KR20200067885A - Anti-glyco-MUC1 antibodies and uses thereof - Google Patents

Abstract

The present disclosure provides anti-glyco-MUC1 antibodies and antigen-binding fragments thereof that specifically bind to cancer-specific glycosylation variants of MUC1, and related fusion proteins and antibody-drug conjugates, as well as nucleic acids encoding such biomolecules It is about. The present disclosure further relates to the use of antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates and nucleic acids for cancer therapy.

Description

Anti-glyco-MUC1 antibodies and uses thereof

1. Sequence Listing

This application contains a sequence listing which is submitted electronically in ASCII form and is hereby incorporated by reference in its entirety. The ASCII copy created on October 24, 2017 has a file name of GOT-001WO_Sequence_Listing.txt and a size of 33,265 bytes.

2. Background

Human mucin MUC1 is a polymorphic transmembrane glycoprotein expressed at the tip surface of monolayer and glandular epithelium (Taylor-Papadimitriou et al., 1999). MUC1 is highly overexpressed in adenocarcinoma and is abnormally O-glycosylated. The extracellular domain of mucin contains a variable number of 20-line repeats (TR) of amino acid residues with 5 potential sites for O-glycosylation (25-125). O-glycans are incompletely processed in cancer cells resulting in the expression of the pan-carcinoma carbohydrate antigen Tn (GalNAcα1-O-Ser/Thr) (Springer, 1984). Tn, a simple mucin-type O-glycan, is widely expressed in adenocarcinomas (including breast and ovarian cancers) and has a limited distribution in normal adult tissues (Springer, 1984). Expression of these O-glycans in cancer correlates with poor prognosis, and natural antibodies to these carbohydrate haptens increase in cancer patients (Miles et al., 1995; Soares et al., 1996; Werther et al., 1996). Treatment modalities using glyco-MUC1 epitopes that are overexpressed in cancer cells are desired in the art.

3. Summary

The present disclosure captures the 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.

The present disclosure provides anti-glyco-MUC1 antibodies and antigen binding fragments thereof that bind to cancer-specific glycosylation variants of MUC1. The present disclosure further provides fusion proteins and antibody-drug conjugates comprising anti-glyco-MUC1 antibodies and antigen-binding fragments, and nucleic acids encoding anti-glyco-MUC1 antibodies, antigen-binding fragments and fusion proteins.

The present disclosure further provides methods of using anti-glyco-MUC1 antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates and nucleic acids for cancer therapy.

In certain aspects, the present disclosure provides cancer-specific glycosylation variants of MUC1 and bispecific and other multispecific anti-glyco-MUC1 antibodies and antigen binding fragments that bind a second epitope. The second epitope may be on MUC1 itself, another protein co-expressed on the cancer cell with MUC1, or on another protein presented on a different cell, such as activated T cells. Additionally, nucleic acids encoding such antibodies are also disclosed, including nucleic acids comprising codon-optimized coding regions and nucleic acids comprising non-codon-optimized coding regions for expression in a particular host cell.

The anti-glyco-MUC1 antibody and binding fragment can be in the form of a fusion protein containing a fusion partner. The fusion partner can be useful to provide a second function, such as the signaling function of the signaling domain of the T cell signaling protein, the peptide modulator of T cell activation or the enzyme component of the labeling system. Exemplary T cell signaling proteins include 4-1BB, CO3C, and fusion peptides such as CD28-CD3-zeta and 4-lBB-CD3-zeta. 4-1BB, or CD137, is a T-cell co-stimulatory receptor, and CD3-zeta is a signal-transmitting component of the T-cell antigen receptor. The moiety that provides the second function can be a modulator of T cell activation, such as an IL-15, IL-15Ra, or IL-15/IL-15Ra fusion, or the extent of binding in vivo or in vitro and And/or may encode a label or enzyme component of a labeling system useful for monitoring location. Constructs encoding such prophylactic and therapeutically active biomolecules placed in the environment of T cells, such as autologous T cells, can be used to prevent T cells delivered in adoption to prevent or treat various cancers in some embodiments of the present disclosure. It provides a powerful platform for mobilization.

In certain aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises heavy and/or light chain variable sequences set forth in Table 1 (or encoded by the nucleotide sequences set forth in Table 1). For clarity, when the term "anti-glyco-MUC1 antibody" is used herein, monospecific and multispecific (including bispecific) anti-glyco-MUC1 antibodies, unless context dictates otherwise. , It is intended to include antigen-binding fragments of monospecific and multispecific antibodies, and fusion proteins and conjugates containing the antibodies and antigen-binding fragments thereof. Similarly, when the term "anti-glyco-MUC1 antibody or antigen-binding fragment" is used, monospecific and multispecific (including bispecific) anti-glyco-MUC1 antibodies, unless context dictates otherwise. And antigen-binding fragments thereof together with fusion proteins and conjugates containing such antibodies and antigen-binding fragments.

In another aspect, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises heavy and/or light chain CDR sequences set forth in Table 1-3 (or encoded by the nucleotide sequences set forth in Table 1-3). do. The CDR sequences listed in Table 1 are IMGT (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77), Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological) for defining CDR boundaries. 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) Defined CDR sequences are included. The CDR sequences set forth in Table 2 are combined overlapping regions for the CDR sequences set forth in Table 1 and are shown in bold, underlined by IMGT, Kabat and Chothia sequences. The CDR sequences listed in Table 3 are common overlapping regions for the CDR sequences shown in Table 1. The framework sequences for these anti-glyco-MUC1 antibodies and antigen-binding fragments can be native murine framework sequences in Table 1, or can be non-natural (eg, humanized or human) framework sequences.

<Table 1>

<Table 2>

<Table 3>

In certain aspects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises a CDR comprising the amino acid sequence of any of the CDR combinations described in numbered embodiments 3 to 17. Thus, in certain embodiments, the anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure is a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, the amino acid of SEQ ID NO: 29 CDR-H2 comprising the sequence, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR comprising the amino acid sequence of SEQ ID NO: 9 -L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:31. In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5, 23, 28, or 32. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6 or 24. In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30 or 26. In some embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10. In another aspect, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 5-7 and a light chain CDR of SEQ ID NO: 8-10. In another aspect, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 23-25 and a light chain CDR of SEQ ID NO: 26, 27, and 10. In another aspect, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises heavy chain CDRs of SEQ ID NOs: 28, 29, and 25 and light chain CDRs of SEQ ID NOs: 30, 9, and 31. . In another aspect, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises heavy chain CDRs of SEQ ID NOs: 32, 24, and 7 and light chain CDRs of SEQ ID NOs: 26, 27, and 10. . In another aspect, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises heavy chain CDRs of SEQ ID NOs: 33, 29, and 25 and light chain CDRs of SEQ ID NOs: 8, 9, and 31. . The antibody or antigen-binding fragment can be a murine, chimeric, humanized or human antibody or antigen-binding fragment.

In a further aspect, the anti-glyco-MUC1 antibody or antigen binding fragment of the present disclosure competes with an antibody or antigen binding fragment comprising heavy and light chain variable regions of SEQ ID NOs: 3 and 4, respectively. In another aspect, the present 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 NOs: 3 and 4, respectively. Gives

In another aspect, the anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure is a single chain variable fragment (scFv). Exemplary scFvs include the N-terminal heavy chain variable fragment of the light chain variable fragment. In some embodiments, the scFv heavy chain variable fragment and light chain variable fragment are covalently linked to 4-15 amino acid linker sequences. The scFv may be in the form of a bispecific T-cellengerger or may be in the chimeric antigen receptor (CAR).

Anti-glyco-MUC1 antibodies and antigen-binding fragments can be in the form of multimers of single chain variable fragments, multispecifics of bispecific single chain variable fragments and multispecifics of bispecific single chain variable fragments. In some embodiments, the multimer of single chain variable fragments is selected from divalent single chain variable fragments, tribody or tetrabody. In some of these embodiments, the multimer of the bispecific single chain variable fragment is a bispecific T-cell engager.

Other aspects of the present disclosure relate to nucleic acids encoding anti-glyco-MUC1 antibodies and antibody-binding fragments of the present disclosure. In some embodiments, a portion of a nucleic acid encoding an anti-glyco-MUC1 antibody or antigen-binding fragment is codon-optimized for expression in human cells. In certain aspects, the present disclosure provides a heavy chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 13 and SEQ ID NO: 12 or SEQ ID NO: Provided are anti-glyco-MUC1 antibodies or antigen binding fragments having heavy and light chain variable regions encoded by a light chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity to No. 14. Vectors comprising such nucleic acids (eg, viral vectors such as lentiviral vectors) and host cells are also within the scope of the present disclosure. The heavy and light chain coding sequences can be on a single vector or on separate vectors.

Another aspect of the present disclosure is an anti-glyco-MUC1 antibody, antigen-binding fragment, nucleic acid (or nucleic acid pair), vector (vector pair) or host cell according to the present disclosure, and a physiologically appropriate buffer, adjuvant or It is a pharmaceutical composition comprising a diluent.

Another aspect of the disclosure is a method of making a chimeric antigen receptor, comprising incubating a cell comprising a nucleic acid or vector according to the present disclosure under conditions suitable for expression of a coding region, and collecting the chimeric antigen receptor. .

Another aspect of the present disclosure includes contacting a cell or tissue sample with an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure and detecting whether the antibody binds to a cell or tissue sample. It is a method to detect.

Another aspect of the disclosure provides a prophylactically or therapeutically effective amount of an anti-glyco-MUC1 antibody, antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition according to the present disclosure to a subject in need of cancer treatment It is a method of treating cancer, including administration.

4. Brief description of drawings
1 : Results of ELISA assay showing specificity of binding of GO2 to glyco-MUC1 compared to MUC1.
Figure 2 : Binding of GO2 to colon cancer tissue. Immunohistochemical labeling of invasive colon carcinoma tissue and adjacent healthy tissue with mAb GO2. mAb GO2 shows clear binding to colon cancer tissues with high reactivity with both intracellular and surface structures on cancer cells. In contrast, there is no reactivity to the surface structure on healthy colon cells.
Figure 3 : Binding of GO2 to pancreatic cancer tissue. Immunohistochemical labeling of pancreatic cancer tissue using mAb GO2. mAb GO2 shows clear binding to pancreatic cancer cells. In contrast, reactivity is not visible or limited to surrounding healthy tissue.
Figure 4 : Binding of GO2 to breast cancer tissue. Immunohistochemical labeling of breast cancer tissue using mAb GO2. mAb GO2 showed clear binding to invasive breast cancer cells.
Figure 5 : Results of antibody dependent cellular cytotoxicity assay with secondary antibody conjugated to antibody GO2 and anti-tubulin agonist monomethyl auristatin F (MMAF).
Figure 6 : Results of ELISA assay to quantify circulating tumor cells using GO2. The X-axis represents the number of cells, and the Y-axis represents the OD450 value.
7A-E : Representative images of MUC1 positive TMA tumor core. Figure 7a: breast cancer; Figure 7b: Non-small cell lung cancer; Figure 7c: Ovarian cancer; 7D: colorectal cancer; Figure 7e: Prostate cancer.
8 : Schematic diagram of exemplary anti-glyco-MUC1 and anti-CD3 T-cell bispecific antibodies (TCB).
9A-B : Undigested patient-derived tumor samples (malignant neoplasm of bronchial and lung: mesenchymal, bronchial or lung, squamous cell carcinoma) and different TCBs of 50 nM (FIG. 9A) or 5 nM (FIG. 9B ). Detroit Jurkat-NFAT Activation Assay.
Figure 10 : Uncategorized patient-derived tumor sample (malignant neoplasm of bronchial and lung; lower lobe, bronchial or lung, non-keratinous squamous cell carcinoma) and Jerkat-NFAT activation assay with 50 nM of different TCBs.
11 : Undigested patient-derived tumor sample (malignant neoplasm of bronchial and lung; upper lobe, bronchial or lung, adenocarcinoma of lung type) and Jerkat-NFAT activation assay with 50 nM of different TCBs.
12A-12B : Binding of GO2 TCB to MUC1 expressed on MCF7 cs (FIG. 12A) and T3M4 pzfv (FIG. 12B) cells measured by flow cytometry.
13A-X : Induction of tumor cell killing with GO2 TCB on T3M4 pzfv in the presence of PBMC from two healthy donors (donor 1 FIGS. 13A-13L; donor 2 FIGS. 13A-13L), and CD4 T cells and CD8 T T cell activation as measured by upregulation of CD25 and CD69 on cells, as well as release of IL6, IL8, IL10, IFNγ, TNFα and Granzyme B. Same legend for each of Figures 13A-13X.
14A-14F : T cell activation measured by induction of tumor cell killing with GO2 TCB on MCF7 cs in the presence of PBMC (FIGS. 14A-14B) and upregulation of CD25 and CD69 on CD8 T cells and CD4 T cells. (Figure 14c-14f respectively). Same legend for each of Figures 14A-14F.
15A-B : Binding of GO2 TCB and HMFG1 TCB to MCF10A (human non-neoplastic breast epithelial cell line) (FIG. 15A) and HBEpiC (human bronchial epithelial cell) (FIG. 15B ).
16A-C : Induction of tumor cell killing with GO2 TCB and HMFG1 TCB on MCF10A cells in the presence of PBMC (FIG. 16A) and upregulation of CD25 on CD4 T cells (FIG. 16B) and CD8 T cells (FIG. 16C ). T cell activation as measured by.
Figure 17 : Illustration of GO2 and GO2 TCB flowing through a glycopeptide coupled flow cell.
18A-B : Sensorgram showing the binding of GO2 (FIG. 18A) and GO2 TCB (FIG. 18B) to human and cynomolgus glycopeptides.
19A-D : Estimates of binding (binding), and “apparent” KD of GO2 antibodies (FIGS. 19A-19B) and GO2 TCB (FIG. 19C-19D) to human and cynomolgus glycopeptides.

5. Detailed Description

5.1 Antibodies

The inventors have developed a novel antibody directed against glycoform MUC1 present on tumor cells. This is illustrated by antibody 5F7 referred to herein as “GO2”. One of the serial repeats present in MUC1, glycosylated with recombinant human glycosyltransferase polypeptides GalNAc-T2, GalNAc-T4, and GalNAc-T1 purified to mimic the glycosylation pattern of MUC1 present on tumor cells. GO2 was identified on the screen for an antibody binding to a glycosylated 60-mer showing three copies of VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 50).

Anti-glyco-MUC1 antibodies of the present disclosure exemplified by antibody GO2 are useful as tools in cancer diagnosis and therapy.

Thus, in certain aspects, the present disclosure provides for GUC1 in the form of a glycoform present on tumor cells (referred to herein as “Glyco-MUC1”), preferably GalNAc-T2, GalNAc as described in US Pat. No. 6,465,220. Antibodies and antigen-binding fragments that bind to the -T4, and a 60-mer peptide glycosylated with GalNAc-T1 (VTSAPDTRPAPGSTAPPAHG) ₃ (SEQ ID NO: 47) are provided.

Anti-glyco-MUC1 antibodies of the present disclosure include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, primate antibodies, single chain antibodies, bispecific antibodies, bi-variable domain antibodies, and the like. Antibodies, monoclonal antibodies, genetically engineered antibodies, and/or antibodies with altered properties in other ways. In various embodiments, the antibody comprises all or part of the constant region of the antibody. In some embodiments, the constant region is an isotype selected from IgA (eg, IgA ₁ or IgA ₂ ), IgD, IgE, IgG (eg, IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ), and IgM to be. In a specific embodiment, an anti-glyco-MUC1 antibody of the present disclosure comprises an IgG ₁ constant region isotype.

The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies are derived from a single clone, including any eukaryotic, prokaryotic or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be made using a wide variety of techniques known in the art, including using hybridoma, recombinant, and phage display techniques, or combinations thereof. For many uses of the present disclosure, including in vivo use of anti-glyco-MUC1 antibodies in humans, chimeric, primate, humanized or human antibodies can be used as appropriate.

The term “chimeric” antibody as used herein 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 a human immunoglobulin template. do. Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, 1985, Science 229(4719):1202-7; [Oi et al., 1986, BioTechniques 4:214-221]; [Gillies et al., 1985, J. Immunol. Methods 125:191-202]; U.S. Patent No. 5,807,715; 4,816,567; And 4,816397, which are incorporated herein by reference in their entirety.

A "humanized" form of a non-human (eg, murine) antibody is a chimeric immunoglobulin containing minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, where all or substantially all CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all FRs. The region is from the human immunoglobulin sequence. Humanized antibodies may also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of humanizing antibodies are known in the art. See, eg, Riechmann et al., 1988, Nature 332:323-7; U.S. Patent No. 5,530,101; 5,585,089; 5,693,761; 5,693,762; And 6,180,370 (Queen et al.); EP239400; PCT Publication WO 91/09967; U.S. Patent No. 5,225,539; EP592106; EP519596; [Padlan, 1991, Mol. Immunol., 28:489-498]; [Studnicka et al., 1994, Prot. Eng. 7:805-814]; [Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973]; And US Patent No. 5,565,332, all of which are incorporated herein by reference in their entirety.

“Human antibody” includes an antibody having an amino acid sequence of human immunoglobulin, and includes an antibody isolated from a human immunoglobulin library or from an animal that is transgenic against one or more human immunoglobulins and does not express endogenous immunoglobulin. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. U.S. Patent Nos. 4,444,887 and 4,716,111; And PCT Publication 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 cannot express functional endogenous immunoglobulins, but can also be produced using transgenic mice capable of expressing human immunoglobulin genes. For example, PCT Publication WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Patent No. 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. Using a technique referred to as "induction selection", fully humanoid antibodies can be generated that recognize the selected epitope. In this approach, selected non-human monoclonal antibodies, e.g., mouse antibodies, are used to induce the selection of fully humanoid antibodies that recognize the same epitope (Jespers et al., 1988, Biotechnology 12:899- 903).

"Primitized antibodies" include monkey variable regions and human constant regions. Methods for producing primate antibodies are known in the art. For example, US Patent No. 5,658,570; 5,681,722; And 5,693,780, which are incorporated herein by reference in their entirety.

Anti-glyco-MUC1 antibodies of the present disclosure include both full-length (intact) antibody molecules, as well as antigen-binding fragments capable of binding glyco-MUC1. Examples of antigen-binding fragments include, for example and without limitation, Fab, Fab', F (ab') ₂ , Fv fragment, single chain Fv fragment and single domain fragment.

The Fab fragment contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Fab' fragments differ from Fab fragments by adding a small number of residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond of the hinge cysteine of the F(ab') ₂ pepsin digestion product. Additional chemical couplings 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, so that they are cleared more rapidly from the animal's circulation and may have less non-specific tissue binding than the intact antibody (eg, See Wahl et al., 1983, J. Nucl. Med. 24:316).

“Fv” fragment is the minimum fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain that is tightly non-covalently associated (V _H -V _L dimer). In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the V _H -V _L dimer. Often, six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv that contains only three CDRs specific for a target) may have the ability to recognize and bind a target, although it has a lower affinity than the entire binding site. .

“Single chain Fv” or “scFv” antigen-binding fragments include the V _H and V _L domains of an antibody, where these domains are in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker that allows scFvs to form the desired structure for target binding between the V _H domain and the V _L domain.

A “single domain antibody” consists of a single V _H or V _L domain that exhibits sufficient affinity for glyco-MUC1. In specific embodiments, the single domain antibody is a camel antibody (see, eg, Riechmann, 1999, Journal of Immunological Methods 231:25-38).

Anti-glyco-MUC1 antibodies of the present disclosure can also be bispecific and other multispecific antibodies. Bispecific antibodies are monoclonal antibodies with binding specificities for two different epitopes on the same or different antigens, often human or humanized antibodies. In the present disclosure, one of the binding specificities can be directed towards glyco-MUC1 and the other in any other antigen, eg, cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virus It may be for a derived protein, an envelope protein encoded by a virus, a protein derived from bacteria, or a bacterial surface protein. In certain preferred embodiments, the bispecific and other multispecific anti-glyco-MUC1 antibodies and antigen binding fragments are second MUC1 epitopes, epitopes on another protein co-expressed with MUC1 on cancer cells, or different cells, such as It specifically binds to an epitope on another protein presented on activated T cells. Bispecific antibodies of the present disclosure include IgG modal bispecific antibodies and single chain-based bispecific antibodies.

IgG modal bispecific antibodies of the present disclosure may be any of various types of IgG mod bispecific antibodies known in the art, such as quadroma bispecific antibodies, "knobs-knobs- in-holes)" bispecific antibodies, CrossMab bispecific antibodies, charge-pair bispecific antibodies, conventional light chain bispecific antibodies, 1-arm single chain Fab-immunoglobulin gamma bispecific Red antibody, disulfide stabilized Fv bispecific antibody, DuetMab, controlled Fab-arm exchange bispecific antibody, strand-exchange engineer domain body bispecific antibody, 2-arm leucine zipper heterodimeric monoclo Raw bispecific antibodies, κλ-body bispecific antibodies, bivariate domain bispecific antibodies, and cross bivariate domain bispecific antibodies. See, eg, Koehler 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., 2015 Nature Commun 6:6113]; [Schanzer et al., 2014, J Biol Chem 289:18693-706]; [Metz et al., 2012 Protein Eng Des Sel 25:571-80]; [Mazor et al., 2015 MAbs 7:377-89]; [Labrijn et al., 2013 Proc Natl 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., 2017, Int. J. Mol. Sci. 18:48, the entire contents of which are incorporated herein by reference.

In some embodiments, the bispecific antibody of the present disclosure is crossmab. CrossMab technology is described 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, the entire contents of which are incorporated herein by reference. Briefly, CrossMab technology is based on domain crossing between heavy and light chains in one Fab-arm of bispecific IgG, which promotes correct chain association. The crossmab bispecific antibody of the present disclosure can be a “crossmab ^FAB ” antibody in which 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 antibodies of the present disclosure can be “crossmab ^VH-VL ” antibodies in which only the variable domains of the heavy and light chains of the Fab portion of one arm of the bispecific IgG antibody have been exchanged. In another embodiment, the crossmab bispecific antibody of the present disclosure can be a “crossmab ^CH1-CL ” antibody in which only the constant domains of the heavy and light chains of the Fab portion of one arm of the bispecific IgG antibody are exchanged. . The Crossmab ^CH1-CL antibody has no expected by-products, in contrast to Crossmab ^FAB and Crossmab ^VH-VL , therefore, in some embodiments a Crossmab ^CH1-CL bispecific antibody is preferred. See Klein et al., 2016, mAbs, 8(6):1010-1020. Additional embodiments of the crossmab of the present disclosure are described in Section 5.2 below.

In some embodiments, the bispecific antibodies of the present disclosure are controlled Fab-arm exchange bispecific antibodies. Methods of making Fab-arm exchange bispecific antibodies are described in PCT Publication No. WO2011/131746 and Labrijn et al., 2014 Nat Protoc. 9(10):2450-63. Briefly, two parental IgG1s containing a single matched point mutation in the CH3 domain are separately expressed, and parental IgG1s are mixed in vitro under redox conditions to enable half-molecule recombination and to reduce reducing agents. By allowing re-oxidation of interchain disulfide bonds, controlled Fab-arm exchange bispecific antibodies can be produced by forming bispecific antibodies.

Bispecific antibodies of the present disclosure may include an Fc domain composed of first and second subunits. In one embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG ₁ Fc domain. In another embodiment, the Fc domain is an IgG ₄ Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), in particular the amino acid substitution S228P. Such amino acid substitutions reduce Fab arm exchange of IgG ₄ antibodies in vivo (see Stubenrauch et al., 2010, Drug Metabolism and Disposition 38:84-91). In a further specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG ₁ Fc domain. An exemplary sequence of the human IgG ₁ Fc region is provided in SEQ ID NO: 42.

In certain embodiments, the Fc domain comprises modifications that promote association of 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 within the CH3 domain. Thus, in one embodiment, the modification is within the CH3 domain of the Fc domain.

In a specific embodiment, said modification that promotes association of the first and second subunits of the Fc domain is a “knob” modification in one of the two subunits of the Fc domain and the other in the other of the two subunits of the Fc domain” A so-called "knob-into-hole" variant, including a "hole" variant. Knob-in-to-hole technology is 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. In general, this method promotes heterodimer formation and ridges at the interface of the first polypeptide (“knobs”) and corresponding in the interface of the second polypeptide so that the ridges can be located within the cavity to prevent homodimer formation and interfere with homodimer formation. It involves introducing a cavity ("hole"). Elevation is established by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (eg tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (eg alanine or threonine), a compensating cavity of the same or smaller size as the ridge is created at the interface of the second polypeptide.

Thus, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue with a larger side chain volume, whereby the first can be located in a cavity within the CH3 domain of the second subunit. A ridge in the CH3 domain of the subunit is generated, and the amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue with a smaller side chain volume, thereby raising the ridge in the CH3 domain of the first subunit. A cavity in the CH3 domain of the second subunit that can be located is created. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). Elevations and cavities can be made, for example, by altering the nucleic acid encoding the polypeptide by site-specific mutagenesis, or by peptide synthesis. An exemplary substitution is Y470T.

In a specific such embodiment, the threonine residue at position 366 in the first subunit of the Fc domain is replaced with a tryptophan residue (T366W), and the tyrosine residue at position 407 in the second subunit of the Fc domain is replaced by a valine residue (Y407V ), optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first subunit of the Fc domain, a serine residue at position 354 is replaced with a cysteine residue (S354C) or a glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (especially a serine residue at position 354). Is replaced with a cysteine residue), a tyrosine residue at position 349 is additionally replaced with a cysteine residue in the second subunit of the Fc domain (Y349C) (numbering according to the Kabat EU index). In certain embodiments, 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 (numbers according to the Kabat EU Index) Pagination).

In some embodiments, electrostatic steering (eg, as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) is the first and second subunit of the Fc domain. It can be used to promote the association.

In some embodiments, an Fc domain comprises one or more amino acid substitutions that bind to an Fc receptor and/or reduce effector function.

In certain embodiments, 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 specific embodiments, 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 of complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

Typically, the same one or more 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.

In one embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group of L234, L235 and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG ₁ Fc domain, particularly a human IgG ₁ Fc domain. In one embodiment, the Fc domain comprises amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, especially P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain comprises amino acid substitutions at P329 and additional amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In certain embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in certain embodiments, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain, The leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (in the Kabat EU Index). Numbering according to). In one such embodiment, the Fc domain is an IgG ₁ Fc domain, particularly a human IgG ₁ Fc domain.

Single chain-based bispecific antibodies of the present disclosure are various types of single chain-based bispecific antibodies known in the art, such as bispecific T-cell engger (BiTE), diabodies, inline diabodies. (Tandab), bi-affinity retargeting molecule (DART), and bispecific killer cell initiator. See, eg, Loeffler 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, the entire contents of which are incorporated herein by reference.

In some embodiments, the bispecific antibody of the present disclosure is a bispecific T-cell engager (BiTE). BiTE is a single polypeptide chain molecule with two antigen-binding domains, one binding to a T-cell antigen and the second to an antigen present on the surface of the target (PCT Publication WO 05, incorporated herein by reference in its entirety). /061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; see Bargou, et al., 2008, Science 321:974-977). Thus, the BiTE of the present disclosure has an antigen binding domain that binds a T-cell antigen, and a second antigen binding domain directed against glyco-MUC1.

In some embodiments, the bispecific antibody of the present disclosure is a bi-affinity retargeting molecule (DART). DART comprises at least two polypeptide chains that are associated (especially through covalent interactions) to form at least two epitope binding sites capable of recognizing the same or different epitopes. Each polypeptide chain of the DART contains an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form epitope binding sites. Rather, the epitope binding site interacts with an immunoglobulin light chain variable region of a different (e.g., second) DART™ polypeptide chain where the immunoglobulin heavy chain variable region of one of the DART polypeptide chains (e.g., the first chain) is different. To form. Similarly, the immunoglobulin heavy chain variable region of one of the DART polypeptide chains (e.g., first chain) interacts with an immunoglobulin heavy chain variable region of a different (e.g., second) DART polypeptide chain to establish an epitope binding site. To form. The DART can be monospecific, bispecific, trispecific, etc., thus simultaneously binding to 1, 2, 3 or more numbers of different epitopes (which can be of the same antigen or different antigens) Can be. DART can additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., and thus 1, 2, 3, 4, 5, 6 or more molecules Can be combined at the same time. These two properties of DART (i.e., specificity) such that bispecific antibodies (i.e., capable of binding to two sets of epitopes), such as tetravalent (i.e., capable of binding to four sets of epitopes), are produced. And degree of bonding value). DART molecules are disclosed in PCT publications WO 2006/113665, WO 2008/157379, and WO 2010/080538, which are incorporated herein by reference in their entirety.

In some embodiments of the bispecific antibodies of the present disclosure, one of the binding specificities is directed against glyco-MUC1 and the other is directed against the antigen expressed on immune effector cells. The term “immune effector cell” or “effector cell” as used herein refers to a cell in a natural cell repertoire in a mammalian immune system that can be activated to affect the viability of a target cell. Immune effector cells include cells of the lymphoid lineage such as natural killer (NK) cells, T cells including cytotoxic T cells, or B cells, but cells of the bone marrow lineage such as monocytes or macrophages, dendritic cells and neutrophil granulocytes It can also be considered as an immune effector cell. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells or neutrophil granulocytes. Immobilization of effector cells to abnormal cells means that the effector cells are brought close to the abnormal target cells so that the effector cells can directly kill the mobilized abnormal cells or indirectly kill their killing. To avoid non-specific interactions, it is preferred that the bispecific antibodies of the present disclosure specifically recognize antigens on immune effector cells that are at least overexpressed by immune effector cells compared to other cells in the body. Target antigens present on immune effector cells can include CD3, CD8, CD16, CD25, CD28, CD64, CD89, NKG2D and NKp46. Preferably, the antigen on immune effector cells is CD3 expressed on T cells.

As used herein, “CD3” refers to mammals such as primates (eg humans), non-human primates (eg cynomolgus monkeys) and rodents (eg mice and rats) unless otherwise indicated. Refers to any natural CD3 from any vertebrate source, including. This term encompasses “full-length” unprocessed CD3, as well as any form of CD3 resulting from processing in cells. These terms also encompass naturally occurring variants of CD3, such as splice variants or allelic variants. The most preferred antigen on immune effector cells is the CD3 epsilon chain. These antigens have been shown to be very effective in mobilizing T cells to abnormal cells. Thus, preferably, bispecific antibodies of the present disclosure specifically recognize CD3 epsilon. The amino acid sequence of human CD3 epsilon is given in 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 [Macaca fascicularis] CD3 epsilon is shown in NCBI GenBank number BAB71849.1. For human therapeutic use, bispecific antibodies are used in which the CD3-binding domain specifically binds human CD3 (eg, human CD3 epsilon chain). For preclinical testing in non-human animals and cell lines, the bispecific specifically binds the CD3-binding domain to CD3 in the species used for preclinical testing (e.g. cynomolgus CD3 for primate testing). Antibodies can be used.

As used herein, a binding domain that “specifically binds” or “specifically recognizes” a target antigen from a particular species does not exclude binding to or recognition of an antigen from another species, Thus, one or more of the binding domains encompass antibodies with cross-reactivity among species. For example, a CD3-binding domain that “specifically binds” to or “specifically recognizes” human CD3 can also bind or recognize cynomolgus CD3 and vice versa.

In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody H2C (described in PCT Publication No. WO2008/119567) for binding to an epitope of CD3. In another embodiment, the bispecific antibodies of the present disclosure are monoclonal antibody V9 (Rodrigues et al., 1992, Int J Cancer Suppl 7:45-50) and US patents for binding to epitopes of CD3. Number 6,054,297). In another embodiment, the bispecific antibodies of the present disclosure are described in the monoclonal antibody FN18 (Nooij et al., 1986, Eur J Immunol 19:981-984) for binding to an epitope of CD3. ). In another embodiment, the bispecific antibody of the present disclosure is a monoclonal antibody SP34 (as described in Pessano et al., 1985, EMBO J 4:337-340) for binding to an epitope of CD3. Can compete with.

Anti-glyco-MUC1 antibodies of the present disclosure include derivatized antibodies. For example, but not limited to, derivatized antibodies are typically glycosylated, acetylated, PEGylated, phosphorylated, amidated, derivatized with known protective/blocking groups, proteolytic cleavage, cellular ligands or other proteins. It is transformed by being connected. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, for example, using ambrx technology, the derivative may contain one or more non-natural amino acids (see, eg, Wolfson, 2006, Chem. Biol. 13(10):1011. -2].

The anti-glyco-MUC1 antibody or binding fragment can be an antibody or fragment whose sequence has been modified to alter at least one constant region-mediated biological effector function. For example, in some embodiments, the anti-glyco-MUC1 antibody reduces binding to, e.g., Fc receptor (FcγR), to reduce at least one constant region-mediated biological effector function compared to an unmodified antibody. It can be modified to let. FcγR binding can be reduced by mutating the immunoglobulin constant region segment of an antibody at a specific region required for FcγR interaction (see, e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491). ; And Lund et al., 1991, J. Immunol. 147:2657-2662). Decreasing the antibody's ability to bind FcγR can also reduce other effector functions that rely on FcγR interactions, such as opsonization, phagocytosis and antigen-dependent cellular cytotoxicity (“ADCC”).

Anti-glyco-MUC1 antibodies or binding fragments described herein are antibodies and/or modified to obtain or improve at least one constant region-mediated biological effector function compared to an unmodified antibody, such as to enhance FcγR interaction Binding fragments (see, eg, US 2006/0134709). For example, the anti-glyco-MUC1 antibody of the present disclosure can have a constant region that binds FcγRIIA, FcγRIIB and/or FcγRIIIA with greater affinity than the corresponding wild-type constant region.

Thus, the antibodies of the present disclosure may have opsonization, phagocytosis, or alteration of biological activity resulting in increased or decreased ADCC. Such changes are known in the art. For example, modifications of antibodies that reduce ADCC activity are described in US Pat. No. 5,834,597. Exemplary ADCC degradation variants correspond to “mutant 3” (shown in FIG. 4 of US Pat. No. 5,834,597) with residues 236 deleted and residues 234, 235 and 237 (using EU numbering) substituted with alanine.

In some embodiments, anti-glyco-MUC1 antibodies of the present disclosure have low fucose levels or lack fucose. Antibodies lacking fucose correlated with enhanced ADCC activity, especially in low dose antibodies. Shields et al., 2002, J. Biol. Chem. 277:26733-26740]; [Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. Methods for making antibodies without fucose include growth in rat myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which encodes the α-1,6-fucosyltransferase, an enzyme required for fucosylation of the polypeptide.

In another aspect, the anti-glyco-MUC1 antibody or binding fragment binds binding affinity to fetal Fc receptor, FcRn, for example, by mutating an immunoglobulin constant region segment in a specific region involved in FcRn interaction. And increasing or decreasing modifications (see, eg, WO 2005/123780). In certain embodiments, the anti-glyco-MUC1 antibody of the IgG class comprises at least one of the amino acid residues 250, 314, and 428 of the heavy chain constant region alone or in any combination thereof, such as at positions 250 and 428, or position 250 And at 314, or at positions 314 and 428, or at positions 250, 314 and 428, and positions 250 and 428 are specific combinations. For position 250, the substituted amino acid residues include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine However, it can be any amino acid residue other than threonine, which is now not limited. For position 314, the substituted amino acid residues include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine However, it can be any amino acid residue other than leucine, which is now not limited. For position 428, the substituted amino acid residues include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. However, it can be any amino acid residue other than methionine, which is now not limited. Specific combinations of suitable amino acid substitutions are identified in Table 1 of US Pat. No. 7,217,797, incorporated herein by reference. This mutation increases binding to FcRn, which protects the antibody from degradation and increases its half-life.

In another aspect, anti-glyco-MUC1 antibodies or antigen-binding fragments of the present disclosure are described, for example, 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 as described in US Patent Application No. 2007/0280931, one or more amino acids are inserted into one or more of its hypervariable regions.

In another aspect, particularly useful for diagnostic use, the anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure is attached to a detectable moiety. Detectable moieties can include radioactive moieties, colorimetric molecules, fluorescent moieties, chemiluminescent moieties, antigens, enzymes, detectable beads (such as magnetic or electron high density (e.g. gold) beads), or other molecules ( For example, biotin or streptavidin).

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

Fluorescent labels include rhodamine, lanthanide phosphorus, fluorescein and derivatives thereof, fluorochrome, GFP (GFP: "green fluorescent protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allopicocyanin , o-phthalaldehyde, and fluorescent amine.

Enzyme labels include horseradish peroxidase, β galaltooxidase, luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxidase, Glucose amylase, anhydrase carbonate, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase.

Chemiluminescent labels or chemiluminescents, such as isoluminol, luminol and dioxetane

Other detectable moieties include molecules such as biotin, deoxygenin or 5-bromodeoxyuridine.

In certain aspects, an anti-glyco-MUC1 antibody or antigen binding fragment of the present disclosure competes with an antibody or antigen binding fragment comprising heavy and light chain variable regions of GO2 or GO2 (SEQ ID NOs: 3 and 4, respectively).

On cells expressing a glyco-MUC1 epitope bound by GO2 or a glycosylated MUC1 peptide containing an epitope bound by GO2, for example GalNAc-T2, GalNAc-T4 and GalNAc-T2 as described in US Pat. No. 6,465,220. Competition can be assayed on a 60-mer peptide glycosylated with GalNAc-T1 (VTSAPDTRPAPGSTAPPAHG) ₃ . Cells that do not express epitopes or unglycosylated peptides can be used as controls.

Cells for which competitive assays can be performed include, but are not limited to, breast cancer cell lines MCF7 or T47D, and recombinant cells engineered to express the glyco-MUC1 epitope. In one non-limiting example, CHO IdID cells lacking UDP-Gal/GalNAc epimerase and lacking GalNAc and GalNAc O-glycosylation and galactosylation, respectively, in the absence of exogenous addition of GalNAc and Gal, were engineered to express MUC1, GalNAc Grown in the absence or presence of, the latter yields cells expressing the Tn glycoform of MUC1 to which GO2 binds. Cells expressing the unglycosylated form of MUC1 can be used as a negative control.

Assays for competition include, but are not limited to, radioactive material labeled immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), sandwich ELISA fluorescence activated cell sorting (FACS) assay and Biacore assay.

In performing antibody competition assays between a reference antibody and a test antibody (regardless of species or isotype), the reference is first detectable with a label, such as a fluorophore, biotin or enzymatic (or even radioactive) to enable subsequent identification ) Can be labeled. In this case, cells expressing glyco-MUC1 are incubated with an unlabeled test antibody, a labeled reference antibody is added, and the strength of the bound label is measured. If the test antibody competes with the labeled reference antibody by binding to the overlapping epitope, the intensity will be reduced compared to the control response performed without the test antibody.

In specific embodiments of this assay, the concentration of the labeled reference antibody (“conc _80% ”) that yields 80% of the maximum binding under assay conditions (eg, specific cell density) is first determined and 10×conc _{80 %} by unlabeled test antibodies and reference antibodies cover of conc _80% of the competitive assay is performed.

The inhibition can be expressed as an inhibition constant, or K _i , which is calculated according to the following formula:

K _i =IC ₅₀ /(1+[reference Ab concentration]/K _d )

[Wherein, IC ₅₀ is the concentration of the test antibody yielding a 50% reduction in binding of the reference antibody, K _d is the dissociation constant of the reference antibody, which is a measure of affinity for glyco-MUC1]. Antibodies competing with the anti-glyco-MUC1 antibodies disclosed herein may have a K _i of 10 pM to 10 nM under the assay conditions described herein.

In various embodiments, the test antibody is capable of binding at least about 20% or more of the binding of the reference antibody at a reference antibody concentration that is 80% of the maximum binding under the specific assay conditions used and at a test antibody concentration that is 10 times higher than the reference antibody concentration, e.g. For example, decreasing the reference antibody by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, or a percentage that falls within any of the above values. It is considered to compete with.

In one example of a competitive assay, the glycosylated MUC1 60-mer peptide is solid surfaced, e.g., by contacting a plate with a solution of the peptide (e.g., at a concentration of 1 μg/mL in PBS overnight at 4° C.). Attach on microwell plate. Plates are washed (e.g., 0.1% Tween 20 in PBS) and blocked (e.g., Superblock, Thermo Scientific, Rockford, Ill.). Semi-saturated amounts of biotinylated GO2 (e.g., concentration of 80 ng/mL) and unlabeled GO2 ("reference" antibody) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in PBS) Alternatively, a mixture of step-diluted competitive anti-glyco-MUC1 antibody (“test” antibody) antibody (eg, a concentration of 2.8 μg/mL, 8.3 μg/mL, or 25 μg/mL) is added to the wells and plate Incubate for 1 hour with gentle shaking. The plate is washed, 1 μg/mL HRP-conjugated streptavidin diluted in ELISA buffer is added to each well and the plate is incubated for 1 hour. Plates were washed and bound antibodies were detected by addition of a substrate (eg, TMB, Biofx Laboratories Inc., Owings Mills, MD). A stop buffer is added (e.g., Bio FX stop reagent, BioF Laboratories Inc., Owings Mills, MD) to terminate the reaction and a microplate reader (e.g., Versamax) (VERSAmax), Molecular Devices, Sunnyvale, Calif., are measured for absorbance at 650 nm.

Variations in this competition assay can also be used to test competition between GO2 and another anti-glyco-MUC1 antibody. For example, in certain aspects, an anti-glyco-MUC1 antibody is used as a reference antibody, and GO2 is used as a test antibody. Additionally, instead of the glycosylated MUC1 60-mer peptide, membrane-bound glyco-MUC1 expressed on the cell surface in culture (eg on the surface of one of the cell types mentioned above) can be used. Generally, about 10 ⁴ to 10 ⁶ transformants are used, for example about 10 ⁵ transformants. Other modalities for competitive assays are known in the art and can be used.

In various embodiments, the anti-glyco-MUC1 antibody has a concentration of 0.08 μg/mL, 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, or any of the above values Anti-glyco-MUC1 antibodies of the present disclosure, when used at concentrations in the range between (e.g., concentrations ranging from 2 μg/mL to 10 μg/mL), at least 40%, at least 40%, of binding of labeled GO2 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by a percentage of the range between any of the above values (e.g., anti-glyco-MUC1 antibodies of the present disclosure Reduces binding of labeled GO2 by 50% to 70%).

In other embodiments, the concentration of GO2 is 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL, 250 μg/mL or a concentration in the range between any of the above values (e.g., When used at concentrations ranging from 2 μg/mL to 10 μg/mL), GO2 is capable of at least 40%, at least 50%, at least 60%, at least 70% binding of the labeled anti-glyco-MUC1 antibody of the present disclosure. , By at least 80%, at least 90%, or by a percentage of the range between any of the above values (e.g., GO2 reduces the binding of the labeled anti-glyco-MUC1 antibody of 50% to 70%) %)).

In this assay, the GO2 antibody can be replaced with a humanized or chimeric counterpart of GO2, such as any antibody or antigen-binding fragment comprising the CDR or heavy and light chain variable regions of GO2.

In certain aspects, the anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises heavy and/or light chain variable sequences set forth in Table 1 (or encoded by the nucleotide sequences set forth in Table 1). In another aspect, an anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure comprises heavy and/or light chain CDR sequences set forth in Table 1 (or encoded by the nucleotide sequences set forth in Table 1). The framework sequences for these anti-glyco-MUC1 antibodies and antigen-binding fragments can be native murine framework sequences in Table 1, or can be non-natural (eg, humanized or human) framework sequences.

In another aspect, the present disclosure provides anti-MUC1 antibody or antigen binding having heavy and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 3 and 4, respectively. Provide a fragment.

In another aspect, the anti-glyco-MUC1 antibody or antigen-binding fragment of the present disclosure is a single chain variable fragment (scFv). Exemplary scFvs include the N-terminal heavy chain variable fragment of the light chain variable fragment. In some embodiments, the scFv heavy chain variable fragment and light chain variable fragment are covalently linked to 4-15 amino acid linker sequences. The scFv can be in the form of a bispecific T-Cell Ingerser or in the chimeric antigen receptor (CAR).

5.2 Anti-glyco-MUC1 and anti-CD3 bispecific antibodies

In some aspects, bispecific antibodies of the present disclosure are specific for a first antigen binding domain that specifically binds CD3 (eg, including the CDRs or VH and VLs listed in Table 4), and glyco-MUC1 It may include a second antigen-binding domain that binds. The second antigen binding domain may comprise the features described above for the glyco-MUC1 antibody alone or in combination (eg, a combination of CDRs identified in Tables 1-3, eg, the following number CDRs comprising the amino acid sequence of any of the CDR combinations described in embodiments 3 to 17, or the VH and VL sequences identified in Table 1.).

<Table 4>

In some embodiments, the first antigen binding domain comprises a heavy chain variable region comprising a heavy chain CDR-H1 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 35, and a CDR-H3 of SEQ ID NO: 36; And a light chain variable region comprising the light chain CDR-L1 of SEQ ID NO: 37, the CDR-L2 of SEQ ID NO: 38 and the CDR-L3 of SEQ ID NO: 39.

In some embodiments, the second antigen binding domain is, for example, a CDR comprising the amino acid sequence of any of the CDR combinations set forth in numbered embodiments 3 to 17, e.g. (i) SEQ ID NO: 5 A heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 6, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; And a light chain variable region comprising light chain CDR (CDR-L) 1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9 and CDR-L3 of SEQ ID NO: 10.

In certain embodiments, bispecific antibodies include:

(i) CDR-H1 specifically binding to CD3 and comprising the amino acid sequence of SEQ ID NO: 34, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and amino acid sequence of SEQ ID NO: 36 Heavy chain variable region comprising CDR-H3 comprising a; And CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39 A first antigen binding domain comprising a region; And

(ii) a specific binding to glyco-MUC1, (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, more preferably CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, more preferably CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, More preferably a heavy chain variable region comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO:7; And CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31, more preferably A second antigen binding domain comprising a light chain variable region comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the first antigen binding domain is a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and SEQ ID NO: : At least about 95%, 96%, 97%, 98%, 99% or 100% identical light chain variable region sequences to the amino acid sequence of 41.

In some embodiments, the first antigen binding domain comprises a heavy chain variable region sequence of SEQ ID NO: 40 and a light chain variable region sequence of SEQ ID NO: 41.

In some embodiments, the second antigen binding domain is a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and SEQ ID NO: : At least about 95%, 96%, 97%, 98%, 99% or 100% identical light chain variable region sequences to the amino acid sequence of 4.

In some embodiments, the second antigen binding domain comprises a heavy chain variable region sequence of SEQ ID NO: 3 and a light chain variable region sequence of SEQ ID NO: 4.

In some embodiments, the first and/or second antigen binding domain is a Fab molecule. In some embodiments, the first antigen binding domain is a cross Fab molecule in which the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged. In this embodiment, the second antigen binding domain is preferably a conventional Fab molecule.

The first and second antigen binding domains of the bispecific antibody are both Fab molecules, and in one of the antigen binding domains (particularly the first antigen binding domain), the variable domains VL and VH of the Fab light chain and the Fab heavy chain are exchanged with each other. In some embodiments,

i) in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbered according to Kabat), and in the constant domain CH1 of the first antigen binding domain, at position 147 The amino acid or amino acid at position 213 is replaced by a negatively charged amino acid (numbered according to the Kabat EU index); or

ii) in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbered according to Kabat), and at the constant domain CH1 of the second antigen binding domain, at position 147 The amino acid or amino acid at position 213 is replaced by a negatively charged amino acid (numbering according to the Kabat EU index).

Bispecific antibodies do not include modifications of both mentioned under i) and ii). The constant domains CL and CH1 of the antigen binding domain with VH/VL exchange are not exchanged with each other (ie, they remain unexchanged).

In a more specific embodiment,

i) In the constant domain CL of the first antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), the first antigen In the constant domain CH1 of the binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E), or aspartic acid (D) (numbering according to the Kabat EU index); or

ii) In the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), the second antigen In the constant domain CH1 of the binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In one such embodiment, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) , In the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In a further embodiment, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), In the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In certain embodiments, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), The amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat), and in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 is Independently substituted with glutamic acid (E), or aspartic acid (D) (numbered according to the Kabat EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E), or aspartic acid (D) (Numbering according to the Kabat EU Index).

In a more specific embodiment, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is replaced by lysine (K) (numbered according to Kabat), and is replaced by the amino acid lysine (K) at position 123 (Numbering according to Kabat), in the constant domain CH1 of the second antigen binding domain, substituted by glutamic acid (E) of the amino acid at position 147 (numbering according to the Kabat EU index), and the amino acid at position 213 Substituted by glutamic acid (E) (numbering according to the Kabat EU index).

In an even more specific embodiment, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is replaced by lysine (K) (numbered according to Kabat), and the amino acid at position 123 is assigned to arginine (R). Substituted (numbered according to Kabat), in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 is substituted by glutamic acid (E) (numbered according to the Kabat EU index), and at position 213 Amino acids are substituted by glutamic acid (E) (numbering according to the Kabat EU index).

In certain embodiments, amino acid substitutions according to the above embodiments are made in the constant domain CL and constant domain CH1 of the second antigen binding domain, and the constant domain CL of the second antigen binding domain is a kappa isotype.

In some embodiments, the first and second antigen binding domains are fused to each other, optionally via a peptide linker.

In some embodiments, the first and second antigen binding domains are each Fab molecules, and (i) the second antigen binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain , Or (ii) the first antigen binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain.

In some embodiments, a bispecific antibody provides monovalent binding to CD3.

In certain embodiments, the bispecific antibody comprises a single antigen binding domain that specifically binds CD3, and two antigen binding domains that specifically bind glyco-MUC1. Thus, in some embodiments, the bispecific antibody comprises a third antigen binding domain that specifically binds glyco-MUC1. In some embodiments, the third antigenic moiety is identical to the first antigen binding domain (eg, is also a Fab molecule and comprises the same amino acid sequence).

In certain embodiments, the bispecific antibody further comprises an Fc domain consisting of first and second subunits. In one embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG ₁ Fc domain. In another embodiment, the Fc domain is an IgG ₄ Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), in particular the amino acid substitution S228P. In a further specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG ₁ Fc domain. An exemplary sequence of the human IgG ₁ Fc region is provided in SEQ ID NO: 42.

In some embodiments where the first, second, and third antigen binding domains, if present, are each Fab molecules, (a) (i) the second antigen binding domain is the first antigen binding domain at the C-terminus of the Fab heavy chain. Is fused to the N-terminus of the Fab heavy chain, and the first antigen binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, or (ii) the first antigen binding domain is Fab The C-terminus of the heavy chain is fused to the N-terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain; (b) The third antigen binding domain, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

In certain embodiments, the Fc domain comprises modifications that promote association of the first and second subunits of the Fc domain, eg, as described in Section 5.1.

In some embodiments, an Fc domain comprises one or more amino acid substitutions that bind to an Fc receptor and/or reduce effector function, eg, as described in Section 5.1.

In certain embodiments, bispecific antibodies are

(i) a first antigen binding domain that specifically binds CD3, wherein the first antigen binding domain is a cross Fab molecule in which the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged;

(ii) a heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; And a light chain variable region comprising the light chain CDR-L1 of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9, and the CDR-L3 of SEQ ID NO: 10, which specifically bind to glyco-MUC1. Second and third antigen binding domains, which are the second and third antigen binding domains, the second and third antigen binding domains being Fab molecules, particularly conventional Fab molecules, respectively;

(iii) Fc domain composed of first and second subunits capable of stably associating

Including,

Wherein the second antigen binding domain is fused to the N-terminus of the Fab heavy chain at the C-terminus of the Fab heavy chain, and the first antigen binding domain is the first subunit of the Fc domain at the C-terminus of the Fab heavy chain Fused to the N-terminus of, and the third antigen binding domain is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

In one embodiment, the first antigen binding domain comprises a heavy chain variable region comprising a heavy chain CDR-H1 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 35, and a CDR-H3 of SEQ ID NO: 36; And a light chain variable region comprising the light chain CDR-L1 of SEQ ID NO: 37, the CDR-L2 of SEQ ID NO: 38 and the CDR-L3 of SEQ ID NO: 39.

In one embodiment, the first antigen binding domain is a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40 and SEQ ID NO: A light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of 41.

In one embodiment, the first antigen binding domain comprises a heavy chain variable region sequence of SEQ ID NO: 40 and a light chain variable region sequence of SEQ ID NO: 41.

In one embodiment, the second and third antigen binding domains are heavy chain variable region sequences and sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3 Light chain variable region sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4. Preferably, the antigen binding domain comprises a CDR comprising the amino acid sequence of any of the CDR combinations described in numbered embodiments 3 to 17. In one embodiment, the second and third antigen binding domains comprise a heavy chain variable region of SEQ ID NO: 3 and a light chain variable region of SEQ ID NO: 4.

The Fc domain according to the above embodiment may include all the features described above alone or in combination with respect to the Fc domain.

In some embodiments, the antigen binding domain and Fc region are fused to each other by a peptide linker, eg, a peptide linker as in SEQ ID NO: 45 and SEQ ID NO: 46.

In one embodiment, in the constant domain CL of the second and third Fab molecules of (ii), the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat), and the amino acid at position 123 is lysine. (K) or arginine (R), especially substituted by arginine (R) (numbered according to Kabat), in the constant domain CH1 of the second and third Fab molecules of (ii), the amino acid at position 147 is glutamic acid Substituted by (E) (numbering according to the Kabat EU index), and the amino acid at position 213 by glutamic acid (E) (numbering according to the Kabat EU index).

In one embodiment, the bispecific antibody is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 43 (preferably a sequence CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31). Polypeptide comprising, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 44 (preferably, the CD3 heavy chain set forth in Table 4) And light chain CDR sequences) A polypeptide comprising a sequence, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 45 ( Preferably, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25 A polypeptide comprising a sequence, and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 46 (preferably sequence identification) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, CDR-H3 of the amino acid sequence of SEQ ID NO: 25, and the amino acid sequence of SEQ ID NO: 37. CDR-L1 comprising, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39).

In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 43 (especially two polypeptides), a polypeptide comprising the sequence of SEQ ID NO: 44, a sequence comprising the sequence of SEQ ID NO: 45 Polypeptides, and polypeptides comprising the sequence of SEQ ID NO:46.

5.3 Antibody-Drug Conjugates

Another aspect of the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-glyco-MUC1 antibody and an antigen-binding fragment of the present disclosure. ADCs generally include anti-glyco-MUC1 antibodies and/or binding fragments as described herein to which one or more cytotoxic and/or cytostatic agents are linked by one or more linkers. In a specific embodiment, the ADC is a compound according to structural formula (I) or a salt thereof:

[DL-XY] _n -Ab

[Wherein each “D” independently of one another represents a cytotoxic and/or cytostatic agent (“drug”); Each “L” independently of one another represents a linker; “Ab” refers to an anti-glyco-MUC1 antigen binding domain, such as the anti-glyco-MUC1 antibody or binding fragment described herein; Each “XY” represents the linkage formed between the functional group R ^x on the linker and the “complementary” functional group R ^y on the antibody, where n represents the number of drugs linked to the ADC or the drug-to-antibody ratio (DAR)].

Specific embodiments of the various antibodies (Ab) that can make the ADC include various embodiments of the anti-glyco-MUC1 antibodies and/or binding fragments described above.

In some specific embodiments of the ADCs and/or salts of structure (I), each D is the same and/or each L is the same.

Specific examples of cytotoxic and/or cytostatic agents (D) and linkers (L) that can achieve anti-glyco-MUC1 ADCs of the present disclosure, as well as the number of cytotoxic and/or cytostatic agents linked to ADC In more detail.

5.3.1. Cytotoxic and/or cytostatic agents

The cytotoxic and/or cytostatic agent can be any agent known to inhibit and/or kill the growth and/or replication of cells, particularly cancer and/or tumor cells. A number of agents with cytotoxic and/or cytostatic properties are known in the literature. Non-limiting examples of classes of cytotoxic and/or cytostatic agents include, but are not limited to, radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA intercalating agents (Eg, groove binding agents such as minor groove binding agents), RNA/DNA anti-metabolites, cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondrial inhibitors, and antimitotic agents. .

Specific non-limiting examples within some of these various classes are provided below.

Alkylating agent : asali ((L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-, ethyl ester; NSC 167780; CAS registration number 3577897) ); AZQ ((1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester; NSC 182986; CAS registration number 57998682)); BCNU ((N,N'-bis(2-chloroethyl)-N-nitrosourea; NSC 409962; CAS Registry Number 154938)); Busulfan (1,4-butanediol dimethanesulfonate; NSC 750; CAS registry number 55981); (Carboxyphthalato) Platinum (NSC 27164; CAS Registry Number 65296813); CBDCA ((cis-(1,1-cyclobutanedicarboxylato)diamine platinum(II)); NSC 241240; CAS Registry Number 41575944)); CCNU ((N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; NSC 79037; CAS registry number 13010474)); CHIP (iproplatin; NSC 256927); Chlorambucil (NSC 3088; CAS Registry Number 305033); Chlorozotocin ((2-[[[(2-chloroethyl) nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC 178248; CAS Registry Number 54749905)); Cis-platinum (cisplatin; NSC 119875; CAS registry number 15663271); Clomeson (NSC 338947; CAS registry number 88343720); Cyanomorpholino doxorubicin (NCS 357704; CAS Registry Number 88254073); Cyclodisone (NSC 348948; CAS registry number 99591738); Dianhydrogalactitol (5,6-diepoxydulcitol; NSC 132313; CAS registry number 23261203); Fluorodopan ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil; NSC 73754; CAS Registry Number 834913); Hepsulfan (NSC 329680; CAS Registry Number 96892578 ); Hycantone (NSC 142982; CAS Registry Number 23255938); Melphalan (NSC 8806; CAS Registry Number 3223072); Methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)) -1-Nitrosourea; NSC 95441; 13909096); Mitomycin C (NSC 26980; CAS Registry Number 50077); Mitozolamide (NSC 353451; CAS Registry Number 85622953); Nitrogen Mustard ((Bis(2-chloroethyl) Methylamine hydrochloride; NSC 762; CAS Registry Number 55867); PCNU ((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea; NSC 95466; CAS Registry No. 13909029)); piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; NSC 344007)); piperazinedione (NSC 135758; CAS Registry No. 41109802); piperoman ((N,N-bis(3-bromopropionyl) piperazine; NSC 25154; CAS Registry Number 54911)); porphyromycin (N-methylmitomycin C; NSC 56410 ; CAS Registration No. 801525); Spirohydantoin Mustard (NSC 172112; CAS Registration No. 56605164); Theroxon (triglycidylisocyanurate; NSC 296934; CAS Registration No. 2451629); Tetraplatin (NSC 363812; CAS Registration No. 62816982); thio-tepa (N,N',N"-tri-1,2-ethanediylthio phosphoramide; NSC 6396; CAS registry number 52244); triethylenemelamine (NSC 9706; CAS registry number 51183) ; Uracil nitrogen mustard (desmethyldopan; NSC 34462; CAS registration number 667 51); Yoshi-864 ((bis(3-mesyloxypropyl)amine hydrochloride; NSC 102627; CAS Registry No. 3458228).

Topoisomerase I inhibitors : camptothecin (NSC 94600; CAS registry number 7689-03-4); Various camptothecin derivatives and analogs (e.g., NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028, NSC 176323, NSC 295501, NSC 606172, NSC 606173 , NSC 610458, NSC 618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830, and NSC 606497); Morpholine isoxrubicin (NSC 354646; CAS registration number 89196043); SN-38 (NSC 673596; CAS registration number 86639-52-3).

Topoisomerase II inhibitors : doxorubicin (NSC 123127; CAS registry number 25316409); Amonafide (benzisoquinolinedione; NSC 308847; CAS registry number 69408817); m-AMSA ((4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide; NSC 249992; CAS Registry Number 51264143)); Anthrapyrazole derivatives ((NSC 355644); etoposide (VP-16; NSC 141540; CAS registry number 33419420); pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2 (6H)-propanamine, 9-methoxy-N, N-dimethyl-5-nitro-, monomethanesulfonate; NSC 366140; CAS registry number 99009219); bisanthrene hydrochloride (NSC 337766; CAS registry number 71439684 ); Daunorubicin (NSC 821151; CAS Registry Number 23541506); Deoxydoxorubicin (NSC 267469; CAS Registry Number 63950061); Mitoxantrone (NSC 301739; CAS Registry Number 70476823); Menogril (NSC 269148; CAS Registry Number) 71628961); N,N-dibenzyl daunomycin (NSC 268242; CAS Registry Number 70878512); Oxanthrazol (NSC 349® CAS Registry Number 105118125); Rubidazone (NSC 164011; CAS Registry Number 36508711); Teniposide (VM-26; NSC 122819; CAS registration number 29767202).

DNA intercalating agent : anthramycin (CAS accession number 4803274); Kycamycin A (CAS Registration No. 89675376); Tomeimycin (CAS registration number 35050556); DC-81 (CAS Registration No. 81307246); Siviromycin (CAS registration number 12684332); Pyrrolobenzodiazepine derivatives (CAS Registration 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]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepine-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]diazepine-5(11aH)-one); NSC 694501; CAS registration number 232931576).

RNA/DNA anti-metabolite : L-alanosine (NSC 153353; CAS registration number 59163416); 5-azacytidine (NSC 102816; CAS Registry Number 320672); 5-fluorouracil (NSC 19893; CAS registry number 51218); Acibicin (NSC 163501; CAS registry number 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]L-aspartic acid (NSC 184692); Aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate (NSC 134033); Antipo ((N ^α -(4-amino-4-deoxyphteroyl)-N ⁷ -hemiphthaloyl-L-ornithine; NSC 623017)); Baker Soluble Antipol (NSC 139105; CAS Registry Number 41191042); Dichlorallyl Lawson ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone; NSC 126771; CAS Registry Number 36417160); Brequinar (NSC 368390; CAS Registry Number 96201886 ); ptorurafur ((prodrug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil; NSC 148958; CAS registry number 37076689); 5,6-dihydro-5-azacytidine ( NSC 264880; CAS registry number 62402317); methotrexate (NSC 740; CAS registry number 59052); methotrexate derivatives (N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino] -1-naphthalenyl]carbonyl]L-glutamic acid; NSC 174121); PALA ((N-(phosphonoacetyl)-L-aspartate; NSC 224131; CAS registration number 603425565); pyrazofurine (NSC 143095 ; CAS Registry Number 30868305); Trimetrexate (NSC 352122; CAS Registry Number 82952645).

DNA anti-metabolite : 3-HP (NSC 95678; CAS registration number 3814797); 2'-deoxy-5-fluorouridine (NSC 27640; CAS registry number 50919); 5-HP (NSC 107392; CAS Registry Number 19494894); α-TGDR (α-2′-deoxy-6-thioguanosine; NSC 71851 CAS Registry No. 2133815); Apidicholine glycinate (NSC 303812; CAS registry number 92802822); Ara C (cytosine arabinoside; NSC 63878; CAS registry number 69749); 5-aza-2'-deoxycytidine (NSC 127716; CAS registry number 2353335); β-TGDR (β-2'-deoxy-6-thioguanosine; NSC 71261; CAS registry number 789617); Cyclocytidine (NSC 145668; CAS registry number 10212256); Guanazol (NSC 1895; CAS registry number 1455772); Hydroxyurea (NSC 32065; CAS registry number 127071); Inosine glycodialdehyde (NSC 118994; CAS registry number 23590990); Macvesin II (NSC 330500; CAS registry number 73341738); Pyrazoloimidazole (NSC 51143; CAS registry number 6714290); Thioguanine (NSC 752; CAS registry number 154427); Thiopurine (NSC 755; CAS registration number 50442).

Cell cycle modulator : silivinin (CAS registration number 22888-70-6); Epigallocatechin gallate (EGCG; CAS registry number 989515); Procyanidine derivatives (eg, procyanidine A1 [CAS registry number 103883030], procyanidine B1 [CAS registry number 20315257], procyanidine B4 [CAS registry number 29106512], arecatanin B1 [CAS registry number 79763283]); Isoflavones (eg, genistein [4%5,7-trihydroxyisoflavone; CAS registry number 446720], daidzein [4',7-dihydroxyisoflavone, CAS registration number 486668]; indole- 3-Carbinol (CAS Registry No. 700061); Quercetin (NSC 9219; CAS Registry Number 117395); Estramustine (NSC 89201; CAS Registry Number 2998574); Nocodazole (CAS Registry Number 31430189); Gradophyllotoxin (CAS Registration number 518285); vinorelbine tartrate (NSC 608210; CAS registration number 125317397); cryptophycin (NSC 667642; CAS registration number 124689652).

Kinase inhibitors : afatinib (CAS registration number 850140726); Axitinib (CAS registration number 319460850); ARRY-438162 (Binimetinib) (CAS Registration No. 606143899); Bosutinib (CAS registration number 380843754); Carbozatinib (CAS Registry No. 1140909483); Ceritinib (CAS accession number 1032900256); Crizotinib (CAS accession number 877399525); Dabrafenib (CAS accession number 1195765457); Dasatinib (NSC 732517; CAS Registry Number 302962498); Erlotinib (NSC 718781; CAS registry number 183319699); Everolimus (NSC 733504; CAS Registry Number 159351696); Fostamatinib (NSC 745942; CAS registry number 901119355); Gefitinib (NSC 715055; CAS registry number 184475352); Ibrutinib (CAS Registration No. 936563961); Imatinib (NSC 716051; CAS registry number 220127571); Lapatinib (CAS registration number 388082788); Renbatinib (CAS accession number 857890392); Mumbritinib (CAS 366017096); Nilotinib (CAS accession number 923288953); Nintdanib (CAS registration number 656247175); Palbosiclip (CAS Registration No. 571190302); Pazopanib (NSC 737754; CAS Registry Number 635702646); Pegabtanib (CAS registration number 222716861); Ponatinib (CAS accession number 1114544318); Rapamycin (NSC 226080; CAS registration number 53123889); Regorafenib (CAS registration number 755037037); AP 23573 (Lidaporolimus) (CAS Registration No. 572924540); INCB018424 (Luxolitinib) (CAS Registration No. 1092939177); ARRY-142886 (celumetinib) (NSC 741078; CAS Registry Number 606143-52-6); Sirolimus (NSC 226080; CAS Registry Number 53123889); Sorafenib (NSC 724772; CAS Registry Number 475207591); Sunitinib (NSC 736511; CAS registry number 341031547); Tofacitinib (CAS Registration No. 477600752); Temsirolimus (NSC 683864; CAS registry number 163635043); Trametinib (CAS accession number 871700173); Vandetanib (CAS accession number 443913733); Bemurafenib (CAS accession number 918504651); SU6656 (CAS Registration No. 330161870); CEP-701 (resautinib) (CAS accession number 111358884); XL019 (CAS registration number 945755566); PD-325901 (CAS Registration No. 391210109); PD-98059 (CAS Registration No. 167869218); PI-103 (CAS registration number 371935749), PP242 (CAS registration number 1092351671), PP30 (CAS registration number 1092788094), Thorin 1 (CAS registration number 1222998368), LY294002 (CAS registration number 154447366), XL-147 ( CAS registration number 934526893), CAL-120 (CAS registration number 870281348), ETP-45658 (CAS registration number 1198357797), PX 866 (CAS registration number 502632668), GDC-0941 (CAS registration number 957054307), BGT226 (CAS registration number 1245537681), BEZ235 (CAS registration number 915019657), ATP-competitive TORC1/TORC2 inhibitors including XL-765 (CAS registration number 934493762).

Protein synthesis inhibitors : acriflavin (CAS registration number 65589700); Amikacin (NSC 177001; CAS registry number 39831555); Arbecasin (CAS registration number 51025855); Astromycin (CAS Registration No. 55779061); Azithromycin (NSC 643732; CAS registry number 83905015); Becanamycin (CAS registration number 4696768); Chlortetracycline (NSC 13252; CAS Registry Number 64722); Clarithromycin (NSC 643733; CAS registry number 81103119); Clindamycin (CAS registration number 18323449); Clomocycline (CAS accession number 1181540); Cycloheximide (CAS accession number 66819); Dactinomycin (NSC 3053; CAS Registry Number 50760); Dalphopristin (CAS Registry No. 112362502); Demeclocycline (CAS registration number 127333); Dibecacin (CAS registration number 34493986); Dihydrostreptomycin (CAS accession number 128461); Dilithromycin (CAS accession number 62013041); Doxycycline (CAS registration number 17086281); Emetin (NSC 33669; CAS registry number 483181); Erythromycin (NSC 55929; CAS registry number 114078); Flurithromycin (CAS accession number 83664208); Pramicetin (neomycin B; CAS registry number 119040); Gentamicin (NSC 82261; CAS registration number 1403663); Glycylcycline, such as tigecycline (CAS Registry No. 220620097); Hygromycin B (CAS registration number 31282049); Isepamycin (CAS registration number 67814760); Irradiation mycin (NSC 122223; CAS registration number 16846245); Kanamycin (CAS registration number 8063078); Ketolides such as telithromycin (CAS registry number 191114484), cetromycin (CAS registry number 205110481), and solithromycin (CAS registry number 760981837); Lincomycin (CAS registration number 154212); Limecycline (CAS Registration No. 992212); Meclocycline (NSC 78502; CAS registration number 2013583); Metacycline (Rondomycin; NSC 356463; CAS Registry Number 914001); Medecamycin (CAS registration number 35457808); Minocycline (NSC 141993; CAS registry number 10118908); Myokamycin (CAS registration number 55881077); Neomycin (CAS registration number 119040); Netylmycin (CAS registration number 56391561); Oleandromycin (CAS registration number 3922905); Oxazolidinones such as eperezolide (CAS registry number 165800044), linezolid (CAS registry number 165800033), posizolide (CAS registry number 252260029), radezolide (CAS registry number 869884786), lanbezolide ( CAS Registry Number 392659380), Sutezolide (CAS Registry Number 168828588), Teddyzolide (CAS Registry Number 856867555); Oxytetracycline (NSC 9169; CAS Registry Number 2058460); Paromomycin (CAS registration number 7542372); Penimepiccycline (CAS accession number 4599604); Peptidyl transferase inhibitors such as chloramphenicol (NSC 3069; CAS registry number 56757) and derivatives such as azidamfenicol (CAS registry number 13838089), florfenicol (CAS registry number 73231342), and thiamphenicol (CAS Accession number 15318453), and pleuromutinline such as retafamuline (CAS accession number 224452668), thiamuline (CAS accession number 55297955), valnemullin (CAS accession number 101312929); Pirmycin (CAS accession number 79548735); Puromycin (NSC 3055; CAS Registry Number 53792); Quinupristin (CAS accession number 120138503); Ribostamicin (CAS accession number 53797356); Locomycin (CAS registration number 74014510); Lolitetracycline (CAS registration number 751973); Oxythromycin (CAS accession number 80214831); Cysomycin (CAS registration number 32385118); Spectinomycin (CAS accession number 1695778); Spiramycin (CAS accession number 8025818); Streptograms such as Pristinamycin (CAS Registry No. 270076603), Quinupristin/Dalphopristin (CAS Registry No. 126602899), and Virginiamycin (CAS Registry No. 11006761); Streptomycin (CAS accession number 57921); Tetracycline (NSC 108579; CAS Registry Number 60548); Tobramycin (CAS accession number 32986564); Troledomomycin (CAS registration number 2751099); Tyrosine (CAS registration number 1401690); Verdamycin (CAS registration number 49863481).

Histone deacetylase inhibitor : abecinostat (CAS registration number 783355602); Bellinostat (NSC 726630; CAS registration number 414864009); Kidamid (CAS Registration No. 743420022); Entinostat (CAS registration number 209783802); Ginostat (CAS registration number 732302997); Captinostat (CAS registration number 726169739); Panobinostat (CAS registration number 404950807); Quicinostat (CAS registration number 875320299); Resminostat (CAS registration number 864814880); Romidepsin (CAS accession number 128517077); Sulfolapan (CAS registration number 4478937); Thioureidobutyronitrile (Kevetrin™; CAS registry number 6659890); Valproic acid (NSC 93819; CAS registry number 99661); Vorinostat (NSC 701852; CAS registration number 149647789); ACY-1215 (Rosilinostat; CAS Registry Number 1316214524); CUDC-101 (CAS Registration No. 1012054599); CHR-2845 (Tefinostat; CAS Registry Number 914382608); CHR-3996 (CAS Registration No. 1235859138); 4SC-202 (CAS Registration No. 910462430); CG200745 (CAS Registration No. 936221339); SB939 (prasinostat; CAS registration number 929016966).

Mitochondrial inhibitors : Pancratisstatin (NSC 349156; CAS Registry Number 96281311); Rhodamine-123 (CAS registration number 63669709); Edelfosine (NSC 324368; CAS Registry Number 70641519); d-alpha-tocopherol succinate (NSC 173849; CAS registry number 4345033); Compound 11β (CAS Registry No. 865070377); Aspirin (NSC 406186; CAS registry number 50782); Ellipticine (CAS registration number 519233); Berberine (CAS registration number 633658); Cerulenin (CAS registration number 17397896); GX015-070 (Obatoclax®; 1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrole-2-yl)methylene)-3-methoxy-2H-pyrrole-5 -Day)-; NSC 729280; CAS registration number 803712676); Cellastrol (trypterin; CAS registry number 34157830); Metformin (NSC 91485; CAS registry number 1115704); Brilliant green (NSC 5011; CAS registry number 633034); ME-344 (CAS registration number 1374524556).

Antimitotic agent : allocolchicine (NSC 406042); Auristatins such as MMAE (monomethyl auristatin E; CAS registry number 474645-27-7) and MMAF (monomethyl auristatin F; CAS registry number 745017-94-1; Haricondrin B (NSC 609395); colchicine (NSC 757; CAS Registry Number 64868); Colchicine Derivatives (N-benzoyl-deacetyl benzamide; NSC 33410; CAS Registry Number 63989753); Dolastatin 10 (NSC 376128; CAS Registry Number 110417-88-4); May Tansin (NSC 153858; CAS Registry Number 35846-53-8); Lorizine (NSC 332598; CAS Registry Number 90996546); Taxol (NSC 125973; CAS Registry Number 33069624); Taxol Derivative ((2'-N-[3- (Dimethylamino)propyl]glutaramate taxol; NSC 608832); thiocolchicine (3-demethylthiocolchicine; NSC 361792); trityl cysteine (NSC 49842; CAS registry number 2799077); vinblastine sulfate ( NSC 49842; CAS Registry Number 143679); Vincristine Sulfate (NSC 67574; CAS Registry Number 2068782).

Any such agent that can include or be modified to include an attachment site for an antibody can be included in the ADCs disclosed herein.

In specific embodiments, the cytotoxic and/or cytostatic agent is an anti-mitotic agent.

In another specific embodiment, the cytotoxic and/or cytostatic agent is auristatin, eg, monomethyl auristatin E (“MMAE”) or monomethyl auristatin F (“MMAF”).

5.3.2. Linker

In the anti-glyco-MUC1 ADCs of the present disclosure, cytotoxic and/or cytostatic agents are linked to antibodies by linkers. The linkers linking the cytotoxic and/or cytostatic agent to the antibodies of the ADC can be short, long, hydrophobic, hydrophilic, flexible or rigid, or each independently such that the linker comprises segments with different properties. It can be composed of segments having one or more of the above-mentioned properties. The linker can be multivalent to covalently link more than one agent to a single site on the antibody, or can be monovalent to covalently link a single agent to a single site on the antibody.

As will be understood by one of ordinary skill in the art, a linker can bind a cytotoxic and/or cytostatic agent to an antibody by forming a covalent linkage to a cytotoxic and/or cytostatic agent at one location and a covalent linkage to an antibody at another location. Connect. Covalent linkages are formed by reactions between functional groups on the linker and functional groups on the agent and antibody. As used herein, the expression "linker" includes (i) an unconjugated form comprising a functional group capable of covalently linking a linker to a cytotoxic and/or cytostatic agent and a functional group capable of covalently linking a linker to an antibody. Linker; (ii) a linker in a partially conjugated form comprising a functional group capable of covalently linking a linker to an antibody, and covalently linked to a cytotoxic and/or cytostatic agent, or vice versa; And (iii) a cytotoxic and/or cytostatic agent and a fully conjugated linker in covalent linkage to both antibodies. In some specific embodiments of the linkers and anti-glyco-MUC1 ADCs of the present disclosure, as well as the syntons used to conjugate the linker-agonist to the antibody, a moiety comprising functional groups on the linker and a covalent linkage formed between the linker and the antibody These are represented by R _x and XY, respectively.

The linker is preferably chemically stable in an environment outside the cell, but is not necessarily, can be designed to be cleaved inside the cell and/or destroyed and/or specifically degraded in other ways. Alternatively, linkers not specifically designed to be cleaved or degraded inside the cell can be used. The choice of a stable versus unstable linker may depend on the cytotoxicity and/or the cytotoxic agent's toxicity. For agents that are toxic to normal cells, a stable linker is preferred. Selective or targeted, agents with low toxicity to normal cells can be used, and the chemical stability of the linker to the extracellular environment is less important. A wide range of linkers useful in linking drugs to antibodies in the context of ADCs are known in the art. Any of these linkers, as well as other linkers, can be used to link cytotoxic and/or cytostatic agents to the antibodies of the anti-glyco-MUC1 ADCs of the present disclosure.

Exemplary multivalent linkers that can be used to link multiple cytotoxic and/or cytostatic agents to a single antibody molecule include, for example, 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; 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 enable high-DAR ADCs with excellent physicochemical properties. As shown below, Musana technology is based on the incorporation of drug molecules into a solubilizing poly-acetal backbone through the sequence of ester linkages. This method provides a highly loaded ADC (DAR up to 20) while maintaining good physicochemical properties.

Additional examples of dendritic type linkers are described in US 2006/116422; US 2005/271615; De Groot 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.

Exemplary monovalent linkers that can be used include, for example, Nolting, 2013, Antibody-Drug Conjugates, 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 No. 7,223,837; U.S. Patent No. 8,568,728; U.S. Patent No. 8,535,678; And WO2004010957, each of which is incorporated herein by reference.

For example, and without limitation, some cleavable linkers and non-cleavable linkers that may be included in the anti-glyco-MUC1 ADCs of the present disclosure are described below.

5.3.3. Cleavable linker

In certain embodiments, the selected linker is cleavable in vivo. Cleavable linkers can include chemically or enzymatically unstable or degradable linkages. Generally, cleavable linkers rely on intracellular processes to release the drug, such as reduction in the cytoplasm, exposure to an acidic environment within the lysosome, or cleavage by specific proteases or other enzymes inside the cell. Cleavable linkers incorporate one or more chemical bonds that can be cleaved chemically or enzymatically, while the rest of the linker is not cleavable. In certain embodiments, the linker comprises chemically labile groups such as hydrazone and/or disulfide groups. Linkers that contain chemically labile groups utilize differential properties between plasma and some cellular compartments. In the case of a hydrazone-containing linker, the intracellular conditions that facilitate drug release are the acidic environments of endosomes and lysosomes, while the disulfide-containing linkers are reduced in cytosols containing high concentrations of thiols, such as glutathione. In certain embodiments, plasma stability of a linker comprising a chemically labile group can be increased by introducing a steric hindrance using a substituent near the chemically labile group.

Acid-labile groups, such as hydrazone, remain intact in the blood's neutral pH environment (pH 7.3-7.5) during systemic circulation and endosomes (pH 5.0-6.5) and lysosomes of cells in which the ADC is moderately acidic ( pH 4.5-5.0) When internalized into the compartment, it undergoes hydrolysis and releases the drug. This pH dependent release mechanism has been associated with non-specific release of drugs. To increase the stability of the linker's hydrazone groups, the linker can be altered by chemical modification, e.g., substitution, which allows adjustments to achieve more efficient release in the lysosome while minimizing loss in circulation. do.

Hydrazone-containing linkers may contain additional cleavable sites, such as additional acid-labile cleavable sites and/or enzymatically labile cleavable sites. ADCs comprising exemplary hydrazone-containing linkers include the following structures:

[Wherein D and Ab represent cytotoxic and/or cytostatic agent (drug) and Ab, respectively, n represents the number of drug-linkers linked to the antibody]. In certain linkers, such as the linker (Ig), the linker comprises two cleavable groups, disulfide and hydrazone moieties. For such linkers, effective release of the unmodified free drug requires acidic pH or disulfide reduction and acidic pH. Linkers such as (Ih) and (Ii) have been shown to be effective as single hydrazone cleavage sites.

Additional linkers that remain intact during systemic circulation, undergo hydrolysis and release the drug when the ADC is internalized into the acidic cell compartment, include carbonate. Such linkers can be useful when cytotoxic and/or cytostatic agents can be covalently attached via oxygen.

Other acid-labile groups that may be included in the linker include cis-aconitil-containing linkers. Cis-aconitil chemistry utilizes carboxylic acids juxtaposed to amide bonds to accelerate amide hydrolysis under an acidic environment.

Cleavable linkers may also include disulfide groups. Disulfide is thermodynamically stable at physiological pH and is designed to release the drug upon internalization within the cell, where the cytosolic fluid provides a significantly more reducible environment compared to the extracellular environment. The disulfide bond generally requires the presence of a cytosolic thiol cofactor, such as (reduced) glutathione (GSH), so that the disulfide-containing linker is reasonably stable in circulation, thereby selectively releasing the drug from the cytosol. do. Intracellular enzyme protein disulfide isomerase, or a similar enzyme capable of cleaving disulfide bonds, may also contribute to preferential cleavage of disulfide bonds inside cells. Compared to a significantly lower concentration of GSH or cysteine (the most abundant low molecular weight thiol) in a cycle of about 5, GSH is reported to be present in cells in a concentration range of 0.5-10 mM. Tumor cells whose irregular blood flow reaches hypoxia result in enhanced activity of the reductase, and thus higher glutathione concentration. In certain embodiments, the in vivo stability of the disulfide-containing linker can be enhanced by chemical modification of the linker, eg, the use of a steric hindrance adjacent to the disulfide bond.

ADCs comprising exemplary disulfide-containing linkers include the following structures:

[Wherein, D and Ab each represents a drug and an antibody, n represents the number of drug-linkers linked to the antibody, and R in each case is independently selected from, for example, hydrogen or alkyl]. In certain embodiments, increasing the steric hindrance adjacent to the disulfide bond increases the stability of the linker. Structures such as (Ij) and (Il) exhibit increased in vivo stability when one or more R groups are selected from lower alkyl such as methyl.

Another type of cleavable linker that can be used is a linker that is specifically cleaved by an enzyme. Such linkers are typically peptide-based or contain peptide regions that serve as substrates for enzymes. Peptide based linkers tend to be more stable in plasma and extracellular environments than chemically unstable linkers. Since lysosomal proteolytic enzymes have very low activity in the blood due to the unfavorably high pH value of blood compared to endogenous inhibitors and lysosomes, peptide binding is generally excellent in plasma stability. Drug release from antibodies occurs specifically due to the action of lysosomal proteases, such as cathepsin and plasmin. These proteases are present at elevated levels in certain tumor cells.

In exemplary embodiments, the cleavable peptide may be a tetrapeptide such as Gly-Phe-Leu-Gly (SEQ ID NO: 128), Ala-Leu-Ala-Leu (SEQ ID NO: 129) or a dipeptide 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)Asp 5, 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 certain embodiments, dipeptides are preferred over longer polypeptides due to the hydrophobicity of the longer peptides.

Various dipeptide-based cleavable linkers have been described that are useful for linking drugs such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepine, thalimycin, and auristatin/orstatin family members to antibodies (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., 2003, Blood 102:1458-1465, each of which is incorporated herein by reference). Both such dipeptide linkers, or modified versions of these dipeptide linkers, can be used in the anti-glyco-MUC1 ADCs of the present disclosure. Other dipeptide linkers that can be used are Brentuximab Vendotin SGN-35 from Seattle Genetics (Adcetris™), Seattle Genetics SGN-75 (anti-CD-70) , Val-Cit-monomethyl auristatin F (MMAF), Seattle Genetics SGN-CD33A (anti-CD-33, Val-Ala-(SGD-1882)), Celldex Therapeutics Glembatumab ( CDX-011) (anti-NMB, Val-Cit-monomethyl auristatin E (MMAE), and cytogen PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE) Includes those found in the same ADC.

Enzymatically cleavable linkers can include self-destructive spacers to spatially separate the drug from the enzyme cleavage site. The direct attachment of a drug to a peptide linker can result in a proteolytic release of the amino acid adduct of the drug, thereby impairing its activity. The use of self-destructive spacers allows for the removal of chemically unmodified drugs that are fully active upon amide bond hydrolysis.

One self-destructive spacer is a bifunctional para-aminobenzyl alcohol group, which is linked to the peptide via an amino group to form an amide bond, while the amine-containing drug is attached to the linker's benzyl hydroxyl group via carbamate functionality Can be (PABC). The resulting prodrug is activated upon protease-mediated cleavage, leading to a 1,6-elimination reaction that releases the remainder of the unmodified drug, carbon dioxide and linker groups. The following scheme depicts fragmentation and drug release of p-amidobenzyl ether:

[Wherein, X-D represents an unmodified drug].

Heterocyclic variants of these self-destructive groups have also been described. See, for example, US Pat. No. 7,989,434, incorporated herein by reference.

In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. Easy drug release can be realized through cleavage of the β-glucuronide glycoside bond by the lysosomal enzyme β-glucuronidase. While these enzymes are abundant in lysosomes and are overexpressed in some tumor types, the enzyme activity outside the cell is low. β-glucuronic acid-based linkers can be used to bypass the tendency of the ADC to undergo aggregation due to the hydrophilic nature of β-glucuronide. In some embodiments, β-glucuronic acid-based linkers are preferred as linkers to ADCs linked to hydrophobic drugs. The following scheme depicts drug release from ADCs containing β-glucuronic acid-based linkers:

Various cleavable β-glucuronic acid-based linkers useful for linking drugs such as auristatin, camptothecin and doxorubicin analogs, CBI minor-groove binders, and cymberins have been described (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., 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-glyco-MUC1 ADCs of the present disclosure.

Additionally, cytotoxic and/or cytostatic agents containing phenolic groups can be covalently linked to the linker via phenolic oxygen. One such linker described in WO 2007/089149 relies on how diamino-ethane "SpaceLink" is used in conjunction with traditional "PABO"-based self-destructive groups to deliver phenol. Cleavage of the linker is schematically shown below, where D represents a cytotoxic and/or cytostatic agent with a phenolic hydroxyl group.

A cleavable linker can include a non-cleavable portion or segment, and/or a cleavable segment or portion can be included in a non-cleavable linker to make it cleavable if different. By way of example only, polyethylene glycol (PEG) and related polymers can include cleavable groups within the polymer backbone. For example, a polyethylene glycol or polymer linker can include one or more cleavable groups such as disulfide, hydrazone or dipeptide.

Other degradable bonds that may be included in the linker are ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on biologically active agents, where such ester groups are generally under physiological conditions. Hydrolyzes and releases agents that are biologically active. The linkages that can be broken down by hydrolysis include carbonate linkages; Imine linkages resulting from the reaction of amines and aldehydes; Phosphate ester linkages formed by reacting alcohol and phosphate groups; Acetal linkages, which are reaction products of aldehydes and alcohols; Orthoester linkage which is the reaction product of formate and alcohol; And oligonucleotide linkages formed by phosphoramidite groups and 5'hydroxyl groups of oligonucleotides, including but not limited to those at the ends of the polymer.

In certain embodiments, a linker, e.g., a linker comprising structural formula (IVa) or (IVb), or a salt thereof, comprises an enzymatically cleavable peptide moiety:

는 링커가 세포독성 및/또는 세포정지성 작용제에 부착되는 지점을 나타내며; *는 나머지 링커의 부착 지점을 나타낸다].[Wherein, the peptide refers to a peptide that is cleavable by a lysosomal enzyme (illustrated as C→N, not showing carboxy and amino “terminals”); T represents a polymer or alkylene chain comprising one or more ethylene glycol units, or a combination thereof; R ^a is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

Represents the point at which the linker is attached to a cytotoxic and/or cytostatic agent; * Indicates the point of attachment of the remaining linkers].

In certain embodiments, the peptide is selected from tripeptides or dipeptides. In certain embodiments, the dipeptide is 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 certain embodiments, the dipeptide is Cit-Val; And Ala-Val.

Specific exemplary embodiments of the linker according to structural formula (IVa) that can be included in the anti-glyco-MUC1 ADC of the present disclosure include a linker illustrated below (as illustrated, the linker covalently links the linker to the antibody) Includes groups suitable for making):

Specific exemplary embodiments of a linker according to structural formula (IVb) that can be included in the anti-glyco-MUC1 ADC of the present disclosure include a linker illustrated below (as illustrated, the linker covalently links the linker to the antibody) Includes groups suitable for making):

In certain embodiments, a linker, e.g., a linker comprising structural formula (IVc) or (IVd), or a salt thereof, comprises an enzymatically cleavable peptide moiety:

는 링커가 세포독성 및/또는 세포정지성 작용제에 부착되는 지점을 나타내며; *는 나머지 링커의 부착 지점을 나타낸다].[Wherein, the peptide refers to a peptide that is cleavable by a lysosomal enzyme (illustrated as C→N, not showing carboxy and amino “terminals”); T represents a polymer or alkylene chain comprising one or more ethylene glycol units, or a combination thereof; R ^a is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

Represents the point at which the linker is attached to a cytotoxic and/or cytostatic agent; * Indicates the point of attachment of the remaining linkers].

Specific exemplary embodiments of the linker according to structural formula (IVc) that can be included in the anti-glyco-MUC1 ADC of the present disclosure include a linker illustrated below (as illustrated, the linker covalently links the linker to the antibody) Includes groups suitable for making):

Specific exemplary embodiments of the linker according to structural formula (IVd) that can be included in the anti-glyco-MUC1 ADC of the present disclosure include a linker illustrated below (as illustrated, the linker covalently links the linker to the antibody) Includes groups suitable for making):

In certain embodiments, linkers comprising structural formulas (IVa), (IVb), (IVc), or (IVd) further comprise a carbonate moiety that is cleavable by exposure to an acidic medium. In certain embodiments, the linker is attached to the cytotoxic and/or cytostatic agent via oxygen.

5.3.4. Non-cleavable linker

Although a cleavable linker may provide certain advantages, the linker constituting the anti-glyco-MUC1 ADC of the present disclosure need not be cleavable. In the case of non-cleavable linkers, drug release is not dependent on the differential nature between plasma and some cytoplasmic compartments. It is hypothesized that drug release will occur after internalization of the ADC through antigen-mediated endocytosis and delivery to the lysosome compartment, where the antibody is degraded to the amino acid level through intracellular proteolytic degradation. This process releases the drug derivative, which is formed by the drug, linker, and amino acid residues to which the linker is covalently attached. Compared to a conjugate with a cleavable linker, the amino acid drug metabolite from the conjugate with a non-cleavable linker is more hydrophilic, generally less membrane permeable, which has less bystander effect and less non-specificity. It leads to toxicity. In general, ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleavable linkers. The non-cleavable linker may be an alkylene chain, or may be polymeric in nature, for example based on a polyalkylene glycol polymer, an amide polymer, or an alkylene chain, polyalkylene glycol And/or segments of amide polymers.

Various non-cleavable linkers used to link 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 can be included in the anti-glyco-MUC1 ADC of the present disclosure.

In certain embodiments, a linker, e.g., a linker according to the structural formulas (VIa), (VIb), (VIc) or (VId) or salts thereof is not cleavable in vivo (as illustrated, the linker is a linker Include groups suitable for covalent linkage to antibodies):

는 링커가 세포독성 및/또는 세포정지성 작용제에 부착되는 지점을 나타낸다].[Wherein R ^a is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; R ^x is a moiety containing a functional group capable of covalently linking the linker to the antibody;

Indicates the point at which the linker is attached to the cytotoxic and/or cytostatic agent].

는 세포독성 및/또는 세포정지성 작용제에 부착되는 지점을 나타낸다):Specific exemplary embodiments of linkers according to structural formulas (VIa)-(VId) that may be included in the anti-glyco-MUC1 ADCs of the present disclosure include linkers illustrated below (as illustrated, the linkers linker Includes groups suitable for covalent linkage to antibodies,

Indicates the point of attachment to the cytotoxic and/or cytostatic agent):

5.3.5. Group used to attach linker to antibody

Various groups can be used to yield ADCs by attaching linker-drug synthons to the antibody. The attachment groups can be electrophilic in nature and include maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, haloformates, acid halides, alkyl benzyl halides such as haloacetamide. As discussed below, technologies related to “self-stabilizing” maleated and “crosslinked disulfide” that can be used in accordance with the present disclosure are also emerging. The specific group used will depend, in part, on the site attached to the antibody.

An example of a “self-stabilizing” maleimide group that spontaneously hydrolyzes under antibody conjugation conditions to provide an ADC species with improved stability is shown in the scheme below. See US20130309256 A1; See also Lyon et al., Nature Biotech published online, doi:10.1038/nbt.2968.

Polytherics has disclosed a method of crosslinking a pair of sulfhydryl groups derived from reduction of natural hinge disulfide bonds. Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. The reaction is shown in the scheme below. The advantage of this method is the ability to synthesize concentrated DAR4 ADCs by reaction with 4 equivalents of alkylating agent followed by complete reduction of IgG (which provides 4 pairs of sulfhydryls). ADCs containing crosslinked “disulfide” are also claimed to have increased stability.

Similarly, as shown below, maleimide derivatives (1, below) that can crosslink a pair of sulfhydryl groups have been developed. See WO2013/085925.

5.3.6. Linker selection considerations

As is known to those skilled in the art, a linker selected for a particular ADC can be used to determine the site of attachment to the antibody (e.g., lys, cys or other amino acid residues), the structural constraints of the drug's pharmacological group and the drug's affinity. It can be influenced by a variety of factors, including but not limited to. The specific linker chosen for the ADC should seek to balance these different factors for specific antibody/drug combinations. For a review of factors influenced by linker selection in ADCs, see Nutting, 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.

For example, it has been observed that ADCs result in the killing of bystander antigen-negative cells present adjacent to antigen-positive tumor cells. The mechanism of bystander cell death by ADC has shown that metabolic products formed during the intracellular processing of ADC can play a role. Neutral cytotoxic metabolites produced by metabolism of ADCs in antigen-positive cells appear to play a role in bystander cell killing, while charged metabolites can be prevented from spreading across the membrane into the medium and thus bystander killing Can not affect. In certain embodiments, the linker is selected to attenuate the bystander killing effect caused by the cellular metabolites of the ADC. In certain embodiments, the linker is selected to increase bystander killing effects.

The nature of the linker can also affect the aggregation of the ADC under use and/or storage conditions. Typically, ADCs reported in the literature contain 3-4 or fewer drug molecules per antibody molecule (see, eg, Chari, 2008, Acc Chem Res 41:98-107). Attempts to obtain higher drug-to-antibody ratios (“DARs”) often failed due to aggregation of ADCs, especially when both drug and linker were hydrophilic (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, DARs higher than 3-4 can be beneficial as a means to increase efficacy. If the cytotoxic and/or cytostatic agent is hydrophobic in nature, it may be desirable to select a relatively hydrophilic linker as a means of reducing ADC aggregation, especially if DAR in excess of 3-4 is desired. . Thus, in certain embodiments, chemical moieties that reduce aggregation of the ADC during storage and/or use are incorporated into the linker. Polar or hydrophilic groups such as charged groups or groups charged under physiological pH can be incorporated into the linker to reduce aggregation of the ADC. For example, charged groups such as salts or groups that deprotonate at physiological pH, such as carboxylates, or protonated salts or groups, such as amines, can be incorporated into the linker.

Exemplary multivalent linkers reported to yield DARs as high as 20 that can be used to link multiple cytotoxic and/or cytostatic agents 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; WO 2014/093640, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, the aggregation of ADCs during storage or use is less than about 10% as determined by size-exclusion chromatography (SEC). In certain embodiments, aggregation of the ADC during storage or use is less than 10%, such as less than about 5%, less than about 4%, less than about 3%, less than about 2% as determined by size-exclusion chromatography (SEC) , Less than about 1%, less than about 0.5%, less than about 0.1%, or even lower.

5.3.7. Method of making anti-glyco-MUC1 ADC

The anti-glyco-MUC1 ADCs of the present disclosure can be synthesized using well-known chemistry. The chemistry chosen will depend, inter alia, on the identity of the cytotoxic and/or cytostatic agent(s), linker, and groups used to attach the linker to the antibody. In general, ADCs according to formula (I) can be prepared according to the following scheme:

DLR ^x +Ab-R ^y →[DL-XY] _n -Ab (I)

[Wherein, D, L, Ab, XY and n are as previously defined, and R ^x and R ^y represent complementary groups capable of forming a covalent link with each other as discussed above].

The identity of the R ^x and R ^y groups will depend on the chemistry used to link the Sinton DLR ^x to the antibody. In general, the chemistry used should not alter the integrity of the antibody, eg its ability to bind its target. Preferably, the binding properties of the conjugated antibody will be very similar to that of the unconjugated antibody. Various chemistries and techniques for conjugating molecules to biological molecules, such as antibodies, are known in the art and are particularly well known for antibodies. See, eg, 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 Cancer Detection And Therapy, Baldwin et al. , Eds., Academic Press, 1985]; [Thorpe et al. , 1982, Immunol. Rev. 62:119-58]; See PCT Publication WO 89/12624. Any of these chemistries can be used to link syntones to antibodies.

A number of functional groups R ^x and chemistries useful for linking syntones to accessible lysine residues are known and include, for example and without limitation NHS-esters and isothiocyanates.

A number of functional groups R ^x and chemistry that are useful for linking syntones to accessible free sulfhydryl groups of cysteine residues are known and include, for example and without limitation haloacetyl and maleimide.

However, the conjugation chemistry is not limited to the side chain groups available. By linking suitable small molecules to the amine, side chains such as amines can be converted to other useful groups, such as hydroxyl. Using this strategy, the number of available linkage sites on the antibody can be increased by conjugating the multifunctional small molecule to the side chain of an accessible amino acid residue of the antibody. Then, a suitable functional group R ^x for covalently linking the syntone to this "converted" functional group is included in the syntone.

Antibodies can also be engineered to include amino acid residues for conjugation. Approaches for engineering antibodies to include genetically unencoded amino acid residues useful for conjugating drugs in the context of ADCs are described in Axup et al., 2012, Proc Natl Acad Sci USA. 109(40):16101-16106, as well as chemical and functional groups useful for linking syntones to uncoding amino acids.

Typically, the synton is linked to the side chain of the amino acid residue of the antibody, for example comprising the primary amino group of the accessible lysine residue or the sulfhydryl group of the accessible cysteine residue. Free sulfhydryl groups can be obtained by reducing interchain disulfide bonds.

In the case of a linkage where R ^y is a sulfhydryl group (eg, when R ^x is maleimide), generally the antibody is first fully or partially reduced to break the interchain disulfide bridge between cysteine residues.

Cysteine residues that do not participate in the disulfide bridge can be engineered into the antibody by mutation of one or more codons. Reduction of this unpaired cysteine yields sulfhydryl groups suitable for conjugation. Preferred positions for incorporating engineered cysteines include, for example and without limitation, positions S112C, S113C, A114C, S115C, A176C, 5180C, S252C, V286C, V292C, S357C, A359C, S398C, on human IgG ₁ heavy chain. S428C (Kabat numbering) and positions on human Ig kappa light chains V110C, S114C, S121C, S127C, S168C, V205C (Kabat numbering) (e.g., U.S. Pat.No. 7,521,541, U.S. Pat.No. 7,855,275 and U.S. Pat. See Patent No. 8,455,622).

As will be apparent to those skilled in the art, the number of cytotoxic and/or cytostatic agents linked to the antibody molecule may be different, such that the selection of ADCs may be heterogeneous in nature where some antibodies are one and some antibodies are two. , Some antibodies contain three or more linked agents (some do not). The degree of heterogeneity will depend, inter alia, on the chemistry used to link cytotoxic and/or cytostatic agents. For example, when the antibody is reduced to yield sulfhydryl groups for attachment, heterogeneous mixtures of antibodies linked to 0, 2, 4, 6 or 8 agents per molecule are often produced. In addition, by limiting the molar ratio of the adhering compound, antibodies with 0, 1, 2, 3, 4, 5, 6, 7 or 8 agonists per molecule are often produced. Thus, it will be understood that, depending on the context, the DARs mentioned may be averages for antibody selection. For example, “DAR4” has not been applied to purification to isolate specific DAR peaks, and different numbers of cytostatic and/or cytotoxic agents per antibody (eg, 0, 2, 4, 6, 8 per antibody) Dog agonists) may be attached, but may refer to ADC preparations with an average drug-to-antibody ratio of 4. Similarly, in some embodiments, “DAR2” refers to a heterogeneous ADC formulation with an average drug-to-antibody ratio of 2.

If a concentrated preparation is desired, a limited number of cytotoxic and/or cytostatic agents linked antibodies can be obtained via purification of a heterogeneous mixture, for example, column chromatography, e.g., hydrophobic interaction chromatography. Can be.

Purity can be assessed by a variety of methods as known in the art. As a specific example, ADC formulations can be analyzed by HPLC or other chromatography, and purity can be assessed by analyzing the area under the curve of the resulting peak.

5.4 chimeric antigen receptor

The present disclosure provides a chimeric antigen receptor (CAR) comprising an anti-glyco-MUC1 antibody or antigen-binding fragment described herein.

The CAR of the present disclosure typically comprises an extracellular domain operably linked to a transmembrane domain, in turn the transmembrane domain is operably linked to an intracellular domain for signaling.

The extracellular domain of the CAR of the present disclosure comprises the sequence of an anti-glyco-MUC1 antibody or antigen-binding fragment (eg, as described in Section 5.1 or embodiments 1 to 90).

Exemplary transmembrane domain sequences and intracellular domain sequences are described in Sections 5.4.1 and 5.4.2, respectively.

Several fusion proteins described herein (eg, embodiments 92 and 94-96) are CARs, and CAR-related disclosures apply to these fusion proteins.

5.4.1. Transmembrane domain

With respect to the transmembrane domain, the CAR can be designed to include a transmembrane domain (eg, fused) operably linked to the extracellular domain of the CAR.

The transmembrane domain can be derived from natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present disclosure include alpha, beta or zeta chains of T-cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, CD154 (ie, may include at least its transmembrane region(s)). In some cases, various human hinges can also be used, including the human Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain is synthetic (ie, non-naturally occurring). Examples of synthetic transmembrane domains are peptides containing predominantly hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2 to 10 amino acids in length, can form a link between the transmembrane domain of the CAR and the cytoplasmic signaling domain. Glycine-serine doublets provide particularly suitable linkers.

In one embodiment, the transmembrane domain in the CAR of the present disclosure is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence YLHLGALGRDLWGPSPVTGYHPLL.

In one embodiment, the transmembrane domain in the CAR of the present disclosure is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV.

In some cases, the transmembrane domain in the CAR of the present disclosure comprises a CD8a hinge domain. In one embodiment, the CD8a hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC.

5.4.2. Intracellular domain

The intracellular signaling domain of the CAR of the present disclosure is responsible for activation of at least one of the normal effector functions of immune cells in which the CAR is expressed. The term “effector function” refers to a cell's special function. The effector function of T cells can be, for example, cytolytic activity or helper activity including cytokine secretion. Thus, the term “intracellular signaling domain” refers to the portion of a protein that carries effector function signals and directs cells to perform special functions. Generally, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. When the truncated portion of the intracellular signaling domain is used, it can be used instead of the intact chain as long as this truncated portion carries an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to deliver effector function signals.

Preferred examples of intracellular signaling domains for use in CARs of the present disclosure include the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that work cooperatively to initiate signal transduction after antigen receptor engagement, as well as of these sequences. Any derivative or variant, and any synthetic sequence having the same functional capacity.

Signals generated through TCR alone may be insufficient for full activation of T cells, and secondary or co-stimulation signals are also required. Thus, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: initiating antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequence) and secondary or co- -Acting in an antigen-dependent manner to provide a stimulatory signal (secondary cytoplasmic signaling sequence).

The primary cytoplasmic signaling sequence modulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain an immunoreceptor tyrosine-based activation motif or a signaling motif known as ITAM.

Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly useful in CARs of the present disclosure are from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Includes what is derived. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the present disclosure comprises a cytoplasmic signaling sequence from CD3-zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR is a primary cytoplasmic signaling sequence domain (e.g., of CD3-zeta) independently or any other preferred cytoplasmic domain(s) useful in the context of the CAR of the present disclosure. It is designed to contain ITAM containing in combination with. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region.

The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of the costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigenic receptor or a ligand thereof required for efficient response of lymphocytes to an antigen. Examples of such molecules are 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 a ligand specifically binding to CD83.

Cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present disclosure can be linked to each other randomly or in a specific order. Optionally, a short oligo- or polypeptide linker, preferably 2 to 10 amino acids in length, can form a link. Glycine-serine doublets provide particularly suitable linkers.

In one embodiment, the cytoplasmic domain comprises the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain comprises the signaling domain of CD3-zeta and the signaling domain of 4-1BB.

5.5 nucleic acids, recombinant vectors and host cells

The present disclosure includes nucleic acid molecules encoding immunoglobulin light and heavy chain genes for anti-glyco-MUC1 antibodies, vectors comprising such nucleic acids, and host cells capable of producing anti-glyco-MUC1 antibodies of the present disclosure. do. In certain aspects, the nucleic acid molecule is an anti-glyco-MUC1 antibody and antibody-binding fragment of the present disclosure (e.g., as described in Section 5.1 and embodiments 1 to 90), as well as fusion proteins containing it ( For example, as described in embodiments 91-96) and chimeric antigen receptors (e.g. as described in section 5.4 and embodiments 97-98), the host cell can express it. . Exemplary vectors of the present disclosure are described in embodiments 111-113, and exemplary host cells are described in embodiments 114-117.

Anti-glyco-MUC1 antibodies of the present disclosure can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To recombinantly express the antibody, the host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the antibody's immunoglobulin light and heavy chains, such that the light and heavy chains are expressed in the host cell, optionally The host cell is secreted into the cultured medium, and the antibody can be recovered from the medium. Standard recombinant DNA methods such as 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 using those described in US Pat. No. 4,816,397 to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells.

To generate nucleic acids encoding such anti-glyco-MUC1 antibodies, DNA fragments encoding light and heavy chain variable regions are first obtained. Such DNA can be obtained by amplification and modification of germline DNA or cDNA encoding light and heavy chain variable sequences, for example, using polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see, for example, the "VBASE" human germline sequence database, Kabat et al., 1991, Sequences of Proteins of Immunological Interest) , Fifth Edition, US Department 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).

Once DNA fragments encoding anti-glyco-MUC1 antibody-related V _H and V _L segments have been obtained, such DNA fragments can be converted, for example, to convert the variable region gene into a full-length antibody chain gene, Fab fragment gene or scFv gene. It can be further manipulated by standard recombinant DNA techniques. In this manipulation, the V _H -or V _L -encoding DNA fragment is operably linked to another protein, such as an antibody constant region or another DNA fragment encoding a flexible linker. The term “operably linked” as used in this context is intended to mean that the two DNA fragments are joined such that the amino acid sequence encoded by the two DNA fragments remains in-frame.

V _H - heavy chain constant region coding _{DNA (CH 1, CH 2,} CH 3 and optionally the CH ₄₎ the isolated encoding the V _H region by operatively connected to another DNA molecule encoding DNA is full-length heavy chain gene Can be converted to The sequence of human heavy chain constant region genes is known in the art (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA, IgE, IgM or IgD constant region, but in certain embodiments is an IgG ₁ or IgG ₄ constant region. In the case of the Fab fragment heavy chain gene, the V _H -encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH ₁ constant region.

V _L - is by operatively connecting the encoding DNA to another DNA molecule encoding the light chain constant domain CL is an isolated DNA encoding the V _L region can be converted to full-length light chain gene (as well as Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but in certain embodiments is a kappa constant region.

To generate the scFv gene, the V _H -and V _L -encoding DNA fragments are such that the V _H and V _L sequences can be expressed as a continuous single chain protein while the V _H and V _L regions are linked by a flexible linker. It may be operably linked to another fragment encoding a flexible linker, for example, the amino acid sequence (Gly4-Ser) ₃ (see, eg, Bird et al., 1988, Science 242:423 -426]; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; see McCafferty et al., 1990, Nature 348:552-554).

To express the anti-glyco-MUC1 antibodies of the present disclosure, DNA encoding partial or full-length light and heavy chains, obtained as described above, into expression vectors such that genes are operatively linked to transcriptional and translational control sequences. Is inserted. In this context, the term “operably linked” is intended to mean that the antibody gene is ligated into the vector such that the transcriptional and translational control sequences in the vector serve 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 expression host cell used. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or, more typically, both genes are inserted into the same expression vector.

The antibody gene is inserted into the expression vector by standard methods (eg, ligation of complementary restriction sites of antibody gene fragments and vectors, or blunt end ligation if restriction sites are not present). Before insertion of the anti-glyco-MUC1 antibody-related light or heavy chain sequence, the expression vector may already retain the antibody constant region sequence. For example, one approach to converting anti-glyco-MUC1 monoclonal antibody-related V _H and V _L sequences to full length antibody genes is that they are operatively linked to the C _H segment(s) in the V _H segment. .., The heavy chain constant and light chain constant regions, respectively, are inserted into an expression vector already encoding such that the V _L segment is operatively linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode signal peptides that facilitate secretion of antibody chains from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain gene, the recombinant expression vector of the present disclosure carries 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 (eg, polyadenylation signals) that control transcription or translation of antibody chain genes. 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 understand that the design of the expression vector, including selection of regulatory sequences, may depend on factors such as the choice of host cells to be transformed, the level of expression of the desired protein, and the like. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV) (such as CMV promoter/enhancer), simian virus 40 (SV40) ( Such as SV40 promoter/enhancer), adenovirus, (eg, adenovirus major late promoter (AdMLP)) and promoters and/or enhancers derived from polyomas. For further explanation of viral regulatory elements, and their sequences, see, for example, U.S. Pat.No. 5,168,062 to Stinski, U.S. Pat.No. 4,510,245 to Bell et al., and U.S. Pat. See Patent No. 4,968,615.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the present disclosure can carry additional sequences, such as sequences that regulate replication of vectors in host cells (eg, origins of replication) and selectable marker genes. . Selectable marker genes facilitate the selection of host cells into which vectors have been introduced (see, e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017 (all Axel et al.)). For example, typically, a selectable marker gene confers resistance to a drug, such as G418, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR ^- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). For the expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transduced into host cells by standard techniques. The various forms of the term "transfection" are a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc. It is intended to include.

Antibodies of the present disclosure can be expressed in prokaryotic or eukaryotic host cells. In certain embodiments, the expression of the antibody is performed in eukaryotic cells, eg, mammalian host cells, that are correctly folded and optimally secreted immunologically active antibodies. Exemplary mammalian host cells for expressing recombinant antibodies of the present disclosure are described in Chinese hamster ovary (CHO cells) (e.g., Kaufman and Sharp, 1982, Mol. Biol. 159:601-621). NSO myeloma cells, including DHFR ^- CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), used with DHFR selectable markers, as , COS cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is cultured by culturing the host cell for a period sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the grown culture medium. Is produced. Antibodies can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations of the above procedure fall within the scope of the present disclosure. For example, it may be desirable to transfect the host cell with DNA encoding the light or heavy chain (but not both) of the anti-glyco-MUC1 antibody of the present disclosure.

For expression of CARs of the present disclosure as described, for example, in Section 5.4 and Embodiments 97 and 98, it is preferred that the host cells are T cells, preferably human T cells. In some embodiments, the host cell exhibits anti-tumor immunity when the cell is cross-linked with MUC1 on tumor cells. Detailed methods for producing T cells of the present disclosure are described in Section 5.5.1.

Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that are not essential for binding to glyco-MUC1. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the present disclosure.

For recombinant expression of the anti-glyco-MUC1 antibodies of the present disclosure, host cells can be co-transfected with two expression vectors, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. have. The two vectors can contain the same selectable marker, or each can contain a separate selectable marker. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used.

If nucleic acids encoding one or more portions of an anti-glyco-MUC1 antibody have been generated, for example, generating antibodies with different CDR sequences, antibodies with reduced affinity for Fc receptors, or nucleic acids encoding antibodies of different subclasses To that end, additional alterations or mutations can be introduced into the coding sequence.

The anti-glyco-MUC1 antibodies of the present disclosure are chemically synthesized (e.g., Solid Phase Peptide Synthesis, 2nd ed., 1984) (The Pierce Chemical Co (Rockford, Ill.) ). Variant antibodies may also be generated using a cell-free platform (e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al. ., 2013, Current Opinion in Chemical Biology, 17:420-426).

Once the anti-glyco-MUC1 antibody of the present disclosure has been produced by recombinant expression, by any method known in the art for purification of immunoglobulin molecules, for example, chromatography (e.g., ion exchange , Affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Additionally, anti-glyco-MUC1 antibodies and/or binding fragments of the present disclosure can be fused to heterologous polypeptide sequences described in the art or otherwise known in the art to facilitate purification.

Once isolated, if desired, see, for example, by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980), or The anti-glyco-MUC1 antibody can be further purified by gel filtration chromatography on Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

5.5.1. Recombinant production of CAR in T cells

In some embodiments, nucleic acids encoding anti-glyco-MUC1 CARs of the present disclosure are delivered into cells using retroviral or lentiviral vectors. CAR-expressing retroviral and lentiviral vectors are delivered into different types of eukaryotic cells, as well as transduced cells or encapsulated as a carrier, or cell-free local or systemic delivery of naked vectors into tissues and whole organisms. Can be. The method used may be for any purpose where stable expression is required or sufficient.

In other embodiments, the CAR sequence is delivered into cells using mRNA transcribed in vitro. MRNA CARs transcribed in vitro can be delivered into different types of eukaryotic cells, as well as transduced cells as carriers or into tissues and whole organisms using cell-free local or systemic delivery of encapsulated, bound or naked mRNA. have. The method used may be for any purpose where temporary expression is required or sufficient.

In another embodiment, the desired CAR can be expressed in cells by transposon.

One advantage of the RNA transfection method of the present disclosure is that RNA transfection is essentially transient and vector-free: as a minimal expression cassette without the need for any additional viral sequence, the RNA transgene will be delivered to lymphocytes. Can be expressed there after simple in vitro cell activation. Under these conditions, integration of the transgene into the host cell genome is nearly impossible. Cell cloning is not required due to the efficiency of RNA transfection and its ability to uniformly transform the entire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA) uses two different strategies, both tested sequentially in various animal models. Cells are transfected with in vitro-transcribed RNA by lipofection or electroporation. Preferably, it is desirable to stabilize the IVT-RNA using various modifications to achieve long-term expression of the delivered IVT-RNA.

Some IVT vectors genetically modified in a manner that is used as a template for in vitro transcription in a standardized manner and in which a stabilized RNA transcript is produced are known in the literature. The protocol currently used in the art is based on a plasmid vector of the following structure: a 5'RNA polymerase promoter that enables RNA transcription, followed by an untranslated region (UTR) flanked by 3'and/or 5'interest 3'polyadenyl cassette containing the gene, and 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized by a type II restriction enzyme downstream of the polyadenyl cassette (recognition sequence corresponds to the cleavage site). Thus, the polyadenyl cassette corresponds to the poly(A) sequence in subsequent transcripts. As a result of this procedure, some nucleotides are retained as part of the enzyme cleavage site after linearization, and the poly(A) sequence is extended or masked at the 3'end. It is not clear whether this non-physiological overhang affects the amount of protein produced in cells from these constructs.

RNA has several advantages over traditional plasmid or viral approaches. Gene expression from an RNA source does not require transcription, and protein products are rapidly produced after transfection. Additionally, because RNA only needs to access the cytoplasm rather than the nucleus, the typical transfection method thus results in an extremely high proportion of transfection. Additionally, plasmid-based approaches require that the promoter driving the expression of the gene of interest is active in the cell under study.

In another aspect, RNA constructs can be delivered into cells by electroporation. See, for example, the formulation of nucleic acid constructs as taught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US 2004/0059285A1, US 2004/0092907A1 and electroporation into mammalian cells. Various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in the relevant study literature, as well as in numerous patents and applications in the field. See, for example, US Patent No. 6,678,556, US Patent No. 7,171,264, and US Patent No. 7,173,116. Devices for therapeutic application of electroporation, commercially available, for example, MedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, San Diego, CA) Possible, US Patent No. 6,567,694; US Patent No. 6,516,223, US Patent No. 5,993,434, US Patent No. 6,181,964, US Patent No. 6,241,701, and US Patent No. 6,233,482; Electroporation can also be used for transfection of cells in vitro, for example as described in US20070128708A1. Electroporation can also be used to deliver nucleic acids into cells in vitro. Thus, electroporation-mediated administration of nucleic acids into cells with expression constructs using any of a number of available devices and electroporation systems known to those skilled in the art is interesting for delivering RNA of interest to target cells. New means are proposed.

5.5.1.1 Sources of T cells

Prior to expansion and genetic modification, a source of T cells is obtained from the subject. The term “subject” is intended to include living organisms (eg, mammals) capable of eliciting an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human.

T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, a number of T cell lines available in the art can be used. In certain embodiments of the present disclosure, T cells can be obtained from blood units collected from a subject using a number of techniques known to those skilled in the art, such as Ficoll™ separation. In one preferred embodiment, cells from circulating blood from an individual are obtained by apheresis. Typically, apheresis products contain lymphocytes, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets, including T cells. In one embodiment, cells collected by agglutination can be washed to remove plasma fractions and place the cells in a suitable buffer or medium for subsequent processing steps. In one embodiment of the present disclosure, cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the cleaning solution lacks calcium, may lack magnesium, or, if not all, a number of divalent cations. Again, surprisingly, the initial activation phase in the absence of calcium leads to augmentation of activation. As those skilled in the art will readily understand, by methods known to those skilled in the art, such as semi-automated "perfusion" centrifuges (eg, Cove 2991 cell processor, Baxter Cytomate (Baxter) A cleaning step can be achieved by using CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of apheresis samples can be removed and cells can be resuspended directly in culture medium.

In another embodiment, T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugation purification. Certain subpopulations of T cells, such as CD3 ⁺ , CD28', CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and CD45RO ⁺ T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, incubation with anti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a period sufficient for positive selection of the desired T cells. By doing so, T cells are isolated. In one embodiment, the period of time is about 30 minutes. In a further embodiment, the period of time ranges from 30 minutes to 36 hours or more and all integer values in between. In further embodiments, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In another preferred embodiment, the period of time is 10 to 24 hours. In one preferred embodiment, the incubation period is 24 hours. For isolation of T cells from leukemia patients, using a longer incubation time, such as 24 hours, may increase cell yield. Longer incubation times can be used to isolate T cells in any situation where there are fewer T cells compared to other cell types, such as isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Additionally, using a longer incubation time can increase the capture efficiency of CD8+ T cells. Thus, simply by shortening or extending the time allowed for T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), initiation of culture. A subpopulation of T cells can be preferentially screened at or at different time points during the process, or can be screened against it. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, a subpopulation of T cells can be preferentially screened at the beginning of the culture or at other desired time points or against it. Can be selected. Those skilled in the art will recognize that multiple rounds of selection may also be used in the context of the present disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use “unselected” cells in the activation and expansion process. “Unselected” cells can also be applied to additional rounds of selection.

Enhancement of the T cell population by negative selection can be achieved with a combination of antibodies directed against surface markers unique to cells that are negatively selected. One method is cell sorting and/or selection by negative autoimmune adsorption or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on cells that are negatively selected. For example, to enhance CD4 ⁺ cells by negative selection, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enhance or positively select for regulatory T cells that typically express CD4 ⁺ , CD25 ⁺ , CD62L ^hi , GITR ⁺ , and FoxP3 ⁺ . Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

For isolation of a desired cell population by positive or negative selection, concentrations of cells and surfaces (eg, particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, more than 100 million cells/ml is used. In a further embodiment, a cell concentration of 10 million, 50 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells/ml is used. In another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells/ml is used. In a further embodiment, a concentration of 125 million or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation and cell expansion. Additionally, using high cell concentrations may result in more efficient capture of cells capable of weakly expressing the target antigen of interest, such as CD28-negative T cells, or samples in which multiple tumor cells are present (eg, leukemia blood, tumor tissue). Etc.). This population of cells may be of therapeutic value and would be desirable to obtain. For example, using high concentrations of cells generally allows more efficient selection of CD8 ⁺ T cells with weaker CD28 expression.

In related embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surfaces (eg particles such as beads), the interaction between particles and cells is minimized. This selects cells that express a high amount of the desired antigen to be bound to the particles. For example, CD4 ⁺ T cells express higher levels of CD28 and are captured more efficiently than CD8 ⁺ T cells at dilute concentrations. In one embodiment, the concentration of cells used is 5×10 ⁶ cells/ml. In other embodiments, the concentration of cells used can be from about 1×10 ⁵ cells/ml to 1×10 ⁶ cells/ml, and any integer value in between.

In other embodiments, cells can be incubated on a rotator at 2-10° C. or room temperature at various rates for various lengths of time.

T cells for stimulation may be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes within the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a frozen solution. A number of freeze solutions and parameters are known in the art and will be useful in this context, but one method is PBS with 20% DMSO and 8% human serum albumin, or 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% plasmalight-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO Using a culture medium containing, or other suitable cell freezing medium containing, for example, Hespan and Plasmalite A, after which the cells were frozen at -80°C at a rate of 1° per minute. Store in the gas phase of the liquid nitrogen storage tank. Other controlled freezing methods, as well as non-controlled freezing at -20°C or in liquid nitrogen can be used immediately.

In certain embodiments, cryopreserved cells are thawed and washed as described herein, rested at room temperature for 1 hour, and then activated using the methods of the present disclosure.

It is also envisioned in the context of the present disclosure to collect blood samples or agglutination products from a subject prior when expanded cells as described herein may be needed. As such, a source of cells to be expanded can be collected at any required time point, and the desired cells, such as T cells, are subsequently followed in T cell therapy for a number of diseases or conditions that would benefit from T cell therapy, such as those described herein. It is isolated and frozen for use in. In one embodiment, a blood sample or apheresis is generally taken from a healthy subject. In certain embodiments, blood samples or apheresis are taken from generally healthy subjects who are at risk of developing the disease but have not yet developed the disease, and cells of interest are isolated and frozen for future use. In certain embodiments, T cells can be expanded, frozen and used later. In certain embodiments, samples are collected from patients immediately after diagnosis of a particular disease as described herein, but prior to any treatment. In a further embodiment, natalizumab, epalizumab, antiviral agents, chemotherapy, irradiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoremoving agents such as CAMPATH, Prior to a number of related treatment modalities, including but not limited to treatment with agents such as anti-CD3 antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation Cells are isolated from blood samples or apheresis from the subject. These drugs inhibit calcium-dependent phosphatase calcineurin (cyclosporin and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin). (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 a further embodiment, the cells are isolated from the patient, and in conjunction with bone marrow or stem cell transplantation, or chemotherapy agents such as fludarabine, external-radiation therapy (XRT), T cell removal therapy with cyclophosphamide (e.g. For example, before, together or after) it is frozen for later use.

In a further embodiment of the present disclosure, T cells are obtained from the patient immediately after treatment. In this regard, immediately after treatment for a specific cancer treatment, especially for a period during which the patient is normally recovering from treatment, after treatment with drugs that compromise the immune system, the quality of the T cells obtained will be optimal for the ability to expand in vitro. It could be observed that it could or could be improved. Likewise, after ex vivo manipulation using the methods described herein, these cells may be in a preferred state for engraftment and enhanced in vivo expansion. Thus, it is envisioned in the context of the present disclosure to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage, during this recovery phase. Additionally, in certain embodiments, mobilization (e.g., GM-CSF) to create a condition in a subject where reproliferation, recycling, regeneration and/or expansion of a particular cell type is preferred, particularly during a limited time window after therapy. Mobilization) and conditioning therapy can be used. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

5.5.1.2 Activation and expansion of T cells

For example, US Patent No. 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 the method as described in US Patent Application Publication No. 20060121005, generally T cells are activated and expanded.

In general, T cells of the present disclosure are expanded by contact with an agent that stimulates a CD3/TCR complex associated signal and a ligand attached surface that stimulates a co-stimulatory molecule on the T cell surface. In particular, as described herein, protein kinase C activators (e.g., by surface-immobilized anti-CD3 antibodies or antigen-binding fragments thereof, or by contact with anti-CD2 antibodies, or with calcium ion transporters (e.g. For example, the T cell population can be stimulated by contact with bryostatin. For co-stimulation of an auxiliary molecule on the T cell surface, a ligand that binds the auxiliary molecule is used. For example, a population of T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable to stimulate proliferation of T cells. To stimulate the proliferation of CD4 ⁺ T cells or CD8 ⁺ T cells, anti-CD3 antibodies and anti-CD28 antibodies are used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and can be used as with other methods commonly known in the art (Berg et al. , Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp.Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2 ):53-63, 1999).

In certain embodiments, the primary stimulation signal and the co-stimulation signal for T cells can be provided by different protocols. For example, an agent that provides each signal can be in solution or can be coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (ie, “cis” large) or to a separate surface (ie, “trans” large). Alternatively, one agent can be coupled to the surface and the other agent can be in solution. In one embodiment, an agent that provides a co-stimulatory signal is bound to the cell surface, and an agent that provides a primary activation signal is in solution or coupled to the surface. In certain embodiments, both agents may be in solution. In another embodiment, the agent can be in a soluble form, and then crosslinked to a surface such as a cell expressing an Fc receptor or antibody or other binding agent that will bind the agent. In this regard, see, for example, US Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) envisioned for use in activating and expanding T cells of the present disclosure.

In one embodiment, two agents are immobilized on the same bead, ie “cis”, or on separate beads, ie “trans”. For example, an agent that provides a primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and an agent that provides a co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof, both of which act I co-fix onto the same beads in equimolar amounts. In one embodiment, a 1:1 ratio of each antibody bound to beads is used for CD4 ⁺ T cell expansion and T cell growth. In certain aspects of the present disclosure, a ratio of anti-CD3:CD28 antibody bound to beads is used such that an increase in T cell expansion is observed compared to expansion observed using a ratio of 1:1. In one particular embodiment, an increase of about 1 to about 3 times is observed compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to beads ranges from 100:1 to 1:100 and all integer values in between. In one aspect of the present disclosure, more anti-CD28 antibodies are bound to the particles than the anti-CD3 antibodies, ie, the ratio of CD3:CD28 is less than 1. In certain embodiments of the present disclosure, the ratio of anti CD28 antibody to anti CD3 antibody bound to beads is greater than 2:1. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, an antibody at a 1:30 CD3:CD28 ratio bound to beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to beads is used.

The ratio of particles to cells of 1:500 to 500:1 and any integer value therebetween can be used to stimulate T cells or other target cells. As those skilled in the art will readily appreciate, the ratio of particles to cells can depend on the particle size relative to the target cells. For example, small-sized beads can bind only small cells, while larger beads can bind many. In certain embodiments, the ratio of cells to particles ranges from 1:100 to 100:1 and any integer value therebetween, and in further embodiments 1:9 to 9:1 and any integer value between them. Including ratios can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupling particles to T cells resulting in T cell stimulation may differ as mentioned above, but certain preferred values are 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 of at least 1:1 It is a particle/T cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used. In one particular embodiment, the preferred particle:cell ratio is 1:5. In further embodiments, the ratio of particles to cells can vary depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is 1:1 to 10:1 on the first day, and then 1:1 to 1:10 (based on the number of cells on the day of addition) Additional particles are added to the cells daily, or every other day up to 10 days. In one particular embodiment, the ratio of particles to cells is 1:1 on the first stimulation day and adjusted to 1:5 on the third fifth stimulation day. In another embodiment, particles are added on a daily or every other day basis at a final ratio of 1:1 of the first stimulation day and 1:5 of the third and fifth stimulation days. In another embodiment, the ratio of particles to cells is 2:1 on the first stimulation day and adjusted to 1:10 on the third and fifth stimulation days. In another embodiment, particles are added on a daily or every other day basis at a final ratio of 1:1 of the first stimulation day and 1:10 of the third and fifth stimulation days. Those skilled in the art will understand that various other ratios may be suitable for use in the present disclosure. In particular, the ratio will vary depending on particle size and cell size and type.

In further embodiments of the present disclosure, cells, such as T cells, are combined with agonist-coated beads, and then the beads and cells are isolated, and then the cells are cultured. In an alternative embodiment, before incubation, the agent-coated beads and cells are not isolated, but cultured together. In a further embodiment, beads and cells are first concentrated by application of a force, such as magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

For example, cell surface proteins can be ligated by allowing paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached to contact T cells. In one embodiment, cells (eg, 10 ⁴ to 10 ⁹ T cells) and beads (eg, a 1:1 ratio of DYNABEADS® M-450 CD3/CD28 T paramagnetic beads) are buffers, preferably Combined in PBS (without divalent cations such as calcium and magnesium). Again, those skilled in the art will understand that any cell concentration can be used. For example, target cells can be very rare in a sample, and can make up only 0.01% of the sample, or the entire sample (ie, 100%) can include target cells of interest. Accordingly, any cell number is within the context of the present disclosure. In certain embodiments, to ensure maximum contact of cells and particles, it may be desirable to significantly reduce (ie, increase cell concentration) the volume in which the particles and cells are mixed together. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, more than 100 million cells/ml is used. In a further embodiment, a cell concentration of 10 million, 50 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells/ml is used. In another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells/ml is used. In a further embodiment, a concentration of 125 million or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation and cell expansion. Additionally, using high cell concentrations allows for more efficient capture of cells that can weakly express the target antigen of interest, such as CD28-negative T cells. Such cell populations may be of therapeutic value, and in certain embodiments, it will be desirable to obtain them. For example, using a high concentration of cells generally allows more efficient selection of CD8+ T cells with weaker CD28 expression.

In one embodiment of the present disclosure, the mixture can be incubated for hours (about 3 hours) to about 14 days or any time integer value in between. In another embodiment, the mixture can be incubated for 21 days. In one embodiment of the present disclosure, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Multiple stimulation cycles may also be desirable so that the culture time of T cells can be 60 days or longer. Conditions suitable for T cell culture include suitable media that may contain factors necessary for proliferation and survival (e.g., minimal essential media or RPMI media 1640 or X-vivo 15 (Lonza)) And the factor is serum (eg, human or fetal bovine 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 for cell growth known to those skilled in the art Other additives for cell growth include surfactants, plasmanates, and reducing agents such as N-acetyl- Cysteine and 2-mercaptoethanol, but are not limited to, the medium is serum free or an appropriate amount of serum (or plasma) or a sufficient set of cytokines for growth and bowel growth of a limited set of hormones and/or T cells. Supplemented with amino acids, sodium pyruvate and vitamins added RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer Antibiotics, such as penicillin and streptomycin, are only included in the experimental culture and not in the cell culture to be injected into the subject Conditions necessary to support growth, such as suitable temperature (e.g. , 37° C.) and under the atmosphere (eg air + 5% CO ₂ ), the target cells are maintained.

T cells exposed to various stimulation times may exhibit different properties. For example, a typical blood or apheresis peripheral blood mononuclear cell product has more helper T cell populations (T _H , CD4 ⁺ ) than cytotoxic or inhibitor T cell populations (T _C , CD8 ⁺ ). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells predominantly composed of T _H cells approximately on days 8-9, while approximately 8--9 After one day, the T cell population contains an increasing number of T _C cell populations. Therefore, depending on the purpose of treatment, it may be advantageous to inject a T cell population predominantly containing T _H cells into the subject. Similarly, if an antigen-specific subset of T _C cells has been isolated, it may be advantageous to expand this subset even further.

Additionally, in addition to CD4 and CD8 markers, other phenotypic markers change significantly, but mostly reproducibly, during the course of the cell expansion process. Thus, this reproducibility enables the ability to tailor activated T cell products to specific purposes.

5.6 composition

The anti-glyco-MUC1 antibody and/or anti-glyco-MUC1 ADC of the present disclosure may be in the form of a composition comprising an anti-glyco-MUC1 antibody and/or ADC and one or more carriers, excipients and/or diluents. The composition can be formulated for a specific use, such as for veterinary use or pharmaceutical use in humans. The form of the composition (eg, dry powder, liquid formulation, etc.) and excipients, diluents and/or carriers will depend on the intended use of the antibody and/or ADC and, in the case of therapeutic use, the mode of administration.

For therapeutic use, the composition can be supplied as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. Such compositions can be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition can be administered to the patient by various routes, such as by oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local or topical routes. The most appropriate route of administration in any given case will depend on the nature and severity of the particular antibody and/or ADC, the subject, and the subject's disease and physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

The pharmaceutical composition can conveniently be presented in unit dosage form containing a predetermined amount of anti-glyco-MUC1 antibody and/or anti-glyco-MUC1 ADC of the present disclosure per dose. The amount of antibody and/or ADC included in a unit dose will depend on the disease being treated, as well as other factors as well known in the art. Such unit doses may be in the form of a lyophilized dry powder or liquid containing an appropriate amount of antibody and/or ADC for single administration. The dry powder unit dosage form can be packaged in a kit with a syringe, an appropriate amount of diluent and/or other ingredients useful for administration. Unit doses in liquid form can be conveniently supplied in the form of a syringe pre-filled with an appropriate amount of antibody and/or ADC for a single administration.

The pharmaceutical composition may also be supplied in bulk form containing an appropriate amount of ADC for multiple administration.

Any pharmaceutically acceptable carrier, excipient or stabilizer (all referred to herein as a "carrier"), ie, a buffer, a stabilizer, which is typically used in the art for antibodies and/or ADCs having the desired degree of purity, Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing with preservatives, isotonic agents, non-ionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). These additives should be non-toxic to the recipient at the dosage and concentration used.

Buffers help maintain the pH in a range close to physiological conditions. It can be present in a wide range of concentrations, but will typically be present in concentrations ranging from about 2 mM to about 50 mM. Suitable buffers for use with the present disclosure are both organic and inorganic acids and salts thereof such as citrate buffers (e.g. monosodium citrate-sodium citrate mixture, trisodium citrate-trisodium citrate mixture, monosodium citrate-sodium citrate mixture) Etc.), succinate buffers (e.g., succinic acid-sodium succinic acid mixture, succinic acid-sodium hydroxide mixture, succinic acid-sodium succinic acid mixture, etc.), tartrate buffer (e.g. tartaric acid-sodium tartrate mixture, tartaric acid-tartaric acid) Potassium mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffer (e.g., fumaric acid-sodium fumarate mixture, fumaric acid-sodium fumarate mixture, sodium fumarate-sodium fumarate mixture, etc.), gluconate buffer (e.g. For example, gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc., oxalate buffers (e.g. oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-oxalic acid Potassium mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide) Mixtures, etc.). Additionally, phosphate buffers, histidine buffers, trimethylamine salts such as Tris can be used.

Preservatives can be added to retard microbial growth and can be added in an amount ranging from about 0.2%-1% (w/v). Suitable preservatives for use with the present disclosure are phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halide (e.g. chloride, bromide, iodide) , Hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as “stabilizers” can be added to ensure the isotonicity of the liquid compositions of the present disclosure, and polyhydric sugar alcohols, such as trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, Xylitol, sorbitol and mannitol. Stabilizers refer to a broad category of excipients that can range in function from additives to additives that help to solubilize the therapeutic agent or prevent it from denatured or adhere to the container wall. Typical stabilizers include polyhydric sugar alcohols (listed above); Amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine and the like; Organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like (including cyclitol such as inositol); Polyethylene glycol; Amino acid polymers; Sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thio sulfate; Low molecular weight polypeptides (ie, 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, glucose; Disaccharides such as lactose, maltose, sucrose and trehalose; And trisaccharides such as raffinose; And polysaccharides such as dextran. Stabilizers may be present in an amount ranging from 0.5 to 10% by weight per ADC weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) can be added to help solubilize glycoproteins, as well as to protect glycoproteins against agitation-induced agglomeration, which also allows the formulation to Allow exposure to the pressured shear surface without causing protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), pluronic polyols. The non-ionic surfactant can be present in a range of about 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL to about 0.2 mg/mL.

Additional various excipients include bulking agents (eg starch), chelating agents (eg EDTA), antioxidants (eg ascorbic acid, methionine, vitamin E), and cosolvents.

5.7 How to use

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

Anti-glyco-MUC1 antibodies or binding fragments described herein are administered to a subject, preferably into the bloodstream, wherein the antibody is labeled with a detectable moiety such as a radio-opaque agent or radioisotope, and the presence of the labeled antibody in the host. And radiation in vivo imaging for location verification. This imaging technique is useful in staging and treating malignant tumors.

Anti-glyco-MUC1 antibodies or binding fragments, ADCs and CARs described herein are useful in the treatment of glyco-MUC1 expressing cancers, particularly epithelial cancers such as breast cancer, ovarian cancer, pancreatic cancer, and lung cancer.

When the CAR of the present disclosure is used for therapy, a method of treatment of the present disclosure is directed to a CAR of the present disclosure (e.g., Section 5.4 or Embodiment 97 or Embodiment 98) in a subject with a glyco-MUC1-expressing tumor. Administering an effective amount of genetically modified cells engineered to express). Methods of modifying cells, particularly T cells, to express CAR are described in Section 5.5.1.

6. Examples

6.1 Example 1: Identification of anti-glyco-Muc1 antibody

6.1.1. survey

Chemoenzymatic synthesis of multi-repeat MUC1 glycopeptides of different O-glycan density and Tn (GalNAcα1-O-Ser/Thr) glycoforms was developed using recombinant glycosyltransferases. Different polypeptide GalNAc-transferase isoforms were used to indicate O-glycan occupancy sites (Bennett et al., 1998). The optimal vaccine design was found to be in the Tn glycoform with high O-glycan density, and the glycopeptide conjugated to KLH was found to overcome resistance. In wild-type Balb/c mice, a complete O-glycan-occupied glycopeptide caused the strongest antibody response to react with MUC1 expressed in a breast cancer cell line, thus indicating the most effective vaccine design. The induced humoral immune response showed remarkable specificity for cancer cells.

6.1.2. Materials and methods

6.1.2.1 Chemoenzymatic synthesis of multimeric Tn MUC1 glycopeptides

As originally reported in Fontenot et al., 1993, the peptide MUC1 60 (VTSAPDTRPAPGSTAPPAHG) n = 3 (SEQ ID NO: 47) was synthesized. The control peptide used was derived from a serial repeat (TR) of MUC2 (PTTTPISTTTMVTPTPTPTC) (SEQ ID NO: 51) and MUC4 (CPLPVTDTSSASTGHATPLPV) (SEQ ID NO: 52). Using purified recombinant human glycosyltransferase polypeptides GalNAc-T2, GalNAc-T4, and GalNAc-T1 (Bennett et al., 1998; Schwientek et al., 2002) as described in U.S. Pat.No. 6,465,220. The peptide was glycosylated in vitro. GalNAc glycosylation of peptides in a reaction mixture (1 mg peptide/mL) containing 25 mM cacodylate buffer (pH 7.4), 10 mM MnCl2, 0.25% Triton X-100, and 2 mM UDP-GalNAc. Was performed. Glycosylation of 1 mg 60-mer peptide with 2 GalNAc (MUC160Tn6) per TR was obtained using GalNAc-T1. Incorporation of 3 GalNAc per TR (MUC160Tn9) was obtained using GalNAc-T2. Substitution of all five putative O-glycosylation sites (MUC160Tn15) in MUC1 TR was performed using MUC160Tn9 as a substrate in reaction with GalNAc-T4. Glycosylation was monitored using a nano-scale reverse phase column (Poros R3, PerSeptive Biosystems, Framingham, Mass.) and MALDI-TOF mass spectrometry. Zorbax 300SB-C3 column (9.4 mm ×) in a 1100 Hewlett Packard system (Avondale, Pa.) using a gradient of 0.1% trifluoroacetic acid (TFA) and 0-80% acetonitrile. The glycopeptide was purified by high performance liquid chromatography (HPLC) on (25 cm) (Agilent Technologies, Palo Alto, Calif.). Quantitative and yield estimates of glycosyl reactions were performed by comparison of HPLC peaks by UV 210 absorbance using 10 μg weight peptide as a standard. GalNAc glycosylation of the peptide generally yielded 80-90% recovery. Purified glycopeptides were characterized by MALDI-TOF mass spectrometry on a Voyager DE or Voyager DE Pro MALDI-TOF mass spectrometer (Perceptive Biosystems) equipped with delayed extraction. The MALDI matrix was 10 g/L of 2,5-dihydroxybenzoic acid (Aldrich, Milwaukee, Wis.) dissolved in a 2:1 mixture of 0.1% TFA in 30% aqueous acetonitrile. Samples dissolved in 0.1% TFA at a concentration of ˜1 pmol/μl were prepared for analysis by placing 1 μl of the sample solution on the probe tip and then placing 1 μl of the matrix. All mass spectra were obtained in linear mode. Data processing was performed using GRAMS/386 software (Galactic Industries, Salem, Hampshire).

6.1.2.2 Immunization Protocol

Glyphalaldehyde was used to couple the glycopeptide to KLH (Pierce, Rockford, Ill.). Conjugation efficiency was assessed by analyzing the reaction by size exclusion chromatography on a PD-10 column using an anti-MUC1 ELISA of fractions. Essentially all reactivity was identified as the exclusion fraction, and a significant reactivity in the inclusion fraction expected to contain the peptide was identified. Further evaluation included a comparative titration analysis of the corresponding glycopeptide and KLH conjugate in ELISA. Analysis of both showed that the conjugation was almost complete, which should result in a KLH to glycopeptide ratio of 1:300. Female Balb/c wild-type mice were injected subcutaneously with a total volume of 200 μl of 10 or 15 μg of (glyco)peptide (Freund's adjuvant (1: 1 mixture with Sigma). Meeting immunity was provided and blood samples were obtained by tail or eye bleeding one week after the third and fourth immunizations.

6.1.2.3 Generation of mouse MAb anti-Tn-MUC1

MAb was produced from wild-type Balb/c mice immunized with fully GalNAc-glycosylated 60-mer MUC1 glycopeptide coupled to KLH. Screening was based on immunocytology into breast cancer cell lines (MCF7 and T47D) after glycopeptide ELISA and immunohistology into cancer tissue. Screening was based on reactivity patterns similar to total serum from the same mouse.

6.1.2.4 ELISA

ELISA was performed using a 96-well MaxiSorp plate (Nunc, Denmark). Plates were coated overnight with 4 μg/mL glycopeptide in bicarbonate-carbonate buffer (pH 9.6) at 4° C. and blocked with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Then, incubated with serum (diluted in PBS) or MAb for 2 hours at room temperature. The bound antibody is a peroxidase-conjugated rabbit anti-mouse immunoglobulin (DakoCytomation, Glosstrop, Denmark) or an isotype-specific antibody peroxidase-conjugated goat anti-mouse IgM, IgG1, IgG2a , IgG2b, or IgG3 (Southern Biotechnology Associates, Birmingham, Alabama). Plates were developed with O-phenylenedimine purification (DakoCytomation) and read at 492 nm. Control antibodies included anti-MUC1 antibodies HMFG2 and SM3 (Burchell et al., 1987) and anti-carbohydrate antibodies 5F4 (Tn) and 3F1 (STn) (Mandel et al., 1991). Control serum included mice immunized with MUC4 mucin peptide linked to KLH.

6.1.3. result

A glycopeptide specific mAb was produced for GalNAc-MUC1 using GalNAc-MUC1 60-mer glycopeptide conjugated to KLH as an immunogen. MUC1 serial repeats glycosylated in vitro using recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4 with purified mAb GO2 (5F7) generated using ELISA assay (VTSAPDTRPAPGSTAPPAHG) ₃ (SEQ ID NO: 47) and no corresponding MUC1 peptide without GalNAc-residue or unrelated glycopeptide of the same type of Tn glycoform. The results of the ELISA assay are presented in FIG. 1.

6.2 Example 2: Characterization of anti-glyco-Muc1 antibody

6.2.1. survey

The monoclonal antibody GO2 (5F7) was characterized for its specificity binding to the Tn glycoform of MUC1 associated with cancer cells.

6.2.2. Materials and methods

6.2.2.1 Immunocytochemistry

Cell lines were fixed in ice-cold acetone for 10 minutes or in methanol:acetone. Immobilized cells were incubated overnight with mouse serum (1:200/1:400/1:800) or MAb at 5° C., followed by fluorescein isothiocyanate (FITC)-conjugated rabbits at room temperature for 45 minutes. Incubated with anti-mouse immunoglobulin (Dakocytomation). Slides were mounted on glycerol containing p-phenylenediamine and examined on a Zeiss fluorescence microscope (FluoresScience, Halbergmos, Germany).

6.2.2.2 Immunohistochemistry

Formalin-fixed, paraffin wax-embedded tissue of breast carcinoma was obtained. All cases were conventionally classified by histological type. The avidin-biotin peroxidase complex method was used for immunostaining. The wax was removed from the paraffin section, rehydrated and treated with 0.5% H ₂ O ₂ in methanol for 30 minutes. Sections were rinsed in TBS and incubated with rabbit non-immune serum for 20 minutes. Sections were rinsed and incubated overnight at 5° C. with primary antibody. The sections are rinsed, incubated for 30 minutes with biotin-labeled rabbit anti-mouse serum (Dakocytomation) diluted 1:200 in TBS, rinsed with TBS, and avidin-Biotin peroxidase complex (Dacocation) for 1 hour. ). Sections were rinsed with TBS and developed with 0.05% 3,3'-diaminobenzidine tetrahydrochloride freshly prepared in 0.05 M TBS containing 0.1% H ₂ O ₂ . Sections were stained with hematoxylin, dehydrated and mounted.

6.2.3. result

Immunohistochemistry of colorectal carcinoma, pancreatic carcinoma and invasive breast adenocarcinoma was performed with GO2. Staining of colorectal cancer tissue (FIG. 2) demonstrated strong intracellular and surface structures on most cancer cells. In contrast, no reactivity was seen or significantly lower for healthy columnar epithelial cells. Labeling in healthy cells was limited to the intracellular structure, which is expected to be attributed to the presence of large amounts of biosynthetic intermediates (GalNAc-modified glycoproteins) in cells with high secretion capacity, such as colon columnar epithelium. Similar patterns were observed with the pancreatic (FIG. 3) and breast cancer tissue (FIG. 4 ), with high reactivity with cancer cells and with or without limitations with intracellular structures in surrounding healthy epithelial or connective tissue cells.

6.3 Example 3: Sequence analysis of anti-glyco-Muc1 antibody

MRNA from hybridomas producing monoclonal antibody GO2 (5F7) was prepared, reverse transcribed, and sequenced.

The nucleotide sequences encoding heavy and light chain variable regions together with their signal sequences are described in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The heavy and light chain variable regions encoded by SEQ ID NO: 11 and SEQ ID NO: 12 are described in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Expected mature variable region sequences (after truncation of the signal peptide) are described in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and are encoded by SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Expected heavy chain CDR sequences (IMGT definition) are described in SEQ ID NO: 5-7, respectively, and expected light chain CDR sequences (IMGT definition) are described in SEQ ID NO: 8-10, respectively.

6.4 Example 4: Drug delivery to cancer cells with anti-glyco-Muc1 antibody

6.4.1. survey

The monoclonal antibody GO2 (5F7) was tested for its ability to deliver cytotoxic agents to target cells.

6.4.2. Materials and methods

OVCar human ovarian carcinoma cells were added to a 24-well cell culture plate at 1,000 cells/well. Secondary antibody (anti-mFc-NC-MMAF) conjugated to monoclonal antibody GO2 and anti-tubulin agonist monomethyl auristatin F (MMAF) to provide the following concentrations (in μg/ml) of GO2 and ADC: (Moradec catalog number AM-101-AF) was added to the plate:

<Table 2>

Plates were incubated at 37° C. for 48 hours. After 48 hours incubation, AlamarBlue® (Invitrogen) was added to each well and fluorescence at 600 nm was measured.

6.4.3. result

The results are presented in FIG. 5. The results show that cytotoxicity is dependent on primary antibody (GO2) concentration and presence of antibody, and secondary ADC conjugated antibody concentration and presence. In other words, GO2 induces cytotoxicity of these cancer cell lines when coupled with secondary antibodies carrying the cytotoxic agent MMAF.

6.5 Example 5: Quantification of circulating tumor cells with anti-Muc1 antibody

6.5.1. survey

The monoclonal antibody GO2 (5F7) was tested for its ability to be used to quantify circulating tumor cells.

6.5.2. Materials and methods

GO2 was conjugated to magnetically isolated beads and allowed to interact with samples of different concentrations of tumor cells. Cells and beads were removed from the solution with a magnet and washed several times to remove unbound material. Thereafter, GO2 conjugated to horseradish peroxidase was applied to magnetic separation beads containing bound cancer cells, and after incubation, unbound conjugated GO2 was washed. Colorimetric reactions were performed using TNB as a substrate. After the reaction was terminated with sulfuric acid, an OD 450 reading was taken from the sample.

6.5.3. result

Results are presented in FIG. 6. The assay results demonstrated GO2 binding of tumor cells.

6.6 Example 6: Immunohistochemical staining of tumor tissue with anti-glyco-Muc1 antibody

6.6.1. Materials and methods

Sections from six formalin fixed, paraffin embedded (FFPE) tissue microarrays (TMA) were cut to a thickness of 2.5 μm. TMA from breast cancer (BC), colorectal cancer (CRC), ovarian cancer (OVC), non-small cell lung cancer (NSCLC) and prostate cancer (PrC) tumors was used in the study. 25-47 tumor tissue cores per TMA were available for evaluation. The core size was 1 mm, 2 mm or 3 mm depending on TMA. Each tissue core represented 1 patient.

Staining was performed on a Discovery XT autostainer (Ventana Medical Systems). After antigen recovery with Cell Condition 1 (CC1) solution (Ventana Medical Systems), GO2 was applied at a concentration of 1 μg/mL in a Dako green medium antibody diluent and incubated at 37° C. for 60 minutes. Binding of GO2 to tumor cells was detected using the Optiview DAB IHC detection kit (Ventana Medical Systems) visualized in DAB (brown precipitate).

6.6.2. result

Representative images of MUC1-positive TMA tumor cores are presented in FIG. 7. In BC and OVC TMA, most spots (>90%) indicated that GO2 binds moderately or strongly to tumor cells. 70% and 51% of the cases of NSCLC and CRC showed that the antibody binds moderately or strongly to tumor cells, respectively. In prostate cancer, it appeared that the antigen was less expressed. When GO-2 was applied, only 28% of the spots in TMA 1 were of moderate or strong staining intensity. The staining pattern was always cytoplasmic and membrane-bound in many cases. A tip membrane staining pattern was observed in several cores.

6.7 Example 7: Production and purification of anti-MUC1 antibodies in T-cell bispecific (TCB) format

6.7.1. Materials and methods

6.7.1.1 Expression vector production

Knob-into-hole in the Fc region and P329G/L234A/L235A (“PGLALA”) mutation and charged residues in the MUC1 CH1 (147E/213E; “EE”) and CL (123R/124K; “RK”) regions Including, GO2 antibody was converted to TCB format (see SEQ ID NOs: 43-46). The TCB antibody is illustrated in Figure 8. Briefly, variable heavy and variable light chains of GO2 mAb were synthesized (Geneart, Regensburg, Germany) and inserted into an appropriate expression vector, where they are fused to a suitable human constant heavy chain or human constant light chain. The expression cassette in this vector consists of the CMV promoter, intron A with 5'UTR, and the BGH polyadenylation site. Additionally, the plasmid contains the oriP region from Epstein Barr virus for stable maintenance in HEK293 cells carrying the EBV nuclear antigen (EBNA).

6.7.1.2 Transient transfection and production

Antibodies were temporarily produced in HEK293 EBNA cells using the following PEI mediated transfection procedure. HEK293 EBNA cells are cultured in serum-free suspension in an Excell culture medium containing 6 mM L-glutamine. For antibody production in a 500 ml shake flask, 300 million HEK293 EBNA cells are seeded 24 hours prior to transfection (for alternative scale, all amounts are adjusted accordingly). For transfection, cells are centrifuged at 210 xg for 10 minutes, and the supernatant is replaced with pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After the addition of 540 μl PEI, the solution is vortexed for 15 seconds and then incubated for 10 minutes at room temperature. The cells are then mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated at 37° C. for 3 hours in an incubator with 5% CO ₂ atmosphere. After incubation, 160 ml Ex-cell® medium (Sigma-Aldrich) containing 6 mM glutamine, 1.25 mM Valproic acid and 12.5% Pespoy was added and cells were added. Incubate for 24 hours. One day after transfection, 12% Feed 7 (48 mL) + 3 g/L glucose is added. After 7 days, the culture supernatant is collected for purification by centrifugation at 3000×g for 45 minutes. The solution is sterile filtered (0.22 μm filter) and sodium azide with a final concentration of 0.01% w/v is added. Thereafter, the solution is stored at 4°C.

6.7.1.3 Antibody Purification

The secreted protein was purified by affinity chromatography using Protein A followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded onto a Protein A MapSelect SuRe column (GE Healthcare) equilibrated with 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unbound protein was removed by washing with equilibration buffer. Bound protein was eluted using step (standard IgG) or gradient (bispecific antibody) elution generated with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. The pH of the collected fractions was adjusted by adding 1/10 (v/v) of 0.5 M sodium phosphate pH 8.0. Proteins were concentrated, filtered, and loaded onto a HiLoad Superdex 200 column (GE Healthcare) flattened to 20 mM histidine, 140 mM sodium chloride, pH 6.0.

The aggregate content of the eluted fraction was analyzed by analytical size exclusion chromatography. Then, 30 μl of each fraction was applied to a TSK G3000 SWXL column (TOSOH, 7.8 mm×30 cm) equilibrated with 200 mM arginine, 25 mM K ₂ PO ₄ , 125 mM sodium chloride, 0.02% NaN ₃ , pH 6.7. Did. Fractions containing less than 2% oligomer were pooled and final concentration of 1-1.5 mg/ml using a centrifugal concentrator (Millipore, Amicon® ULTRA-15, 30k MWCO) Concentrated with. The purified protein was stored at -80 °C.

6.7.2. result

The production yield and quality of GO2 TCB antibodies are presented in Table 5.

<Table 5>

6.8 Example 8: Jerkat-NFAT reporter assay for monitoring in vitro target expression in undigested patient-derived samples

6.8.1. survey

Using the Jurkat NFAT reporter assay, ex vivo target expression (glyco-MUC1) in undigested primary human tumor samples was monitored using GO2 TCB.

6.8.2. Materials and methods

6.8.2.1 Substance

GO2 TCB (실시예 7 참조)

GO2 TCB (see Example 7)

DP47 TCB (비-표적화, 음성 대조군)

DP47 TCB (non-targeting, negative control)

매트리겔(Matrigel) (아이템 번호 734-1101, 코닝(Corning)/VWR, 스위스)

Matrigel (Item No. 734-1101, Corning/VWR, Switzerland)

코닝® 코스타(Costar)® 울트라-로우(Ultra-Low) 부착 멀티웰 플레이트 (아이템 번호 CLS7007-24EA, 시그마)

Corning® Costar® Ultra-Low Multiwell Plate (Item No. CLS7007-24EA, Sigma)

세포 배양 마이크로플레이트, 96웰 (아이템 번호 655098, 그라이너 바이오-원(Greiner Bio-one), 스위스)

Cell culture microplate, 96 well (Item No. 655098, Greiner Bio-one, Switzerland)

RPMI1640 배지 (아이템 번호 42401-018, 피셔사이언티픽(FisherScientific), 스위스)

RPMI1640 badge (Item No. 42401-018, FisherScientific, Switzerland)

저캣 배지: RPMI1640 배지 + 2 g/l D-글루코스, 2 g/l NaHCO₃, 10 % FCS, 25 mM HEPES, 2 mM L-글루타민, 1× NEAA, 1× 피루브산나트륨, 200 ㎍/ml 히그로마이신 B

Jerkat medium: RPMI1640 medium + 2 g/l D-glucose, 2 g/l NaHCO ₃ , 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1× NEAA, 1× sodium pyruvate, 200 μg/ml hygro Mycin B

저캣 NFAT 루시퍼라제 리포터 세포 (프로메가(Promega))

Jerkat NFAT Luciferase Reporter Cell (Promega)

독일의 인디부메드 게엠베하(Indivumed GmbH)에서 종양 샘플을 받았다. 샘플을 철야로 운반 배지에서 운송하였다. 수술하고 나서 약 24시간 후에 샘플을 소형 조각으로 절단하였다.

Tumor samples were obtained from Indivumed GmbH, Germany. Samples were shipped overnight in the transport medium. Samples were cut into small pieces about 24 hours after surgery.

6.8.2.2 Method

96-well cell culture microplates were prepared by adding 17 μl of cold Matrigel. After incubating the plate for 2 minutes at 37° C., tumor fragments were added (triple). 33 μl of cold Matrigel per well was added and the plate was incubated again at 37° C. for 2 minutes. 100 μl (50 nM or 5 nM) of TCB antibody dilution per well (without hygromycin but diluted in Jerkat medium with 2× penicillin/streptomycin) was added. Jerkat-NFAT reporter cells were harvested and viability was assessed using ViCell. Cells were centrifuged at 350×g for 7 minutes, then resuspended in Jerkat medium without hygromycin. 50 μl of cell suspension per well was added (50,000 cells/well). Plates were incubated in a humidified incubator at 37° C. for 4-5 hours, and then removed for luminescence readings. To each well, 50 μl of ONE-Glo solution was added and incubated for 10 minutes at room temperature in the dark. Luminescence was detected using a Wallac Victor3 ELISA reader (PerkinElmer2030) with a 5 second/well detection time.

6.8.3. result

Results from tumor samples from 3 patients are presented in Figures 9-11. The results presented in Figure 9 are from tumor samples obtained from patients with malignant neoplasms of the bronchi and lungs: mesenchymal, bronchial or lung, squamous cell carcinoma. The results presented in FIG. 10 are from tumor samples obtained from patients with malignant neoplasms of the bronchi and lungs: lower lobe, bronchial or lung, non-keratinous squamous cell carcinoma. The results presented in FIG. 11 are from tumor samples obtained from patients with malignant neoplasms of the bronchi and lungs: adenocarcinoma of the upper lobe, bronchial or lung, sore type. Each bar in Figures 9-11 represents the average of the triplicates. Standard error is indicated by the error bar. Dotted lines indicate luminescence for Jerkat NFAT cells incubated with tumor samples without any TCB. Two-sided non-responsive t-tests were used for statistical analysis. P-values less than 0.05 were considered significant and indicated with an asterisk (* P ≤ 0.05; ** P ≤ 0.001; *** P ≤ 0.001). In each of FIGS. 9-11, tumor samples incubated with GO2 TCB showed significantly more luminescence than samples incubated with DP47 negative control TCB.

6.9 Example 9: In vitro characterization of GO2 TCB

6.9.1. survey

GO2 TCB (Example 7) that recognizes tumor-specific abnormally glycosylated MUC1 (Example 7) was functionally characterized in tumor cells expressing MUC1.

6.9.2. Materials and methods

6.9.2.1 Cell lines and PBMCs

T3M4 pfzv and MCF7 cs engineered tumor cell lines were cultured in DMEM + 10% FCS and 2 mM glutamine. MCF10A is a human non-neoplastic breast epithelial cell line (ATCC® CRL-10317). HBEpiC is a human bronchial epithelial cell (Sciencell #3210). PBMCs were isolated by gradient centrifugation using whole blood from healthy volunteers.

6.9.2.2 Target binding by flow cytometry

Target cells as indicated were harvested with cell dissociation buffer, washed with PBS and resuspended in FACS buffer. Antibody staining was performed in 96 well round bottom plates. 200,000 cells per well were seeded. The plate was centrifuged at 400 g for 4 minutes and the supernatant was removed. Test antibodies were diluted in FACS buffer and 30 μl of the antibody solution was added to the cells at 4° C. for 30 minutes. To remove unbound antibody, cells were washed twice with FACS buffer, followed by diluted secondary antibody (PE-conjugated Affineure F(ab')2 fragment goat anti-human IgG Fcγ fragment specific , Jackson ImmunoResearch #109-116-170). After incubation at 4°C for 30 minutes, the unbound secondary antibody was washed. Prior to measurement, cells were resuspended in 200 μl FACS buffer and analyzed by flow cytometry using BD Fortessa. The assay was performed in triplicate.

6.9.2.3 T cell mediated tumor cell killing and T cell activation

Target cells were harvested with trypsin/EDTA, counted and viability checked. Cells were resuspended in their respective media to a final concentration of 300,000 cells/ml. Thereafter, 100 μl of the target cell suspension was transferred into each well of a 96-flat bottom plate. The plates were incubated overnight at 37° C. in an incubator to attach them to the cells. The next day, PBMCs were isolated from whole blood. Blood was diluted 2:1 with PBS, covered on 15 ml Hisopaque-1077 (#10771, Sigma-Aldrich) in Leucosep tubes and centrifuged at 450 g for 30 minutes without braking. After centrifugation, the band containing the cells was collected with a 10 ml pipette and transferred into a 50 ml tube. The tube was filled with PBS up to 50 ml and centrifuged (400 g, 10 min, room temperature). The supernatant was removed and the pellet was resuspended in PBS. After centrifugation (300 g, 10 min, room temperature), the supernatant was discarded, the two tubes were pooled, and the washing step was repeated (this time centrifugation at 350 g, 10 min, room temperature). Cells were then resuspended and pellets were pooled in 50 ml PBS for cell counting. After counting, cells were centrifuged (350 g, 10 min, room temperature) and resuspended with 6 million cells per ml RPMI + 2% FCS and 2 nM glutamine. Media was removed from the plated target cells and test antibody diluted in RPMI + 2% FCS and 2 nM glutamine was added. An effector cell solution of 300,000 cells was transferred to each well resulting in an E:T ratio of 10:1. To determine maximal release, target cells were lysed with Triton X-100. LDH release was determined after 24 and 48 hours using a cytotoxicity detection kit (1644793, Roche Applied Science). Activation marker upregulation on T cells after tumor cell killing was measured by flow cytometry. Briefly, PBMCs were harvested, transferred into 96-well round-bottom plates, CD4 APC diluted in FACS buffer (300514, BioLegend), CD8 FITC (344704, Bio Legend), CD25 BV421 (302630, Bio Legend) , CD69 PE (310906, Bio Legend) antibody. After incubation at 4°C for 30 minutes, cells were washed twice with FACS buffer. Cells were resuspended in 200 μl FACS buffer prior to measuring fluorescence using BD Fortessa II. The assay was performed in triplicate.

6.9.2.4 Cytokine/chemokine release by cytometric bead arrays

Cytokine/chemokine secretion in the supernatant was measured by flow cytometry using a cytometric bead array (CBA) according to the manufacturer's instructions. Supernatants from the T cell mediated killing assay were collected and stored at -20°C. The supernatant was then thawed and tested according to the manufacturer's instructions. The following CBA kit (BD Biosciences) was used: CBA Human Interferon Gamma (IFNγ) Flex Set (E7), CBA Human Granzyme B Flex Set (D7), CBA Human IL6 Flex Set (A7), CBA human IL8 flex set (A9), CBA human IL10 flex set (B7) and CBA human tumor necrosis factor (TNF) flex set (D9). Samples were measured using BD FACS Canto II, and analysis was performed using Diva Software (BD Biosciences). The assay was performed in triplicate.

6.9.3. result

Binding of GO2 TCB to breast cancer cell line MCF7 and pancreatic cancer cell line T3M4 engineered to both express abnormally glycosylated MUC1 was confirmed (FIG. 12 ). The activity of GO2 TCB was then tested in tumor cell lines of both MCF7 and T3M4 using freshly isolated PBMCs (Figures 13 and 14). Tumor cell killing of both cell lines was detected after 24 hours, and even stronger after 48 hours. This was accompanied by activation of CD4 T cells and CD8 T cells determined by upregulation of two activation markers CD25 and CD69, and release of IL6, IL8, IL10, IFNγ, TNFα and Granzyme B into the supernatant. As a negative control, each untargeted TCB was included.

To demonstrate that GO2 TCB does not bind normally glycosylated MUC1 on epithelial cells, binding to human non-oncogenic mammary epithelial cell line MCF10A and primary human bronchial epithelial cell HBEpiC was tested. As a positive control, HMFG1 TCB, which does not distinguish MUC1 expressed on normal cells and tumor cells, was included. While HFMG1 TCB was found to bind to both tested cells, expression of MUC1 was confirmed, but GO2 TCB was unable to bind to these cells (FIG. 15 ). Additionally, GO2 TCB was tested to see if it would induce killing or T cell activation in the presence of normal epithelial cells expressing MUC1. It was tested on MCF10A cells and there was no killing or T cell activation detectable with GO2 TCB, but HMFG1 TCB induced killing as well as T cell activation (FIG. 16 ).

6.10 Example 10: Functional characterization of GO2 and GO2 TCB antibodies by surface plasmon resonance

6.10.1. survey

GO2 and GO2 TCB (Example 7) were characterized by surface plasmon resonance.

6.10.2. Materials and methods

6.10.2.1 Binding of GO2 and GO2 TCB to immobilized glycopeptides

The binding of GO2 antibody and GO2 TCB to human and cynomolgus glycopeptides (Table 6) was evaluated by surface plasmon resonance (SPR). All SPR experiments were performed using HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, Biacore (Freiburg, Germany)) as a running buffer, Biacore at 25°C. T200.

Table 6.

Biotinylated glycopeptide was dissolved in PBS and the final concentration was 0.9-1.8 mg/ml (Table 6). Biotinylated glycopeptide was coupled directly to the flow cell of a streptavidin (SA) sensor chip. A fixed level of up to 880 resonance units (RU) was used. GO2 antibody or GO2 TCB was injected over a flow of 30 μl/min through a flow cell at a concentration of 1000 nM over 240 seconds (FIG. 17 ). Dissociation was monitored for 500 seconds. The difference in bulk refractive index was corrected by subtracting the reaction obtained in a reference flow cell in which the protein was not immobilized.

6.10.2.2 Adhesion of GO2 and GO2 TCB to immobilized glycopeptides

The binding power of GO2 and GO2 TCB was evaluated by surface plasmon resonance (SPR). All SPR experiments were performed on Biacore T200 at 25° C. using HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, Biacore (Freiburg, Germany)) as a running buffer. . Biotinylated glycopeptide (Table 6) was coupled directly to the flow cell of a streptavidin (SA) sensor chip. A fixed level of up to 200 resonance units (RU) was used.

GO2 antibody or GO2 TCB was injected over a flow of 30 μl/min through a flow cell at a concentration of 3.9 to 1000 nM (1:2 dilution) over 120 seconds. Dissociation was monitored for 400 seconds. The difference in bulk refractive index was corrected by subtracting the reaction obtained in a reference flow cell in which the protein was not immobilized. KD was derived despite the bivalentity of the interaction by fitting the curve to a 1:1 Langmuir binding using Bieval software (GE Healthcare). Thus, the "apparent" KD can only be used for comparison purposes.

6.10.3. result

As can be seen in the sensorgram of Figure 18, GO2 antibodies (Figure 18A) and GO2 TCB (Figure 18B) bind to both human and cynomolgus glycopeptides. GO2 antibody and GO2 TCB bind to cynomolgus with higher binding force than human glycopeptide.

As can be seen in Figure 19, the binding of the divalent GO2 binding agent (IgG, TCB) to human glycopeptides is tridentate nanomolar (Figures 19A and 19C), while the binding to cynomolgus glycopeptide is bidentate nanomolar ( Figures 19b and 19d).

6.11 Example 11: Exploratory single dose pharmacokinetics and tolerance study of GO2 TCB in cynomolgus monkeys

6.11.1. survey

The purpose of this study was to determine the pharmacokinetics and resistance of GO2 TCB described in Example 7 when given by cynomolgus monkey by single intravenous injection.

6.11.2. Materials and methods

6.11.2.1 Preparation of GO2 TCB

Frozen mother liquors of GO2 TCB (2.12 mg/mL) and formulation buffer (20 mM histidine, 140 mM NaCl, 0.01% Tween 20; pH 6.0) are thawed overnight in a refrigerator set to maintain at 4°C. Test article dosing formulations are prepared under sterile conditions at suitable concentrations that meet dose level requirements by dilution with formulation buffer.

Dosage formulations are prepared within 2 hours prior to injection and stored at room temperature until use. Polypropylene containers are used to prepare and store the dosage form to prevent adsorption. Dosage formulations should not be filtered, stirred or shaken. All mixing is done by gentle pipetting or gentle swinging of the container.

6.11.2.2 animals

Cynomolgus monkeys (Macaca fascicularis), 2-4 years old and weighing less than 4 kg, are used in the study. Animals are allowed to acclimatize to the test facility primate toxicology facility for at least 6 weeks prior to initiation of administration.

During the week prior to initiation of dosing, animals are approved for participation in the trial based on satisfactory veterinary examination (performed immediately after arrival), clinical observation record, weight profile, and clinical pathology investigation.

Animals selected for study are randomly assigned to cages based on group compatibility information provided, and then individual study numbers are assigned. Animals are assigned to cages of up to 5 groups.

6.11.2.3 Breeding

Animals are socially inhabited in groups of up to five animals, if possible, in a two-story group fence measuring 1.61 × 1.66 × 2.5 m on the first floor and 1.61 × 1.66 × 2.03 m on the second floor. Bedding material is wood shavings. Bedding is free of known contaminants that will interfere with research objectives.

The target conditions of the animal room environment are as follows:

Temperature: 18-24℃

Humidity: 40-70%

Ventilation: At least 10 air exchanges per hour

Lighting cycle: 12 hours bright and 12 hours dark (unless interrupted by study procedure/activity).

Temperature and humidity are automatically controlled and continuously monitored and recorded. The lighting cycle is automatically controlled.

Special Food Service (SDS) MP(E) Short-term SQC diets are provided as daily rations throughout the study. A ration of about 200 grams per animal is provided once a day. There are no known contaminants in the feed that would interfere with the research objectives. Animals have free access to public watering. There are no known contaminants in the water that would interfere with research objectives.

The animal's home environment is strengthened to promote social interaction, play and exploration. Animals have perch, and materials such as plastic toys, balls, climbing frames and stainless steel mirrors. Frequently exchange them to reduce familiarity. Prior to replacement, all toys and climbing frames are thoroughly cleaned to prevent cross-contamination. In addition, various other treats such as food mixtures, vegetables, nuts, biscuits and fruits are generally provided to animals on a daily basis.

Veterinary care is available throughout the study process and animals are examined by veterinary staff as warranted by clinical signs or other changes.

6.11.2.4 Experimental design

GO2 TCB is administered to 1 male and 1 female animal.

<Table 7>

GO2 TCB is administered to suitable animals by a single intravenous slow bolus injection (1-2 min) in a saphenous vein or tail vein at least 8 days apart. Only one male and one female were staggered to provide a new dose level on any single day. Based on observations from previous dose levels (including clinical pathology data), doses are increased or decreased for the next dose group. Naive animals are used for each dose level. The dose is provided using a syringe with a Vygon injection needle attached.

Animals are necrosed approximately 72 hours after dosing (after taking the last scheduled sample). For all animals that must be terminated before the due date due to severe clinical symptoms, a complete panel of clinical pathology (if feasible, additional sampling prior to termination) and histopathology are analyzed.

The intravenous route of administration was chosen for this study because it is a possible clinical application route. Low and high dose levels were selected to cover a range of clinically relevant doses and to minimize potential harm to animals. The low dose was selected based on experience in cynomolgus monkeys with similar efficacy of similar T-cell bispecific antibodies, and the high dose represents a 3-fold increment thereof.

6.11.3. result

GO2 TCB is allowed at the dose tested.

7. Reference to specific embodiments and references

While various specific embodiments have been described and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). The present disclosure is illustrated by the numbered embodiments described below.

1. a. Preferentially binds to the glyco-MUC1 epitope overexpressed in cancer cells compared to normal cells;

b. Competing with an antibody or antigen binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to the breast cancer cell line MCF7 or T47D

Anti-glyco-MUC1 antibody or antigen binding fragment.

2. a. Binding to a glycosylated MUC1 serial repeat (VTSAPDTRPAPGSTAPPAHG) ₃ (hereinafter referred to as “first epitope”) using purified recombinant human glycosyltransferase GalNAc-T1, GalNAc-T2, and GalNAc-T4. and;

b. Competing with an antibody or antigen binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to the breast cancer cell line MCF7 or T47D

Anti-glyco-MUC1 antibody or antigen binding fragment.

3. The complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, according to the embodiment 1 or 2, SEQ ID NO: CDR-H3 comprising the amino acid sequence of 25, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO: 31 Anti-glyco-MUC1 antibody or antigen-binding fragment comprising CDR-L3 comprising a.

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

5. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO:23.

6. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO:28.

7. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO:32.

8. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 7, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:6.

9. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 7, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:24.

10. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 9, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:7.

11. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 10, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30.

12. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 10, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:26.

13. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 12, wherein CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27.

14. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 3 to 13, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10.

15. The anti-glyco- of embodiment 1 or embodiment 2, wherein the VH comprises the complementarity determining region (CDR) of SEQ ID NO: 5-7, and the VL comprises the CDR of SEQ ID NO: 8-10. MUC1 antibody or antigen-binding fragment.

16. The term of embodiment 1 or 2, wherein VH comprises the complementarity determining region (CDR) of SEQ ID NOs: 23-25, and VL comprises the CDRs of SEQ ID NOs: 26, 27, and 10. -Glyco-MUC1 antibody or antigen-binding fragment.

17. The method of embodiment 1 or embodiment 2, wherein the VH comprises the complementarity determining regions (CDRs) of SEQ ID NOs: 28, 29, and 25, and the VL comprises the CDRs of SEQ ID NOs: 30, 9, and 31. Containing anti-glyco-MUC1 antibody or antigen-binding fragment.

18. The anti-glyco-MUC1 antibody or antigen-binding fragment according to any of embodiments 1 to 17, which is a chimeric or humanized antibody.

19. The method of any one of embodiments 1 to 18, wherein the VH comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3, and the VL has at least 95% sequence identity to SEQ ID NO:4. Anti-glyco-MUC1 antibody or antigen-binding fragment comprising a phosphorus amino acid sequence.

20. The method of any one of embodiments 1 to 18, wherein the VH comprises an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 3, and the VL has at least 97% sequence identity to SEQ ID NO:4. Anti-glyco-MUC1 antibody or antigen-binding fragment comprising a phosphorus amino acid sequence.

21. The method of any one of embodiments 1 to 18, wherein the VH comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 3, and the VL has at least 99% sequence identity to SEQ ID NO:4. Anti-glyco-MUC1 antibody or antigen-binding fragment comprising a phosphorus amino acid sequence.

22. The anti-glyco-MUC1 antibody or antigen of any one of embodiments 1 or 2, wherein VH comprises the amino acid sequence of SEQ ID NO: 3 and VL comprises the amino acid sequence of SEQ ID NO: 4. -Combined fragments.

23. The multivalent anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 22.

24. The anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 1 to 22, which is in the form of a single chain variable fragment (scFv).

25. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 24, wherein the scFv comprises the N-terminal heavy chain variable fragment of the light chain variable fragment.

26. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 24, wherein the scFv heavy chain variable fragment and light chain variable fragment are covalently linked to a linker sequence of 4-15 amino acids.

27. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 22, which is in the form of a multispecific antibody.

28. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 27, wherein the multispecific antibody is a bispecific antibody that binds a second epitope different from the first epitope.

29. The anti-glyco-, according to embodiment 28, wherein the bispecific antibody is a crossmab, a Fab-arm exchange antibody, a bispecific T-cell engager (BiTE), or a bi-affinity retargeting molecule (DART). MUC1 antibody or antigen-binding fragment.

30. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is crossmab.

31. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 30, wherein the bispecific antibody is CrossMab ^FAB .

32. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 30, wherein the bispecific antibody is Crossmab ^VH-VL .

33. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 30, wherein the bispecific antibody is Crossmab ^CH1-CL .

34. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is a Fab-arm exchange antibody.

35. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is a bi-affinity retargeting molecule (DART).

36. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is a bispecific T-cell Engerer (BiTE).

37. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 28 to 35, wherein the second epitope is a MUC1 epitope.

38. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 28 to 35, wherein the second epitope is a MUC1 epitope overexpressed in cancer cells compared to normal cells.

39. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 28 to 36, wherein the second epitope is a T-cell epitope.

40. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 39, wherein the T-cell epitope comprises a CD3 epitope, CD8 epitope, CD 16 epitope, CD25 epitope, CD28 epitope, or NKG2D epitope.

41. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 40, wherein the T-cell epitope comprises a CD3 epitope, and optionally it is an epitope present on human CD3.

42. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 41, wherein the CD3 epitope comprises a CD3 gamma epitope, CD3 delta epitope, CD3 epsilon epitope, or CD3 zeta epitope.

43. The anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of embodiments 1 to 42, which is conjugated to a detectable moiety.

44. The anti-glyco-MUC1 antibody or antigen binding fragment of embodiment 43, wherein the detectable marker is an enzyme, radioisotope, or fluorescent label.

45. A bispecific antibody comprising a first antigen binding domain that binds CD3 (optionally human CD3) and a second antigen binding domain that binds glyco-MUC1, wherein the bispecific antibody is the breast cancer cell line MCF7 or T47D Competing with an antibody or antigen-binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to, the first antigen binding domain is sequenced A heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 34, CDR-H2 of SEQ ID NO: 35, and CDR-H3 of SEQ ID NO: 36; And a light chain variable region comprising a light chain CDR-L1 of SEQ ID NO: 37, a CDR-L2 of SEQ ID NO: 38 and a CDR-L3 of SEQ ID NO: 39.

46. The method of embodiment 45, wherein the second antigen binding domain comprises (i) CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7 Heavy chain variable region; And a light chain variable region comprising a light chain CDR-L1 of SEQ ID NO: 8, a CDR-L2 of SEQ ID NO: 9 and a CDR-L3 of SEQ ID NO: 10.

47. The heavy chain variable of embodiment 45 or 46, wherein the first antigen binding domain is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40. A bispecific antibody comprising a region sequence, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.

48. The bispecific antibody of embodiment 47, wherein the first antigen binding domain comprises a heavy chain variable region sequence of SEQ ID NO: 40 and a light chain variable region sequence of SEQ ID NO: 41.

49. The heavy chain of any one of embodiments 45 to 48, wherein the second antigen binding domain is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3. A bispecific antibody comprising a variable region sequence and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4.

50. The bispecific antibody of embodiment 49, wherein the second antigen binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 4.

51. The bispecific antibody of any one of embodiments 45-50, wherein the first and/or second antigen binding domain is a Fab molecule.

52. The bispecific antibody of embodiment 51, wherein the first antigen binding domain is a cross Fab molecule in which the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged.

53. The variable of Fab light chain and Fab heavy chain of embodiment 52, wherein the first and second antigen binding domains of the bispecific antibody are both Fab molecules and in one of the antigen binding domains (particularly the first antigen binding domain). The domains VL and VH are exchanged with each other, where

a. In the constant domain CL of the first antigen binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbered according to Kabat), and in the constant domain CH1 of the first antigen binding domain, the amino acid at position 147, or The amino acid at position 213 is replaced by a negatively charged amino acid (numbered according to the Kabat EU index); or

b. In the constant domain CL of the second antigen binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbered according to Kabat), and in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147, or The amino acid at position 213 is replaced by a negatively charged amino acid (numbered according to the Kabat EU index),

The constant domains CL and CH1 of the antigen binding domain with VH/VL exchange are not exchanged with each other

Bispecific antibodies.

54. The method of embodiment 53,

a. In the constant domain CL of the first antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), the first antigen binding domain In the constant domain CH1 of, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E), or aspartic acid (D) (numbered according to the Kabat EU index); or

b. In the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), the second antigen binding domain In the constant domain CH1 of, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index)

Bispecific antibodies.

55. The light of embodiment 54, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (number according to Kabat) Numbering), in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index ) Bispecific antibodies.

56. The light of embodiment 55, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (number according to Kabat) Numbering), in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) bispecific antibody .

57. The light of embodiment 55, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (number according to Kabat) Numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), in the constant domain CH1 of the second antigen binding domain, position 147 The amino acid at is independently substituted by glutamic acid (E), or aspartic acid (D) (numbered according to the Kabat EU index), and the amino acid at position 213 is independently of glutamic acid (E), or aspartic acid (D) Bispecific antibody (numbered according to the Kabat EU index).

58. The light chain of embodiment 57, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is replaced by lysine (K) (numbered according to Kabat), and at amino acid lysine (K) at position 123 Substituted (numbered according to Kabat), in the constant domain CH1 of the second antigen binding domain, substituted by glutamic acid (E) of the amino acid at position 147 (numbered according to the Kabat EU index), at position 213 Bispecific antibody in which the amino acid is substituted by glutamic acid (E) (numbered according to the Kabat EU index).

59. The light chain of embodiment 57, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is replaced by lysine (K) (numbered according to Kabat), and the amino acid at position 123 is arginine (R). Substituted (numbered according to Kabat), in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 is substituted by glutamic acid (E) (numbered according to the Kabat EU index), position 213 Bispecific antibody in which the amino acids of are substituted by glutamic acid (E) (numbered according to the Kabat EU index).

60. The bispecific antibody of any one of embodiments 53 to 59, wherein the constant domain CL of the second antigen binding domain is a kappa isotype.

61. The bispecific antibody of any one of embodiments 45-60, wherein the first and second antigen binding domains are fused to each other, optionally via a peptide linker.

62. The embodiment 61, wherein the first and second antigen binding domains are each Fab molecules, and (i) the second antigen binding domain is the C-terminus of the Fab heavy chain to the N-terminal of the Fab heavy chain of the first antigen binding domain. , Or (ii) a bispecific antibody wherein the first antigen binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain.

63. The bispecific antibody of any one of embodiments 45 to 62, wherein the bispecific antibody provides monovalent binding to CD3.

64. The bispecific antibody of embodiment 63, comprising two antigen binding domains that specifically bind glyco-MUC1.

65. The bispecific antibody of embodiment 64, wherein the two antigen binding domains specifically binding glyco-MUC1 comprise the same amino acid sequence.

66. The bispecific antibody of any one of embodiments 45 to 65, wherein the bispecific antibody further comprises an Fc domain consisting of first and second subunits.

67. The bispecific antibody of embodiment 66, wherein the Fc domain is an IgG Fc domain.

68. The bispecific antibody of embodiment 67, wherein the Fc domain is an IgG ₁ Fc domain.

69. The bispecific antibody of embodiment 67, wherein the Fc domain is an IgG ₄ Fc domain.

70. The bispecific antibody of embodiment 69, wherein the IgG ₄ Fc domain comprises an amino acid substitution at position S228 (Kabat EU index numbering), preferably the amino acid substitution S228P.

71. The bispecific antibody of embodiment 66, wherein the Fc domain is a human Fc domain.

72. The bispecific antibody of embodiment 71, wherein the Fc domain is a human IgG ₁ Fc domain, which optionally comprises SEQ ID NO: 42.

73. The method of any one of embodiments 66 to 72, wherein the first, second, and third antigen binding domains, if present, are each Fab molecules, and (a) (i) the second antigen binding domain is of a Fab heavy chain. Is fused to the N-terminus of the Fab heavy chain of the first antigen binding domain at the C-terminus, and the first antigen binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, or ( ii) the first antigen binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is the first sub of the Fc domain at the C-terminus of the Fab heavy chain Fused to the N-terminus of the unit; (b) A bispecific antibody wherein the third antigen binding domain, if present, is fused from the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

74. The bispecific antibody of any one of embodiments 66 to 73, wherein the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

75. The bispecific antibody of any one of embodiments 66 to 74, wherein the Fc domain comprises one or more amino acid substitutions that bind to an Fc receptor and/or reduce effector function.

76. The method of embodiment 45,

a. A first antigen binding domain that specifically binds CD3, wherein the first antigen binding domain is a cross Fab molecule in which the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged;

b. A heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; And a light chain variable region comprising the light chain CDR-L1 of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9, and the CDR-L3 of SEQ ID NO: 10, which specifically bind to glyco-MUC1. Second and third antigen binding domains, wherein the second and third antigen binding domains are Fab molecules, respectively;

c. Fc domain composed of first and second subunits that can stably associate

Including,

Wherein the second antigen binding domain is fused to the N-terminus of the Fab heavy chain at the C-terminus of the Fab heavy chain, and the first antigen binding domain is the first subunit of the Fc domain at the C-terminus of the Fab heavy chain Fused to the N-terminus of, the third antigen binding domain fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain

Bispecific antibodies.

77. The heavy chain variable of embodiment 77, wherein the first antigen binding domain comprises a heavy chain CDR-H1 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 35, and a CDR-H3 of SEQ ID NO: 36. domain; And a light chain variable region comprising a light chain CDR-L1 of SEQ ID NO: 37, a CDR-L2 of SEQ ID NO: 38 and a CDR-L3 of SEQ ID NO: 39.

78. The heavy chain variable region sequence and sequence of embodiment 77, wherein the first antigen binding domain is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40. A bispecific antibody comprising a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41.

79. The bispecific antibody of embodiment 78, wherein the first antigen binding domain comprises a heavy chain variable region sequence of SEQ ID NO: 40 and a light chain variable region sequence of SEQ ID NO: 41.

80. The method of any one of embodiments 76-79, wherein the second and third antigen binding domains are at least about 95%, 96%, 97%, 98%, 99% or 100 relative to the amino acid sequence of SEQ ID NO:3. % A bispecific antibody comprising the same heavy chain variable region sequence and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4.

81. The bispecific antibody of embodiment 80, wherein the second and third antigen binding domains comprise the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4.

82. The bispecific antibody of any one of embodiments 76-81, wherein the Fc domain comprises all of the features described in Sections 5.1 and 5.2 alone or in combination with respect to the Fc domain.

83. The bispecific antibody of any one of embodiments 76-82, wherein the antigen binding domain and Fc region are fused to each other by a peptide linker.

84. The bispecific antibody of embodiment 83, wherein the peptide linker comprises a peptide linker as in SEQ ID NO: 45 and/or SEQ ID NO: 46.

85. The amino acid of position 124 is substituted by lysine (K) in the constant domain CL of the second and third Fab molecules according to any one of embodiments 76 to 84 (numbering according to Kabat), position 123. The amino acid of is substituted by lysine (K) or arginine (R), preferably arginine (R) (numbered according to Kabat), in the constant domain CH1 of the second and third Fab molecules of (ii), Bispecific where the amino acid at position 147 is substituted by glutamic acid (E) (numbered according to the Kabat EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the Kabat EU index) Antibodies.

86. The sequence of any one of embodiments 76 to 85 that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 43. Polypeptide comprising, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 44, SEQ ID NO: 45 A polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of, and at least 80% to the sequence of SEQ ID NO: 46, A bispecific antibody comprising a polypeptide comprising 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical sequence.

87. The polypeptide of embodiment 86, wherein the bispecific antibody comprises the sequence of SEQ ID NO: 43, the polypeptide comprising the sequence of SEQ ID NO: 44, the polypeptide comprising the sequence of SEQ ID NO: 45, and A bispecific antibody comprising a polypeptide comprising the sequence of SEQ ID NO: 46.

88. The bispecific antibody of embodiment 87, comprising two polypeptides comprising the sequence of SEQ ID NO: 43.

89. The bispecific antibody of any of embodiments 45-88 conjugated to a detectable moiety.

90. The bispecific antibody of embodiment 89, wherein the detectable marker is an enzyme, radioisotope, or fluorescent label.

91. Anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 44 operably linked to at least a second amino acid sequence or amino acid sequence of the bispecific antibody of any one of embodiments 45 to 90 Fusion protein comprising a.

92. The fusion protein of embodiment 91, wherein the second amino acid sequence is from 4-1BB, CD3-zeta, or a fragment thereof.

93. The fusion protein of embodiment 91, wherein the second amino acid sequence is of a fusion peptide.

94. The fusion protein of embodiment 93, wherein the fusion peptide is a CD28-CD3-zeta or 4-lBB (CD137)-CD3-zeta fusion peptide.

95. The fusion protein of embodiment 91, wherein the second amino acid sequence is a modulator of T cell activation or a fragment thereof.

96. The fusion protein of embodiment 95, wherein the modulator of T cell activation is IL-15 or IL-15Ra.

97. A chimeric antigen receptor (CAR) comprising the scFv of any one of embodiments 24-26.

98. The CAR of embodiment 97, comprising a human CD8 leader peptide, scFv, human CD8 hinge domain, human CD8 transmembrane domain, and CD3-zeta signaling domain in amino-terminal to carboxy-terminal order.

99. Anti-glyco-MUC1 antibody of any one of embodiments 1 to 44 conjugated to a cytotoxic agent or antigen-binding fragment or bispecific antibody of any one of embodiments 45 to 90 or any of embodiments 91 to 96 Antibody-drug conjugate comprising one fusion protein.

100. The antibody-drug according to embodiment 99, wherein the cytotoxic agent is auristatin, DNA minor groove binding agent, alkylating agent, endynein, lexitroxine, duocarmycin, taxane, dolastatin, maytansinoid, or vinca alkaloid. Conjugate.

101. The antibody-drug conjugate of embodiment 100, wherein the anti-glyco-MUC1 antibody or antigen-binding fragment or bispecific antibody is conjugated to a cytotoxic agent via a linker.

102. The antibody-drug conjugate of embodiment 101, wherein the linker is cleavable under intracellular conditions.

103. The antibody-drug conjugate of embodiment 102, wherein the cleavable linker is cleavable by an intracellular protease.

104. The antibody-drug conjugate of embodiment 103, wherein the linker comprises a dipeptide.

105. The antibody-drug conjugate of embodiment 104, wherein the dipeptide is val-cit or phe-lys.

106. The antibody-drug conjugate of embodiment 102, wherein the cleavable linker is hydrolysable at a pH of less than 5.5.

107. The antibody-drug conjugate of embodiment 106, wherein the hydrolyzable linker is a hydrazone linker.

108. The antibody-drug conjugate of embodiment 102, wherein the cleavable linker is a disulfide linker.

109. Anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 1 to 44 or bispecific antibody of any of embodiments 45 to 90, fusion protein of any of embodiments 91 to 96, or A nucleic acid comprising the coding region for the CAR of embodiment 97 or 98.

110. The nucleic acid of embodiment 109, wherein the coding region is codon-optimized for expression in human cells.

111. A vector comprising the nucleic acid of embodiment 109 or embodiment 110.

112. The vector of embodiment 111, which is a viral vector.

113. The vector of embodiment 112, wherein the viral vector is a lentiviral vector.

114. A host cell engineered to express the nucleic acid of embodiment 109 or embodiment 110.

115. The host cell of embodiment 114, which is a human T-cell engineered to express the CAR of embodiment 97 or 98.

116. A host cell comprising the vector of any of embodiments 111-113.

117. The host cell of embodiment 116, which is T-cell and the vector encodes the CAR of embodiment 97 or embodiment 98.

118. (a) Anti-glyco-MUC1 antibody or antigen binding fragment of any one of embodiments 1 to 44, bispecific antibody of any one of embodiments 45 to 90, fusion protein of any of embodiments 91 to 96 , CAR of embodiment 97 or 98, antibody-drug conjugate of any of embodiments 99 to 108, nucleic acid of embodiment 109 or embodiment 110, vector of any of embodiments 111 to 113, or embodiment 114 A pharmaceutical composition comprising the host cell of any one of embodiments to 117, and (b) a physiologically appropriate buffer, adjuvant or diluent.

119. An effective amount of an anti-glyco-MUC1 antibody or antigen binding fragment of any one of embodiments 1 to 44 to a subject in need of cancer treatment, a bispecific antibody of any one of embodiments 45 to 90, embodiments 91 to Fusion protein of any one of 96, CAR of embodiment 97 or 98, antibody-drug conjugate of any of embodiments 99 to 108, nucleic acid of embodiment 109 or embodiment 110, any of embodiments 111 to 113 A method of treating cancer, comprising administering a vector of, a host cell of any one of embodiments 114-117, or a pharmaceutical composition of embodiment 118.

120. The method of embodiment 119, wherein the subject is suffering from breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer or colorectal cancer.

121. Biological, comprising contacting a sample with an anti-glyco-MUC1 antibody or antigen-binding fragment according to any of embodiments 1 to 44, and detecting binding of the anti-glyco-MUC1 antibody or antigen-binding fragment. How to detect cancer in a sample.

122. The method of embodiment 121, further comprising quantifying the binding of the anti-glyco-MUC1 antibody or antigen-binding fragment.

123. The method of embodiment 121 or embodiment 122, wherein binding is compared to a normal tissue control as a negative/baseline control and/or a cancerous tissue control as a positive control.

All publications, patents, patent applications and other documents cited in this application are hereby for all purposes to the same extent as individually indicated that each individual publication, patent, patent application or other document is incorporated by reference for all purposes. The full text is incorporated by reference. In the event of a discrepancy between one or more of the references contained herein and the teachings of the present disclosure, the teachings herein are intended.

8. References

SEQUENCE LISTING <110> GO Therapeutics, Inc. <120> ANTI-GLYCO-MUC1 ANTIBODIES AND THEIR USES <130> GOT-001WO <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 136 <212> PRT <213> Mus musculus <400> 1 Met Gly Trp Ser Gly Ile Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp His Ala Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Ile His Tyr Asn 65 70 75 80 Glu Lys Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Lys Arg Ser Tyr Asp Lys Asp Phe Asp Cys Trp Gly Gln 115 120 125 Gly Thr Thr Leu Thr Val Ser Ser 130 135 <210> 2 <211> 140 <212> PRT <213> Mus musculus <400> 2 Met Val Leu Ile Leu Leu Leu Leu Trp Val Ser Gly Thr Cys Gly Asp 1 5 10 15 Ile Val Met Ser Gln Ser Pro Ser Ser Leu Gly Val Ser Val Gly Glu 20 25 30 Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser Thr 35 40 45 Asn Gln Lys Asn Tyr Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn 50 55 60 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu 65 70 75 80 Ile Tyr Trp Val Ser Asn Arg Lys Ser Gly Val Pro Asp Arg Phe Thr 85 90 95 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys 100 105 110 Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Arg Tyr Pro 115 120 125 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 130 135 140 <210> 3 <211> 117 <212> PRT <213> Mus musculus <400> 3 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Ile His Tyr Asn Glu Lys Phe 50 55 60 Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Lys Arg Ser Tyr Asp Lys Asp Phe Asp Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 4 <211> 125 <212> PRT <213> Mus musculus <400> 4 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Gly Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Thr Asn Gln Lys Asn Tyr Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys 35 40 45 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu 50 55 60 Leu Ile Tyr Trp Val Ser Asn Arg Lys Ser Gly Val Pro Asp Arg Phe 65 70 75 80 Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val 85 90 95 Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Arg Tyr 100 105 110 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 <210> 5 <211> 8 <212> PRT <213> Mus musculus <400> 5 Gly Tyr Thr Phe Thr Asp His Ala 1 5 <210> 6 <211> 8 <212> PRT <213> Mus musculus <400> 6 Phe Ser Pro Gly Asn Asp Asp Ile 1 5 <210> 7 <211> 10 <212> PRT <213> Mus musculus <400> 7 Lys Arg Ser Tyr Asp Lys Asp Phe Asp Cys 1 5 10 <210> 8 <211> 12 <212> PRT <213> Mus musculus <400> 8 Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr 1 5 10 <210> 9 <211> 3 <212> PRT <213> Mus musculus <400> 9 Trp Val Ser One <210> 10 <211> 9 <212> PRT <213> Mus musculus <400> 10 Gln Gln Tyr Tyr Arg Tyr Pro Leu Thr 1 5 <210> 11 <211> 408 <212> DNA <213> Mus musculus <400> 11 atgggatgga gcgggatctt tctcttcttc ctgtcagtaa ctacaggtgt ccactcccag 60 gttcagctgc agcagtctga cgcggagttg gtgaaacctg gggcttcagt gaagatatcc 120 tgcaaggctt ctggctacac tttcactgac catgctattc actgggtgaa gcagaggcct 180 gaacagggcc tggaatggat tggatatttt tctcccggaa atgatgacat tcactacaat 240 gagaagttcg agggcaaggc cacactgact gcagacaaat cctccagcac tgcctacatg 300 cagctcaaca gcctgacatc tgaagattct gcagtgtatt tctgtaaaag atcttacgac 360 aaggactttg actgctgggg ccaaggcacc actctcacag tctcctca 408 <210> 12 <211> 384 <212> DNA <213> Mus musculus <400> 12 atggttctta tcttactgct gctatgggta tctggtacct gtggggacat tgtgatgtca 60 cagtctccat cctccctagg tgtgtcagtt ggagagaagg ttactatgag ctgcaagtcc 120 agtcagagcc ttttatacag taccaatcaa aagaactacc tggcctggta ccagcagaaa 180 ccagggcagt ctcctaagtt gctgatttac tgggtatcta ataggaaatc tggggtccct 240 gatcgcttca caggcagtgg atctgggaca gatttcactc tcaccatcag tagtgtgaag 300 gctgaagacc tggcagttta ttactgtcag caatattata ggtatccgct cacgttcggt 360 gctgggacca agctggagct gaaa 384 <210> 13 <211> 351 <212> DNA <213> Mus musculus <400> 13 caggttcagc tgcagcagtc tgacgcggag ttggtgaaac ctggggcttc agtgaagata 60 tcctgcaagg cttctggcta cactttcact gaccatgcta ttcactgggt gaagcagagg 120 cctgaacagg gcctggaatg gattggatat ttttctcccg gaaatgatga cattcactac 180 aatgagaagt tcgagggcaa ggccacactg actgcagaca aatcctccag cactgcctac 240 atgcagctca acagcctgac atctgaagat tctgcagtgt atttctgtaa aagatcttac 300 gacaaggact ttgactgctg gggccaaggc accactctca cagtctcctc a 351 <210> 14 <211> 339 <212> DNA <213> Mus musculus <400> 14 gacattgtga tgtcacagtc tccatcctcc ctaggtgtgt cagttggaga gaaggttact 60 atgagctgca agtccagtca gagcctttta tacagtacca atcaaaagaa ctacctggcc 120 tggtaccagc agaaaccagg gcagtctcct aagttgctga tttactgggt atctaatagg 180 aaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagtagtg tgaaggctga agacctggca gtttattact gtcagcaata ttataggtat 300 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339 <210> 15 <211> 25 <212> PRT <213> Mus musculus <400> 15 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25 <210> 16 <211> 17 <212> PRT <213> Mus musculus <400> 16 Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Tyr <210> 17 <211> 38 <212> PRT <213> Mus musculus <400> 17 His Tyr Asn Glu Lys Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp 20 25 30 Ser Ala Val Tyr Phe Cys 35 <210> 18 <211> 11 <212> PRT <213> Mus musculus <400> 18 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 19 <211> 26 <212> PRT <213> Mus musculus <400> 19 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Gly Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser 20 25 <210> 20 <211> 17 <212> PRT <213> Mus musculus <400> 20 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 21 <211> 36 <212> PRT <213> Mus musculus <400> 21 Asn Arg Lys Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 22 <211> 10 <212> PRT <213> Mus musculus <400> 22 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 1 5 10 <210> 23 <211> 5 <212> PRT <213> Mus musculus <400> 23 Asp His Ala Ile His 1 5 <210> 24 <211> 17 <212> PRT <213> Mus musculus <400> 24 Tyr Phe Ser Pro Gly Asn Asp Asp Ile His Tyr Asn Glu Lys Phe Glu 1 5 10 15 Gly <210> 25 <211> 8 <212> PRT <213> Mus musculus <400> 25 Ser Tyr Asp Lys Asp Phe Asp Cys 1 5 <210> 26 <211> 17 <212> PRT <213> Mus musculus <400> 26 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 27 <211> 7 <212> PRT <213> Mus musculus <400> 27 Trp Val Ser Asn Arg Lys Ser 1 5 <210> 28 <211> 7 <212> PRT <213> Mus musculus <400> 28 Gly Tyr Thr Phe Thr Asp His 1 5 <210> 29 <211> 6 <212> PRT <213> Mus musculus <400> 29 Ser Pro Gly Asn Asp Asp 1 5 <210> 30 <211> 13 <212> PRT <213> Mus musculus <400> 30 Ser Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr 1 5 10 <210> 31 <211> 7 <212> PRT <213> Mus musculus <400> 31 Tyr Tyr Arg Tyr Pro Leu Thr 1 5 <210> 32 <211> 10 <212> PRT <213> Mus musculus <400> 32 Gly Tyr Thr Phe Thr Asp His Ala Ile His 1 5 10 <210> 33 <211> 2 <212> PRT <213> Mus musculus <400> 33 Asp His One <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 CDR-H1 peptide <400> 34 Thr Tyr Ala Met Asn 1 5 <210> 35 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 CDR-H2 peptide <400> 35 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly <210> 36 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 CDR-H3 peptide <400> 36 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 37 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 CDR-L1 peptide <400> 37 Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 38 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 CDR-L2 peptide <400> 38 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 CDR-L2 peptide <400> 39 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 40 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 VH <400> 40 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 41 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 VL <400> 41 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 42 <211> 225 <212> PRT <213> Artificial Sequence <220> <223> synthetic hIgG1 Fc region <400> 42 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro 225 <210> 43 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> synthetic MUC1 VL-CL(RK) <400> 43 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Gly Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Thr Asn Gln Lys Asn Tyr Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys 35 40 45 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu 50 55 60 Leu Ile Tyr Trp Val Ser Asn Arg Lys Ser Gly Val Pro Asp Arg Phe 65 70 75 80 Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val 85 90 95 Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Arg Tyr 100 105 110 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val 115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys 130 135 140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 44 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD3 VH-CL <400> 44 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val 115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 45 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> MUC1 VH-CH1(EE)-Fc (hole, P329G LALA) <400> 45 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Ile His Tyr Asn Glu Lys Phe 50 55 60 Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Lys Arg Ser Tyr Asp Lys Asp Phe Asp Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 46 <211> 672 <212> PRT <213> Artificial Sequence <220> <223> synthetic MUC1 VH-CH1(EE)-CD3 VL-CH1-Fc (knob, P329G LALA) <400> 46 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Ile His Tyr Asn Glu Lys Phe 50 55 60 Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Lys Arg Ser Tyr Asp Lys Asp Phe Asp Cys Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro Ser 225 230 235 240 Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser 245 250 255 Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys 260 265 270 Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala 275 280 285 Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala 290 295 300 Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr 305 310 315 320 Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys 325 330 335 Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 340 345 350 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 355 360 365 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 370 375 380 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 385 390 395 400 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 405 410 415 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 420 425 430 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 435 440 445 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 450 455 460 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 465 470 475 480 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 485 490 495 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 500 505 510 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 515 520 525 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 530 535 540 Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 545 550 555 560 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 565 570 575 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 580 585 590 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 595 600 605 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 610 615 620 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 625 630 635 640 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 645 650 655 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665 670 <210> 47 <211> 60 <212> PRT <213> Homo sapiens <400> 47 Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro 1 5 10 15 Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly 20 25 30 Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg 35 40 45 Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 50 55 60 <210> 48 <211> 21 <212> PRT <213> Homo sapiens <400> 48 Pro Asp Thr Ser Ala Ala Pro Gly Ser Thr Ala Pro Pro Ala His Val 1 5 10 15 Val Thr Ser Ala Pro 20 <210> 49 <211> 21 <212> PRT <213> Macaca fascicularis <400> 49 Pro Asp Thr Ser Ala Ala Pro Gly Ser Thr Gly Pro Pro Ala His Val 1 5 10 15 Val Thr Ser Ala Pro 20 <210> 50 <211> 20 <212> PRT <213> Homo sapiens <400> 50 Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro 1 5 10 15 Pro Ala His Gly 20 <210> 51 <211> 20 <212> PRT <213> Homo sapiens <400> 51 Pro Thr Thr Thr Pro Ile Ser Thr Thr Thr Met Val Thr Pro Thr Pro 1 5 10 15 Thr Pro Thr Cys 20 <210> 52 <211> 21 <212> PRT <213> Homo sapiens <400> 52 Cys Pro Leu Pro Val Thr Asp Thr Ser Ser Ala Ser Thr Gly His Ala 1 5 10 15 Thr Pro Leu Pro Val 20

Claims (77)

a. Preferentially binds to the glyco-MUC1 epitope overexpressed in cancer cells compared to normal cells;
b. Competing with an antibody or antigen binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to the breast cancer cell line MCF7 or T47D doing
Anti-glyco-MUC1 antibody or antigen binding fragment. a. Binding to a glycosylated MUC1 serial repeat (VTSAPDTRPAPGSTAPPAHG) ₃ (hereinafter referred to as “first epitope”) using purified recombinant human glycosyltransferase GalNAc-T1, GalNAc-T2, and GalNAc-T4. and;
b. Competing with an antibody or antigen binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to the breast cancer cell line MCF7 or T47D
Anti-glyco-MUC1 antibody or antigen binding fragment. 3. The complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 25 of claim 1 or 2. CDR-H3 comprising the amino acid sequence, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO: 31 Anti-glyco-MUC1 antibody or antigen-binding fragment comprising CDR-L3. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO:5. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO:23. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO:28. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO:32. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO:6. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein the CDR-H2 comprises the amino acid sequence of SEQ ID NO:24. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein the CDR-H3 comprises the amino acid sequence of SEQ ID NO:7. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO:26. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein the CDR-L2 comprises the amino acid sequence of SEQ ID NO:27. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 3, wherein the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10. The anti-glyco- of claim 1 or 2, wherein the VH comprises the complementarity determining region (CDR) of SEQ ID NO: 5-7, and the VL comprises the CDR of SEQ ID NO: 8-10. MUC1 antibody or antigen-binding fragment. The term of claim 1 or 2, wherein the VH comprises the complementarity determining regions (CDRs) of SEQ ID NOs: 23-25, and the VL comprises the CDRs of SEQ ID NOs: 26, 27, and 10. -Glyco-MUC1 antibody or antigen-binding fragment. 3. The method of claim 1 or 2, wherein the VH comprises the complementarity determining regions (CDRs) of SEQ ID NOs: 28, 29, and 25, and the VL comprises the CDRs of SEQ ID NOs: 30, 9, and 31. Anti-glyco-MUC1 antibody or antigen-binding fragment. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1 or 2, which is a chimeric or humanized antibody. The amino acid sequence of claim 1 or 2, wherein VH comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3, and VL has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:4. Anti-glyco-MUC1 antibody or antigen-binding fragment comprising a. The amino acid sequence of claim 1 or 2, wherein the VH comprises an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 3, and the VL has an amino acid sequence with at least 97% sequence identity to SEQ ID NO:4. Anti-glyco-MUC1 antibody or antigen-binding fragment comprising a. The amino acid sequence of claim 1 or 2, wherein the VH comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 3, and the VL has an amino acid sequence with at least 99% sequence identity to SEQ ID NO:4. Anti-glyco-MUC1 antibody or antigen-binding fragment comprising a. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 or 2, wherein VH comprises the amino acid sequence of SEQ ID NO: 3, and VL comprises the amino acid sequence of SEQ ID NO:4. . The multivalent anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 or 2. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1 or 2, which is in the form of a single chain variable fragment (scFv). 25. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 24, wherein the scFv comprises the N-terminal heavy chain variable fragment of the light chain variable fragment. 25. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 24, wherein the scFv heavy chain variable fragment and light chain variable fragment are covalently linked to 4-15 amino acid linker sequences. The anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1 or 2, which is in the form of a multispecific antibody. 28. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 27, wherein the multispecific antibody is a bispecific antibody that binds a second epitope different from the first epitope. The bispecific antibody of claim 28 is CrossMab, Fab-arm exchange antibody, bispecific T-cellengerger (BiTE), or bi-affinity retargeting molecule (DART). Anti-glyco-MUC1 antibody or antigen-binding fragment. 30. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 29, wherein the bispecific antibody is crossmab. 31. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 30, wherein the bispecific antibody is CrossMab ^FAB . 31. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 30, wherein the bispecific antibody is CrossMab ^VH-VL . 31. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 30, wherein the bispecific antibody is Crossmab ^CH1-CL . 30. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 29, wherein the bispecific antibody is a Fab-arm exchange antibody. 30. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 29, wherein the bispecific antibody is a bi-affinity retargeting molecule (DART). 30. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 29, wherein the bispecific antibody is a bispecific T-cell engager (BiTE). 30. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 28, wherein the second epitope is a MUC1 epitope. 29. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 28, wherein the second epitope is an MUC1 epitope overexpressed in cancer cells compared to normal cells. 29. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 28, wherein the second epitope is a T-cell epitope. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 39, wherein the T-cell epitope comprises a CD3 epitope, a CD8 epitope, a CD 16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope. 41. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 40, wherein the T-cell epitope comprises a CD3 epitope, and optionally it is an epitope present on human CD3. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 41, wherein the CD3 epitope comprises a CD3 gamma epitope, CD3 delta epitope, CD3 epsilon epitope, or CD3 zeta epitope. The anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 or 2, which is conjugated to a detectable moiety. 44. The anti-glyco-MUC1 antibody or antigen binding fragment of claim 43, wherein the detectable marker is an enzyme, radioisotope, or fluorescent label. A fusion protein comprising the amino acid sequence of the anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 or 2 operably linked to at least a second amino acid sequence. 46. The fusion protein of claim 45, wherein the second amino acid sequence is from 4-1BB, CD3-zeta, or a fragment thereof. 46. The fusion protein of claim 45, wherein the second amino acid sequence is of a fusion peptide. The fusion protein of claim 47, wherein the fusion peptide is a CD28-CD3-zeta or 4-lBB (CD137)-CD3-zeta fusion peptide. 46. The fusion protein of claim 45, wherein the second amino acid sequence is a modulator of T cell activation or a fragment thereof. 50. The fusion protein of claim 49, wherein the modulator of T cell activation is IL-15 or IL-15Ra. A chimeric antigen receptor (CAR) comprising the scFv of claim 24. 52. The CAR of claim 51, comprising a human CD8 leader peptide, scFv, human CD8 hinge domain, human CD8 transmembrane domain, and CD3-zeta signaling domain in amino-terminal to carboxy-terminal order. An antibody-drug conjugate comprising the anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 or 2 conjugated to a cytotoxic agent. 54. The antibody-drug conjugate of claim 53, wherein the cytotoxic agent is auristatin, DNA minor groove binding agent, alkylating agent, endynein, lexitroxine, duocarmycin, taxane, dolastatin, maytansinoid, or vinca alkaloid. 55. The antibody-drug conjugate of claim 54, wherein the anti-glyco-MUC1 antibody or antigen-binding fragment is conjugated to a cytotoxic agent via a linker. The antibody-drug conjugate of claim 55, wherein the linker is cleavable under intracellular conditions. The antibody-drug conjugate of claim 56, wherein the cleavable linker is cleavable by an intracellular protease. The antibody-drug conjugate of claim 57, wherein the linker comprises a dipeptide. The antibody-drug conjugate of claim 58, wherein the dipeptide is val-cit or phe-lys. The antibody-drug conjugate of claim 56, wherein the cleavable linker is hydrolysable at a pH of less than 5.5. The antibody-drug conjugate of claim 60, wherein the hydrolyzable linker is a hydrazone linker. The antibody-drug conjugate of claim 56, wherein the cleavable linker is a disulfide linker. A nucleic acid comprising the coding region for the anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 or 2. 64. The nucleic acid of claim 63, wherein the coding region is codon-optimized for expression in human cells. A vector comprising the nucleic acid of claim 63. 66. The vector of claim 65, which is a viral vector. 67. The vector of claim 66, wherein the viral vector is a lentiviral vector. A host cell engineered to express the nucleic acid of claim 63. A host cell that is a human T-cell engineered to express the CAR of claim 51. A host cell comprising the vector of claim 65. A host cell that is a T-cell comprising the vector encoding the CAR of claim 51. A pharmaceutical composition comprising (a) the anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 or 2, and (b) a physiologically appropriate buffer, adjuvant or diluent. A method of treating cancer, comprising administering an effective amount of the anti-glyco-MUC1 antibody or antigen-binding fragment of claim 1 to a subject in need thereof. The method of claim 73, wherein the subject is suffering from breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer or colorectal cancer. Cancer in a biological sample comprising contacting the sample with an anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1 or 2 and detecting binding of the anti-glyco-MUC1 antibody or antigen-binding fragment. How to detect. The method of claim 75, further comprising quantifying the binding of the anti-glyco-MUC1 antibody or antigen-binding fragment. The method of claim 75, wherein the binding is compared to a normal tissue control as a negative/baseline control and/or a cancerous tissue control as a positive control.

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