-chain variable fragment is selected from a divalent single-chain variable fragment, a tribody, or a tetrabody. In some of these embodiments, the multimer of the bispecific single-chain variable fragment is a bispecific T-cell adaptor.

[0041] Other aspects of this disclosure relate to nucleic acids encoding the anti-glyco-MUC1 antibody and antigen-binding fragment of this disclosure. In some embodiments, the portion of the nucleic acid encoding the anti-glyco-MUC1 antibody or antigen-binding fragment is codon-optimized for expression in human cells. In some aspects, this disclosure provides an anti-glyco-MUC1 antibody or antigen-binding fragment having variable regions of heavy and light chains, said variable regions being encoded by a heavy chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:21 or SEQ ID NO:43 and a light chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:22 or SEQ ID NO:44. Vectors containing nucleic acids (e.g., viral vectors, such as lentiviral vectors) and host cells are also within the scope of this disclosure. The heavy and light chain coding sequences may be present in a single vector or separate vectors.

[0042] Another aspect of this disclosure is a pharmaceutical composition comprising an anti-glucan-MUC1 antibody according to this disclosure, an antigen-binding fragment, a nucleic acid (or a pair of nucleic acids), a carrier (or a pair of carriers) or a host cell and a physiologically suitable buffer, adjuvant or diluent.

[0043] Another aspect of this disclosure is a method for preparing a chimeric antigen receptor, which includes incubating cells containing a nucleic acid or vector according to this disclosure under conditions suitable for expression of the coding region, and collecting the chimeric antigen receptor.

[0044] Another aspect of this disclosure is a method for detecting cancer, which includes contacting a cell or tissue sample with an anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure and detecting whether the antibody binds to the cell or tissue sample.

[0045] Another aspect of this disclosure is the use of anti-glycemic-MUC1 antibodies or antigen-binding fragments according to this disclosure for the detection of cancer.

[0046] Another aspect of this disclosure is a method of treating cancer, comprising administering to a subject in need a preventive or therapeutically effective amount of an anti-glycemic-MUC1 antibody, antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition according to this disclosure.

[0047] Another aspect of this disclosure is the use of anti-glycemic-MUC1 antibodies, antigen-binding fragments, nucleic acids, vectors, host cells, or pharmaceutical compositions according to this disclosure for the treatment of cancer.

[0048] Another aspect of this disclosure is the use of an anti-glycemic-MUC1 antibody, antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition according to this disclosure in the preparation of a medicament for treating cancer.

[0049] 5. Brief description of the attached drawings

[0050] Figure 1A -EF: Results of ELISA assays showed the specificity of 1AG and 4AG binding to glycosylated and non-glycosylated MUC1 peptides. Figure 1A : Peptide C2; Figure 1B : Peptide C3; Figure 1C Peptide C4; Figure 1D Peptide 70; Figure 1E GST; Figure 1F : BSA closed well (control).

[0051] Figure 2: Results of ELISA assay, showing 1AG ( Figure 2A ) and 4AG ( Figure 2B The specificity of binding to different glycosylated and non-glycosylated MUC1 peptides.

[0052] Figure 3 Results of antibody titration assays against antigen GSTA using 1AG and 4AG.

[0053] Figure 4 Results of immunohistochemical screening of antibody 1AG using tumor microarray BCN721a.

[0054] Figure 5 Results of antibody 4AG immunohistochemical screening using tumor microarray BCN721a.

[0055] Figure 6 Results of immunohistochemical screening of antibody 1AG using tumor microarray OV241c.

[0056] Figure 7 Results of antibody 4AG immunohistochemical screening using tumor microarray OV241c.

[0057] Figure 8 Results of immunohistochemical screening of antibody 1AG using tumor microarray BC000119.

[0058] Figure 9 Results of antibody 4AG immunohistochemical screening using tumor microarray BC000119.

[0059] Figure 10 Results of alanine scanning using antibody 5E5.

[0060] Figure 11 Results of alanine scanning using antibody 1AG.

[0061] Figure 12 Results of alanine scanning using antibody 4AG. 6. Detailed Description of the Invention

[0063] 6.1 Antibody

[0064] This disclosure provides novel antibodies targeting the glycoform of MUC1 present on tumor cells. These antibodies are exemplified by antibodies 1AG and 4AG. 1AG and 4AG were identified in antibody screening; these antibodies bind to the glycosylated 15-mer present in MUC1, i.e. It is glycosylated by GalNAc on the serine and threonine residues shown in bold underlined text to mimic the glycosylation pattern of MUC1 present on tumor cells.

[0065] The anti-glyco-MUC1 antibodies disclosed herein, taking antibodies 1AG and 4AG as examples, can be used as tools in cancer diagnosis and therapy.

[0066] Therefore, in some respects, this disclosure provides for binding to the glycoform of MUC1 present on tumor cells (referred to herein as "glyco-MUC1"), and preferably to the 15-mer peptide. The bound antibody and antigen-binding fragment, the 15-mer peptide is glycosylated with GalNAc on the serine and threonine residues shown in bold underlined text.

[0067] The anti-glyco-MUC1 antibody disclosed herein may be polyclonal, monoclonal, genetically engineered, and / or otherwise modified in nature, including but not limited to chimeric antibodies, humanized antibodies, human antibodies, primate antibodies, single-chain antibodies, bispecific antibodies, and antibodies with bivariate domains. In various embodiments, the antibody comprises all or part of the antibody constant region. In some embodiments, the constant region is an isotype selected from: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. In a specific embodiment, the anti-glyco-MUC1 antibody of this disclosure comprises an IgG1 constant region isotype.

[0068] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies are derived from a single clone by any means available or known in the art, including any eukaryotic, prokaryotic, or phage clone. A variety of techniques known in the art can be used to prepare monoclonal antibodies usable in this disclosure, including hybridoma technology, recombinant technology, and phage display technology, or combinations thereof. Among the various uses of this disclosure, including in vivo use of anti-glyco-MUC1 antibodies in humans, chimeric antibodies, primate-derived antibodies, humanized antibodies, or human antibodies may be suitably used.

[0069] As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region typically selected from a human immunoglobulin template. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gilles et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, all of which are incorporated herein by reference in their entirety.

[0070] "Humanized" forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins containing minimal sequences derived from non-human immunoglobulins. Typically, humanized antibodies will contain at least one, and usually two, substantially entire variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin sequences. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically a portion of the common sequence of human immunoglobulins. Methods for antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; Queen et al., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370; EP239400; PCT Publication WO 91 / 09967; U.S. Patent Nos. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al. al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332, the entire contents of which are incorporated herein by reference.

[0071] "Human antibodies" include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from human immunoglobulin libraries or from animals transgenic against one or more human immunoglobulins that do not express endogenous immunoglobulins. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98 / 46645; WO 98 / 50433; WO 98 / 24893; WO 98 / 16654; WO 96 / 34096; WO 96 / 33735; and WO 91 / 10741, each of which is incorporated herein by reference in its entirety. Human antibodies can also be generated using transgenic mice that do not express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT publications WO98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated herein by reference in their entirety. A technique known as “guided selection” can be used to generate fully human antibodies that recognize selected epitopes. In this approach, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (see, Jespers et al., 1988, Biotechnology 12:899-903).

[0072] "Primate-like antibodies" comprise monkey variable regions and human constant regions. Methods for generating primate-like antibodies are known in the art. See, for example, U.S. Patent Nos. 5,658,570; 5,681,722; and 5,693,780, which are incorporated herein by reference in their entirety.

[0073] The disclosed anti-glyco-MUC1 antibody comprises both a full-length (intact) antibody molecule and an antigen-binding fragment capable of binding to glyco-MUC1. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments, and single-domain fragments.

[0074] The Fab fragment contains a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. The F(ab') fragment is generated by cleaving the disulfide bond at the hinge cysteine ​​residue of the F(ab')2 pepsin digestion product. Other chemical conjugations of antibody fragments are known to those skilled in the art. The Fab and F(ab')1 fragments lack the Fc fragment of the intact antibody, are cleared from animal circulation more quickly, and may exhibit less nonspecific tissue binding than the intact antibody (see, for example, Wahl et al., 1983, J. Nucl. Med. 24:316).

[0075] The “Fv” fragment is the smallest fragment of an antibody containing complete target recognition and binding sites. This region consists of a dimer (V) of a tightly nonvalently associated heavy chain variable domain and a light chain variable domain. H -V L It consists of a dimer. In this configuration, the three CDRs of each variable domain interact to form a V H -V L The surface of the dimer defines the target binding site. Typically, the six CDRs confer target-antibody binding specificity. However, in some cases, even a single variable domain (or half the Fv of only three target-specific CDRs) can recognize and bind to the target, although its affinity is lower than that of the entire binding site.

[0076] A "single-chain Fv" or "scFv" antigen-binding fragment contains the antibody's V. H and V L Domains, which are present within a single polypeptide chain. Typically, Fv polypeptides also contain domains located in V... H and V L The peptide linkers between the domains enable scFv to form the desired structure for target binding.

[0077] "Single-domain antibody" consists of a single V that exhibits sufficient affinity for sugar-MUC1. H or V L Domain composition. In a specific implementation, the single-domain antibody is a camel-like antibody (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0078] The anti-glyco-MUC1 antibody disclosed herein can also be a bispecific antibody and other multispecific antibodies. A bispecific antibody is a monoclonal antibody that has binding specificity to two different epitopes on the same or different antigens, typically a human antibody or a humanized antibody. In this disclosure, one binding specificity may target glyco-MUC1, and the other may target any other antigen, such as a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virus-derived protein, virus-encoded envelope protein, bacterial-derived protein, or bacterial surface protein. In some preferred embodiments, the bispecific and other multispecific anti-glyco-MUC1 antibodies and antigen-binding fragments specifically bind to a second MUC1 epitope, an epitope on another protein co-expressed with MUC1 on cancer cells, or an epitope on another protein presented on different cells, such as activated T cells. The bispecific antibodies disclosed herein include bispecific antibodies in the form of IgG and single-chain-based bispecific antibodies.

[0079] The IgG bispecific antibody disclosed herein can be any of the various types of IgG bispecific antibodies known in the art, such as tetravalent hybridoma bispecific antibodies, "knobs-in-holes" bispecific antibodies, CrossMab bispecific antibodies, charge-paired bispecific antibodies, common light chain bispecific antibodies, single-arm single-chain Fab-immunoglobulin γ bispecific antibodies, disulfide-stabilized Fv bispecific antibodies, DuetMab, controlled Fab arm exchange bispecific antibodies, chain exchange engineered domain body bispecific antibodies, two-arm leucine zipper heterodimer monoclonal bispecific antibodies, κλ body bispecific antibodies, bivariate domain bispecific antibodies, and cross-bivariate domain bispecific antibodies. See, for example, and Milstein, 1975, Nature 256:495-497; Milstein and Cuello, 1983, Nature 305:537-40; Ridgway et al., 1996, Protein Eng. 9: 617-621; Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92; Gunasekaran et al., 2010, J Biol Chem 285:19637-46; Fischer et al., 2015Nature Commun 6:6113; Schanzer et al., 2014, J Biol Chem 289:18693–706; Metz et al., 2012 Protein EngDes Sel 25:571–80; Mazor et al., 2015 MAbs7:377–89; Labrijn et al., 2013 ProcNatl Acad Sci USA 110:5145–50; Davis et al., 2010 Protein Eng Des Sel 23:195–202; Wranik et al., 2012, J Biol Chem 287:43331–9; Gu et al., 2015, PLoS One 10(5):e0124135; Steinmetz et al., 2016, MAbs 8(5):867-78; Klein et al., 2016, mAbs, 8(6): 1010-1020; Liu et al., 2017, Front. Immunol. 8:38; and Yang et al. al., 2017, Int. J. Mol. Sci. 18:48, which is incorporated herein by reference in its entirety.

[0080] In some embodiments, the bispecific antibody of this disclosure is CrossMab. CrossMab technology is described in detail in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2013 / 026833, WO 2016 / 020309 and Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92, which are incorporated herein by reference in their entirety. In short, CrossMab technology is based on the cross-linking of domains between the heavy and light chains within one Fab arm of a bispecific IgG, thereby promoting proper chain association. The CrossMab bispecific antibody of this disclosure may be “CrossMab…” FAB "Antibody, wherein the heavy and light chains of the Fab portion of one arm of a bispecific IgG antibody are exchanged. In other embodiments, the CrossMab bispecific antibody of this disclosure may be a "CrossMab..." VH-VL "Antibody, wherein the variable domains of the heavy and light chains of the Fab portion of only one arm of the bispecific IgG antibody are exchanged. In other embodiments, the CrossMab bispecific antibody of this disclosure may be "CrossMab..." CH1-CL "Antibodies in which the constant domains of the heavy and light chains of the Fab portion of only one arm of a bispecific IgG antibody are exchanged. With CrossMab..." FAB and CrossMab VH-VL In comparison, CrossMab CH1-CL The antibody does not produce predicted byproducts; therefore, in some implementations, CrossMab... CH1-CL Bispecific antibodies are preferred. See Klein et al., 2016, mAbs, 8(6): 1010-1020.

[0081] In some embodiments, the bispecific antibody of this disclosure is a controlled Fab arm exchange bispecific antibody. Methods for preparing Fab arm exchange bispecific antibodies are described in PCT Publication No. WO2011 / 131746 and Labrijn et al., 2014 Nat Protoc. 9(10):2450-63, which are incorporated herein by reference in their entirety. In short, a controlled Fab arm exchange bispecific antibody can be prepared by expressing two parental IgG1 molecules containing a single matching point mutation in the CH3 domain, mixing the parental IgG1 molecules under in vitro redox conditions to allow half-molecule recombination, and removing the reducing agent to allow for re-oxidation of interchain disulfide bonds, thereby forming a bispecific antibody.

[0082] The bispecific antibody disclosed herein may comprise an Fc domain consisting of a first subunit and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228 (Kabat EU index number), specifically amino acid substitution S228P. Unless otherwise stated herein, the amino acid residues in the Fc domain or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. This amino acid substitution reduces in vivo Fab arm exchange of the IgG4 antibody (see Stubenrauch et al., 2010, Drug Metabolism and Disposition 38:84-91). In a more specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of the human IgG1 Fc region is given in SEQ ID NO:42.

[0083] In a specific embodiment, the Fc domain includes modifications that promote association between the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain. Therefore, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0084] In a specific implementation, the modification promoting association between the first and second subunits of the Fc domain is a so-called "protrusion-in-pore" modification, which includes a "protrusion" modification in one of the two subunits of the Fc domain and a "pore" modification in the other of the two subunits of the Fc domain. Protrusion-in-pore techniques are described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621; and Carter, J, 2001, Immunol Meth 248:7-15. Typically, this method involves introducing a protrusion ("protrusion") at the interface of a first polypeptide and a corresponding cavity ("pore") at the interface of a second polypeptide, such that the protrusion can be placed within the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine), a compensation cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide.

[0085] Therefore, in some embodiments, amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit. This protrusion can be positioned within a cavity in the CH3 domain of the second subunit. Conversely, amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit. The protrusion in the CH3 domain of the first subunit can be positioned within this cavity. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis.

[0086] In specific embodiments of this kind, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (according to Kabat EU index number). In other embodiments, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine ​​residue (E356C) (specifically, the serine residue at position 354 is replaced with a cysteine ​​residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C) (according to Kabat EU index number). In a specific implementation, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (according to Kabat EU index numbers).

[0087] In some implementations, electrostatic manipulation (e.g., as described in Gunasekaran et al., 2010, J BiolChem 285(25):19637-46) can be used to promote the association of the first and second subunits of the Fc domain.

[0088] In some implementations, the Fc domain contains one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0089] In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0090] Typically, one or more identical amino acid substitutions are present in each of the two subunits of the Fc domain. In one embodiment, one or more amino acid substitutions reduce the binding affinity of the Fc domain to the Fc receptor. In one embodiment, one or more amino acid substitutions reduce the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.

[0091] In one embodiment, the Fc domain contains an amino acid substitution at a position selected from E233, L234, L235, N297, P331, and P329 (according to Kabat EU index numbers). In a more specific embodiment, the Fc domain contains an amino acid substitution at a position selected from L234, L235, and P329 (according to Kabat EU index numbers). In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A (according to Kabat EU index numbers). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly the human IgG1 Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (according to Kabat EU index numbers). In one embodiment, the Fc domain comprises an amino acid substitution at position P329, and other amino acid substitutions selected from positions E233, L234, L235, N297, and P331 (according to Kabat EU index numbers). In a more specific embodiment, the other amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (according to Kabat EU index numbers). In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”). Specifically, in a particular embodiment, each subunit of the Fc domain contains amino acid substitutions L234A, L235A, and P329G (Kabat EU index number), that is, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (according to the Kabat EU index number). In one such embodiment, the Fc domain is the IgG1 Fc domain, particularly the human IgG1 Fc domain.

[0092] The single-chain-based bispecific antibody disclosed herein can be any of the various types of single-chain-based bispecific antibodies known in the art, such as bispecific T-cell adaptors (BiTE), biantibodies, tandem biantibodies (tandab), dual-affinity retargeting molecules (DART), and bispecific cytotoxic cell adaptors. See, for example, et al., 2000, Blood 95:2098–103; Holliger et al., 1993, Proc Natl Acad Sci USA, 90:6444–8; Kipriyanov et al., 1999, Mol Biol 293:41–56; Johnson et al., 2010, Mol Biol 399:436–49; Wiernik et al., 2013, Clin Cancer Res 19:3844–55; Liu et al., 2017, Front. Immunol. 8:38; and Yang et al., 2017, Int. J. Mol. Sci. 18:48, which are incorporated herein by reference in their entirety.

[0093] In some embodiments, the bispecific antibody of this disclosure is a bispecific T-cell adaptor (BiTE). A BiTE is a single polypeptide chain molecule having two antigen-binding domains, one of which binds to a T-cell antigen, and the second of which binds to an antigen present on a target surface (see PCT disclosure WO 05 / 061547; Baeuerle et al., 2008, Drugs of the Future 33:137-147; Bargou, et al., 2008, Science 321:974-977, which are incorporated herein by reference in their entirety). Therefore, the BiTE of this disclosure has an antigen-binding domain that binds to a T-cell antigen and a second antigen-binding domain targeting glyco-MUC1.

[0094] In some embodiments, the bispecific antibody of this disclosure is a dual-affinity retargeting molecule (DART). A DART comprises at least two polypeptide chains that bind (particularly through covalent interactions) to form at least two epitope-binding sites that can recognize the same or different epitopes. Each polypeptide chain of a DART contains a variable region of an immunoglobulin light chain and a variable region of an immunoglobulin heavy chain, but these regions do not interact to form epitope-binding sites. Instead, the variable region of the immunoglobulin heavy chain of one (e.g., the first) DART polypeptide chain binds to the variable region of a different (e.g., the second) DART polypeptide chain. TMThe variable regions of the immunoglobulin light chains of polypeptide chains interact to form epitope binding sites. Similarly, the variable region of one (e.g., the first) immunoglobulin light chain of a DART polypeptide chain interacts with the variable regions of the immunoglobulin heavy chains of a different (e.g., the second) DART polypeptide chain to form epitope binding sites. DARTs can be monospecific, bispecific, trispecific, etc., and therefore can bind one, two, three, or more different epitopes (which can be the same or different antigens) simultaneously. DARTs can also be monovalent, divalent, trivalent, tetravalent, pentavalent, hexavalent, etc., and therefore can bind one, two, three, four, five, six, or more molecules simultaneously. These two properties of DARTs (i.e., specificity and titer) can be combined, for example, to produce tetravalent (i.e., capable of binding four sets of epitopes) bispecific antibodies (i.e., capable of binding two epitopes), etc. The DART molecule is disclosed in PCT Publications WO2006 / 113665, WO 2008 / 157379 and WO 2010 / 080538, which are incorporated herein by reference in their entirety.

[0095] In some embodiments of the bispecific antibodies disclosed herein, one binds specifically to glyco-MUC1, while the other binds to an antigen expressed on immune effector cells. As used herein, the term "immune effector cell" or "effector cell" refers to cells in the natural pool of cells in the mammalian immune system that can be activated to influence the viability of target cells. Immune effector cells include lymphoid lineage cells, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, but myeloid lineage cells can also be considered immune effector cells, such as monocytes or macrophages, dendritic cells, and neutrophils. Therefore, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophils. Recruitment of effector cells to abnormal cells means that the immune effector cells are brought near the abnormal target cells, allowing the effector cells to directly kill the recruited abnormal cells or indirectly induce the killing of the recruited abnormal cells. To avoid nonspecific interactions, preferably, the bispecific antibody of this disclosure specifically recognizes antigens on immune effector cells that are at least overexpressed by these immune effector cells compared to other cells in the body. Target antigens present on immune effector cells may include CD3, CD8, CD16, CD25, CD28, CD64, CD89, NKG2D, and NKp46. Preferably, the antigen on immune effector cells is CD3 expressed on T cells.

[0096] As used herein, unless otherwise stated, “CD3” means any naturally occurring CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers “full-length” unprocessed CD3 as well as any form of CD3 produced through cellular processing. The term also covers naturally occurring variants of CD3, such as splice variants or allelic variants. The most preferred antigen on immune effector cells is the CD3ε chain. This antigen has shown to be highly effective in recruiting T cells to abnormal cells. Therefore, the bispecific antibody of this disclosure preferably specifically recognizes CD3ε. The amino acid sequence of human CD3ε is shown as UniProt (www.uniprot.org) accession number P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey (Macaca fascicularis) CD3ε is shown in NCB GenBank no. BAB71849.1. For therapeutic use in humans, a bispecific antibody in which the CD3-binding domain specifically binds to human CD3, such as the human CD3ε chain, is used. For preclinical testing in non-human animals and cell lines, a bispecific antibody in which the CD3-binding domain specifically binds to CD3 in the species used for preclinical testing (e.g., cynomolgus monkey CD3 used for primate testing) can be used.

[0097] As used herein, “specific binding” or “specific recognition” means that the binding domain of a target antigen from a specific species does not preclude binding or recognition of antigens from other species, and therefore encompasses antibodies with one or more binding domains exhibiting interspecies cross-reactivity. For example, the CD3 binding domain of human CD3, which “specifically binds” or “specifically recognizes”, can also bind to or recognize cynomolgus monkey CD3, and vice versa.

[0098] In some embodiments, the bispecific antibody of this disclosure can compete with monoclonal antibody H2C (described in PCT Publication No. WO2008 / 119567) for binding to the CD3 epitope. In other embodiments, the bispecific antibody of this disclosure can compete with monoclonal antibody V9 (described in Rodrigues et al., 1992, Int J Cancer Suppl 7:45-50 and U.S. Patent No. 6,054,297) for binding to the CD3 epitope. In other embodiments, the bispecific antibody of this disclosure can compete with monoclonal antibody FN18 (described in Nooij et al., 1986, Eur J Immunol 19:981-984) for binding to the CD3 epitope. In other embodiments, the bispecific antibody of this disclosure can compete with monoclonal antibody SP34 (described in Pessano et al., 1985, EMBO J 4:337-340) for binding to the CD3 epitope.

[0099] The anti-glyco-MUC1 antibodies disclosed herein include derivatized antibodies. For example, but not as a limitation, derivatized antibodies are typically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. Any of the various chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, the derivatives may contain one or more non-natural amino acids, for example, using the ambrx technique (see, for example, Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0100] Anti-glyco-MUC1 antibodies or binding fragments may be antibodies or fragments whose sequences have been modified to alter the function of at least one constant region-mediated biological effector. For example, in some embodiments, anti-glyco-MUC1 antibodies may be modified to reduce the function of at least one constant region-mediated biological effector relative to unmodified antibodies, such as reducing binding to the Fc receptor (FcγR). FcγR binding can be reduced by mutating the immunoglobulin constant region fragment of the antibody in a specific region necessary for FcγR interaction (see, for example, Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662). Reduction in the FcγR binding capacity of the antibody may also reduce other effector functions dependent on FcγR interaction, such as opsonization, phagocytosis, and antigen-dependent cytotoxicity (“ADCC”).

[0101] The anti-glyco-MUC1 antibodies or binding fragments described herein include antibodies and / or binding fragments that have been modified to acquire or improve biological effector functions mediated by at least one constant region relative to unmodified antibodies, such as enhancing FcγR interactions (see, for example, US 2006 / 0134709). For example, the anti-glyco-MUC1 antibodies of this disclosure may have constant regions that bind FcγRIIA, FcγRIIB, and / or FcγRIIIA with higher affinity than the corresponding wild-type constant regions.

[0102] Therefore, the antibodies of this disclosure may have altered biological activity, resulting in an increase or decrease in opsonization, phagocytosis, or ADCC. Such alterations are known in the art. For example, antibody modifications that reduce ADCC activity are described in U.S. Patent No. 5,834,597. Exemplary variants that reduce ADCC correspond to "Mutant 3" (shown in U.S. Patent No. 5,834,597). Figure 4 In this variant, residue 236 is deleted, and residues 234, 235, and 237 (using EU numbers) are replaced with alanine. Another exemplary variant reducing ADCC comprises amino acid mutations L234A, L235A, and P329G (“P329G LALA”). The amino acid-substituted “P329G LALA” combination almost completely eliminates Fcγ receptor (and its complementor) binding to the human IgG1 Fc domain, as described in PCT Publication No. WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties, such as Fc receptor binding or effector function.

[0103] In some embodiments, the anti-glycan-MUC1 antibody of this disclosure has low levels of fucose or is fucose-free. Fucose-free antibodies are associated with enhanced ADCC activity, especially at low doses. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. A method for preparing fucose-free antibodies involves growth in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme essential for peptide fucosylation.

[0104] In some embodiments, the anti-glyco-MUC1 antibody or binding fragment comprises a bisected oligosaccharide, for example, wherein a biantennary oligosaccharide attached to the Fc domain is bisected by GlcNAc. As described above, such variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in Umana et al., 1999, Nat Biotechnol 17:176-180; Ferrara et al., 2006, BiotechnBioeng 93:851-861; WO 99 / 54342; WO 2004 / 065540; and WO 2003 / 011878.

[0105] On the other hand, the anti-glyco-MUC1 antibody or binding fragment includes modifications that increase or decrease its binding affinity to the fetal Fc receptor FcRn, for example, by mutating a segment of the immunoglobulin constant region involved in FcRn interaction (see, for example, WO 2005 / 123780). In a specific embodiment, the anti-glyco-MUC1 antibody of the IgG class is mutated such that at least one of amino acid residues 250, 314, and 428 in the heavy chain constant region is substituted individually, or in any combination thereof, for example at positions 250 and 428, or at positions 250 and 314, or at positions 314 and 428, or at positions 250, 314, and 428, wherein positions 250 and 428 are a specific combination. For position 250, the substituted amino acid residue can be any amino acid residue other than threonine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. For position 314, the substituted amino acid residue can be any amino acid residue other than leucine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. For position 428, the substituted amino acid residue can be any amino acid residue other than methionine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. Specific combinations of suitable amino acid substitutions are identified in Table 1 of U.S. Patent No. 7,217,797, which is incorporated herein by reference. Such mutations increase binding to FcRn, thereby protecting the antibody from degradation and prolonging its half-life.

[0106] In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure has one or more amino acids inserted into one or more of its hypervariable regions, for example, as described in Jung and Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Patent Application No. 2007 / 0280931.

[0107] In other respects, particularly useful for diagnostic applications, it is possible to attach the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure to a detectable portion. The detectable portion includes a radioactive portion, a colorimetric molecule, a fluorescent portion, a chemiluminescent portion, an antigen, an enzyme, a detectable bead (e.g., magnetic beads or electrode beads (e.g., gold)), or a molecule bound to another molecule (e.g., biotin or streptavidin).

[0108] Radioactive isotopes or radionuclides may include 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0109] Fluorescent labels may include rhodamine, lanthanide phosphors, fluorescein and its derivatives, fluorescent dyes, GFP (GFP stands for "green fluorescent protein"), dansyl sulfonyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescent amine.

[0110] Enzyme labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase (“G6PDH”), α-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase.

[0111] Chemiluminescent labels or chemiluminescent agents include isoluminol, luminol, and dioxane.

[0112] Other detectable components include molecules such as biotin, digitoxin, or 5-bromodeoxyuridine.

[0113] In some respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure competes with antibody or antigen-binding fragments containing 1AG or heavy chain and light chain variable regions (SEQ ID NO: 1 and 2, respectively).

[0114] In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure competes with antibody or antigen-binding fragments containing 4AG or heavy chain and light chain variable regions (SEQ ID NO: 23 and 24, respectively).

[0115] This can be applied to cells expressing glyco-MUC1 epitopes that are bound by 1AG or 4AG, or to glycosylated MUC1 peptides containing epitopes bound by 1AG or 4AG, such as 15-mer peptides glycosylated with GalNAc on serine and threonine residues shown in bold and underlined text. Determine competitiveness. Cells that do not express the epitope or unglycosylated peptides can be used as controls.

[0116] Cells suitable for competitive assays include, but are not limited to, breast cancer cell lines MCF7 or T47D, and recombinant cells engineered to express the glyco-MUC1 epitope. In a non-limiting example, CHO IdID cells lacking UDP-Gal / GalNAc epimerase and lacking GalNAc O-glycosylation and galactosylation respectively in the absence of exogenous GalNAc and Gal were engineered to express MUC1 and cultured in the absence or presence of GalNAc, resulting in cells expressing the Tn glycoform of MUC1 bound to 1AG and 4AG. Cells expressing the unglycosylated form of MUC1 can be used as a negative control.

[0117] Competitive assays include, but are not limited to, radiolabeled immunosorbent assay (RIA), enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescence activated cell sorting (FACS) assay, and surface plasmon resonance (e.g., Biacore) assay.

[0118] In antibody competitive assays between reference and test antibodies (regardless of species or isotype), the reference antibody can be pre-labeled with a detectable marker such as a fluorophore, biotin, or enzyme (or even radioactivity) to enable subsequent identification. In this case, cells expressing glyco-MUC1 are incubated with an unlabeled test antibody, the labeled reference antibody is added, and the strength of the binding to the label is measured. If the test antibody competes with the labeled reference antibody by binding to overlapping epitopes, the strength will be reduced compared to a control reaction without the test antibody.

[0119] In a specific implementation of this assay, the maximum binding 80% (“conc”) is first determined under the assay conditions (e.g., a specified cell density). 80％ The concentration of the reference antibody was marked with ') and measured using 10 x conc 80％ Unlabeled test antibodies and conc 80％ The labeled reference antibody was used for competitive assay.

[0120] Inhibition can be expressed as the inhibition constant or K. i It is calculated according to the following formula:

[0121] K i =IC 50 / (1+[reference antibody concentration] / K) d ),

[0122] IC 50 The concentration of the test antibody is reduced by 50% when the binding of the reference antibody is produced, and K d This is the dissociation constant of the reference antibody, a measure of its affinity for sugar-MUC1. Under the assay conditions described herein, antibodies competing with the anti-sugar-MUC1 antibody disclosed herein can have a Kc of 10 pM to 10 nM. i .

[0123] In various implementations, under the specific assay conditions used, when the reference antibody concentration is 80% of the maximum binding and the test antibody concentration is 10 times higher than the reference antibody concentration, the test antibody is considered to compete with the reference antibody if the test antibody reduces the binding of the reference antibody by at least about 20% or more, for example, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, or by a percentage between any of the above values.

[0124] In one example of a competitive assay, glycosylated MUC1 15-mer peptides are adhered to a solid surface, such as a microplate, by contacting the plate with a peptide solution (e.g., 1 μg / mL in PBS, overnight at 4°C). The plate is washed (e.g., 0.1% Tween 20 in PBS) and blocked (e.g., in Superblock, Thermo Scientific, Rockford, IL). A mixture of subsaturated amounts of biotinylated 1AG or 4AG (e.g., 80 ng / mL) and unlabeled 1AG or 4AG (“reference” antibody) or a competitive anti-glyco-MUC1 antibody (“test” antibody) in serially diluted (e.g., 2.8 μg / mL, 8.3 μg / mL, or 25 μg / mL) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in PBS) is added to the wells and the plate is incubated with gentle agitation for 1 hour. Wash the plate, add 1 μg / mL HRP-conjugated streptavidin diluted in ELISA buffer to each well, and incubate the plate for 1 hour. Wash the plate and detect bound antibodies by adding substrate (e.g., TMB, Biofx Laboratories Inc., Owings Mills, MD). Terminate the reaction by adding stop buffer (e.g., BioFX stop reagent, Biofx Laboratories Inc., Owings Mills, MD) and measure absorbance at 650 nm using a microplate reader (e.g., VERSAmax, Molecular Devices, Sunnyvale, CA).

[0125] This variant of the competition assay can also be used to test the competition between 1AG or 4AG and another anti-glyco-MUC1 antibody. For example, in some respects, the anti-glyco-MUC1 antibody is used as a reference antibody, while 1AG or 4AG is used as the test antibody. Alternatively, instead of the glycosylated MUC1 15-mer peptide, membrane-bound glyco-MUC1 expressed in culture on the cell surface (e.g., on the surface of one of the aforementioned cell types) can be used. Typically, about 10 4 Up to 10 6 Transfectants, for example, about 10 5 One transfectant. Other forms of competition assays are known in the art and can be employed.

[0126] In various embodiments, when the concentration of the anti-glycemic-MUC1 antibody used is 0.08 μg / mL, 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, or a concentration ranging from any of the above values ​​(e.g., a concentration ranging from 2 μg / mL to 10 μg / mL), the anti-glycemic-MUC1 antibody of this disclosure reduces the binding of labeled 1AG or 4AG by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or a percentage reduction ranging from any of the above values ​​(e.g., the anti-glycemic-MUC1 antibody of this disclosure reduces the binding of labeled 1AG or 4AG by 50% to 70%).

[0127] In other embodiments, when the concentration of 1AG or 4AG used is 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 250 μg / mL, or a concentration ranging from any of the above values ​​(e.g., a concentration ranging from 2 μg / mL to 10 μg / mL), 1AG or 4AG reduces the binding of the labeled anti-glyco-MUC1 antibody of this disclosure by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or a percentage reduction ranging from any of the above values ​​(e.g., 1AG or 4AG reduces the binding of the labeled anti-glyco-MUC1 antibody of this disclosure by 50% to 70%).

[0128] In the aforementioned assay, any antibody or antigen-binding fragment containing a CDR or variable regions of heavy and light chains (e.g., humanized or chimeric counterparts of 1AG or 4AG) can be used to replace the 1AG or 4AG antibody.

[0129] In some respects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure has epitopes that are identical or similar to those of 1AG or 4AG. The epitopes of the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure can be characterized by, for example, an alanine scan as described in Example 9. Example 9 describes a library of 12 glycopeptides (SEQ ID NO: 106-117), each glycopeptide associated with a MUC1 peptide. The difference lies in a point mutation of alanine at one of positions 13-24 (or, when the MUC1 peptide has alanine, the difference lies in a point mutation of glycine). Epitopes of the antibody or antigen-binding fragment can be located by measuring the binding of the antibody or antigen-binding fragment to each peptide using ELISA.

[0130] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0131] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of the two is shown in bold and underlined text, where serine and threonine residues represent residues glycosylated by GalNAc.

[0132] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0133] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0134] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0135] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0136] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0137] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0138] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0139] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0140] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0141] In some embodiments, such as those measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure binds to the peptide. The affinity for binding is greater than that for peptides. and The affinity of each binding, where the serine and threonine residues shown in bold and underlined text represent residues glycosylated by GalNAc.

[0142] In some aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain and / or light chain variable region sequences (or encoded by nucleotide sequences) listed in Table 1A or Table 1B. In other aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of this disclosure comprises the heavy chain and / or light chain CDR sequences (or encoded by nucleotide sequences) listed in Tables 1A, 1B, 1C, 1D, 1E, 2A, 2B, 2C, 3A, or 3B. The frame sequence of such anti-glyco-MUC1 antibody and antigen-binding fragment may be a natural mouse frame sequence of the VH and VL sequences listed in Table 1A or Table 1B, or may be a non-natural (e.g., humanized or human) frame sequence.

[0143] In other respects, this disclosure provides anti-MUC1 antibody or antigen-binding fragments having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1 and 2, respectively.

[0144] In other respects, this disclosure provides anti-MUC1 antibody or antigen-binding fragments having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 23 and 24, respectively.

[0145] In other respects, the anti-glyco-MUC1 antibody or antigen-binding fragment disclosed herein is a single-chain variable fragment (scFv). An exemplary scFv comprises a heavy chain variable fragment located at the N-terminus of a light chain variable fragment. In some embodiments, the scFv heavy chain variable fragment and light chain variable fragment are covalently bound to an adapter sequence of 4-15 amino acids. The scFv may be in the form of a bispecific T-cell adaptor or within a chimeric antigen receptor (CAR).

[0146] 6.2 Antibody-Drug Conjugates

[0147] Another aspect of this disclosure relates to antibody-drug conjugates (ADCs) comprising the anti-glyco-MUC1 antibody of this disclosure and an antigen-binding fragment. ADCs typically comprise an anti-glyco-MUC1 antibody and / or a binding fragment as described herein, which are linked to one or more cytotoxic agents and / or cell inhibitors via one or more linkers. In a specific embodiment, the ADC is a compound according to structural formula (I) or a salt thereof:

[0148] [DL-XY] n -Ab

[0149] Each “D” independently represents a cytotoxic agent and / or cell inhibitor (“drug”); each “L” independently represents a linker; “Ab” represents an anti-glyco-MUC1 antigen-binding domain, such as the anti-glyco-MUC1 antibody or binding fragment described herein; each “XY” represents a functional group R on the linker. x Complementary functional group R on the antibody y The connection formed between them, and n represents the number of drugs connected to the ADC or the drug-to-antibody ratio (DAR) of the ADC.

[0150] Specific embodiments of various antibodies (Abs) that may contain ADCs include the various embodiments of the above-described anti-glyco-MUC1 antibody and / or binding fragments.

[0151] In some specific implementations of the ADC and / or salt of structural formula (I), each D is the same and / or each L is the same.

[0152] The following describes in more detail specific embodiments of the cytotoxic agent and / or cell inhibitor (D) and adapter (L) of the anti-glycemic-MUC1 ADC that may include the present disclosure, as well as the number of cytotoxic agents and / or cell inhibitors linked to the ADC.

[0153] 6.2.1. Cytotoxic agents and / or cell inhibitors

[0154] Cytotoxic agents and / or cell inhibitors can be any agent known to inhibit the growth and / or replication of cells, particularly cancer cells and / or tumor cells, and / or kill cells, particularly cancer cells and / or tumor cells. Many agents with cytotoxic and / or cell-inhibiting properties are known in the literature. Non-limiting examples of various cytotoxic agents and / or cell inhibitors include, for example, but not limited to, radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA intercalating agents (e.g., groove-binding agents, such as minor groove binders), RNA / DNA antimetabolites, cell cycle regulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondrial inhibitors, and antimitotic agents.

[0155] Below are specific, non-limiting examples of reagents within certain categories of these various classes.

[0156] Alkylating agents: asaley ((L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-, ethyl ester; NSC 167780; CAS Registry No. 3577897)); AZQ ((1,4-cyclohexadiene-1,4-diaminocarboxylic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester; NSC 182986; CAS Registry No. 57998682)); BCNU ((N,N'-bis(2-chloroethyl)-N-nitrosourea; NSC 409962; CAS Registry No. 154938)); busulfan (1,4-butanediol dimethanesulfonate; NSC 750; CAS Registry No. 55981); (carboxyphthalic acid)platinum (NSC 27164; CAS Registry No. 65296813); CBDCA ((cis-(1,1-cyclobutanedicarboxylic acid)diamineplatinum(II); NSC 241240; CAS Registry No. 41575944)); CCNU ((N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; NSC 79037; CAS Registry No. 13010474)); CHIP (isopropylplatinum; NSC 256927); chlorambucil (NSC 3088; CAS Registry No. 305033); chloramphenicol ((2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC 178248; CAS Registry No. 54749905)); cisplatin (NSC 119875; CAS Registry No. 15663271); Ethyl chloride (NSC 338947; CAS Registry No. 88343720); Cyanomorpholine doxorubicin (NCS 357704; CAS Registry No. 88254073); Ethylene glycol methyl disulfonate (cyclodisone) (NSC 348948; CAS Registry No. 99591738); Dehydroeugenol (5,6-diepoxyeugenol; NSC132313; CAS Registry No. 23261203); Fluorodiphenyl ether ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil; NSC 73754; CAS Registry No. 834913); hepsulfam (NSC 329680; CAS Registry No. 96892578); Heinzone (NSC142982; CAS Registry No. 23255938); Melphalan (NSC 8806; CAS Registry No. 3223072); Methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea; NSC 95441; 13909096); Mitomycin C (NSC26980; CAS Registry No. 50077); Mitozolam (NSC 353451; CAS Registry No. 85622953);Nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride; NSC 762; CAS Registry No. 55867); PCNU ((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidinyl)-1-nitrosourea; NSC 95466; CAS Registry No. 13909029)); piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; NSC 344007)); piperazine dione (NSC 135758; CAS Registry No. 41109802); piperobromo ((N,N-bis(3-bromopropionyl)piperazine; NSC 25154; CAS Registry No. 54911)); pofibromycin (N-methylmitomycin C; NSC 56410; CAS Registry No. 801525); Spirohydantoin Mustard (NSC 172112; CAS Registry No. 56605164); Tiroxiron (Triglycidyl Isocyanurate; NSC 296934; CAS Registry No. 2451629); Tetraplatin (NSC 363812; CAS Registry No. 62816982); Thiotepa (N,N',N”-Tri-1,2-Ethyldithiophosphoramide; NSC 6396; CAS Registry No. 52244); Tratamide (NSC 9706; CAS Registry No. 51183); Uracil Mustard (Desmethyldopan; NSC 34462; CAS Registry No. 66751); Yoshi-864 (Bis(3-Methanesulfonyloxypropyl)amine Hydrochloride; NSC 102627; CAS Registry No. 3458228);

[0157] Topoisomerase I inhibitors Camptothecin (NSC 94600; CAS Registry No.: 7689-03-4); various camptothecin derivatives and analogues (e.g., NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC295500, NSC 249910, NSC 606985, NSC 74028, NSC 176323, NSC 295501, NSC 606172, NSC606173, NSC 610458, NSC 618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC364830 and NSC...). 606497); Moroxydacrylamide (NSC 354646; CAS Registry No. 89196043); SN-38 (NSC673596; CAS Registry No. 86639-52-3).

[0158] Topoisomerase II inhibitorsDoxorubicin (NSC 123127; CAS Registry No. 25316409); Aminofibril (Benzoquinolinedione; NSC 308847; CAS Registry No. 69408817); m-AMSA ((4'-(9-acridylamino))-3'-methoxymethanesulfonylaniline; NSC 249992; CAS Registry No. 51264143); Anthrapyrazole derivatives (NSC 355644); Etoposide (VP-16; NSC 141540; CAS Registry No. 33419420); Pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2(6H)-propylamine, 9-methoxy-N,N-dimethyl-5-nitro-,monomethylsulfonate; NSC 366140; CAS Registry No. 99009219); Bismuth subcitrate hydrochloride (NSC 337766; CAS Registry No. 71439684); Daunorubicin (NSC 821151; CAS Registry No. 23541506); Doxorubicin (NSC 267469; CAS Registry No. 63950061); Mitoxantrone (NSC 301739; CAS Registry No. 70476823); Minoril (NSC 269148; CAS Registry No. 71628961); N,N-Dibenzyldanomycin (NSC268242; CAS Registry No. 70878512); Oxanthrazole (NSC 349174; CAS Registry No. 105118125); Rubidazone (NSC 366140; CAS Registry No. 99009219); 164011; CAS Registry No. 36508711); Teniposide (VM-26; NSC 122819; CAS Registry No. 29767202).

[0159] DNA intercalating agentAnthramycin (CAS Registry No. 4803274); Chicamycin A (CAS Registry No. 89675376); Tomamycin (CAS Registry No. 35050556); DC-81 (CAS Registry No. 81307246); Sibiromycin (CAS Registry No. 12684332); Pyrrolobenzodiazepine derivatives (CAS Registry No. 945490095); SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3-4(S)-7-methoxy-2-(4-methoxyphenyl) )-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5(11aH)-one); SG2000(SJG-136; (11aS, 11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5(11aH)-one); NSC 694501; CAS Registry No. 232931576).

[0160] RNA / DNA antimetabolites L-Aranoxin (NSC 153353; CAS Registry No. 59163416); 5-azacytidine (NSC 102816; CAS Registry No. 320672); 5-Fluorouracil (NSC 19893; CAS Registry No. 51218); Acivitine (NSC 163501; CAS Registry No. 42228922); aminopterin derivative N-[2-chloro-5-[[((2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl-]L-aspartic acid (NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl ... 184692); aminopterin derivative N-[2-chloro-4-[[((2,4-diamino-6-piperidinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate (NSC 134033); antifolic acid agent ((N α -(4-amino-4-deoxypteroyl)-N 7-Hymenphthalyl-L-ornithine; NSC 623017); Baker's soluble antifolate (NSC 139105; CAS Registry No. 41191042); Dichloroallyl henna quinone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone; NSC 126771; CAS Registry No. 36417160); Buquina (NSC 368390; CAS Registry No. 96201886); Tegafur ((prodrug; 5-fluoro-1-(tetrahydro-2-furanyl)-uracil; NSC 148958; CAS Registry No. 37076689); 5,6-dihydro-5-azacytidine (NSC 264880; CAS Registry No. 62402317); Methotrexate (NSC 740; CAS Registry No.: 59052); Methotrexate derivatives (N-[[4-[[(2,4-diamino-6-piperidinyl)methyl]methylamino]-1-naphthalene]carbonyl]L-glutamic acid; NSC 174121); PALA ((N-(phosphonoacetyl)-L-aspartic acid; NSC 224131; CAS Registry No.: 603425565); Pyrazofurantoin (NSC 143095; CAS Registry No.: 30868305); Trimethoprim (NSC 352122; CAS Registry No.: 82952645).

[0161] DNA antimetabolites: 3-HP (NSC 95678; CAS Registry No. 3814797); 2'-deoxy-5-fluorouridine (NSC27640; CAS Registry No. 50919); 5-HP (NSC 107392; CAS Registry No. 19494894); α-TGDR (α-2'-deoxy-6-thioguanosine; NSC 71851 CAS Registry No. 2133815); afedipine glycine salt (NSC 303812; CAS Registry No. 92802822); cytarabine (arabinocytosine; NSC 63878; CAS Registry No. 69749); 5-aza-2'-deoxycytidine (NSC 127716; CAS Registry No. 2353335); β-TGDR (β-2'-deoxy-6-thioguanosine; NSC 71261; CAS Registry No. 789617); Cyclocytosine (NSC 145668; CAS Registry No. 10212256); Guanidine (NSC 1895; CAS Registry No. 1455772); Hydroxyurea (NSC 32065; CAS Registry No. 127071); Inosylglucan dialdehyde (NSC 118994; CAS Registry No. 23590990); Macbecin II (NSC 330500; CAS Registry No. 73341738); Pyrazoimidazole (NSC 51143; CAS Registry No. 6714290); Thioguanine (NSC 752; CAS Registry No. 154427); Thiopurine (NSC 755; CAS Registry No. 50442).

[0162] Cell cycle regulatorsSilymarin (CAS Registry No. 22888-70-6); Epigallocatechin gallate (EGCG; CAS Registry No. 989515); Proanthocyanidin derivatives (e.g., proanthocyanidin A1 [CAS Registry No. 103883030], proanthocyanidin B1 [CAS Registry No. 20315257], proanthocyanidin B4 [CAS Registry No. 29106512], arecatannin B1 [CAS Registry No. 79763283]); Isoflavones (e.g., genistein [4% 5,7-trihydroxyisoflavone; CAS Registry No. 446720], daidzein [4',7-dihydroxyisoflavone, CAS Registry No. 486668]); Indole-3-carbinol (CAS Registry No. 700061); Quercetin (NSC) 9219; CAS Registry No. 117395); Estrogenustine (NSC 89201; CAS Registry No. 2998574); Nocodazole (CAS Registry No. 31430189); Podophyllotoxin (CAS Registry No. 518285); Vinorelbine Tartrate (NSC 608210; CAS Registry No. 125317397); Nostocin (NSC 667642; CAS Registry No. 124689652).

[0163] kinase inhibitorsAfatinib (CAS Registry No. 850140726); Axitinib (CAS Registry No. 319460850); ARRY-438162 (binimetinib) (CAS Registry No. 606143899); Bosutinib (CAS Registry No. 380843754); Cabozantinib (CAS Registry No. 1140909483); Ceritinib (CAS Registry No. 1032900256); Crizotinib (CAS Registry No. 877399525); Dabrafenib (CAS Registry No. 1195765457); Dasatinib (NSC 732517; CAS Registry No. 302962498); Erlotinib (NSC 718781; CAS Registry No. 183319699); Everolimus (NSC 733504; CAS Registry No. 159351696); Fotatinib (NSC 745942; CAS Registry No. 901119355); Gefitinib (NSC 715055; CAS Registry No. 184475352); Ibrutinib (CAS Registry No. 936563961); Imatinib (NSC 716051; CAS Registry No. 220127571); Lapatinib (CAS Registry No. 388082788); Lenvatinib (CAS Registry No. 857890392); Muritinib (CAS Registry No. 159351696); Futatinib (NSC 745942; CAS Registry No. 901119355); Gefitinib (NSC 715055; CAS Registry No. 184475352); Ibrutinib (CAS Registry No. 936563961); Imatinib (NSC 716051; CAS Registry No. 220127571); Lapatinib (CAS Registry No. 388082788); Lenvatinib (CAS Registry No. 857890392); Muritinib (CAS Registry No. 159351696); Fotatinib (NSC 745942; CAS Registry No. 901119355); Gefitinib (NSC 715055; CAS Registry No. 184475352); Ibrutinib (CAS Registry No. 936563961); Imatinib ( 366017096); Nilotinib (CAS Registry No. 923288953); Nintedanib (CAS Registry No. 656247175); Palbociclib (CAS Registry No. 571190302); Pazopanib (NSC 737754; CAS Registry No. 635702646); Pigastanib (CAS Registry No. 222716861); Panatinib (CAS Registry No. 1114544318); Rapamycin (NSC 226080; CAS Registry No. 53123889); Regorafenib (CAS Registry No. 755037037); AP 23573 (ridaforolimus) (CAS Registry No. 572924540); INCB018424 (ruxolitinib) (CAS Registry No. 1092939177); ARRY-142886 (selmetinib) (NSC 741078; CAS Registry No. 606143-52-6); sirolimus (NSC 226080; CAS Registry No. 53123889); sorafenib (NSC 724772; CAS Registry No. 475207591); sunitinib (NSC 736511; CAS Registry No. 341031547); tofacitinib (CAS Registry No. 477600752); tesirolimus (NSC 23573) (CAS ... 683864; CAS Registry No. 163635043); Trametinib (CAS Registry No. 871700173);Vandetanib (CAS No. 443913733); Vemurafenib (CAS No. 918504651); SU6656 (CAS No. 330161870); CEP-701 (Lesatinib) (CAS No. 111358884); XL019 (CAS No. 945755566); PD-325901 (CAS No. 391210109); PD-98059 (CAS No. 167869218); ATP-competitive TORC1 / TORC2 inhibitors, including PI-103 (CAS No. 371935749), PP242 (CAS No. 1092351671), PP30 (CAS No. 1092788094), Torin 1 (CAS Registry No. 1222998368), LY294002 (CAS Registry No. 154447366), XL-147 (CAS Registry No. 934526893), CAL-120 (CAS Registry No. 870281348), ETP-45658 (CAS Registry No. 1198357797), PX 866 (CAS Registry No. 502632668), GDC-0941 (CAS Registry No. 957054307), BGT226 (CAS Registry No. 1245537681), BEZ235 (CAS Registry No. 915019657), XL-765 (CAS Registry No. 934493762).

[0164] Protein synthesis inhibitorsAcetaminophen (CAS Registry No. 65589700); Amikacin (NSC 177001; CAS Registry No. 39831555); Abakacin (CAS Registry No. 51025855); Asmicin (CAS Registry No. 55779061); Azithromycin (NSC 643732; CAS Registry No. 83905015); Bekanasamycin (CAS Registry No. 4696768); Chlortetracycline (NSC13252; CAS Registry No. 64722); Clarithromycin (NSC 64722); 643733; CAS Registry No. 81103119); Clindamycin (CAS Registry No. 18323449); Chlorocycline (CAS Registry No. 1181540); Cycloheximide (CAS Registry No. 66819); Actinomycin (NSC 3053; CAS Registry No. 50760); Dafospudin (CAS Registry No. 112362502); Desclocycline (CAS Registry No. 127333); Dibekacin (CAS Registry No. 34493986); Dihydrostreptomycin (CAS Registry No. 128461); Erythromycin (CAS Registry No. 62013041); Doxycycline (CAS Registry No. 17086281); Emetine (NSC 33669; CAS Registry No. 483181); Erythromycin (NSC 33669; CAS Registry No. 483181); 55929; CAS Registry No. 114078); Fluoromycin (CAS Registry No. 83664208); Freund's mycelium (Neomycin B; CAS Registry No. 119040); Gentamicin (NSC 82261; CAS Registry No. 1403663); Glycylcycline, e.g., tigecycline (CAS Registry No. 220620097); Hygromycin B (CAS Registry No. 31282049); Isapamicin (CAS Registry No. 67814760); Josamycin (NSC 82261; CAS Registry No. 1403663); 122223; CAS Registry No. 16846245); Kanamycin (CAS Registry No. 8063078); Ketones, such as Telithromycin (CAS Registry No. 191114484), Quinoerythromycin (CAS Registry No. 205110481) and Solithromycin (CAS Registry No. 760981837); Lincomycin (CAS Registry No. 154212); Lemmicin (CAS Registry No. 992212); Meclocycline (NSC78502; CAS Registry No. 2013583); Meclocycline (Rotomycin; NSC 356463; CAS Registry No. 914001); Midecamycin (CAS Registry No. 35457808); Minocycline (NSC 141993; CAS Registry No. 10118908); Micardic acid (CAS Registry No. 55881077); Neomycin (CAS Registry No. 119040); Netilmicin (CAS Registry No. 56391561); Prunus ceramide (CAS Registry No. 3922905);Oxazolidinones, such as ipilazolamide (CAS Registry No. 165800044), linezolid (CAS Registry No. 165800033), preszolid (CAS Registry No. 252260029), radizolid (CAS Registry No. 869884786), ranbezolid (CAS Registry No. 392659380), sutezolid (CAS Registry No. 168828588), terdizole (CAS Registry No. 856867555); oxytetracycline (NSC 9169; CAS Registry No. 2058460); paromomycin (CAS Registry No. 7542372); penicillin (CAS Registry No. 4599604); peptidyl transferase inhibitors, such as chloramphenicol (NSC 9169; CAS Registry No. 2058460); paromomycin (CAS Registry No. 7542372); penicillin (CAS Registry No. 4599604); peptidyl transferase inhibitors, such as chloramphenicol (NSC 9169; CAS Registry No. 2058460); 3069; CAS Registry No. 56757) and derivatives, such as chloramphenicol azidophos (CAS Registry No. 13838089), florfenicol (CAS Registry No. 73231342) and thiamphenicol (CAS Registry No. 15318453), and pleuromyopiclin, such as retapalline (CAS Registry No. 224452668), thimolin (CAS Registry No. 55297955), vanemoline (CAS Registry No. 101312929); pirizine (CAS Registry No. 79548735); puromyopiclin (NSC) 3055; CAS Registry No. 53792); Quinupordin (CAS Registry No. 120138503); Ribomycin (CAS Registry No. 53797356); Rotamycin (CAS Registry No. 74014510); Rolicycline (CAS Registry No. 751973); Roxithromycin (CAS Registry No. 80214831); Sisomicin (CAS Registry No. 32385118); Spectinomycin (CAS Registry No. 1695778); Spiramycin (CAS Registry No. 8025818); Streptomycin, such as Punamycin (CAS Registry No. 270076603), Quinupordin / Dalfopristin (CAS Registry No. 126602899) and Virginiamycin (CAS Registry No. 11006761); Streptomycin (CAS Registry No. 57921); Tetracycline (NSC 108579; CAS Registry No. 60548); Tobramycin (CAS Registry No. 32986564); Acetylosin (CAS Registry No. 2751099); Tylosin (CAS Registry No. 1401690); Verdamicin (CAS Registry No. 49863481).

[0165] Histone deacetylases inhibitorsabexinostat (CAS Registry No. 783355602); belistat (NSC) 726630; CAS Registry No. 414864009); Chidamide (CAS Registry No. 743420022); Entecavir (CAS Registry No. 209783802); Givinostat (CAS Registry No. 732302997); Mocetinostat (CAS Registry No. 726169739); Papirostat (CAS Registry No. 404950807); Quisinostat (CAS Registry No. 875320299); Resminostat (CAS Registry No. 864814880); Romidexin (CAS Registry No. 128517077); Sulforaphane (CAS Registry No. 4478937); Kevetrin TM ; CAS Registry No. 6659890); Valproic acid (NSC 93819; CAS Registry No. 99661); Vorinostat (NSC 701852; CAS Registry No. 149647789); ACY-1215 (rocilinostat; CAS Registry No. 1316214524); CUDC-101 (CAS Registry No. 1012054599); CHR-2845 (tefinostat; CAS Registry No. 914382608); CHR-3996 (CAS Registry No. 1235859138); 4SC-202 (CAS Registry No. 910462430); CG200745 (CAS Registry No. 936221339); SB939 (pracinostat; CAS Registry No. 929016966).

[0166] Mitochondrial inhibitors : Hydnocarbazine (NSC 349156; CAS Registry No. 96281311); Rhodamine 123 (CAS Registry No. 63669709); Edifosine (NSC 324368; CAS Registry No. 70641519); d-α-tocopherol succinate (NSC173849; CAS Registry No. 4345033); Compound 11β (CAS Registry No. 865070377); Aspirin (NSC 406186; CAS Registry No. 50782); Rosacetic acid (CAS Registry No. 519233); Berberine (CAS Registry No. 633658); Chloramphenicol (CAS Registry No. 17397896); GX015-070 ( 1H-Indole, 2-(2-((3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-; NSC729280; CAS Registry No. 803712676); Tripterygium wilfordii (CAS Registry No. 34157830); Metformin (NSC 91485; CAS Registry No. 1115704); Brilliant Green (NSC5011; CAS Registry No. 633034); ME-344 (CAS Registry No. 1374524556).

[0167] Antimitotic agents Colchicine (NSC 406042); Auristatin, such as MMAE (methylauristatin E; CAS Registry No. 474645-27-7) and MMAF (methylauristatin F; CAS Registry No. 745017-94-1); Soft sponge B (NSC 609395); Colchicine (NSC 757; CAS Registry No. 64868); Colchicine derivatives (N-benzoyl-deacetylated benzamide; NSC 33410; CAS Registry No. 63989753); Tail slug 10 (NSC 376128; CAS Registry No. 110417-88-4); Maytansine (NSC 153858; CAS Registry No. 35846-53-8); Rhozoxin (NSC 332598; CAS Registry No. 90996546); Paclitaxel (NSC 125973; CAS Registry No. 33069624); Paclitaxel derivatives ((2'-N-[3-(dimethylamino)propyl]glutamic acid paclitaxel; NSC 608832); Thiocolchicine (3-demethylthiocolchicine; NSC 361792); Triphenylmethylcysteine ​​(NSC 49842; CAS Registry No. 2799077); Vincristine sulfate (NSC 49842; CAS Registry No. 143679); Vincristine sulfate (NSC 67574; CAS Registry No. 2068782).

[0168] Any of these reagents, including or potentially modified to include sites for attachment to antibodies, may be included in the ADCs disclosed herein.

[0169] In specific implementation schemes, cytotoxic agents and / or cell inhibitors are antimitotic agents.

[0170] In another specific implementation, the cytotoxic agent and / or cell inhibitor is aureatin, such as methylaureatin E (“MMAE”) or methylaureatin F (“MMAF”).

[0171] 6.2.2. Connector

[0172] In the anti-glycemic-MUC1 ADC of this disclosure, cytotoxic agents and / or cell inhibitors are linked to antibodies via adapters. The adapters linking the cytotoxic agents and / or cell inhibitors to the antibody in the ADC can be short, long, hydrophobic, hydrophilic, flexible, or rigid, or can be composed of segments each independently possessing one or more of the aforementioned properties, such that the adapters can include segments with different properties. The adapters can be multivalent, such that they covalently link more than one reagent to a single site on the antibody, or they can be monovalent, such that they covalently link a single reagent to a single site on the antibody.

[0173] Those skilled in the art will understand that the adapter connects the cytotoxic agent and / or cell inhibitor to the antibody by forming a covalent link at one location with the cytotoxic agent and / or cell inhibitor and a covalent link at another location with the antibody. The covalent link is formed through a reaction between functional groups on the adapter and functional groups on the reagent and the antibody. As used herein, the term "adapter" is intended to include (i) an unconjugated adapter comprising functional groups enabling covalent linking of the adapter to the cytotoxic agent and / or cell inhibitor, and functional groups enabling covalent linking of the adapter to the antibody; (ii) a partially conjugated adapter comprising functional groups enabling covalent linking of the adapter to the antibody, and which is also covalently linked to the cytotoxic agent and / or cell inhibitor, and vice versa; and (iii) a fully conjugated adapter covalently linked to both the cytotoxic agent and / or cell inhibitor and the antibody. In certain specific embodiments of the adapters and anti-glyco-MUC1ADCs disclosed herein, and syntheticons for conjugating adapter reagents to antibodies, the portion comprising functional groups on the adapter and the covalent link formed between the adapter and the antibody are specifically represented as R. x And XY.

[0174] The adapter is preferably, but not necessarily, chemically stable to extracellular conditions and may be designed to be specifically cleaved, destroyed, and / or otherwise degraded within the cell. Alternatively, adapters not designed to be specifically cleaved or degraded within the cell may be used. The choice between a stable and an unstable adapter may depend on the cytotoxicity, i.e., and / or the toxicity of the cell inhibitor. For agents toxic to normal cells, a stable adapter is preferred. For selective or targeted agents with low toxicity to normal cells, the chemical stability of the adapter to the extracellular environment is less important. In the context of ADCs, various adapters that can be used to conjugate drugs to antibodies are known in the art. Any of these adapters, as well as others, can be used to conjugate cytotoxic agents and / or cell inhibitors to antibodies of the anti-glyco-MUC1 ADC of this disclosure.

[0175] Exemplary multivalent linkers that can be used to link many cytotoxic agents and / or cell inhibitors to a single antibody molecule are found in WO 2009 / 073445, WO 2010 / 068795, WO 2010 / 138719, WO 2011 / 120053, WO 2011 / 171020, WO 2013 / 096901, WO 2014 / 008375, WO 2014 / 093379, WO 2014 / 093394, and WO 2014 / 093640, the contents of which are incorporated herein by reference in their entirety. For example, the Fleximer linker technology developed by Mersana et al. has the potential to impart favorable physicochemical properties to high-DAR ADCs. As shown below, the Mersana technology is based on incorporating drug molecules into a soluble polyacetal backbone via a series of ester bonds. This method enables high-load ADCs (DAR up to 20) while maintaining good physicochemical properties.

[0176] Other examples of dendritic connectors can be found in: US 2006 / 116422; US 2005 / 271615; deGroot et al. (2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al. (2003) Angew. Chem. Int. Ed. 42:4494-4499; Shamis et al. (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King et al. (2002) Tetrahedron Letters 43:1987-1990, each of which is incorporated herein by reference.

[0177] Exemplary multivalent adapters that may be used are described, for example, in Nolting, 2013, Antibody-DrugConjugates, Methods in Molecular Biology 1045:71-100; Kitson et al., 2013, CROs / CMOs--Chemica Oggi--Chemistry Today 31(4):30-38; Ducry et al., 2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J.Med.Chem. 54:3606-3623; U.S. Patent Nos. 7,223,837; 8,568,728; 8,535,678; and WO2004010957, each of which is incorporated herein by reference.

[0178] As examples and not limitations, the following describes some cuttable and non-cuttable connectors that may be included in the anti-glycemic-MUC1 ADC of this disclosure.

[0179] 6.2.3. Cuttable connector

[0180] In some embodiments, the selected linker is in vivo cleavable. A cleavable linker may include chemically or enzymatically unstable or degradable linkages. Cleavable linkers typically rely on intracellular processes to release drugs, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific intracellular proteases or other enzymes. Cleavable linkers typically incorporate one or more chemically or enzymatically cleavable chemical bonds, while the remainder of the linker is not cleavable. In some embodiments, the linker contains chemically unstable groups, such as hydrazones and / or disulfide groups. Linkers containing chemically unstable groups utilize the differences between plasma and certain cytoplasmic compartments. Intracellular conditions that promote drug release from hydrazone-containing linkers are acidic environments in endosomes and lysosomes, while disulfide-containing linkers are reduced in cytosols containing high concentrations of thiols (e.g., glutathione). In some embodiments, the plasma stability of linkers containing chemically unstable groups can be improved by introducing steric hindrance using substituents near the chemically unstable groups.

[0181] Acid-labile groups, such as hydrazones, remain intact during systemic circulation in the neutral pH environment of blood (pH 7.3–7.5) and undergo hydrolysis to release the drug after the ADC is internalized into the weakly acidic endosomes (pH 5.0–6.5) and lysosomal compartments (pH 4.5–5.0) of the cell. This pH-dependent release mechanism is associated with non-specific drug release. To increase the stability of the hydrazone groups at the linker, the linker can be modified chemically, such as by substitution, thereby allowing for regulation to achieve more efficient release in lysosomes while minimizing loss during circulation.

[0182] The hydrazone-containing linker may contain additional cleavage sites, such as additional acid-labile cleavage sites and / or enzyme-labile cleavage sites. An ADC including an exemplary hydrazone-containing linker comprises the following structure:

[0183]

[0184] Where D and Ab represent cytotoxic agents and / or cell inhibitors (drugs) and Ab, respectively, and n represents the number of drug linkers linked to the antibody. In some linkers, such as linker (Ig), the linker contains two cleavable groups: a disulfide and an hydrazone moiety. For such linkers, efficient release of the unmodified free drug requires an acidic pH or the reduction of the disulfide and an acidic pH. Linkers such as (Ih) and (Ii) have been shown to be effective for single hydrazone cleavage sites.

[0185] Other linkers that remain intact during systemic circulation and undergo hydrolysis to release the drug as the ADC is internalized into the acidic cellular compartment include carbonates / esters. Such linkers can be useful where cytotoxic agents and / or cell inhibitors can be covalently linked via oxygen.

[0186] Other acid-labile groups that can be included in the linker include those containing a cis-aconitol. Cis-aconitol chemistry uses a carboxylic acid parallel to the amide bond to accelerate the hydrolysis of the amide under acidic conditions.

[0187] Cleavable linkers can also include disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release drugs after internalization within cells, where the cytosol provides a significantly more reducing environment compared to the extracellular environment. The breaking of disulfide bonds typically requires the presence of cytosolic thiol cofactors, such as (reduced) glutathione (GSH), to make the disulfide-containing linker relatively stable in circulation, thereby selectively releasing drugs into the cytosol. Intracellular enzymatic proteins disulfide isomerases or similar enzymes capable of cleaving disulfide bonds can also contribute to the preferential cleavage of intracellular disulfide bonds. GSH has been reported to be present in cells at concentrations ranging from 0.5 to 10 mM, while in contrast, the concentrations of GSH or cysteine ​​(the most abundant low-molecular-weight thiol) in circulation are significantly lower, around 5. Tumor cells (where irregular blood flow leads to a hypoxic state) result in enhanced reductase activity, leading to even higher glutathione concentrations. In some implementations, the in vivo stability of disulfide-containing joints can be enhanced by chemical modification of the joints, such as by using steric hindrance adjacent to the disulfide bonds.

[0188] An ADC including an exemplary disulfide-containing junction comprises the following structure:

[0189]

[0190] Where D and Ab represent the drug and antibody, respectively, n represents the number of drug linkers linked to the antibody, and R is independently selected, for example, from hydrogen or alkyl groups each time it appears. In some embodiments, increasing the steric hindrance of the adjacent disulfide bond increases the stability of the linker. When one or more R groups are selected from lower alkyl groups such as methyl, for example, the structures (Ij) and (II) show increased in vivo stability.

[0191] Another type of cleavable linker that can be used is the enzyme-specific cleavage linker. These linkers are typically peptide-based or include peptide regions that act as enzyme substrates. Compared to chemically unstable linkers, peptide-based linkers tend to be more stable in plasma and extracellular environments. Peptide bonds generally exhibit good serum stability because lysosomal proteases have very low activity in the blood due to endogenous inhibitors and the unfavorable high blood pH compared to lysosomes. Drug release from antibodies occurs primarily due to the action of lysosomal proteases such as cathepsins and plasmin. These proteases may be present at elevated levels in some tumor cells.

[0192] In an exemplary embodiment, the cleavable peptide is selected from tetrapeptides, such as Gly-Phe-Leu-Gly (SEQ ID NO:100), Ala-Leu-Ala-Leu (SEQ ID NO:101) or dipeptides, such as Val-Cit, Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, AM Met-(D)Lys, Asn-(D)Lys, AW Met-(D)Lys, and Asn-(D)Lys. In some embodiments, dipeptides are preferred over longer polypeptides due to the hydrophobicity of longer peptides.

[0193] Several dipeptide-based cleavable linkers have been described for linking drugs such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepine, tamethasone, and aurestatin / aurestatin family members to antibodies (see Dubowchik et al., 1998, J.Org.Chem.67:1866-1872; Dubowchik et al., 1998, Bioorg.Med.Chem.Lett.8(21):3341-3346; Walker et al., 2002, Bioorg.Med.Chem.Lett.12:217-219; Walker et al., 2004, Bioorg.Med.Chem.Lett.14:4323-4327; Sutherland et al., 2013, Blood 122:1455-1463; and Francisco et al.). (al., 2003, Blood 102:1458-1465, each of which is incorporated herein by reference). All of these dipeptide linkers, or modified forms of these dipeptide linkers, can be used in the anti-glycemic-MUC1 ADC of this disclosure. Other dipeptide linkers that can be used include those found in ADCs, such as Seattle Genetics' Brentuximab Vendotin SGN-35 (Adcetris TMSeattle Genetics SGN-75 (anti-CD-70, Val-Cit-methylaurestatin F (MMAF)), Seattle Genetics SGN-CD33A (anti-CD-33, Val-Ala- (SGD-1882)), Celldex Therapeutics glembatumumab (CDX-011) (anti-NMB, Val-Cit-methylaurestatin E (MMAE)), and Cytogen PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

[0194] Enzymatically cleavable linkers may include self-immolative spacers to spatially separate the drug from the enzymatic cleavage site. Direct attachment of the drug to the peptide linker can lead to the proteolytic release of the drug's amino acid adducts, thereby diminishing its activity. The use of self-immolative spacers allows for the elimination of fully active, chemically unmodified drugs after amide bond hydrolysis.

[0195] A self-degrading spacer is a bifunctional p-aminobenzyl alcohol group that links to a peptide via an amino group, forming an amide bond. The amine-containing drug can then be attached to the benzyl hydroxyl group of the linker via a carbamate functional group (PABC). The resulting prodrug is activated upon protease-mediated cleavage, leading to a 1,6-elimination reaction that releases the unmodified drug, carbon dioxide, and the residues of the linker group. The following scheme describes the fragmentation of p-aminobenzyl ether and the release of the drug:

[0196]

[0197] XD represents the unmodified drug.

[0198] Heterocyclic variants of this self-degrading group are also described. See, for example, U.S. Patent No. 7,989,434, which is incorporated herein by reference.

[0199] In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. The easy release of the drug can be achieved by cleaving the β-glucuronic acid glycosidic bond with the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and overexpressed in certain tumor types, while its extracellular enzyme activity is low. The β-glucuronic acid-based linker can be used to avoid the tendency of ADCs to aggregate due to the hydrophilic nature of β-glucuronic acid. In some embodiments, the β-glucuronic acid-based linker is preferably used as a linker for ADCs that attach to hydrophobic drugs. The following schemes describe drug release from an ADC and ADCs comprising a β-glucuronic acid-based linker:

[0200]

[0201] Several cleavable β-glucuronic acid-based linkers have been described for linking drugs such as auristatin, camptothecin and doxorubicin analogs, CBI small groove binders and psymberin to antibodies (see Nolting, Chapter 5 "Linker Technology in Antibody-Drug Conjugates," In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Springer Science & Business Medica, LLC, 2013; Jeffrey et al., 2006, Bioconjug. Chem. 17: 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17: 2278-2280; and Jiang et al.). al., 2005, J.Am.Chem.Soc. 127:11254-11255, each of which is incorporated herein by reference. All of these β-glucuronic acid-based linkers can be used in the anti-glucose-MUC1 ADC of this disclosure.

[0202] Additionally, phenolic cytotoxic agents and / or cell inhibitors can be covalently bonded to the linker via phenol-oxygen bonding. One such linker described in WO 2007 / 089149 relies on a method in which a diaminoethane “SpaceLink” is used in conjunction with a conventional “PABO”-based self-degrading group to deliver phenol. The linker cleavage is illustrated in the figure below, where D represents a cytotoxic agent and / or cell inhibitor containing a phenolic hydroxyl group.

[0203]

[0204] A cuttable joint may include an uncuttable portion or segment, and / or a cuttable segment or portion may be included in another uncuttable joint to make it cuttable. By way of example only, polyethylene glycol (PEG) and related polymers may include cuttable groups in the polymer backbone. For example, a PEG or polymer joint may contain one or more cuttable groups, such as disulfides, hydrazones, or dipeptides.

[0205] Other degradable bonds that may be included in the linker include ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Hydrolyzable bonds include, but are not limited to, carbonate bonds; imine bonds resulting from the reaction of amines and aldehydes; phosphate ester bonds formed by reacting an alcohol with a phosphate group; acetal bonds as products of the reaction of aldehydes and alcohols; orthoester bonds as products of the reaction of formate esters and alcohols; and oligonucleotide bonds formed by a phosphoramidite group (including, but not limited to, a phosphoramidite group at the polymer terminus) and the 5' hydroxyl group of an oligonucleotide.

[0206] In some embodiments, the adapter comprises an enzyme-cleavable peptide moiety, for example, an adapter comprising the structure (IVa) or (IVb) or a salt thereof:

[0207]

[0208] Wherein: peptide represents a peptide that can be cleaved by lysosomal enzymes (shown as C→N, carboxyl and amino "termini" are not displayed); T represents a polymer containing one or more ethylene glycol units or alkylene chains or combinations thereof; R a Selected from hydrogen, alkyl, sulfonate / ester and methanesulfonate / ester; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; * indicates the attachment point of the connector to the cytotoxic agent and / or cell inhibitor; and * indicates the attachment point of the rest of the connector.

[0209] In some embodiments, the peptide is selected from tripeptides or dipeptides. In specific embodiments, the dipeptide is selected from Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Val-Lys; Ala-Lys; Phe-Cit; Leu-Cit; Ile-Cit; Phe-Arg and Trp-Cit. In some embodiments, the dipeptide is selected from Cit-Val and Ala-Val.

[0210] Specific exemplary embodiments of the adapter according to structural formula (IVa) that may be included in the anti-glycan-MUC1 ADC of this disclosure include the adapter shown below (as shown, the adapter includes groups suitable for covalently linking the adapter to the antibody):

[0211]

[0212]

[0213] Specific exemplary embodiments of the adapter according to structural formula (IVb) that may be included in the anti-glycan-MUC1 ADC of this disclosure include the adapter shown below (as shown, the adapter includes groups suitable for covalently linking the adapter to the antibody):

[0214]

[0215]

[0216]

[0217]

[0218] In some embodiments, the adapter comprises an enzyme-cleavable peptide moiety, for example, an adapter comprising the structure (IVc) or (IVd) or a salt thereof:

[0219]

[0220] Wherein: peptide represents a peptide that can be cleaved by lysosomal enzymes (shown as C→N, carboxyl and amino "termini" are not displayed); T represents a polymer containing one or more ethylene glycol units or alkylene chains or combinations thereof; R a Selected from hydrogen, alkyl, sulfonate / ester, and methanesulfonate / ester; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; x * indicates the attachment point of the connector to the cytotoxic agent and / or cell inhibitor; and * indicates the attachment point of the rest of the connector.

[0221] Specific exemplary embodiments of the adapter according to structural formula (IVc) that may be included in the anti-glycemic-MUC1 ADC of this disclosure include adapters as shown below (as shown, the adapter includes groups suitable for covalently linking the adapter to the antibody):

[0222]

[0223]

[0224] Specific exemplary embodiments of the linker according to the structural formula (IVd) that may be included in the anti-glycan-MUC1 ADC of this disclosure include the linker shown below (as shown, the linker includes groups suitable for covalently linking the linker to the antibody):

[0225]

[0226]

[0227]

[0228] In some embodiments, the connector comprising structures (IVa), (IVb), (IVc), or (IVd) further includes a carbonate portion that can be cleaved by exposure to an acidic medium. In specific embodiments, the connector is attached to a cytotoxic agent and / or a cell inhibitor via oxygen attachment.

[0229] 6.2.4. Non-cuttable joints

[0230] While cleavable linkers can offer certain advantages, the linker comprising the anti-glyco-MUC1 ADC of this disclosure does not necessarily need to be cleavable. With non-cleavable linkers, drug release is not dependent on differential properties between plasma and certain cytoplasmic compartments. It is assumed that drug release occurs after the ADC is internalized via antigen-mediated endocytosis and delivered to a lysosomal compartment, where the antibody is degraded to the amino acid level via intracellular proteolytic degradation. This process releases a drug derivative consisting of the drug, the linker, and amino acid residues covalently attached to the linker. Compared to conjugates with cleavable linkers, amino acid drug metabolites from conjugates with non-cleavable linkers exhibit greater hydrophilicity and generally lower membrane permeability, resulting in fewer bystander effects and less nonspecific toxicity. Typically, ADCs with non-cleavable linkers exhibit greater stability in circulation than ADCs with cleavable linkers. The non-cleavable linker can be an alkylene chain or can be essentially a polymer, such as a polyalkylene glycol-based polymer, an amide polymer, or may include segments of alkylene chains, polyalkylene glycols, and / or amide polymers.

[0231] Various non-cleavable linkers for linking drugs to antibodies have been described. See Jeffrey et al., 2006, Bioconjug. Chem. 17: 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17: 2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. 127: 11254-11255, each of which is incorporated herein by reference. All of these linkers may be included in the anti-glycan-MUC1 ADC disclosed herein.

[0232] In some embodiments, the adapter is non-cleavable in vivo, for example, an adapter according to structural formula (VIa), (VIb), (VIc), or (VId) (as shown, the adapter includes a group suitable for covalently linking the adapter to an antibody) or a salt thereof:

[0233]

[0234] Where: R a Selected from hydrogen, alkyl, sulfonate / ester, and methanesulfonate / ester; Rx is a moiety including a functional group capable of covalently linking the linker to the antibody; and This indicates the connection point between the connector and the cytotoxic agent and / or cell inhibitor.

[0235] Specific exemplary embodiments of the adapters according to structural formulas (VIa)-(IVd) that may be included in the anti-glycemic-MUCl ADC of this disclosure include adapters as shown below (as shown, the adapters include groups suitable for covalently linking the adapters to the antibody, and...) (Indicates the attachment point to cytotoxic agents and / or cell inhibitors):

[0236]

[0237] 6.2.5. Groups used to attach linkers and antibodies

[0238] Various groups can be used to attach linker drug synthons to antibodies to generate ADCs. Attachment groups can be electrophilic in nature and include: maleimide groups, activated disulfides, active esters, and amides. Emerging techniques related to “self-stabilizing” maleimides and “bridging disulfides” as described below also exist, which can be used according to this disclosure. The specific groups used will depend in part on the antibody attachment site.

[0239] The schematic diagram below illustrates an example of a “self-stabilizing” maleimide group that spontaneously hydrolyzes under antibody conjugation conditions to provide an ADC material with improved stability. See US20130309256 A1; and Lyon et al., Nature Biotech published online, doi:10.1038 / nbt.2968.

[0240]

[0241]

[0242] This leads to "DAR loss" over time.

[0243] SGN MalDPR (maleimide dipropylamino) system:

[0244]

[0245] Polytherics discloses a method for bridging a pair of thiol groups derived from the reduction of natural hinge disulfide bonds. See Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. The reaction is shown in the figure below. The advantage of this method is that it enables the synthesis of enriched DAR4 ADCs by completely reducing IgG (producing 4 pairs of thiol groups) and then reacting it with 4 equivalents of an alkylating agent. It is also claimed that ADCs containing “bridging disulfide bonds” have increased stability.

[0246]

[0247] Similarly, as described below, maleimide derivatives capable of bridging a pair of thiol groups have been developed (1, hereinafter). See WO2013 / 085925.

[0248]

[0249] 6.2.6. Factors to consider when selecting a connector

[0250] As is known to those skilled in the art, the linker selected for a particular ADC can be influenced by a variety of factors, including but not limited to the antibody attachment site (e.g., lys, cys, or other amino acid residues), the structural constraints of the drug pharmacophore, and the lipophilicity of the drug. The selection of a specific linker for an ADC should aim to balance these different factors of a particular antibody / drug combination. For a review of factors influencing linker selection in ADCs, see Nolting, Chapter 5, “Linker Technology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Springer Science & Business Medicine, LLC, 2013.

[0251] For example, ADCs have been observed to kill bystander antigen-negative cells present near antigen-positive tumor cells. The mechanism of bystander cell killing by ADCs suggests that metabolites formed during the intracellular processing of ADCs may play a role. Neutral cytotoxic metabolites produced from ADC metabolism in antigen-positive cells appear to play a role in bystander cell killing, while charged metabolites prevent transmembrane diffusion into the mediator and therefore do not affect bystander killing. In some embodiments, selector junctions are chosen to attenuate the bystander killing effect induced by cellular metabolites of ADCs. In some embodiments, selector junctions are chosen to enhance the bystander killing effect.

[0252] The nature of the linker can also affect ADC aggregation under conditions of use and / or storage. Typically, ADCs reported in the literature contain no more than 3–4 drug molecules per antibody molecule (see, for example, Chari, 2008, Acc Chem Res 41:98–107). Attempts to achieve a higher drug-to-antibody ratio (“DAR”) often fail due to ADC aggregation, especially if both the drug and linker are hydrophobic (King et al., 2002, J Med Chem 45:4336–4343; Hollander et al., 2008, Bioconjugate Chem 19:358–361; Burke et al., 2009 Bioconjugate Chem 20:1242–1250). In many cases, a DAR higher than 3–4 can be beneficial as a means of increasing potency. In cases where cytotoxic agents and / or cell inhibitors are inherently hydrophobic, it may be desirable to select relatively hydrophilic linkers as a means of reducing ADC aggregation, especially when a DAR greater than 3-4 is required. Therefore, in some embodiments, the linker incorporates a chemical component that reduces ADC aggregation during storage and / or use. The linker may incorporate polar or hydrophilic groups, such as charged groups or groups charged at physiological pH, to reduce ADC aggregation. For example, the linker may incorporate charged groups, such as salts, or groups that deprotonate, such as carboxylates, or protonate, such as amines, at physiological pH.

[0253] Exemplary multivalent adapters that generate up to 20 DARs and can be used to bind a variety of cytotoxic agents and / or cell inhibitors to antibodies are described in WO 2009 / 073445, WO 2010 / 068795, WO 2010 / 138719, WO 2011 / 120053, WO 2011 / 171020, WO 2013 / 096901, WO 2014 / 008375, WO 2014 / 093379, WO 2014 / 093394, and WO 2014 / 093640, the contents of which are incorporated herein by reference in their entirety.

[0254] In specific implementations, as determined by size exclusion chromatography (SEC), the aggregation of the ADC during storage or use is less than about 10%. In specific implementations, as determined by size exclusion chromatography (SEC), the aggregation of the ADC during storage or use is less than 10%, for example, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, or even less.

[0255] 6.2.7. Method for preparing anti-glycation-MUC1 ADC

[0256] The disclosed anti-glycan-MUC1 ADC can be synthesized using well-known chemical methods. The chosen chemicals will depend in particular on the identity of the cytotoxic agent and / or cell inhibitor, the linker, and the group used to attach the linker to the antibody. Typically, the ADC according to formula (I) can be prepared according to the following scheme:

[0257] DLR x +Ab-R y →[DL-XY] n -Ab(I)

[0258] Where D, L, Ab, XY, and n are as previously defined, and R x and R y This refers to complementary groups that, as described above, can form covalent bonds with each other.

[0259] R x and R y The identity of the group will depend on the DLR used to synthesize the synthetic group. xChemical methods for conjugating antibodies. Generally, the chemical methods used should not alter the integrity of the antibody, such as its ability to bind to a target. Preferably, the binding properties of the conjugated antibody will be very similar to those of the unconjugated antibody. Various chemical methods and techniques for conjugating molecules to biomolecules such as antibodies are known in the art, and are particularly well-known for antibodies. See, for example, Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. Eds., Alan R. Liss, Inc., 1985; Hellstrom et al., "Antibodies For Drug Delivery," in: Controlled Drug Delivery, Robinson et al. Eds., Marcel Dekker, Inc., 2nd Ed. 1987; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in: Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al., Eds., 1985; "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in: Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al., Eds., Academic Press, 1985; Thorpe et al., 1982, Immunol. Rev. 62:119-58; PCT Publication WO 89 / 12624. Any of these chemical methods can be used to link synthons to antibodies.

[0260] Many functional groups R used to connect synthons to accessible lysine residues x The chemicals are known, and include, for example, but not limited to, NHS-esters and isothiocyanates.

[0261] Many functional groups R used to link synthons to accessible free thiol groups of cysteine ​​residues x The chemicals are known and include, for example, but not limited to, haloacetyl and maleimide.

[0262] However, conjugation chemistry is not limited to available side-chain groups. By linking a suitable small molecule to an amine, the side chain, such as an amine, can be transformed into other useful groups, such as a hydroxyl group. This strategy can be used to increase the number of available linking sites on the antibody by conjugating a multifunctional small molecule to the side chain of an accessible amino acid residue. Functional groups R suitable for covalently linking synthons to these "transformed" functional groups are... x It is then included in the synthon.

[0263] Antibodies can also be engineered to include amino acid residues for conjugation. Axup et al., 2012, Proc Natl Acad Sci USA. 109(40): 16101-16106 describes methods for engineering antibodies to include non-genetically encoded amino acid residues for conjugation of drugs in the context of ADCs, as well as chemicals and functional groups for linking synthons to non-coding amino acids.

[0264] Typically, the synthon is attached to the side chain of an amino acid residue of the antibody, which includes, for example, a primary amino group of an accessible lysine residue or a thiol group of an accessible cysteine ​​residue. The free thiol group can be obtained by reducing the interchain disulfide bond.

[0265] For R y It is the linkage of the thiol group (e.g., when R...) x When it is maleimide, the antibody is usually first completely or partially reduced to destroy the interchain disulfide bridge between cysteine ​​residues.

[0266] Cysteine ​​residues that do not participate in disulfide bridges can be engineered into antibodies by mutating one or more codons. Reduction of these unpaired cysteine ​​residues produces thiol groups suitable for conjugation. Preferred positions for incorporating engineered cysteine ​​residues include, for example, but not limited to, positions S112C, S113C, A114C, S115C, A176C, 5180C, S252C, V286C, V292C, S357C, A359C, S398C, S428C (Kabat numbers) on the human IgG1 heavy chain and positions V110C, S114C, S121C, S127C, S168C, V205C (Kabat numbers) on the human Igκ light chain (see, for example, U.S. Patent Nos. 7,521,541, 7,855,275, and 8,455,622).

[0267] As those skilled in the art will understand, the number of cytotoxic agents and / or cell inhibitors conjugated to antibody molecules can vary, such that the collection of ADCs can be heterogeneous in nature, with some antibodies containing one conjugate, some containing two, some containing three, etc. (and some antibodies containing no conjugate). The degree of heterogeneity will depend in particular on the chemical method used to conjugate the cytotoxic agents and / or cell inhibitors. For example, in the case of reducing antibodies to produce thiol groups for attachment, a heterogeneous mixture of antibodies with zero, 2, 4, 6, or 8 conjugates per molecule is typically produced. Furthermore, by limiting the molar ratio of the attachment compounds, antibodies with zero, 1, 2, 3, 4, 5, 6, 7, or 8 conjugates per molecule are typically produced. Thus, it will be understood that, depending on the context, the DAR can be an average of the antibody collection. For example, "DAR4" can refer to an ADC formulation that has not been purified to isolate a specific DAR peak and may contain a heterogeneous mixture of ADC molecules, wherein each antibody has a different number of cell inhibitors and / or cytotoxic agents (e.g., 0, 2, 4, 6, 8 reagents per antibody), but the average drug-to-antibody ratio is 4. Similarly, in some embodiments, "DAR2" refers to a heterogeneous ADC formulation in which the average drug-to-antibody ratio is 2.

[0268] When enrichment of a formulation is desired, antibodies with a defined number of linked cytotoxic agents and / or cell inhibitors can be obtained by purifying a heterogeneous mixture, for example by column chromatography, such as hydrophobic interaction chromatography.

[0269] As is known in the art, purity can be assessed using a variety of methods. As a specific example, ADC formulations can be analyzed by HPLC or other chromatographic methods, and purity can be assessed by analyzing the area under the curve of the resulting peak.

[0270] 6.3 Chimeric antigen receptor

[0271] This disclosure provides a chimeric antigen receptor (CAR) comprising an anti-glyco-MUC1 antibody or antigen-binding fragment as described herein.

[0272] The CAR disclosed herein typically includes an extracellular domain operatively connected to a transmembrane domain, which in turn is operatively connected to an intracellular domain for signal transduction.

[0273] The extracellular domain of the CAR disclosed herein contains a sequence of an anti-glyco-MUC1 antibody or an antigen-binding fragment (e.g., as described in Section 6.1 or in Embodiments 1 to 145).

[0274] Exemplary transmembrane domain sequences and intracellular domain sequences are described in Sections 6.3.1 and 6.3.2, respectively.

[0275] The fusion proteins described herein (e.g., embodiments 152 and 153) are CARs, and the disclosures related to CARs apply to such fusion proteins.

[0276] 6.3.1. Transmembrane domain

[0277] Regarding transmembrane domains, CARs can be designed to contain transmembrane domains that are operatively linked (e.g., fused) to the extracellular domains of the CAR.

[0278] The transmembrane domain can be derived from a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. The transmembrane region specifically used in this disclosure can be derived from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., a transmembrane region containing at least the aforementioned chains). In some cases, various human hinges, including human Ig (immunoglobulin) hinges, may also be used.

[0279] In one embodiment, the transmembrane domain is synthetic (i.e., not naturally occurring). Examples of synthetic transmembrane domains are peptides primarily containing hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is present at each end of the synthetic transmembrane domain. Optionally, short oligopeptides or peptide linkers, preferably oligopeptides or peptide linkers with a length of 2 to 10 amino acids, can form a connection between the transmembrane domain of the CAR and the cytoplasmic signaling domain. Glycine-serine duplexes provide particularly suitable linkers.

[0280] In one embodiment, the transmembrane domain in the CAR of this disclosure is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence YLHLGALGRDLWGPSPVTGYHPLL (SEQ ID NO:102).

[0281] In one embodiment, the transmembrane domain in the CAR of this disclosure is the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:103).

[0282] In some embodiments, the transmembrane domain of the CAR disclosed herein comprises a CD8a hinge domain. In one embodiment, the CD8a hinge domain comprises the amino acid sequence TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC (SEQ ID NO: 104).

[0283] 6.3.2. Intracellular domains

[0284] The intracellular signaling domain of the CAR disclosed herein is responsible for activating at least one normal effector function of CAR-expressing immune cells. The term "effector function" refers to a specific function of a cell. For example, the effector function of a T cell could be cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform a specific function. While the entire intracellular signaling domain can generally be used, in many cases, the entire strand is not necessary. Regarding the use of truncated portions of intracellular signaling domains, such truncated portions can be used in place of the complete strand as long as they transduce effector function signals. Therefore, the term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce effector function signals.

[0285] Preferred examples of intracellular signal transduction domains used in the CAR of this disclosure include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that work together to initiate signal transduction upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0286] The signal generated by the TCR alone may not be sufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0287] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, which are referred to as immune receptor tyrosine-based activation motifs or ITAMs.

[0288] Examples of primary cytoplasmic signaling sequences containing ITAM specifically used in the CARs of this disclosure include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Particularly preferred are the cytoplasmic signaling molecules in the CARs of this disclosure that comprise cytoplasmic signaling sequences derived from CD3-ζ.

[0289] In a preferred embodiment, the cytoplasmic domain of the CAR is designed to include, or combine with, any other desired cytoplasmic domain that may be used in the CAR of this disclosure, a primary cytoplasmic signaling sequence domain containing ITAM (e.g., a CD3-ζ cytoplasmic signaling sequence domain). For example, the cytoplasmic domain of the CAR may include a CD3ζ chain portion and a co-stimulatory signaling region.

[0290] The costimulatory signal transduction region refers to the portion of the CAR containing the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective lymphocyte response to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0291] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR disclosed herein can be linked together in a random or specified order. Optionally, short oligopeptide or polypeptide linkers, preferably oligopeptide or polypeptide linkers with a length of 2 to 10 amino acids, can form links. Glycine-serine dinucleotides provide particularly suitable linkers.

[0292] In one embodiment, the cytoplasmic domain comprises a CD3-ζ signal transduction domain and a CD28 signal transduction domain. In another embodiment, the cytoplasmic domain comprises a CD3-ζ signal transduction domain and a 4-1BB signal transduction domain.

[0293] 6.4 Nucleic Acids, Recombinant Vectors, and Host Cells

[0294] This disclosure covers nucleic acid molecules encoding immunoglobulin light and heavy chain genes for anti-glyco-MUC1 antibodies, vectors containing such nucleic acids, and host cells capable of producing the anti-glyco-MUC1 antibodies of this disclosure. In some aspects, the nucleic acid molecules encode and the host cells are capable of expressing the anti-glyco-MUC1 antibodies of this disclosure and antibody-binding fragments (e.g., as described in Section 6.1 and Embodiments 1-145), as well as fusion proteins (e.g., as described in Embodiments 146-151) and chimeric antigen receptors containing them (e.g., as described in Section 6.3 and Embodiments 152-153). Exemplary vectors of this disclosure are described in Embodiments 166-168, and exemplary host cells are described in Embodiments 169-172.

[0295] The anti-glyco-MUC1 antibody of this disclosure can be prepared by recombinantly expressing immunoglobulin light and heavy chain genes in host cells. To recombinantly express the antibody, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, so that the light and heavy chains are expressed in the host cells and optionally secreted into a culture medium from which the antibody can be recovered. The heavy and light chain genes of the antibody are obtained using standard recombinant DNA methods, these genes are integrated into a recombinant expression vector, and the vector is then introduced into host cells, such as those described in: Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associates, 1989), and U.S. Patent No. 4,816,397.

[0296] To generate nucleic acids encoding such anti-glyco-MUC1 antibodies, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplifying and modifying germline DNA or cDNA encoding the light and heavy chain variable sequences, for example using polymerase chain reaction (PCR). Germline DNA sequences of human heavy and light chain variable region genes are known in the art (see, for example, the “VBASE” human germline sequence database; also see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol. 22T: 116-198; and Cox et al., 1994, Eur. J. Immunol. 24: 827-836; the contents of each of which are incorporated herein by reference).

[0297] Once the V-related encoding of anti-glyco-MUC1 antibody is obtained... H and V L These DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the gene encoding V... H or V L The DNA fragments can be operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible adapter. The term "operatively linked" as used herein is intended to mean linking two DNA fragments together such that the amino acid sequences encoded by the two DNA fragments remain within the frame.

[0298] By encoding V H The DNA is operatively linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3, and optional CH4), enabling the encoding V... HThe isolated DNA from the region is converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but in some embodiments are IgG1 or IgG4 constant regions. For Fab fragment heavy chain genes, the region encoding V... H The DNA can be operatively linked to another DNA molecule that encodes only the CH1 constant region of the heavy chain.

[0299] By encoding V L The DNA of the gene is operatively linked to another DNA molecule encoding the light chain constant region CL, enabling the encoding of V... L The isolated DNA from the regions is converted into full-length light chain genes (and Fab light chain genes). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but in some embodiments are κ constant regions.

[0300] To generate the scFv gene, the encoding V can be... H and V L The DNA fragment can be operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4–Ser)3, so that V H and V L The sequence can be expressed as a continuous single-chain protein, where V H and V LThe zones are connected by flexible joints (see, for example, Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0301] To express the anti-glyco-MUC1 antibody of this disclosure, DNA encoding a partial or full-length light and heavy chain, obtained as described above, is inserted into an expression vector such that the gene is operatively linked to transcriptional and translational control sequences. Hereinafter, the term "operatively linked" means linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the host cells used for expression. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes can be inserted into the same expression vector.

[0302] The antibody gene is inserted into the expression vector using standard methods (e.g., ligation of the antibody gene fragment and complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present). The expression vector may already carry the antibody constant region sequence before inserting the light or heavy chain sequence associated with the anti-glyco-MUC1 antibody. For example, the V-chain sequence associated with the anti-glyco-MUC1 monoclonal antibody... H and V L One method to convert sequences into full-length antibody genes is to insert them, respectively, into expression vectors that already encode the heavy chain constant region and the light chain constant region, so that V H The segment is operatively connected to the CH segment within the carrier, while the V segment... L The segment is operatively linked to the CL segment within the vector. Alternatively or concurrently, the recombinant expression vector may encode a signal peptide that promotes the secretion of antibody chains from host cells. The antibody chain gene can be cloned into the vector such that the signal peptide is linked within the frame to the N-terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0303] In addition to the antibody chain gene, the recombinant expression vectors of this disclosure also carry regulatory sequences that control the expression of the antibody chain gene in host cells. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. Those skilled in the art will recognize that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the selection of the host cell to be transformed, the desired protein expression level, etc. Suitable regulatory sequences for expression in mammalian host cells include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyomaviruses. For further description of viral regulatory elements and their sequences, see, for example, U.S. Patent No. 5,168,062 to Stinski, U.S. Patent No. 4,510,245 to Bell et al., and U.S. Patent No. 4,968,615 to Schaffner et al.

[0304] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of this disclosure may also carry other sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells to which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017 to Axel et al.). For example, typically, selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate in host cells to which the vector has been introduced. Suitable selectable marker genes include dihydrofolate reductase (DHFR) genes (for DHFRs with methotrexate selection / amplification). - The host cell contains the light and heavy chains (heavy and neo genes, used for G418 selection). To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into the host cell using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipid transfection, calcium phosphate precipitation, DEAE-glucan transfection, etc.

[0305] It is possible to express the antibodies of this disclosure in prokaryotic or eukaryotic host cells. In some embodiments, antibody expression is carried out in eukaryotic cells, such as mammalian host cells, that optimally secrete antibodies that are properly folded and possess immunogenic activity. Exemplary mammalian host cells for expressing the recombinant antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including DHFR cells). - CHO cells (described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with DHFR optional markers, such as those described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, antibodies are generated by culturing the host cells for a period sufficient to allow antibody expression in the host cells or to allow antibody secretion into the medium in which the host cells grow. Antibodies can be recovered from the medium using standard protein purification methods. Host cells can also be used to generate portions of the complete antibody, such as Fab fragments or scFv molecules. It should be understood that variations of the above process are within the scope of this disclosure. For example, it may be desirable to transfect host cells with DNA encoding the light chain or heavy chain (but not both) of the anti-glyco-MUC1 antibody of this disclosure.

[0306] To express the CAR of this disclosure, as described in Section 6.3 and embodiments 152 and 153, the preferred host cell is a T cell, preferably a human T cell. In some embodiments, the host cell exhibits anti-tumor immunity when it cross-links with MUC1 on tumor cells. Detailed methods for generating the T cells of this disclosure are described in Section 6.4.1.

[0307] Recombinant DNA technology can also be used to remove part or all of the DNA from one or both of the light and heavy chains that are not required to encode the binding sugar MUC1. Molecules expressed by such truncated DNA molecules are also covered by the antibodies disclosed herein.

[0308] To recombinantly express the anti-glyco-MUC1 antibody of this disclosure, host cells can be co-transfected with the two expression vectors of this disclosure, wherein the first vector encodes a heavy chain-derived polypeptide and the second vector encodes a light chain-derived polypeptide. The two vectors may contain the same optional marker, or each may contain a separate optional marker. Alternatively, a single vector encoding both the heavy chain and light chain polypeptides may be used.

[0309] Once a nucleic acid encodes one or more portions of an anti-glyco-MUC1 antibody, further alterations or mutations can be introduced into the coding sequence, for example, to produce nucleic acids encoding antibodies with different CDR sequences, antibodies with reduced affinity for the Fc receptor, or antibodies of different subclasses.

[0310] The anti-glyco-MUC1 antibody of this disclosure can also be produced by chemical synthesis (e.g., by the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also be produced using cell-free platforms (see, for example, Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17:420-426).

[0311] Once the anti-glyco-MUC1 antibody of this disclosure is generated through recombinant expression, it can be purified using any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, the anti-glyco-MUC1 antibody and / or binding fragment of this disclosure can be fused with heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0312] After separation, if necessary, it can be performed, for example, by high-performance liquid chromatography (see, for example, Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, Work and Burdon, eds., Elsevier, 1980) or by Superdex. TM The anti-glyco-MUC1 antibody was further purified by gel filtration chromatography on a 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0313] 6.4.1. Recombination of CARs in T cells

[0314] In some embodiments, nucleic acids encoding the disclosed anti-glyco-MUC1CAR are delivered into cells using retroviral or lentiviral vectors. CAR-expressing retroviral and lentiviral vectors can be delivered to different types of eukaryotic cells, as well as tissues and the whole organism, using transduced cells as delivery vehicles or cell-free local or systemic delivery using encapsulated, bound, or naked vectors. The methods used can be used for any purpose requiring or sufficient for stable expression.

[0315] In other embodiments, the CAR sequence is delivered into cells using in vitro transcribed mRNA. Transfected cells can be used as delivery vehicles, or cell-free local or systemic delivery of encapsulated, bound, or naked mRNA can be used to deliver the in vitro transcribed mRNA CAR to different types of eukaryotic cells, as well as tissues and the whole organism. The methods used can be used for any purpose requiring or sufficient for transient expression.

[0316] In another implementation, the desired CAR can be expressed in cells via transposons.

[0317] One advantage of the RNA transfection method disclosed herein is that RNA transfection is essentially transient and vector-free: RNA transgenes can be delivered to lymphocytes and expressed as a minimal expression cassette after brief in vitro cell activation without requiring any other viral sequences to be expressed in the lymphocytes. Under these conditions, it is impossible for transgenes to integrate into the host cell genome. Due to the efficiency of RNA transfection and its ability to uniformly modify the entire lymphocyte population, cloning cells are not required.

[0318] Genetic modification of T cells using in vitro transcribed RNA (IVT-RNA) employs two distinct strategies, both of which have been serially tested in various animal models. Cells are transfected with the in vitro transcribed RNA via lipid transfection or electroporation. Preferably, various modifications are desired to stabilize the IVT-RNA, thereby achieving prolonged expression of the transferred IVT-RNA.

[0319] Several IVT vectors are known in the literature to be used as templates for in vitro transcription in a standardized manner and have been genetically modified to produce stable RNA transcripts. Currently, the protocol used in this field is based on a plasmid vector with the following structure: a 5' RNA polymerase promoter capable of RNA transcription, followed by a target gene with an untranslated region (UTR) on the 3' and / or 5' side, and a 3' polyadenylated cassette containing 50–70 nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylated cassette using a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylated cassette corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, and the poly(A) sequence is extended or masked at the 3' end. It is unclear whether this non-physiological protrusion affects the amount of protein produced intracellularly from such constructs.

[0320] Compared to more traditional plasmid or viral methods, RNA offers several advantages. Gene expression from RNA sources does not require transcription and produces protein products rapidly after transfection. Furthermore, because RNA only needs to enter the cytoplasm and not the nucleus, typical transfection methods result in extremely high transfection rates. Additionally, plasmid-based methods require that the promoter driving the target gene expression be active in the cells being studied.

[0321] In another aspect, RNA constructs can be delivered into cells via electroporation. See, for example, formulations and methods for delivering nucleic acid constructs into mammalian cells via electroporation, as taught in US2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, and US 2004 / 0092907A1. Various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in relevant research literature and in numerous patents and applications in this field. See, for example, US Patent Nos. 6,678,556, 7,171,264, and 7,173,116. Devices for therapeutic applications of electroporation are commercially available, such as MedPulser. TMThe DNA electroporation therapy system (Inovio / Genetronics, San Diego, Calif.) is described, for example, in U.S. Patent Nos. 6,567,694, 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482; electroporation can also be used for in vitro transfection of cells, as described in US20070128708A1. Electroporation can also be used for the in vitro delivery of nucleic acids into cells. Therefore, by utilizing any of the many available devices and electroporation systems known to those skilled in the art, electroporation-mediated delivery of nucleic acids, including expression constructs, into cells provides an exciting new means of delivering target RNA to target cells.

[0322] 6.4.1.1 Sources of T cells

[0323] The source of T cells is obtained from a subject prior to amplification and genetic modification. The term "subject" is intended to include any living organism (e.g., a mammal) capable of evoking an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is a human.

[0324] T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of this disclosure, any number of T cell lines available in the art may be used. In some embodiments of this disclosure, any number of techniques known to those skilled in the art, such as Ficoll, may be used. TMT cells are isolated from a unit of blood collected from a subject. In a preferred embodiment, cells are obtained from an individual's circulating blood via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected via apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing steps. In one embodiment of this disclosure, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution is deficient in calcium and may be deficient in magnesium, or may be deficient in many (if not all) divalent cations. Again, surprisingly, in the absence of calcium, the initial activation step results in amplified activation. As will be readily understood by those skilled in the art, the washing step can be performed by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., the Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) as per the manufacturer's instructions. After washing, cells can be resuspended in various biocompatible buffers, such as Ca- and Mg-free PBS, PlasmaLyte A, or other salt solutions with or without buffer. Alternatively, unwanted components of the agarose sample can be removed, and the cells can be directly resuspended in culture medium.

[0325] In another embodiment, red blood cells are dissolved and monocytes are consumed, for example by PERCOLL. TM T cells are isolated from peripheral blood mononuclear cells by gradient centrifugation or countercurrent centrifugation. Specific subsets of T cells, such as CD3, can be further separated using positive or negative sorting techniques. + CD28', CD4 + CD8 + CD45RA + and CD45RO + T cells. For example, in one embodiment, via beads conjugated with anti-CD3 / anti-CD28 (i.e., 3×28), for example... Incubation of M-450CD3 / CD28T cells together for a period sufficient for positive selection of desired T cells is used to isolate T cells. In one embodiment, this period is approximately 30 minutes. In another embodiment, the period ranges from 30 minutes to 36 hours or longer, and all integer values ​​between the two. In yet another embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours. In a preferred embodiment, the incubation period is 24 hours. Using a longer incubation time, such as 24 hours, can increase cell yield for isolating T cells from leukemia patients. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as in isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immune-impaired individuals. Furthermore, using a longer incubation time can improve the efficiency of capturing CD8+ T cells. Therefore, by simply shortening or lengthening the time T cells bind to CD3 / CD28 beads and / or by increasing or decreasing the bead-to-T-cell ratio (as further described herein), T-cell subsets can be preferentially selected at the start of culture or at other points in the process. Additionally, by increasing or decreasing the proportion of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T-cell subsets can be preferentially selected at the start of culture or at other desired points in the process. Those skilled in the art will recognize that multi-round selection can also be used in the context of this disclosure. In some embodiments, it may be desirable to perform a selection procedure and use “unselected” cells during activation and expansion. “Unselected” cells may also undergo a further round of selection.

[0326] T cell population enrichment via negative selection can be accomplished using a combination of antibodies targeting surface markers specific to negatively selected cells. One approach is cell sorting and / or sorting via negative magnetic immunoadhesion or flow cytometry (which uses a mixture of monoclonal antibodies targeting cell surface markers present on negatively selected cells). For example, to enrich CD4+ cells via negative selection, a monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, it may be desirable to enrich or positively select cells that typically express CD4. + CD25 + CD62L hi GITR + and FoxP3 + Regulatory T cells. Alternatively, in some embodiments, regulatory T cells are consumed via anti-C25 conjugated beads or other similar selection methods.

[0327] To separate a desired cell population through positive or negative selection, the concentrations of cells and surfaces (e.g., particles, such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume of beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In yet another embodiment, a concentration greater than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In other embodiments, a concentration of 125 million or 150 million cells / ml is used. Using high concentrations can lead to increased cell yield, cell activation, and cell proliferation. Furthermore, high cell concentrations allow for more effective capture of cells that may weakly express the target antigen, such as CD28-negative T cells, or cells from samples containing a large number of tumor cells (i.e., leukemia blood, tumor tissue, etc.). Such cell populations can be therapeutically valuable and are desirable. For example, using high cell concentrations allows for more effective selection of CD8 cells that typically have weak CD28 expression. + T cells.

[0328] In relevant implementations, it may be desirable to use a lower cell concentration. By significantly diluting the mixture of T cells and surfaces (e.g., particles, such as beads), the interaction between particles and cells is minimized. This selects cells expressing a large amount of the desired antigen to bind to the particles. For example, with diluted concentrations of CD8... + Compared to T cells, CD4 + T cells express higher levels of CD28 and are captured more effectively. In one embodiment, the cell concentration used is 5 x 10⁻⁶ cells / cells. 6 / ml. In other embodiments, the concentration used can be approximately 1x10⁻⁶. 5 / ml to 1x10 6 / ml, and any integer value in between.

[0329] In other implementations, cells can be incubated for different durations at different speeds on a rotator at 2-10°C or at room temperature.

[0330] T cells used for stimulation can also be frozen after the washing step. Hoping to be free from theoretical constraints, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and, to some extent, monocytes from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and would be useful in this case, one approach involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a medium containing 10% dextran 40 and 5% dextran, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextran, 0.45% NaCl, 10% dextran 40 and 5% dextran, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1°C per minute and storing them in the gas phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as uncontrolled freezing at -20°C or immediately in liquid nitrogen.

[0331] In some embodiments, as described herein, the cryopreserved cells are thawed and washed, and then allowed to stand at room temperature for one hour before activation using the methods of this disclosure.

[0332] In the context of this disclosure, it is also contemplated that blood samples or apheresis products may be collected from subjects at a time prior to the potential need for cell amplification as described herein. This allows for the collection of the source of cells to be amplified at any necessary point in time, and the isolation and freezing of desired cells, such as T cells, for use in subsequent T-cell therapy for various diseases or conditions, such as those described herein, which benefit from T-cell therapy. In one embodiment, the blood sample or apheresis sample is taken from a generally healthy subject. In some embodiments, the blood sample or apheresis sample is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed one, and the target cells are isolated and frozen for later use. In some embodiments, T cells may be amplified, frozen, and used at a later time. In some embodiments, samples are collected from the patient shortly after diagnosis of a specific disease as described herein, but before any treatment. In another implementation, cells are isolated from a subject's blood or apheresis sample prior to any number of relevant treatment modalities (including, but not limited to, treatment with agents such as natamizumab, efazolin, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunoablation agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, and radiation). These drugs inhibit calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling. (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In another embodiment, cells are isolated from the patient and frozen for subsequent (e.g., before, during, or after) use in combination with bone marrow or stem cell transplantation or T-cell ablation therapy using chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide.

[0333] In another embodiment of this disclosure, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that, following certain cancer treatments, particularly those with drugs that impair the immune system, shortly after treatment within the period when patients typically recover from treatment, the quality of the obtained T cells may be optimal or have improved in vitro expansion capacity. Similarly, after in vitro manipulation using the methods described herein, these cells can be in a preferred state for enhanced transplantation and in vivo expansion. Therefore, in the context of this disclosure, it is contemplated that blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, be collected during the recovery phase. Furthermore, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning protocols may be used to generate in subjects a favorable state in which the reaggregation, recycling, regeneration, and / or expansion of specific cell types is possible, particularly within a timeframe determined post-treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0334] 6.4.1.2 Activation and Expansion of T Cells

[0335] T cells are typically activated and expanded using methods described in, for example, the following documents: U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0336] Typically, the T cells of this disclosure are expanded through surface contact with a reagent to which a signal associated with stimulating the CD3 / TCR complex is attached, and with a ligand stimulating a co-stimulatory molecule on the surface of the T cells. Specifically, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator conjugated to a calcium ionocarrier (e.g., lichenin). For co-stimulation of helper molecules on the T cell surface, a ligand binding to that helper molecule is used. For example, the T cell population can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. For stimulating CD4… + T cells or CD8 +T cell proliferation is achieved using anti-CD3 and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), and other commonly known methods in the art may also be used (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0337] In some embodiments, primary and co-stimulatory signals for T cells can be provided in different ways. For example, the reagent providing each signal can be in solution or coupled to a surface. When coupled to a surface, the reagent can be coupled to the same surface (i.e., in a “cis” form) or to a separate surface (i.e., in a “trans” form). Alternatively, one reagent can be coupled to a surface while another is in solution. In one embodiment, the reagent providing the co-stimulatory signal binds to the cell surface, and the reagent providing the primary activation signal is in solution or coupled to a surface. In some embodiments, both reagents can be in solution. In another embodiment, the reagent can be in a soluble form and then cross-linked to a surface, such as cells or antibodies expressing Fc receptors or other binding agents that will bind the reagent. In this regard, see, for example, the artificial antigen-presenting cells (aAPCs) of U.S. Patent Application Publications 20040101519 and 20060034810, which are contemplated for activating and expanding the T cells of this disclosure.

[0338] In one embodiment, the two reagents are immobilized on a bead, either on the same bead (cis-) or on separate beads (trans-). For example, the reagent providing the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment, and the reagent providing the co-stimulatory signal is an anti-CD28 antibody or its antigen-binding fragment; and the two reagents are co-immobilized on the same bead at equal molecular weights. In one embodiment, targeting CD4... +T cell amplification and T cell growth are achieved using a 1:1 ratio of each bead-bound antibody. In certain aspects of this disclosure, a bead-bound anti-CD3:CD28 antibody ratio is used such that an increase in T cell amplification is observed compared to amplification observed using a 1:1 ratio. In one specific embodiment, an increase of approximately 1 to approximately 3-fold is observed compared to amplification observed using a 1:1 ratio. In one embodiment, the bead-bound CD3:CD28 antibody ratio is in the range of 100:1 to 1:100, and all integer values ​​between them. In one aspect of this disclosure, more anti-CD28 antibody binds to the particles compared to the anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of this disclosure, the bead-bound anti-CD28 antibody to anti-CD3 antibody ratio is greater than 2:1. In one specific embodiment, a bead-bound CD3:CD28 antibody ratio of 1:100 is used. In another embodiment, a bead-bound CD3:CD28 antibody ratio of 1:75 is used. In yet another embodiment, a bead-binding CD3:CD28 antibody ratio of 1:50 is used. In another embodiment, a bead-binding CD3:CD28 antibody ratio of 1:30 is used. In a preferred embodiment, a bead-binding CD3:CD28 antibody ratio of 1:10 is used. In another embodiment, a bead-binding CD3:CD28 antibody ratio of 1:3 is used. In yet another embodiment, a bead-binding CD3:CD28 antibody ratio of 3:1 is used.

[0339] The particle-to-cell ratio is from 1:500 to 500:1, and any integer value between these ratios can be used to stimulate T cells or other target cells. As will be readily understood by those skilled in the art, the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, small beads can bind only a few cells, while larger beads can bind many cells. In some embodiments, the cell-to-particle ratio is from 1:100 to 100:1, and any integer value between these ratios; in other embodiments, the ratio includes 1:9 to 9:1, and any integer value between these ratios can also be used to stimulate T cells. As described above, the ratio of anti-CD3 and anti-CD28 coupled granules to T cells leading to T cell stimulation can vary, but certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 granules per T cell. In one embodiment, a granule-to-cell ratio of 1:1 or smaller is used. In a particular embodiment, a preferred granule-to-cell ratio is 1:5. In other embodiments, the granule-to-cell ratio can vary depending on the number of days of stimulation. For example, in one embodiment, on the first day, the particle-to-cell ratio is 1:1 to 10:1, and thereafter additional particles are added to the cells daily or every other day at a final ratio from 1:1 to 1:10 (based on the cell count on the day of addition) until 10 days. In one specific embodiment, on the first day of stimulation, the particle-to-cell ratio is 1:1, and it is adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added daily or every other day based on the final ratio of 1:1 on the first day of stimulation and the final ratio of 1:5 on the third and fifth days of stimulation. In another embodiment, on the first day of stimulation, the particle-to-cell ratio is 2:1, and it is adjusted to 1:10 on the third and fifth days of stimulation. In yet another embodiment, particles are added daily or every other day based on the final ratio of 1:1 on the first day of stimulation and the final ratio of 1:10 on the third and fifth days of stimulation. Those skilled in the art will recognize that various other ratios may be applied to this disclosure. In particular, the ratio will vary depending on the particle size and the cell size and type.

[0340] In other embodiments of this disclosure, cells, such as T cells, are combined with reagent-coated beads, followed by separation of the beads and cells, and then cell culture. In an alternative embodiment, the reagent-coated beads and cells are not separated prior to culture, but are cultured together. In yet another embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased binding of cell surface markers, thereby inducing cell stimulation.

[0341] For example, cell surface proteins can be linked by contacting T cells with paramagnetic beads (3 x 28 beads) coated with anti-CD3 and anti-CD28. In one embodiment, the cells (e.g., 10 4 Up to 10 9 T cells) and beads (e.g., T cells) and beads M-450CD3 / CD28T paramagnetic beads (in a 1:1 ratio) are mixed in a buffer, preferably PBS (free of divalent cations, such as calcium and magnesium). Similarly, those skilled in the art will readily recognize that any cell concentration can be used. For example, the target cells may be very few in the sample, only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells. Therefore, any number of cells is within the scope of this disclosure. In some embodiments, it may be desirable to significantly reduce the volume of particles and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, a concentration greater than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75 million cells / ml, 80 million cells / ml, 85 million cells / ml, 90 million cells / ml, 95 million cells / ml, or 100 million cells / ml are used. In other embodiments, concentrations of 125 million or 150 million cells / ml are used. Using high concentrations can lead to increased cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations can more effectively capture cells that may weakly express the target antigen, such as CD28-negative T cells. In some embodiments, such cell populations may have therapeutic value and would be desirable. For example, using high cell concentrations can more effectively select CD8 cells that typically have weak CD28 expression. + T cells.

[0342] In one embodiment of this disclosure, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any integer value of hours in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of this disclosure, beads and T cells are cultured together for about eight days. In another embodiment, beads and T cells are cultured together for 2-3 days. Several stimulation cycles may be required so that the culture time of T cells can be 60 days or longer. Suitable conditions for T cell culture include a suitable culture medium (e.g., minimal basal medium or RPMI 1640 or X-vivo 15, (Lonza)) that may contain factors required for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any other additives known to the art for cell growth. Other additives used for cell growth include, but are not limited to, surfactants, plasma products, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, which are fortified with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with sufficient amounts of serum (or plasma) or a defined set of hormones, and / or sufficient amounts of cytokines to induce T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures to be infused into the subject. Target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2).

[0343] T cells exposed to different stimuli for varying durations can exhibit different characteristics. For example, typical blood or apheresis peripheral blood mononuclear cell products contain a population of helper T cells (T cells). H CD4 + The helper T cell population is larger than the cytotoxic or suppressor T cell population (T...). C CD8 + In vitro T cell expansion via stimulation of CD3 and CD28 receptors resulted in the following T cell population: Prior to this population, approximately 8-9 days prior, this population was primarily composed of T cells. H Cellular composition, and after about 8-9 days, this T cell population contains a gradual increase in T cells. C Cell population. Therefore, depending on the therapeutic goal, the infusion administered to the subject primarily consists of T cells. H The T cell population of the cell may be advantageous. Similarly, if T cells have already been isolated... CFor antigen-specific subgroups of cells, it may be beneficial to expand those subgroups to a greater extent.

[0344] In addition to CD4 and CD8 markers, other phenotypic markers also change significantly during cell expansion, but these changes are largely reproducible. This reproducibility allows for the customization of activated T cell products for specific purposes.

[0345] 6.5 Composition

[0346] The anti-glycemic-MUC1 antibody and / or anti-glycemic-MUC1 ADC disclosed herein may be in the form of a composition comprising an anti-glycemic-MUC1 antibody and / or ADC and one or more loaders, excipients, and / or diluents. The composition may be formulated for a specific purpose, such as for veterinary or human pharmaceutical use. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or loads used will depend on the intended use of the antibody and / or ADC, as well as the therapeutic use and route of administration.

[0347] For therapeutic use, the composition may be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the method required to administer it to the patient). The pharmaceutical composition may be administered to the patient via a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical, or local. In any given case, the most suitable route of administration will depend on the specific antibody and / or ADC, the subject, the nature and severity of the disease, and the subject's physical condition. Typically, the pharmaceutical composition is administered intravenously or subcutaneously.

[0348] The pharmaceutical composition can be conveniently available in unit dosage forms containing a predetermined amount of the disclosed anti-glycan-MUC1 antibody and / or anti-glycan-MUC1 ADC per dose. The amount of antibody and / or ADC contained in a unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized powder containing an amount of antibody and / or ADC suitable for a single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit containing a syringe, appropriate amounts of diluent, and / or other components for administration. Liquid unit doses can be conveniently provided in the form of syringes pre-filled with an amount of antibody and / or ADC suitable for a single administration.

[0349] The pharmaceutical composition may also be provided in bulk form containing an amount of ADC suitable for multiple administrations.

[0350] Pharmaceutical compositions for storage in lyophilized or aqueous formulations can be prepared by mixing antibodies and / or ADCs of desired purity with optional pharmaceutically acceptable loads, excipients, or stabilizers (all of which are referred to herein as “loads”), i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and other confounding additives. See Remington’s Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be non-toxic to the recipient at the doses and concentrations employed.

[0351] Buffers help maintain pH within a range close to physiological conditions. They can be present in a variety of concentrations, but are typically present at concentrations from about 2 mM to about 50 mM. Suitable buffers for use in this disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., mixtures of monosodium citrate and disodium citrate, mixtures of citrate and trisodium citrate, mixtures of citrate and monosodium citrate, etc.), succinate buffers (e.g., mixtures of succinic acid and monosodium succinate, mixtures of succinic acid and sodium hydroxide, mixtures of succinic acid and disodium succinate, etc.), tartrate buffers (e.g., mixtures of tartaric acid and sodium tartrate, mixtures of tartaric acid and potassium tartrate, mixtures of tartaric acid and sodium hydroxide, etc.), and fumarate buffers (e.g., fumarate and monosodium fumarate). Mixtures, fumarate-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.; gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.); oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.); lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.); and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0352] Preservatives may be added to inhibit microbial growth, and may be added in an amount of about 0.2% to 1% (w / v). Suitable preservatives for use in this disclosure include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride (e.g., chloride, bromide, and iodide), hexamethylammonium chloride, and alkylparaben esters, such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes referred to as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of this disclosure, and isotonic agents include polyols, such as ternary or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad range of excipients, ranging from fillers to additives that dissolve therapeutic agents or help prevent denaturation or adhesion to container walls. Typical stabilizers can be polyols (listed above); amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclic sugar alcohols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, for example... Examples of stabilizers include urea, glutathione, alpha-lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in amounts from 0.5 to 10% by weight per ADC.

[0353] Nonionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve glycoproteins and protect them from aggregation caused by agitation. This also allows the formulation to be exposed to stressed shear surfaces without protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and Pranic polyols. Nonionic surfactants can be present at concentrations from about 0.05 mg / mL to about 1.0 mg / mL, for example, from about 0.07 mg / mL to about 0.2 mg / mL.

[0354] Other hybrid excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and solubilizers.

[0355] 6.6 How to use

[0356] The anti-glyco-MUC1 antibodies or antigen-binding fragments described herein can be used in a variety of diagnostic assays. For example, antibodies and binding fragments can be used in immunoassays, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS), and Western blotting.

[0357] The anti-glyco-MUC1 antibodies or binding fragments described herein can also be used for in vivo imaging in radiography, where antibodies labeled with detectable fractions (e.g., radiopaque reagents or radioisotopes) are administered to a subject, preferably into the bloodstream, and the presence and location of the labeled antibodies in the host are determined. Such imaging techniques can be used for staging and treatment of malignant tumors.

[0358] The anti-glyco-MUC1 antibodies or binding fragments, ADCs, and CARs described herein can be used to treat cancers expressing glyco-MUC1, including bladder cancer, lung cancer, brain cancer, breast cancer, non-Hodgkin's lymphoma, cervical cancer, ovarian cancer, colorectal cancer, gastric cancer, bile duct cancer, chondrosarcoma, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, uterine cancer, and cervical cancer. In some implementations, the cancer is an epithelial cancer, such as breast cancer, ovarian cancer, pancreatic cancer, or lung cancer.

[0359] Therefore, this disclosure provides anti-glycan-MUC1 antibodies, binding fragments, ADCs, and CARs as described herein, for use as pharmaceuticals, such as for treating cancers, such as any of the cancers mentioned above, for diagnostic assays, and for in vivo imaging in radiography. This disclosure also provides the use of anti-glycan-MUC1 antibodies, binding fragments, ADCs, and CARs as described herein in the preparation of pharmaceuticals, such as for treating cancers, such as any of the cancers mentioned above.

[0360] When using the CAR of this disclosure for treatment, the treatment method of this disclosure includes administering to a subject with a tumor expressing glyco-MUC1 an effective amount of genetically modified cells engineered to express the CAR of this disclosure, such as those described in Section 6.3 or Embodiment 152 or Embodiment 153. Methods for modifying cells, particularly T cells, to express the CAR are described in Section 6.4.1.

[0361] 7. Example

[0362] 7.1 Example 1: Identification of anti-glyco-Muc1 antibody

[0363] 7.1.1. Materials and Methods

[0364] 7.1.1.1 Synthesis of Tn MUC1 Glycopeptide

[0365] GST-MUC115-mer peptide (Biotin) was synthesized using standard synthetic peptide chemistry and F-MOC-GalNAc-Ser / Thr targeting glycosylation sites. A peptide containing GalNAc at the serine and threonine residues shown in bold underline was originally reported, as in Fontenot et al., 1993, Pept. Res. 6:330–336. This glycopeptide is sometimes referred to herein as “GSTA”. Screening was performed using a control peptide 70-MUC1 (Biotin-RPAPGSTAPPAHGVT) (SEQ ID NO: 99) without glycosylation. The purified glycopeptide was characterized by MALDI-TOF mass spectrometry on a Voyager DE or Voyager DE Pro MALDI-TOF mass spectrometer (PerSeptive Biosystems) equipped with delayed extraction. The MALDI matrix was 10 g / L of 2,5-dihydroxybenzoic acid (Aldrich, Milwaukee, WI) dissolved in a 2:1 mixture of 0.1% TFA in 30% acetonitrile aqueous solution. Samples with a concentration of approximately 1 pmol / μL dissolved in 0.1% TFA were analyzed by placing 1 μL of sample solution on the probe tip and then adding 1 μL of matrix. All mass spectra were obtained in linear mode. Data processing was performed using GRAMS / 386 software (Galactic Industries, Salem, NH).

[0366] 7.1.1.2 Immunization regimen

[0367] GST-glycopeptides were conjugated to KLH (Pierce, Rockford, IL) using glutaraldehyde. The efficiency of the conjugation was assessed by size exclusion chromatography on a PD-10 column using an anti-MUC1 ELISA of the fractions. Almost all reactivity was present in the excluded fractions, while the reactivity of the fractions expected to contain the peptides was not apparent. Further evaluation included comparative titration analysis of the KLH conjugates with the corresponding glycopeptides by ELISA. Both analyses indicated that the conjugation was nearly complete, which should result in a KLH to glycopeptide ratio of 1:300. Female Balb / c wild-type mice were subcutaneously injected with 10 or 15 μg of the (glycopeptide) in a total volume of 200 μL (mixed 1:1 with Sigma's Freunds adjuvant). Mice were immunized four times at 14-day intervals, and blood samples were obtained one week after the third and fourth immunizations via tail or eye bleeding.

[0368] 7.1.1.3 Production of mouse monoclonal antibody against Tn-MUC1

[0369] Monoclonal antibodies (mAbs) from wild-type Balb / c mice immunized with the KLH-conjugated GST-MUC1 glycopeptide were screened using a glycopeptide ELISA, followed by immunocytological examination of breast cancer cell lines (MCF7 and T47D) and immunohistochemical examination of cancer tissue. Selection was based on reactivity patterns similar to those of total serum from the same mice. Two antibodies, 1AG and 4AG, were selected for further characterization.

[0370] 7.2 Example 2: Functional characterization of 1AG and 4AG antibodies by ELISA

[0371] 7.2.1. Overview

[0372] 1AG and 4AG were characterized by indirect ELISA to test the reactivity of anti-MUC1mAb to titrated MUC1 peptides (including non-glycosylated peptides) with different glycosylation sites as shown in Table 4.

[0373]

[0374] 7.2.2. Materials and Methods

[0375] ELISA plates were coated overnight with MUC1 peptide titrated overnight in 0.2 M bicarbonate buffer at pH 9.4 at concentrations ranging from 0.08 μg / ml to 10 μg / ml. BSA was used as a control / background measure. The plates were then plated with SuperBlock at room temperature. TM (Thermo Fisher) Block for 1 hour. After washing the plate, incubate the supernatant of 1AG and 4AG hybridomas on the ELISA plate for 1 hour. Then wash the plate and incubate it with the secondary antibody (1 / 3000 goat anti-mouse IgG (H+L) HRP (abcam 62-6520)) for 1 hour. Then wash the plate and use 1-Step TM Develop with Ultra TMB (Thermo Fisher) for 2 minutes. Then stop development with 2N sulfuric acid. Then measure the absorbance at 450 nm.

[0376] 7.2.3. Results

[0377] The results are shown in Figures 1A to 1F and Figures 2A to 2B As shown in the figure, 1AG and 4AG specifically bind to glycosylated peptides C3, C4, and GST, but not to glycosylated peptide C2 or non-glycosylated peptide 70.

[0378] 7.3 Example 3: Functional characterization of 1AG and 4AG antibodies by antibody titration

[0379] 1AG and 4AG were characterized by antibody titration assays against a constant concentration of antigen GSTA.

[0380] The results are shown in Figure 3 .

[0381] 7.4 Example 4: Functional characterization of 1AG and 4AG antibodies by surface plasmon resonance

[0382] 7.4.1. Materials and Methods

[0383] The binding of 1AG and 4AG antibodies to a glycosylated peptide and a non-glycosylated peptide was evaluated by surface plasmon resonance (SPR) (Table 5).

[0384]

[0385] All SPR experiments were performed at 25°C on a Biacore T200 with a CM5 sensor chip and 10,000 RU anti-mouse antibody (GE Healthcare) per channel, using HBS-P+ as the run sample buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween 20 and 1 mg / ml BSA) and 10 mM glycine at pH 1.7 as the regeneration buffer.

[0386] Using standard amine coupling chemistry (EDC / NHS activation), 1AG and 4AG antibodies were immobilized in 10 mM sodium acetate at pH 5.0 as a 30 μg / mL solution. Antibody capture was performed at 5 μL / min in reaction buffer for 7 to 10 minutes using 10X dilution. Immobilization levels ranged from 600 to 1000 RU. Peptide samples were prepared as a two-fold dilution series in running buffer at an initial concentration of 100 nM (6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM) and injected at 50 μL / min in a single cycle for 2 minutes. Dissociation was monitored for 5 to 30 minutes.

[0387] 7.4.2. Results

[0388] The results are shown in Table 6. As shown in Table 6, 1AG has an affinity for glycosylated peptides in the double-digit nanomolar range, while 4AG has an affinity for glycosylated peptides in the single-digit nanomolar range.

[0389]

[0390] 7.5 Example 5: Sequence Analysis of Anti-Glyco-Muc1 Antibody

[0391] Rapid amplification of cDNA ends (RACE) was performed to determine the heavy and light chain nucleotide sequences of 1AG and 4AG.

[0392] The nucleotide sequences encoding the heavy and light chain variable regions of 1AG are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively. The heavy and light chain variable regions encoded by SEQ ID NO:21 and SEQ ID NO:22 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The predicted heavy chain CDR sequences (defined by IMGT) are shown in SEQ ID NO:3-5, and the predicted light chain CDR sequences (defined by IMGT) are shown in SEQ ID NO:6-8. The predicted heavy chain CDR sequences (defined by Kabat) are shown in SEQ ID NO:9-11, and the predicted light chain CDR sequences (defined by Kabat) are shown in SEQ ID NO:12-14. The predicted heavy chain CDR sequences (defined by Chothia) are shown in SEQ ID NO:15-17, and the predicted light chain CDR sequences (defined by Chothia) are shown in SEQ ID NO:18-20.

[0393] The nucleotide sequences encoding the variable regions of the heavy and light chains of 4AG are shown in SEQ ID NO:43 and SEQ ID NO:44, respectively. The variable regions of the heavy and light chains encoded by SEQ ID NO:43 and SEQ ID NO:44 are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively. The predicted heavy chain CDR sequences (defined by IMGT) are shown in SEQ ID NO:25-27, and the predicted light chain CDR sequences (defined by IMGT) are shown in SEQ ID NO:28-30. The predicted heavy chain CDR sequences (defined by Kabat) are shown in SEQ ID NO:31-33, and the predicted light chain CDR sequences (defined by Kabat) are shown in SEQ ID NO:34-36. The predicted heavy chain CDR sequences (defined by Chothia) are shown in SEQ ID NO:37-39, and the predicted light chain CDR sequences (defined by Chothia) are shown in SEQ ID NO:40-42.

[0394] 7.6 Example 6: Immunohistochemical staining of various tumor tissues using anti-glyco-Muc1 antibody

[0395] 7.6.1. Materials and Methods

[0396] Human Tissue Microarray (TMA) BCN721a (Biomax, USA) was used to screen the ability of antibodies 1AG and 4AG to stain various tumor tissues. The TMA contained tissue samples from 12 human organs, including esophagus, stomach, colon, rectum, liver, lung, kidney, breast, cervix, ovary, prostate, and pancreas, each taken from three normal human individuals, with each case represented by a single chip.

[0397] Table 7 shows the identity of each organization in TMA.

[0398]

[0399]

[0400]

[0401] Slides were fixed in cold 10% buffered neutral formalin for 15 minutes. Immunohistochemistry (IHC) was performed by incubating slides with undiluted hybridoma supernatant or purified mAbs (1 AG and 4 AG) for 24 hours at 4°C or room temperature. The bound mAbs were detected using FITC-conjugated rabbit anti-mouse immunoglobulin (1:100; Dako, Denmark) or peroxidase-conjugated rabbit anti-mouse immunoglobulin. Slides were fixed with Prolong Gold anti-fading agent containing DAPI (Invitrogen), and photomicrographs were obtained using a Leica wide-field fluorescence microscope.

[0402] 7.6.2. Results

[0403] The results of 1AG are shown in Figure 4 The results of 4AG are shown in Figure 5 mAb 1AG and mAb 4AG showed specific reactions with most of the examined cancerous tissues. Staining of healthy tissues showed very limited reactivity with surface structures, although some markers of intracellular structures were observed, particularly in gastric tissue.

[0404] 7.7 Example 7: Immunohistochemical staining of ovarian tumor tissue using anti-glyco-Muc1 antibody

[0405] 7.7.1. Materials and Methods

[0406] The human tissue microarray (TMA) OV241c (Biomax, USA) was used to screen the ability of antibodies 1AG and 4AG to stain ovarian tumor tissue.

[0407] Table 8 shows the identity of each organization in TMA.

[0408]

[0409]

[0410] Slides were fixed in cold 10% buffered neutral formalin for 15 minutes. Immunohistochemistry (IHC) was performed by incubating slides with undiluted hybridoma supernatant or purified mAbs (1 AG and 4 AG) for 24 hours at 4°C or room temperature. The bound mAbs were detected using FITC-conjugated rabbit anti-mouse immunoglobulin (1:100; Dako, Denmark) or peroxidase-conjugated rabbit anti-mouse immunoglobulin. Slides were fixed with Prolong Gold anti-fading agent containing DAPI (Invitrogen), and photomicrographs were obtained using a Leica wide-field fluorescence microscope.

[0411] 7.7.2. Results

[0412] The results of 1AG are shown in Figure 6 The results of 4AG are shown in Figure 7 mAb 4AG reacted specifically with most examined tissues from ovarian adenocarcinoma. Staining of adjacent healthy tissues revealed certain markers of intracellular structures. Under these conditions, mAb 1AG was not significantly marked in ovarian cancer and tumor-adjacent tissues.

[0413] 7.8 Example 8: Immunohistochemical staining of tumor tissue using anti-glyco-Muc1 antibody

[0414] 7.8.1. Materials and Methods

[0415] Human tissue microarray (TMA) BC000119 (Biomax, USA) was used to screen the staining ability of antibodies 1AG and 4AG against various tumor tissues. The TMA included 40 cases of invasive ductal carcinoma of the breast, squamous cell carcinoma of the lung, adenocarcinoma of the colon, adenocarcinoma of the prostate, and adenocarcinoma of the pancreas. Healthy control samples were evaluated using a multi-organ normal tissue array.

[0416] Table 9 shows the identity of each organization in TMA.

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423] Slides were fixed in cold 10% buffered neutral formalin for 15 minutes. Immunohistochemistry (IHC) was performed by incubating slides with undiluted hybridoma supernatant or purified mAbs (1 AG and 4 AG) for 24 hours at 4°C or room temperature. The bound mAbs were detected using FITC-conjugated rabbit anti-mouse immunoglobulin (1:100; Dako, Denmark) or peroxidase-conjugated rabbit anti-mouse immunoglobulin. Slides were fixed with Prolong Gold anti-fading agent containing DAPI (Invitrogen), and photomicrographs were obtained using a Leica wide-field fluorescence microscope.

[0424] 7.8.2. Results

[0425] The results of 1AG are shown in Figure 8 The results of 4AG are shown in Figure 9 mAb 1AG showed a specific reaction with a proportion of tumors from most examined cancerous tissues, including those from breast cancer, lung cancer, colon cancer, and pancreatic cancer. mAb 4AG showed a strong specific reaction with a significant proportion of tumors from most examined cancers, including those from breast cancer, lung cancer, colon cancer, prostate cancer, and pancreatic cancer.

[0426] 7.9 Example 9: Epitope mapping of 1AG and 4AG antibodies by alanine scanning

[0427] 7.9.1. Materials and Methods

[0428] 7.9.1.1 Materials

[0429] The following reagents were used in the study described in this embodiment.

[0430]

[0431] 7.9.1.2 Synthesis of alanine library by step

[0432] A walking alanine library was constructed, which contains the 28-mer motif based on the MUC1 protein. The library contains 12 linear glycopeptides (the bolded and underlined residues are glycosylated with GalNAc). Each of these 12 glycopeptides contains a unique alanine point mutation at positions 13-24 of the 28-mer MUC1 protein. If the native amino acid at positions 13-24 is alanine, the residue is mutated to a glycine residue. The amino acid sequences of the library glycopeptides are shown in Table 10A. The glycopeptides were synthesized using the Fmoc chemical method. The Fmoc-GalNAc-serine (GalNac-Ser) and Fmoc-GalNAc-threonine (GalNAc-Thr) constructs were purchased from Sussex Research (Ottawa, Canada). Unglycosylated forms of the 28-mer MUC1 peptide were synthesized together with these glycopeptides as negative controls. After synthesis, the peptides were lyophilized and resuspended in deionized water to a concentration of 1 mg / mL before use.

[0433] 7.9.1.3 Epitope plotting of 5E5, 1AG and 4AG

[0434] This library was used in ELISA format to plot the epitopes of the following MUC1-Tn antibodies: 5E5, 1AG, and 4AG. Unglycosylated MUC1 peptides were used as negative controls, while peptides glycosylated with GalNAc at serine and threonine residues (shown in bold underlined text) were used. Used as a positive control. Unrelated glycopeptides were used to measure GalNAc cross-reactivity. The sequence of the control peptide is shown in Table 10B.

[0435]

[0436]

[0437] Glycopeptides and relevant controls were diluted to 0.25 μg / mL in 0.2 M bicarbonate buffer at pH 9.8, and 100 μl was plated into each well of a high-binding ELISA plate. The plate was then incubated overnight at 4 °C. After discarding the coating buffer, the plate was blocked for 1 hour at room temperature with 300 μl of blocking buffer (2.5% BSA in 1xPBS). The blocking buffer was then discarded, and 100 μl of antibody supernatant (diluted in blocking buffer) for each of the 5E5, 1AG, and 4AG antibodies was added to each well and incubated for 1 hour at room temperature. The antibody supernatant dilutions were optimized beforehand for each antibody (data not shown). Biotinylated VillaVillosa lectin (100 μl per well, 1 μg / mL in blocking buffer) was added to the plate as a control to detect the presence of GalNAc-modified residues on the peptides. Wash the plate three times with 300 μl of 1x TBS / 0.05% Tween-20 (TBST) per well. Dilute the secondary antibody, goat anti-mouse IgG (H+L)-HRP, 1 / 5000 in blocking buffer, add 100 μl of the diluted antibody to each well, and incubate the plate at room temperature for 1 hour. Dilute the secondary antibody, streptavidin-HRP, 1 / 3000 in blocking buffer, and add 100 μl of the diluted streptavidin-HRP to each well containing biotinylated lectin. After two incubations, wash the plate four times with 300 μl of TBST per well, then develop with 75 μl of 1-Step TMB substrate at room temperature. After 1 minute, stop development with 75 μl of 2N sulfuric acid. Then read the plate at 450 nm using a Molecular Device SpectraMax i3 microplate reader.

[0438] 7.9.1.4 Data Analysis

[0439] Analyze the data in Microsoft Excel. Interpret the data by converting the reactivity of each antibody with the "mutant" peptide into a binding percentage compared to the wild-type peptide. Calculate the binding percentage using the formula (OD_mutant peptide / OD_wild-type peptide). Then display the binding percentage as a bar chart with all values ​​between 0 and 1.

[0440] 7.9.2. Results

[0441] The percentages of 5E5, 1AG, and 4AG bound to the peptide are shown in the figures. Figure 10-12Alanine substitutions in the region of residues 17-23 significantly reduced the binding of 1AG and 4AG. The binding of 1AG and 4AG was reduced by more alanine substitutions compared to 5E5. For each of peptides 5-11, the binding of 4AG was significantly reduced compared to the "wild-type" peptide. This indicates that the binding epitope of 4AG is located in the region of residues 17-23. The binding of 1AG was even more restrictive, with significantly reduced binding for each of peptides 3-12 (corresponding to the region of residues 15-24) compared to the wild type. In the region of residues 19-22, the binding of 1AG and 4AG was almost completely inhibited by the Ser-GalNAc substitution on peptide 8 (2.23% and 1.63%, respectively), while 5E5 could still bind (18%). In contrast, the Thr-GalNAc substitution on peptide 9 almost completely inhibited 5E5 binding (2.93%), while 4AG and 1AG could still bind at 14.62% and 6.07%, respectively. This indicates that glycosylated serines play a greater role in the binding of 1AG and 4AG than GalNAc-Thr, which is the opposite for 5E5. Overall, the alanine scanning results suggest that the epitopes of 1AG and 4AG appear to differ from those of 5E5. The differences include a greater number of contacts with sugar-MUC1, which (unbound by theory) is thought to confer a greater degree of selectivity against cancer antigens.

[0442] 8. Specific implementation plan and references

[0443] Although various specific embodiments have been shown and described, it should be understood that various changes may be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by the numbered embodiments shown below.

[0444] 1. A method for competitively binding MUC1 peptide to an antibody or antigen-binding fragment comprising: Anti-glycemic MUC1 antibody or antigen-binding fragment: (i) the heavy chain variable (VH) sequence of SEQ ID NO:1 and the light chain variable (VL) sequence of SEQ ID NO:2 or (ii) the heavy chain variable (VH) sequence of SEQ ID NO:23 and the light chain variable (VL) sequence of SEQ ID NO:24, wherein the MUC1 peptide is glycosylated with GalNAc at serine and threonine residues shown in bold and underlined.

[0445] 2. An anti-glyco-MUC1 antibody or antigen-binding fragment that competes with an antibody or antigen-binding fragment comprising: (i) the heavy chain variable (VH) sequence of SEQ ID NO:1 and the light chain variable (VL) sequence of SEQ ID NO:2 or (ii) the heavy chain variable (VH) sequence of SEQ ID NO:23 and the light chain variable (VL) sequence of SEQ ID NO:24.

[0446] 3. An anti-glyco-MUC1 antibody or antigen-binding fragment comprising a complementarity-determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO:93, a CDR-H2 containing the amino acid sequence of SEQ ID NO:94, a CDR-H3 containing the amino acid sequence of SEQ ID NO:95, a CDR-L1 containing the amino acid sequence of SEQ ID NO:96, a CDR-L2 containing the amino acid sequence of SEQ ID NO:97, and a CDR-L3 containing the amino acid sequence of SEQ ID NO:98.

[0447] 4. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 1 or embodiment 2, comprising a complementarity-determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO:93, a CDR-H2 containing the amino acid sequence of SEQ ID NO:94, a CDR-H3 containing the amino acid sequence of SEQ ID NO:95, a CDR-L1 containing the amino acid sequence of SEQ ID NO:96, a CDR-L2 containing the amino acid sequence of SEQ ID NO:97, and a CDR-L3 containing the amino acid sequence of SEQ ID NO:98.

[0448] 5. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:3.

[0449] 6. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:9.

[0450] 7. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:15.

[0451] 8. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:25.

[0452] 9. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:31.

[0453] 10. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:37.

[0454] 11. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:45.

[0455] 12. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:51.

[0456] 13. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:57.

[0457] 14. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:63.

[0458] 15. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:69.

[0459] 16. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:75.

[0460] 17. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:81.

[0461] 18. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein CDR-H1 contains the amino acid sequence of SEQ ID NO:87.

[0462] 19. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:4.

[0463] 20. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:10.

[0464] 21. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:16.

[0465] 22. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:26.

[0466] 23. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:32.

[0467] 24. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:38.

[0468] 25. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:46.

[0469] 26. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 18, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:52.

[0470] 27. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 26, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:5.

[0471] 28. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 26, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:11.

[0472] 29. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 26, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:27.

[0473] 30. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 26, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:33.

[0474] 31. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 26, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:47.

[0475] 32. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:6.

[0476] 33. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:12.

[0477] 34. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:18.

[0478] 35. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:28.

[0479] 36. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:34.

[0480] 37. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:40.

[0481] 38. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:54.

[0482] 39. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 31, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:60.

[0483] 40. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 39, wherein CDR-L2 comprises the amino acid sequence of SEQ ID NO:13.

[0484] 41. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 40, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO:8.

[0485] 42. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing the complementarity-determining regions (CDRs) of SEQ ID NO:3-5 and a VL containing the CDRs of SEQ ID NO:6-8.

[0486] 43. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing the complementarity-determining region (CDR) of SEQ ID NO: 9-11 and a VL containing the CDR of SEQ ID NO: 12-14.

[0487] 44. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing the complementarity-determining region (CDR) of SEQ ID NO:15-17 and a VL containing the CDR of SEQ ID NO:18-20.

[0488] 45. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing the complementarity-determining region (CDR) of SEQ ID NO:25-27 and a VL containing the CDR of SEQ ID NO:28-30.

[0489] 46. ​​An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO:31-33 and a VL containing a CDR of SEQ ID NO:34-36.

[0490] 47. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO:37-39 and a VL containing a CDR of SEQ ID NO:40-42.

[0491] 48. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO:45-47 and a VL containing a CDR of SEQ ID NO:48-50.

[0492] 49. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO: 51-53 and a VL containing a CDR of SEQ ID NO: 54-56.

[0493] 50. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO: 57-59 and a VL containing a CDR of SEQ ID NO: 60-62.

[0494] 51. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing the complementarity-determining region (CDR) of SEQ ID NO: 63-65 and a VL containing the CDR of SEQ ID NO: 66-68.

[0495] 52. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO: 69-71 and a VL containing a CDR of SEQ ID NO: 72-74.

[0496] 53. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO:75-77 and a VL containing a CDR of SEQ ID NO:78-80.

[0497] 54. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO: 81-83 and a VL containing a CDR of SEQ ID NO: 84-86.

[0498] 55. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH containing a complementarity-determining region (CDR) of SEQ ID NO: 87-89 and a VL containing a CDR of SEQ ID NO: 90-92.

[0499] 56. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 55, wherein the antibody is a chimeric antibody or a humanized antibody.

[0500] 57. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 56, having a VH comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1 and a VL comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2.

[0501] 58. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 56, having a VH comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:1 and a VL comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:2.

[0502] 59. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 56, having a VH comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:1 and a VL comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:2.

[0503] 60. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH comprising the amino acid sequence of SEQ ID NO:1 and a VL comprising the amino acid sequence of SEQ ID NO:2.

[0504] 61. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 56, having a VH comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:23 and a VL comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:24.

[0505] 62. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 56, having a VH comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:23 and a VL comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:24.

[0506] 63. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 56, having a VH comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:23 and a VL comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:24.

[0507] 64. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 4, having a VH comprising the amino acid sequence of SEQ ID NO:23 and a VL comprising the amino acid sequence of SEQ ID NO:24.

[0508] 65. An anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 1 to 4, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0509] 66. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 65, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 5%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0510] 67. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 65, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 3%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0511] 68. An anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 65 to 67, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0512] 69. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 68, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to... The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 20%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0513] 70. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 68, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to... The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 10%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0514] 71. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 68 to 70, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0515] 72. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 71, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 20%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0516] 73. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 71, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 10%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0517] 74. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 71, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 5%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0518] 75. An anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 71 to 74, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0519] 76. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 75, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 10%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0520] 77. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 75, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 5%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0521] 78. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 75 to 77, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0522] 79. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 78, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. 80% of the binding is represented by serine and threonine residues shown in bold and underline, indicating residues glycosylated by GalNAc.

[0523] 80. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 78, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. 70% of the binding is represented by serine and threonine residues shown in bold and underline, indicating residues glycosylated by GalNAc.

[0524] 81. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 78, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 20%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0525] 82. An anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 65 to 81, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0526] 83. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 82, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 5%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0527] 84. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 82, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 3%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0528] 85. An anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 82 to 84, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0529] 86. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 85, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 10%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0530] 87. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 85, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 5%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0531] 88. An anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 85 to 87, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to a peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0532] 89. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 88, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 5%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0533] 90. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 88, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 3%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0534] 91. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 88 to 90, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0535] 92. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 91, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. 80% of the binding is represented by serine and threonine residues shown in bold and underline, indicating residues glycosylated by GalNAc.

[0536] 93. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 91, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 50%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0537] 94. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 91, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 20%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0538] 95. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 91, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 10%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0539] 96. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 91 to 95, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The affinity for binding is greater than that for peptides. The binding affinity, where serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0540] 97. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 96, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. 80% of the binding is represented by serine and threonine residues shown in bold and underline, indicating residues glycosylated by GalNAc.

[0541] 98. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 96, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 50%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0542] 99. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 96, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment is bound to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 20%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0543] 100. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 96, wherein, as measured by ELISA, the anti-glyco-MUC1 antibody or antigen-binding fragment binds to the peptide. The binding is smaller than that of the anti-glyco-MUC1 antibody or antigen-binding fragment and peptide. The binding is 10%, of which the serine and threonine residues shown in bold and underline represent residues glycosylated by GalNAc.

[0544] 101. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 65 to 100, wherein the ELISA comprises an ELISA using the peptide at a concentration of 0.25 μg / ml.

[0545] 102. An anti-glycan-MUC1 antibody or antigen-binding fragment of any one of embodiments 65 to 101, wherein the ELISA comprises an ELISA performed on cell culture supernatant from a cell line expressing the anti-glycan-MUC1 antibody or antigen-binding fragment.

[0546] 103. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 65 to 101, wherein the ELISA comprises an ELISA performed on a diluted cell culture supernatant from a cell line expressing the anti-glyco-MUC1 antibody or antigen-binding fragment.

[0547] 104. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 103, wherein the diluted cell culture supernatant comprises cell culture supernatant diluted 2 to 20 times with buffer.

[0548] 105. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 65 to 104, wherein the concentration of the antibody or antigen-binding fragment in the ELISA is from 0.1 mg / mL to 2 mg / mL, optionally, wherein the concentration of the antibody or antigen-binding fragment in the ELISA is from 0.5 mg / mL to 1 mg / mL.

[0549] 106. An anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 65 to 105, wherein the concentration of the antibody or antigen-binding fragment in the ELISA results in a binding reaction with the MUC1 peptide. The combination of.

[0550] 107. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 65 to 106, wherein the ELISA is performed as described in Example 9.

[0551] 108. A method for competitively binding to the MUC1 peptide against a reference antibody or antigen-binding fragment. The reference antibody or antigen-binding fragment comprises (i) the heavy chain variable (VH) sequence of SEQ ID NO:1 and the light chain variable (VL) sequence of SEQ ID NO:2 or (ii) the heavy chain variable (VH) sequence of SEQ ID NO:23 and the light chain variable (VL) sequence of SEQ ID NO:24, wherein the MUC1 peptide is glycosylated with GalNAc at serine and threonine residues shown in bold and underlined, and the anti-glyc-MUC1 antibody or antigen-binding fragment comprises:

[0552] (a) A VH sequence having a first, second, and third CDR within the VH sequence; and

[0553] (b) A VL sequence having a fourth, fifth, and sixth CDR within the VL sequence.

[0554] The first, second, third, fourth, fifth, and sixth CDRs cooperate to achieve the binding of an anti-glyco-MUC1 antibody or antigen-binding fragment to the MUC1 peptide, which competes with the binding of a reference antibody or antigen-binding fragment.

[0555] 109. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 108, wherein the anti-glyco-MUC1 antibody or antigen-binding fragment competes with a reference antibody or antigen-binding fragment comprising the VH sequence of SEQ ID NO:23 and the VL sequence of SEQ ID NO:24.

[0556] 110. An anti-glycan-MUC1 antibody or antigen-binding fragment of embodiment 108, wherein the anti-glycan-MUC1 antibody or antigen-binding fragment competes with a reference antibody or antigen-binding fragment comprising the VH sequence of SEQ ID NO:1 and the VL sequence of SEQ ID NO:2.

[0557] 111. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 110, which preferentially binds to glyco-MUC1 epitopes overexpressed on cancer cells compared with normal cells.

[0558] 112. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 111, having a binding affinity (K0) of 1 nM to 50 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0559] 113. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0.05) of 3 nM to 50 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0560] 114. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0) of 3 nM to 40 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0561] 115. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0.05) of 3 nM to 25 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0562] 116. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0) of 5 nM to 50 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0563] 117. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0.05) of 5 nM to 25 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0564] 118. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0) of 10 nM to 50 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0565] 119. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0) of 10 nM to 25 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0566] 120. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0.05) of 15 nM to 50 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0567] 121. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity of 15 nM to 25 nM as measured by surface plasmon resonance (K0). D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0568] 122. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity (K0) of 20 nM to 50 nM as measured by surface plasmon resonance. D The MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0569] 123. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 112, having a binding affinity of 25 nM to 50 nM as measured by surface plasmon resonance (K0). DThe MUC1 peptide, which binds to serine and threonine residues glycosylated by GalNAc (shown in bold and underlined), is a key component of this peptide.

[0570] 124. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of implementation schemes 1 to 123, which is multivalent.

[0571] 125. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 123, wherein the antigen is in the form of a single-chain variable fragment (scFv).

[0572] 126. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 125, wherein the scFv comprises a heavy chain variable fragment located at the N-terminus of the light chain variable fragment.

[0573] 127. An anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 125 or embodiment 126, wherein the scFv heavy chain variable fragment and light chain variable fragment are covalently bound to a linker sequence of 4-15 amino acids.

[0574] 128. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 123, which is in the form of a multispecific antibody.

[0575] 129. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 128, wherein the multispecific antibody is a bispecific antibody that binds to a second epitope different from the first epitope.

[0576] 130. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 129, wherein the bispecific antibody is a CrossMab, a Fab arm exchange antibody, a bispecific T-cell adaptor (BiTE), or a dual-affinity retargeting molecule (DART).

[0577] 131. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 130, wherein the bispecific antibody is CrossMab.

[0578] 132. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 131, wherein the bispecific antibody is CrossMab FAB .

[0579] 133. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 131, wherein the bispecific antibody is CrossMab VH-VL .

[0580] 134. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 131, wherein the bispecific antibody is CrossMab CH1-CL.

[0581] 135. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 130, wherein the bispecific antibody is a Fab arm exchange antibody.

[0582] 136. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 130, wherein the bispecific antibody is a dual-affinity retargeting molecule (DART).

[0583] 137. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 130, wherein the bispecific antibody is a bispecific T-cell adaptor (BiTE).

[0584] 138. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 129 to 137, wherein the second epitope is a MUC1 epitope.

[0585] 139. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 129 to 137, wherein the second epitope is a MUC1 epitope overexpressed on cancer cells compared to normal cells.

[0586] 140. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 129 to 137, wherein the second epitope is a T-cell epitope.

[0587] 141. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 140, wherein the T cell epitope includes a CD3 epitope, a CD8 epitope, a CD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope.

[0588] 142. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 141, wherein the T-cell epitope includes a CD3 epitope, which is optionally an epitope present in human CD3.

[0589] 143. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 142, wherein the CD3 epitope includes a CD3γ epitope, a CD3δ epitope, a CD3ε epitope, or a CD3ζ epitope.

[0590] 144. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 143, which is conjugated to a detectable portion.

[0591] 145. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 144, wherein the detectable portion is an enzyme, a radioisotope, or a fluorescent label.

[0592] 146. A fusion protein comprising an amino acid sequence of an anti-glyco-MUC1 antibody or antigen-binding fragment operably linked to a second amino acid sequence according to any one of embodiments 1 to 145.

[0593] 147. The fusion protein of embodiment 146, wherein the second amino acid sequence is an amino acid sequence of 4-1BB, CD3ζ or a fragment thereof.

[0594] 148. The fusion protein of embodiment 146, wherein the second amino acid sequence is the amino acid sequence of the fusion peptide.

[0595] 149. The fusion protein of embodiment 148, wherein the fusion peptide is a CD28-CD3ζ or a 4-1BB(CD137)-CD3ζ fusion peptide.

[0596] 150. The fusion protein of embodiment 146, wherein the second amino acid sequence is an amino acid sequence of a regulator of T cell activation or a fragment thereof.

[0597] 151. The fusion protein of embodiment 150, wherein the regulator of T cell activation is IL-15 or IL-15Ra.

[0598] 152. A chimeric antigen receptor (CAR) comprising the scFv of any one of embodiments 125 to 127.

[0599] 153. The CAR of embodiment 152 comprises, in the order from amino terminus to carboxyl terminus, a human CD8 leader peptide, scFv, a human CD8 hinge domain, a human CD8 transmembrane domain, and a CD3ζ signal transduction domain.

[0600] 154. An antibody-drug conjugate comprising an anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145 conjugated to a cytotoxic agent, or a fusion protein of any one of embodiments 146 to 151.

[0601] 155. The antibody-drug conjugate of embodiment 154, wherein the cytotoxic agent is aurestatin, a DNA minor groove binding agent, an alkylating agent, an enediyne, lexitropsin, duocarmycin, taxane, salicylate, maytansin, or vinca alkaloids.

[0602] 156. The antibody-drug conjugate of embodiment 155, wherein the anti-glyco-MUC1 antibody or antigen-binding fragment or bispecific antibody is conjugated to a cytotoxic agent via a linker.

[0603] 157. The antibody-drug conjugate of embodiment 156, wherein the linker is cleavable under intracellular conditions.

[0604] 158. The antibody-drug conjugate of embodiment 157, wherein the cleavable linker is cleaved by an intracellular protease.

[0605] 159. The antibody-drug conjugate of embodiment 158, wherein the linker comprises a dipeptide.

[0606] 160. The antibody-drug conjugate of embodiment 159, wherein the dipeptide is val-cit or phe-lys.

[0607] 161. The antibody-drug conjugate of embodiment 157, wherein the cleavable linker is hydrolyzable at a pH less than 5.5.

[0608] 162. The antibody-drug conjugate of embodiment 161, wherein the hydrolyzable linker is a hydrazone linker.

[0609] 163. The antibody-drug conjugate of embodiment 157, wherein the cleavable connector is a disulfide connector.

[0610] 164. A nucleic acid comprising an anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, or a fusion protein of any one of embodiments 146 to 151, or a coding region of a CAR of embodiment 152 or embodiment 153.

[0611] 165. The nucleic acid of implementation scheme 164, wherein the coding region is codon-optimized for expression in human cells.

[0612] 166. A vector comprising the nucleic acid of embodiment 164 or embodiment 165.

[0613] 167. The carrier of implementation scheme 166 is a viral carrier.

[0614] 168. The vector of implementation scheme 167, wherein the viral vector is a lentiviral vector.

[0615] 169. A host cell engineered to express the nucleic acid of implementation scheme 164 or implementation scheme 165.

[0616] 170. The host cell of embodiment 169 is a human T cell engineered to express the CAR of embodiment 152 or embodiment 153.

[0617] 171. A host cell comprising a vector according to any one of embodiments 166 to 168.

[0618] 172. The host cell of embodiment 171 is a T cell, and wherein the vector encodes the CAR of embodiment 152 or embodiment 153.

[0619] 173. A pharmaceutical composition comprising (a) an anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, or a host cell of any one of embodiments 169 to 172, and (b) a physiologically suitable buffer, adjuvant, or diluent.

[0620] 174. A method of treating cancer, comprising administering to a subject in need an effective amount of an anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of any one of embodiments 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of any one of embodiments 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173.

[0621] 175. The method of implementation plan 174, wherein the subject has breast cancer, lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, uterine cancer, cervical cancer, bladder cancer, brain cancer, non-Hodgkin's lymphoma, cervical cancer, gastric cancer, bile duct cancer, chondrosarcoma, kidney cancer, skin cancer, leukemia, thyroid cancer, or liver cancer.

[0622] 176. The method of implementation scheme 175, wherein the subject has breast cancer.

[0623] 177. The method of implementation scheme 175, wherein the subject has lung cancer.

[0624] 178. The method of implementation scheme 177, wherein the lung cancer is non-small cell lung cancer.

[0625] 179. The method of implementation scheme 175, wherein the subject has prostate cancer.

[0626] 180. The method of implementation scheme 175, wherein the subject has pancreatic cancer.

[0627] 181. The method of implementation scheme 175, wherein the subject has esophageal cancer.

[0628] 182. The method of implementation scheme 175, wherein the subject has colorectal cancer.

[0629] 183. The method of implementation scheme 175, wherein the subject has ovarian cancer.

[0630] 184. The method of implementation scheme 175, wherein the subject has uterine cancer.

[0631] 185. The method of implementation scheme 175, wherein the subject has cervical cancer.

[0632] 186. The method of implementation scheme 175, wherein the subject has bladder cancer.

[0633] 187. The method of implementation scheme 175, wherein the subject has brain cancer.

[0634] 188. The method of implementation scheme 175, wherein the subject has non-Hodgkin lymphoma.

[0635] 189. The method of implementation scheme 175, wherein the subject has cervical cancer.

[0636] 190. The method of implementation scheme 175, wherein the subject has gastric cancer.

[0637] 191. The method of implementation scheme 175, wherein the subject has bile duct cancer.

[0638] 192. The method of implementation scheme 175, wherein the subject has chondrosarcoma.

[0639] 193. The method of implementation scheme 175, wherein the subject has kidney cancer.

[0640] 194. The method of implementation scheme 175, wherein the subject has skin cancer.

[0641] 195. The method of implementation scheme 175, wherein the subject suffers from leukemia.

[0642] 196. The method of implementation scheme 175, wherein the subject has thyroid cancer.

[0643] 197. The method of implementation scheme 175, wherein the subject has liver cancer.

[0644] 198. A method for detecting cancer in a biological sample, comprising contacting the sample with an anti-glycan-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, and detecting the binding of said anti-glycan-MUC1 antibody or antigen-binding fragment.

[0645] 199. The method of implementation scheme 198 further includes quantifying the binding of anti-glyco-MUC1 antibody or antigen-binding fragment.

[0646] 200. The method of embodiment 198 or embodiment 199, wherein the combination is compared with a normal tissue control as a negative / baseline control and / or with a cancerous tissue control as a positive control.

[0647] 201. The method of any one of 198 to 200, wherein the cancer is breast cancer, lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, uterine cancer, cervical cancer, bladder cancer, brain cancer, non-Hodgkin's lymphoma, cervical cancer, gastric cancer, bile duct cancer, chondrosarcoma, kidney cancer, skin cancer, leukemia, thyroid cancer, or liver cancer.

[0648] 202. The method of implementation scheme 201, wherein the cancer is breast cancer.

[0649] 203. The method of implementation scheme 201, wherein the cancer is lung cancer.

[0650] 204. The method of implementation scheme 203, wherein the lung cancer is non-small cell lung cancer.

[0651] 205. The method of implementation scheme 201, wherein the cancer is prostate cancer.

[0652] 206. The method of implementation scheme 201, wherein the cancer is pancreatic cancer.

[0653] 207. The method of implementation scheme 201, wherein the cancer is esophageal cancer.

[0654] 208. The method of implementation 201, wherein the cancer is colorectal cancer.

[0655] 209. The method of implementation scheme 201, wherein the cancer is ovarian cancer.

[0656] 210. The method of implementation scheme 201, wherein the cancer is uterine cancer.

[0657] 211. The method of implementation scheme 201, wherein the cancer is cervical cancer.

[0658] 212. The method of implementation scheme 201, wherein the cancer is bladder cancer.

[0659] 213. The method of implementation scheme 201, wherein the cancer is brain cancer.

[0660] 214. The method of implementation scheme 201, wherein the cancer is non-Hodgkin lymphoma.

[0661] 215. The method of implementation scheme 201, wherein the cancer is cervical cancer.

[0662] 216. The method of implementation scheme 201, wherein the cancer is gastric cancer.

[0663] 217. The method of implementation scheme 201, wherein the cancer is bile duct cancer.

[0664] 218. The method of implementation scheme 201, wherein the cancer is chondrosarcoma.

[0665] 219. The method of implementation scheme 201, wherein the cancer is kidney cancer.

[0666] 220. The method of implementation scheme 201, wherein the cancer is skin cancer.

[0667] 221. The method of implementation scheme 201, wherein the cancer is leukemia.

[0668] 222. The method of implementation scheme 201, wherein the cancer is thyroid cancer.

[0669] 223. The method of implementation scheme 201, wherein the cancer is liver cancer.

[0670] 224. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, used as a drug.

[0671] 225. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of any one of embodiments 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of any one of embodiments 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of cancer, optionally wherein said cancer is breast cancer, lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, uterine cancer, cervical cancer, bladder cancer, brain cancer, non-Hodgkin's lymphoma, cervical cancer, gastric cancer, bile duct cancer, chondrosarcoma, kidney cancer, skin cancer, leukemia, thyroid cancer, or liver cancer.

[0672] 226. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of breast cancer.

[0673] 227. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of lung cancer.

[0674] 228. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of prostate cancer.

[0675] 229. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of pancreatic cancer.

[0676] 230. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of esophageal cancer.

[0677] 231. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of colorectal cancer.

[0678] 232. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of ovarian cancer.

[0679] 233. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of uterine cancer.

[0680] 234. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of cervical cancer.

[0681] 235. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of bladder cancer.

[0682] 236. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of brain cancer.

[0683] 237. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of non-Hodgkin's lymphoma.

[0684] 238. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of cervical cancer.

[0685] 239. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of gastric cancer.

[0686] 240. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of cholangiocarcinoma.

[0687] 241. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of chondrosarcoma.

[0688] 242. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of renal cell carcinoma.

[0689] 243. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of skin cancer.

[0690] 244. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of leukemia.

[0691] 245. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of thyroid cancer.

[0692] 246. An anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of embodiment 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of embodiment 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173, for the treatment of liver cancer.

[0693] 247. Use of any of the following embodiments in the preparation of a medicament for treating cancer: an anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 145, a fusion protein of any one of embodiments 146 to 151, a CAR of any one of embodiments 152 or 153, an antibody-drug conjugate of any one of embodiments 154 to 163, a nucleic acid of any one of embodiments 164 or 165, a vector of any one of embodiments 166 to 168, a host cell of any one of embodiments 169 to 172, or a pharmaceutical composition of embodiment 173. Optionally, the cancer is breast cancer, lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, uterine cancer, cervical cancer, bladder cancer, brain cancer, non-Hodgkin's lymphoma, cervical cancer, gastric cancer, bile duct cancer, chondrosarcoma, kidney cancer, skin cancer, leukemia, thyroid cancer, or liver cancer.

[0694] 248. According to the use of embodiment 247, the cancer is breast cancer.

[0695] 249. According to the use of embodiment 247, the cancer is lung cancer.

[0696] 250. According to the use of embodiment 247, the cancer is prostate cancer.

[0697] 251. According to the use of embodiment 247, the cancer is pancreatic cancer.

[0698] 252. According to the use of embodiment 247, the cancer is esophageal cancer.

[0699] 253. According to the use of embodiment 247, the cancer is colorectal cancer.

[0700] 254. According to the use of embodiment 247, the cancer is ovarian cancer.

[0701] 255. According to the use of embodiment 247, the cancer is uterine cancer.

[0702] 256. According to the use of embodiment 247, the cancer is cervical cancer.

[0703] 257. According to the use of embodiment 247, the cancer is bladder cancer.

[0704] 258. According to the use of embodiment 247, the cancer is brain cancer.

[0705] 259. According to the use of embodiment 247, the cancer is non-Hodgkin's lymphoma.

[0706] 260. According to the use of embodiment 247, the cancer is cervical cancer.

[0707] 261. According to the use of embodiment 247, the cancer is gastric cancer.

[0708] 262. According to the use of embodiment 247, the cancer is bile duct cancer.

[0709] 263. According to the use of embodiment 247, the cancer is chondrosarcoma.

[0710] 264. According to the use of embodiment 247, the cancer is kidney cancer.

[0711] 265. According to the use of embodiment 247, the cancer is skin cancer.

[0712] 266. According to the use of embodiment 247, the cancer is leukemia.

[0713] 267. According to the use of embodiment 247, the cancer is thyroid cancer.

[0714] 268. According to the use of embodiment 247, the cancer is liver cancer.

[0715] All publications, patents, patent applications and other documents cited in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application or other document were individually incorporated herein by reference for all purposes. If there is any inconsistency between the teachings of one or more references incorporated herein and this disclosure, the teachings of this specification shall prevail. sequence list <110> GO Medical Co., Ltd. <120> Anti-glyco-MUC1 antibodies and their uses <130> GOT-002WO <150> 62 / 691,887 <151> 2018-06-29 <150> 62 / 802,865 <151> 2019-02-08 <160> 118 <170> PatentIn version 3.5 <210> 1 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 1 Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Asp 20 25 3 0 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Lys Asn Tyr Thr Pro Ser Leu 50 55 60 Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 8 5 90 95 Ala Thr Ser His Tyr Tyr Gly Leu Phe Gly Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 2 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 3 Gly Phe Asp Phe Ser Arg Asp Trp 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 4 Ile Asn Pro Asp Ser Ser Thr Lys 1 5 <210> 5 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Ala Thr Ser His Tyr Tyr Gly Leu Phe Gly Tyr 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 7 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 7 Leu Ala Ser 1 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 8 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 9 Arg Asp Trp Met Ser 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 10 Glu Ile Asn Pro Asp Ser Ser Thr Lys Asn Tyr Thr Pro Ser Leu Lys 1 5 10 15 Asp <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Ser His Tyr Tyr Gly Leu Phe Gly Tyr 1 5 <210> 12 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 12 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 13 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 13 Leu Ala Ser Tyr Leu Glu Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 14 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 15 Gly Phe Asp Phe Ser Arg Asp 1 5 <210> 16 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 16 Asn Pro Asp Ser Ser Thr 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 Ser His Tyr Tyr Gly Leu Phe Gly Tyr 1 5 <210> 18 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 19 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Leu Ala Ser 1 <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Ser Arg Glu Leu Pro Arg Thr 1 5 <210> twenty one <211> 708 <212> DNA <213> Artificial sequence <220> <223> Synthesized nucleotide sequences <400> twenty one gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc ctggaggatc cctgaaattg 60 tcctgtgcag cctcaggatt cgattttagt agagactgga tgagttgggt ccggcaggct 120 ccagggaaag ggctagaatg gattggagag attaatccag atagcagtac gaaaaactac 180 acgccatctc taaggata attcatcatt tccagagaca acgccaaaaa tacgctgttc ctgcaaatga gcagcgtgag atctgaggac acagcccttt attactgtgc aacctctcat 360. tactacggcc tgtttggtta ctggggccaa gggactctgg tcactgtctc tgcagaggtg aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa attgtcctgt 420 gcagcctcag gattcgattt tagtagc tggatgagtt gggtccggca ggctccaggg 480 aaagggctag aatggattgg together ccagatagca gtacgaaaaa ctacacgcca tctctaaagg ataattcat catttccaga gacaacgcca aaaatacgct gttcctgca atgagcagcg tgagatctga ggacacagcc ctttattact gtgcaacctc tcattactac ggcctgtttg gttactgggg ccaagggact ctggtcactg tctctgca 708 <210> 22 <211> 333 <212> DNA <213> The snowstorm <220> <223> The snowflake snowflakes <400> 22 60. gacattgtgc tgacacagtc tcctgcttcc ttaactgtat ctctggggca gagggccacc atctcatgca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggtac 120 caacagaaac caggacagcc acccaaactc ctcatctatc ttgcttccta cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acagtaggga gcttcctcgg 300 acgttcggtg gaggcaccaa gctggaaatc aaa 333 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 23 Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Glu Ser Asn Thr Met Asn Tyr Ser Pro Ser Leu 50 55 60 Lys Glu Lys Phe Ile Ile Ser Arg Asp Thr Ala Lys Asn Met Leu Tyr<00020965 70 75 80 Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr Ser His His Tyr Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 24 <211> 111 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 24 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Asn Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys 100 105 110 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 Gly Phe Asp Phe Ser Arg Tyr Trp 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Ile Asn Pro Glu Ser Asn Thr Met 1 5 <210> 27 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Ala Thr Ser His His Tyr Gly Leu Phe Asp Tyr 1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 28 Lys Ser Val Ser Thr Ser Gly Tyr Asn Tyr 1 5 10 <210> 29 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 29 Leu Ala Ser 1 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 30 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 31 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 31 Arg Tyr Trp Met Ser 1 5 <210> 32 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 32 Glu Ile Asn Pro Glu Ser Asn Thr Met Asn Tyr Ser Pro Ser Leu Lys 1 5 10 15 Glu <210> 33 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 33 Ser His His Tyr Gly Leu Phe Asp Tyr 1 5 <210> 34 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 34 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Asn Tyr Ile His 1 5 10 15 <210> 35 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 35 Leu Ala Ser Tyr Leu Glu Ser 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 36 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 37 Gly Phe Asp Phe Ser Arg Tyr 1 5 <210> 38 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 38 Asn Pro Glu Ser Asn Thr 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 39 Ser His His Tyr Gly Leu Phe Asp Tyr 1 5 <210> 40 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 40 Ser Lys Ser Val Ser Thr Ser Gly Tyr Asn Tyr 1 5 10 <210> 41 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 41 Leu Ala Ser 1 <210> 42 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 42 Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 43 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Synthesized nucleotide sequences <400> 43 gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc ctggaggatc cctgaaactc 60 tcctgtgtag cctcaggatt cgattttagt agatactgga tgagttgggt ccggcaggct 120 ccagggaaag ggccagaatg gattggagaa attaatccag aaagcaatac gatgaactat 180 tcgccatctc taaaggaaaa attcatcatc tccagagaca ccgccaaaaa tatgttgtac 240 ctgcaaatga gcaaagtgag atctgaggac acagcccttt attactgtgc aacctctcat 300 cactacggcc tattcgatta ctggggccaa gggactctgg tcactgtctc tgca 354 <210> 44 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Synthesized nucleotide sequences <400> 44 gacattgtgc tgacacagtc tcctgcttcc ttagctgtgt ctctggggca gagggccacc 60 atctcatgca gggccagcaa aagtgtcagt acttctggct ataattatat acactggtac 120 caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccta cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctggggacag acttcaccct caacatccac 240 cctgtggagg aggaggatgc tgcaacctat ttctgtcagc acagtaggga gcttcctcgg 300 acgttcggtg gaggcaccaa gctggaattc aaa 333 <210> 45 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (7)..(7) <223> D or Y <400> 45 Gly Phe Asp Phe Ser Arg Xaa Trp 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (4)..(4) <223> D or E <220> <221> MOD_RES <222> (6)..(6) <223> S or N <220> <221> MOD_RES <222> (8)..(8) <223> K or M <400> 46 Ile Asn Pro Xaa Ser Xaa Thr Xaa 1 5 <210> 47 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (5)..(5) <223> Y or H <220> <221> MOD_RES <222> (10)..(10) <223> G or D <400> 47 Ala Thr Ser His Xaa Tyr Gly Leu Phe Xaa Tyr 1 5 10 <210> 48 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (9)..(9) <223> S or N <400> 48 Lys Ser Val Ser Thr Ser Gly Tyr Xaa Tyr 1 5 10 <210> 49 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 49 Leu Ala Ser 1 <210> 50 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 50 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 51 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (2)..(2) <223> D or Y <400> 51 Arg Xaa Trp Met Ser 1 5 <210> 52 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (5)..(5) <223> D or E <220> <221> MOD_RES <222> (7)..(7) <223> S or N <220> <221> MOD_RES <222> (9)..(9) <223> K or M <220> <221> MOD_RES <222> (12)..(12) <223> T or S <220> <221> MOD_RES <222> (17)..(17) <223> D or E <400> 52 Glu Ile Asn Pro Xaa Ser Xaa Thr Xaa Asn Tyr Xaa Pro Ser Leu Lys 1 5 10 15 Xaa <210> 53 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (3)..(3) <223> Y or H <220> <221> MOD_RES <222> (8)..(8) <223> G or D <400> 53 Ser His Xaa Tyr Gly Leu Phe Xaa Tyr 1 5 <210> 54 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (12)..(12) <223> S or N <220> <221> MOD_RES <222> (14)..(14) <223> M or I <400> 54 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Xaa Tyr Xaa His 1 5 10 15 <210> 55 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 55 Leu Ala Ser Tyr Leu Glu Ser 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 56 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 57 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (7)..(7) <223> D or Y <400> 57 Gly Phe Asp Phe Ser Arg Xaa 1 5 <210> 58 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (3)..(3) <223> D or E <220> <221> MOD_RES <222> (5)..(5) <223> S or N <400> 58 Asn Pro Xaa Ser Xaa Thr 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (3)..(3) <223> Y or H <220> <221> MOD_RES <222> (8)..(8) <223> G or D <400> 59 Ser His Xaa Tyr Gly Leu Phe Xaa Tyr 1 5 <210> 60 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (10)..(10) <223> S or N <400> 60 Ser Lys Ser Val Ser Thr Ser Gly Tyr Xaa Tyr 1 5 10 <210> 61 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 61 Leu Ala Ser 1 <210> 62 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 62 Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 63 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 63 Gly Phe Asp Phe Ser Arg Asp Trp Met Ser 1 5 10 <210> 64 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 64 Glu Ile Asn Pro Asp Ser Ser Thr Lys Asn Tyr Thr Pro Ser Leu Lys 1 5 10 15 Asp <210> 65 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 65 Ala Thr Ser His Tyr Tyr Gly Leu Phe Gly Tyr 1 5 10 <210> 66 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 66 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 67 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 67 Leu Ala Ser Tyr Leu Glu Ser 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 68 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 69 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 69 Gly Phe Asp Phe Ser Arg Tyr Trp Met Ser 1 5 10 <210> 70 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 70 Glu Ile Asn Pro Glu Ser Asn Thr Met Asn Tyr Ser Pro Ser Leu Lys 1 5 10 15 Glu <210> 71 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 71 Ala Thr Ser His His Tyr Gly Leu Phe Asp Tyr 1 5 10 <210> 72 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 72 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Asn Tyr Ile His 1 5 10 15 <210> 73 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 73 Leu Ala Ser Tyr Leu Glu Ser 1 5 <210> 74 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 74 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 75 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (7)..(7) <223> D or Y <400> 75 Gly Phe Asp Phe Ser Arg Xaa Trp Met Ser 1 5 10 <210> 76 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (5)..(5) <223> D or E <220> <221> MOD_RES <222> (7)..(7) <223> S or N <220> <221> MOD_RES <222> (9)..(9) <223> K or M <220> <221> MOD_RES <222> (12)..(12) <223> T or S <220> <221> MOD_RES <222> (17)..(17) <223> D or E <400> 76 Glu Ile Asn Pro Xaa Ser Xaa Thr Xaa Asn Tyr Xaa Pro Ser Leu Lys 1 5 10 15 Xaa <210> 77 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (5)..(5) <223> Y or H <220> <221> MOD_RES <222> (10)..(10) <223> G or D <400> 77 Ala Thr Ser His Xaa Tyr Gly Leu Phe Xaa Tyr 1 5 10 <210> 78 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (12)..(12) <223> S or N <220> <221> MOD_RES <222> (14)..(14) <223> M or I <400> 78 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Xaa Tyr Xaa His 1 5 10 15 <210> 79 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 79 Leu Ala Ser Tyr Leu Glu Ser 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 80 Gln His Ser Arg Glu Leu Pro Arg Thr 1 5 <210> 81 <211> 2 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 81 Arg Asp 1 <210> 82 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 82 Asn Pro Asp Ser Ser Thr 1 5 <210> 83 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 83 Ser His Tyr Tyr Gly Leu Phe Gly Tyr 1 5 <210> 84 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 84 Lys Ser Val Ser Th...

Claims

1. Anti-glyco-MUC1 antibody or antigen-binding fragment, comprising: (a) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:3-5 and VL containing CDRs 1-3 as shown in SEQ ID NO:6-8, as numbered according to the definition of IMGT; (b) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:9-11 and VL containing CDRs 1-3 as shown in SEQ ID NO:12-14, as numbered according to the definition of Kabat; (c) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:15-17 and VL containing CDRs 1-3 as shown in SEQ ID NO:18-20, as numbered according to the definition of Chothia; (d) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:25-27 and VL containing CDRs 1-3 as shown in SEQ ID NO:28-30, as numbered according to the definition of IMGT; (e) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:31-33 and a VL containing CDRs 1-3 as shown in SEQ ID NO:34-36, as numbered according to the Kabat definition; or (f) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:37-39 and VL containing CDRs 1-3 as shown in SEQ ID NO:40-42, as numbered according to the definition of Chothia.

2. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1, comprising: (a) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:3-5 and VL containing CDRs 1-3 as shown in SEQ ID NO:6-8, as numbered according to the definition of IMGT; (b) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO: 9-11 and a VL containing CDRs 1-3 as shown in SEQ ID NO: 12-14, as numbered according to the Kabat definition; or (c) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:15-17 and VL containing CDRs 1-3 as shown in SEQ ID NO:18-20, as numbered according to the definition of Chothia.

3. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1, comprising: VHs containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:3-5 and VLs containing CDRs 1-3 as shown in SEQ ID NO:6-8, as numbered according to the definition of IMGT.

4. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1, comprising: VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:9-11 and VL containing CDRs 1-3 as shown in SEQ ID NO:12-14, as numbered according to the Kabat definition.

5. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1, comprising: VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:15-17 and VL containing CDRs 1-3 as shown in SEQ ID NO:18-20, as numbered according to the definition of Chothia.

6. The anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5, comprising: (a) VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1 and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2; (b) VH comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:1 and VL comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:2; or (c) VH containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO:1 and VL containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

2.

7. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 6, having a VH comprising the amino acid sequence of SEQ ID NO:1 and a VL comprising the amino acid sequence of SEQ ID NO:

2.

8. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1, comprising: (a) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:25-27 and a VL containing CDRs 1-3 as shown in SEQ ID NO:28-30, as numbered according to the definition of IMGT; (b) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:31-33 and a VL containing CDRs 1-3 as shown in SEQ ID NO:34-36, as numbered according to the definition of Kabat; or (c) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:37-39 and VL containing CDRs 1-3 as shown in SEQ ID NO:40-42, as numbered according to the definition of Chothia.

9. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 8, comprising: VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:25-27 and VL containing CDRs 1-3 as shown in SEQ ID NO:28-30, as numbered according to the definition of IMGT.

10. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 8, comprising: VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:31-33 and VL containing CDRs 1-3 as shown in SEQ ID NO:34-36, as numbered according to the Kabat definition.

11. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 8, comprising: VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:37-39 and VL containing CDRs 1-3 as shown in SEQ ID NO:40-42, as numbered according to the definition of Chothia.

12. The anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of claims 8 to 11, comprising: (a) VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO:23 and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO:24; (b) VH comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:23 and VL comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:24; or (c) VH containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO:23 and VL containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

24.

13. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 12, having a VH comprising the amino acid sequence of SEQ ID NO:23 and a VL comprising the amino acid sequence of SEQ ID NO:

24.

14. The anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 11, wherein it is in the form of a single-chain variable fragment (scFv).

15. A nucleic acid comprising a coding region of an anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 11.

16. A vector comprising the nucleic acid according to claim 15.

17. A host cell engineered to express the nucleic acid according to claim 15.

18. A host cell comprising the vector according to claim 16.

19. A pharmaceutical composition comprising (a) an anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 11, and (b) a physiologically suitable buffer, adjuvant, or diluent.

20. Use of the anti-glycemic-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 11 in the preparation of a medicament for treating breast cancer, lung cancer, colon cancer, or pancreatic cancer, wherein said anti-glycemic-MUC1 antibody or antigen-binding fragment comprises: (a) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:3-5 and VL containing CDRs 1-3 as shown in SEQ ID NO:6-8, as numbered according to the definition of IMGT; (b) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO: 9-11 and a VL containing CDRs 1-3 as shown in SEQ ID NO: 12-14, as numbered according to the Kabat definition; or (c) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:15-17 and VL containing CDRs 1-3 as shown in SEQ ID NO:18-20, as numbered according to the definition of Chothia.

21. Use of the anti-glycemic-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 11 in the preparation of a medicament for treating breast cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, or ovarian cancer, wherein said anti-glycemic-MUC1 antibody or antigen-binding fragment comprises: (a) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:25-27 and a VL containing CDRs 1-3 as shown in SEQ ID NO:28-30, as numbered according to the definition of IMGT; (b) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:31-33 and a VL containing CDRs 1-3 as shown in SEQ ID NO:34-36, as numbered according to the definition of Kabat; or (c) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:37-39 and VL containing CDRs 1-3 as shown in SEQ ID NO:40-42, as numbered according to the definition of Chothia.

22. Use of the anti-glycan-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 11 in the preparation of a kit for detecting cancer in a biological sample, wherein the kit is used in a method comprising contacting a sample with the anti-glycan-MUC1 antibody or antigen-binding fragment and detecting binding of the anti-glycan-MUC1 antibody or antigen-binding fragment, wherein the cancer is breast cancer, lung cancer, colon cancer, or pancreatic cancer, and wherein the anti-glycan-MUC1 antibody or antigen-binding fragment comprises: (a) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:3-5 and VL containing CDRs 1-3 as shown in SEQ ID NO:6-8, as numbered according to the definition of IMGT; (b) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO: 9-11 and a VL containing CDRs 1-3 as shown in SEQ ID NO: 12-14, as numbered according to the Kabat definition; or (c) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:15-17 and VL containing CDRs 1-3 as shown in SEQ ID NO:18-20, as numbered according to the definition of Chothia.

23. Use of the anti-glycan-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 11 in the preparation of a kit for detecting cancer in a biological sample, wherein the kit is used in a method comprising contacting a sample with the anti-glycan-MUC1 antibody or antigen-binding fragment and detecting binding of the anti-glycan-MUC1 antibody or antigen-binding fragment, wherein the cancer is breast cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, or ovarian cancer, and wherein the anti-glycan-MUC1 antibody or antigen-binding fragment comprises: (a) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:25-27 and a VL containing CDRs 1-3 as shown in SEQ ID NO:28-30, as numbered according to the definition of IMGT; (b) A VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:31-33 and a VL containing CDRs 1-3 as shown in SEQ ID NO:34-36, as numbered according to the definition of Kabat; or (c) VH containing complementary determination regions (CDRs) 1-3 as shown in SEQ ID NO:37-39 and VL containing CDRs 1-3 as shown in SEQ ID NO:40-42, as numbered according to the definition of Chothia.

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