Antiglyco-MUC1 antibody and its use

Abstract

To provide anti-glyco-MUC1 antibodies and antigen binding fragments thereof that specifically bind to a cancer-specific glycosylation variant of MUC1; to provide related fusion proteins and antibody-drug conjugates; to provide nucleic acids encoding such biomolecules; and to provide use of the antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates and nucleic acids for cancer therapy.SOLUTION: Provided is an anti-glyco-MUC1 antibody or antigen binding fragment, characterized by a. preferentially binding to a glyco-MUC1 epitope that is overexpressed on cancer cells as compared to normal cells; and b. competing with an antibody or antigen binding fragment comprising a heavy chain variable (VH) sequence and a light chain variable (VL) sequence of a specific sequence for binding to the breast cancer cell line MCF7 or T47D.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, thereby The entire text is incorporated by reference. The aforementioned ASCII copy was published on October 24, 2017. It was created and named GOT-001WO_Sequence_Listing.txt It is 33,265 bytes in size. [Background technology]

[0002] 2.Background Human mucin MUC1 is a pleomorphic transmembrane expressed on the apical surface of monolayer epithelium and glandular epithelium. It is a glycoprotein (Taylor-Papadimitriou et al., 1999). MUC1 is elevated in adenocarcinoma. It is overexpressed and abnormally O-glycosylated. The extracellular domain of mucin has five A variable number tandem of 20 amino acid residues with a site for possible O-glycosylation. It contains repeats (TR) (25-125). O-glycans are incomplete in cancer cells. It is processed, resulting in pancarcinoma carbohydrate antigen Tn(GalNAcα Expression of 1-O-Ser / Thr) occurs (Springer, 1984). Simple mucin-type O-glycosyl Tn, a type of cancer, is widely expressed in adenocarcinomas (including breast and ovarian cancers) and in normal adult tissues. These O-glycans exhibit a limited distribution (Springer, 1984). Expression of these O-glycans in cancer. This is correlated with a poor prognosis, and cancer patients have natural antibodies against these carbohydrate haptens. This increases (Miles et al., 1995; Soares et al., 1996; Werther et al., 1996). In the industry, there is a growing interest in therapies that utilize the glycoMUC1 epitope, which is overexpressed in cancer cells. It is necessary. [Overview of the project]

[0003] 3. Summary This disclosure relates to antibodies and antigen-binding agents selective to the cancer-specific epitope of glycoMUC1. By providing therapeutic and diagnostic agents based on lagment, the glycopeptide barrier This acquires tumor specificity for the target.

[0004] The disclosure of this invention relates to an antiglycerid that binds to cancer-specific glycosylated variants of MUC1. The present invention provides an MUC1 antibody and its antigen-binding fragment. Further disclosures of the present invention include an anti-g Lyco-MUC1 antibody and antigen-binding fragment fusion protein and antibody-drug combination Injugates, as well as anti-glycoMUC1 antibodies, antigen-binding fragments, and fusion proteins It provides nucleic acids that code for chromosomes.

[0005] The disclosure of the present invention further relates to anti-glycoMUC1 antibodies, antigen-binding fragments for cancer therapy. This invention provides methods for using nucleotides, fusion proteins, antibody-drug conjugates, and nucleic acids.

[0006] In certain embodiments, this disclosure relates to cancer-specific glycosylated variants of MUC1, Antiglycerides MU with bispecificity and other multispecificity that bind to a second epitope. The C1 antibody and antigen-binding fragment are provided. The second epitope is MUC1 itself Another protein co-expressed in the body, on cancer cells, or other cells such as activated T cells It can be any other protein present on the cell. Furthermore, codon Nucleic acids containing optimized coding regions, and codon optimization for expression in specific host cells. nucleic acids encoding antibodies, such as nucleic acids that include uncoded coding regions, are also disclosed.

[0007] The anti-glycoMUC1 antibody and its binding fragment contain a fusion tangent containing a fusion partner. It can take the form of a protein. The fusion partner has a second function, for example, T cell signaling. Signaling function of protein signaling domains, peptide modules for T cell activation It may be useful in providing an enzymatic element for a labeling system or labeling system. Examples of T cell signaling proteins include 4-1BB, CO3C, and fusion peptides. Examples include CD28-CD3-zeta and 4-1BB-CD3-zeta. 4-1BB, or CD137, is a T cell costimulatory receptor, and CD3-zeta is a T cell receptor. It is a signaling element of cell antigen receptors. The part that provides the second function is T cell activation. modulators such as IL-15, IL-15Ra, or IL-15 / IL-15 It may also be Ra fusion, or the degree of binding in vivo or in vitro and / or It codes for the labeling or enzymatic elements of a labeling system that is useful for monitoring location. This may be done under the conditions of T cells, for example, autologous T cells, and their prophylactic and therapeutic activity. A construct encoding a biomolecule having such a construct is, in some embodiments of this disclosure, A potent drug for supplementing adopted T cells to prevent or treat various cancers. We provide rat forms.

[0008] In certain embodiments, the anti-glycoMUC1 antibody or antigen-binding fragment of the Disclosure is Table 1 includes heavy and / or light chain variable sequences (or nucleotide sequences) (Coded). For clarity, the term “anti-glycoMUC1 antibody” is used in this document. If so, unless otherwise indicated in the context, it refers to single specificity and multiple specificity (double Anti-glycoMUC1 antibodies (including specificity), monospecific and multispecific antibodies, antigen-binding flux A fusion protein containing an antibody and its antigen-binding fragment It is intended to include conjugates. Similarly, the term "anti-glycoMUC1 antibody or When the term "antigen-binding fragment" is used, it also means that, unless otherwise indicated in the context, Monospecific and multispecific (including bispecific) anti-glycoMUC1 antibodies and their antigens In addition to binding fragments, fusions containing such antibody and antigen-binding fragments. It is intended to include proteins and conjugates as well.

[0009] In other embodiments, the anti-glycoMUC1 antibody or antigen-binding fragment of this disclosure is shown in the table. Includes the heavy chain and / or light chain CDR sequences described in 1-3 (or nucleotide sequences) (This is coded). The CDR sequences listed in Table 1 are IM sequences used to define the boundaries of the CDRs. GT (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77, Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Se rvice, National Institutes of Health, Bethesda, Md.), and Chothia (Al-Lazikan CDR sequences defined according to the scheme of i et al., 1997, J. Mol. Biol 273:927-948) Includes. The CDR sequences listed in Table 2 are combinations of the CDR sequences shown in Table 1. This is the wrap region, and the IMGT, Kabat, and Chothia sequences are shown in bold underline. The CDR sequences listed in Table 3 are the common overlaps of the CDR sequences shown in Table 1. This is the region. Such an anti-glycoMUC1 antibody and antigen-binding fragment framework The framework sequence may be a natural mouse framework sequence as listed in Table 1, or a non- This may also be a natural (e.g., humanized or human) framework sequence.

[0010] [Table 1-1]

[0011] [Table 1-2]

[0012] [Table 1-3]

[0013] [Table 1-4]

[0014] [Table 2]

[0015] [Table 3]

[0016] In certain embodiments, the Disclosure may refer to the anti-glycoMUC1 antibody or antigen-binding flag of the Disclosure. The ment is an amino acid from any of the CDR combinations described in the numbered embodiments 3 to 17. It provides that the CDR includes an acid sequence. Therefore, in certain embodiments, this invention The anti-glycoMUC1 antibody or antigen-binding fragment shown is the amino acid sequence of SEQ ID NO: 33. CDR-H1 contains the amino acid sequence of SEQ ID NO: 29, CDR-H2 contains the amino acid sequence of SEQ ID NO: 25 CDR-H3 containing amino acid sequence, CDR-L1 containing amino acid sequence of SEQ ID NO: 8, sequence CDR-L2 containing the amino acid sequence of number 9, and C containing the amino acid sequence of sequence number 31. Includes DR-L3. In some embodiments, CDR-H1 is sequence numbers 5, 23, 28. , or containing a sequence of 32 amino acids. In some embodiments, CDR-H2 is sequence number It contains amino acid sequence number 6 or 24. In some embodiments, CDR-H3 is sequence It contains the amino acid sequence number 7. In some embodiments, CDR-L1 is sequence number 30 or containing a sequence of 26 amino acids. In some embodiments, CDR-L2 is sequence number It contains a sequence of 27 amino acids. In some embodiments, CDR-L3 is the same as in SEQ ID NO: 10 Includes an amino acid sequence. In other embodiments, the anti-glycoMUC1 antibody or antigen binding of the disclosure. The fragment contains heavy chain CDRs of SEQ ID NOs. 5-7 and light chain CDRs of SEQ ID NOs. 8-10. In other embodiments, the anti-glycoMUC1 antibody or antigen-binding fragment of the present disclosure is Heavy chain CDRs of SEQ ID NOs. 23-25 ​​and light chain CDRs of SEQ ID NOs. 26, 27, and 10 This includes, in other embodiments, the anti-glycoMUC1 antibody or antigen-binding fragment of the Disclosure. This includes the heavy chain CDRs of SEQ ID NOs. 28, 29, and 25, and SEQ ID NOs. 30, 9, and 3 Contains 1 light chain CDR. In other embodiments, the anti-glycoMUC1 antibody or antigen of the Disclosure. The combined fragments are the heavy chain CDRs of SEQ ID NOs. 32, 24, and 7, and SEQ ID NO. 26. Includes 27 and 10 light chain CDRs. In other embodiments, the present disclosure contains antiglycoMUC1 anti The body or antigen-binding fragment is the heavy chain CDR of SEQ ID NOs. 33, 29, and 25, as well as It contains the light chain CDRs of SEQ ID NOs. 8, 9, and 31. The antibody or antigen-binding fragment is Mouse antibodies, chimeric antibodies, humanized antibodies, or human antibodies, or their antigen-binding flags. It could also be a comment.

[0017] In a further embodiment, the anti-glycoMUC1 antibody or antigen-binding fragment of the present disclosure is The antibody or antigen-binding filaments containing the heavy chain and light chain variable regions of SEQ ID NOs. 3 and 4, respectively. It competes with the gument. In further embodiments, this disclosure relates to Sequence IDs 3 and 4, respectively. Heavy chains having at least 95%, 98%, 99%, or 99.5% sequence identity The present invention provides an anti-MUC1 antibody or antigen-binding fragment having a light chain variable region.

[0018] In yet another embodiment, the anti-glycoMUC1 antibody or antigen-binding fragment of the present disclosure. This is a single-chain variable fragment (scFv). An example scFv is a light-chain variable fragment. The N-terminal side of the nt contains a heavy chain variable fragment. In some embodiments, the heavy chain of scFv Chain-variable fragments and light-chain-variable fragments have a linker sequence of 4-15 amino acids. They are covalently bonded. scFv may be in the form of a bispecific T cell derivative, or This may also be within a chimeric antigen receptor (CAR).

[0019] Antiglyco-MUC1 antibodies and antigen-binding fragments are multimers of single-chain variable fragments. , bispecific single-chain variable fragments, and polymers of bispecific single-chain variable fragments It may also be in a different form. In some embodiments, the polymer of the single-chain variable fragment is divalent. Select from single-chain variable fragments, tribodies, or tetrabodies. Selected. In some of these embodiments, a multimer of bispecific single-chain variable fragments It is a bispecific T cell derivative.

[0020] Other aspects of this disclosure involve the anti-glycoMUC1 antibody and antigen-binding fragment of this disclosure. We are interested in nucleic acids that are involved in this process. In some embodiments, anti-glycoMUC1 antibodies and The nucleic acid portion that codes for antigen-binding fragments is necessary for expression in human cells. It is optimized. In certain embodiments, this disclosure refers to Sequence ID 11 or Sequence ID 13. and heavy chain nuclei having at least 95%, 98%, 99%, or 99.5% sequence identity The leotide sequence, and at least 95%, 98%, 9% of sequence numbers 12 or 14. Encoded by light chain nucleotide sequences with 9% or 99.5% sequence identity An anti-glycoMUC1 antibody or antigen-binding fragment having heavy chain and light chain variable regions Provides a vector containing nucleic acid (for example, a viral vector such as a lentiviral vector). -) and host cells are also within the scope of this disclosure. The sequences encoding the heavy chain and light chain are single It may exist in the vector, or it may exist in a separate vector.

[0021] Further aspects of this disclosure include anti-glycoMUC1 antibodies, antigen-binding fragments, nucleic acids ( (or pairs of nucleic acids), vectors (or pairs of vectors) or host cells as a result of this disclosure, and A pharmaceutical composition comprising a physiologically suitable buffer, adjuvant, or diluent.

[0022] A further aspect of this disclosure is a method for producing a chimeric antigen receptor, wherein the coding region Incubate cells containing nucleic acids or vectors according to the present disclosure under conditions suitable for expression. The method includes the actions of collecting chimeric antigen receptors.

[0023] Another aspect of this disclosure is a method for detecting cancer, wherein a cell or tissue sample is subjected to this disclosure. Contact with the indicated anti-glycoMUC1 antibody or antigen-binding fragment, and the antibody A method that includes detecting whether or not something is bound to a cell or tissue sample.

[0024] Another aspect of this disclosure is a method for treating cancer, in a prophylactic or therapeutically effective amount This disclosure relates to anti-glycoMUC1 antibodies, antigen-binding fragments, nucleic acids, vectors, and host cells. A method comprising administering cells or a pharmaceutical composition to a subject requiring it. [Brief explanation of the drawing]

[0025] [Figure 1] This figure shows the results of an ELISA assay demonstrating the binding specificity of GO2 to glyco-MUC1 in relation to MUC1. [Figure 2] This figure shows the binding of GO2 to colon cancer tissue. Immunohistochemical labeling of invasive colon cancer tissue and adjacent healthy tissue using GO2 mAbs. GO2 mAbs show distinct binding to colon cancer tissue and exhibit high reactivity both intracellularly and on the surface structure of cancer cells. In contrast, no reactivity is observed on the surface structure of healthy colon cells. [Figure 3]This figure shows the binding of GO2 to pancreatic cancer tissue. Immunohistochemical labeling of pancreatic cancer tissue using mAb GO2. mAb GO2 shows distinct binding to pancreatic cancer cells. In contrast, responsiveness to surrounding healthy tissue was absent or limited. [Figure 4] This figure shows the binding of GO2 to breast cancer tissue. Immunohistochemical labeling of breast cancer tissue using mAb GO2. mAb GO2 showed distinct binding to invasive breast cancer cells. [Figure 5] This figure shows the results of an antibody-dependent cell-mediated cytotoxicity assay using the antibody GO2 and a secondary antibody conjugated with the antitubulin agent monomethyl auristatin F (MMAF). [Figure 6] This figure shows the results of an ELISA assay using GO2 to quantify circulating tumor cells. The X-axis represents the number of cells, and the Y-axis represents the OD450 value. [Figure 7] This figure shows representative images of the core of MUC1-positive TMA tumors. Figure 7A: Breast cancer; Figure 7B: Non-small cell lung cancer; Figure 7C: Ovarian cancer; Figure 7D: Colorectal cancer; Figure 7E: Prostate cancer. [Figure 8] This is a schematic diagram of exemplary anti-glycoMUC1 and anti-CD3 T cell bispecific antibodies (TCBs). [Figure 9A] This figure shows Jurkat-NFAT activation assays using undigested patient-derived tumor samples (malignant neoplasms of the bronchi and lung: middle lobe, bronchi, or lung, squamous cell carcinoma) and different 50 nM TCBs. [Figure 9B] This figure shows Jurkat-NFAT activation assays using undigested patient-derived tumor samples (malignant neoplasms of the bronchi and lung: middle lobe, bronchi, or lung, squamous cell carcinoma) and different 5nM TCBs. [Figure 10] This figure shows Jurkat-NFAT activation assays using undigested patient-derived tumor samples (malignant neoplasms of the bronchi and lung: lower lobe, bronchi, or lung, non-keratinizing squamous cell carcinoma) and different 50 nM TCBs. [Figure 11]This figure shows Jurkat-NFAT activation assays using undigested patient-derived tumor samples (malignant neoplasms of the bronchi and lung: adenocarcinoma with acinar form, upper lobe, bronchi, or lung) and different 50 nM TCBs. [Figure 12A] This figure shows the binding of GO2 TCB to MUC1 expressed on MCF7 cs cells, as measured by flow cytometry. [Figure 12B] This figure shows the binding of GO2 TCB to MUC1 expressed on T3M4 pzfv cells, as measured by flow cytometry. [Figure 13-1] This figure shows the upregulation of CD25 and CD69 on CD4 T cells and CD8 T cells in the presence of PBMCs derived from two healthy donors, as well as the induction of tumor cell lethality and T cell activation, as measured by the release of IL6, IL8, IL10, IFNγ, TNFα, and granzyme B by GO2 TCBs on T3M4 pzfv cells (Donor 1, Figures 13A-13L). The legend is the same for each of Figures 13A-13X. [Figure 13-2] This figure shows the upregulation of CD25 and CD69 on CD4 T cells and CD8 T cells in the presence of PBMCs derived from two healthy donors, as well as the induction of tumor cell lethality and T cell activation, as measured by the release of IL6, IL8, IL10, IFNγ, TNFα, and granzyme B by GO2 TCBs on T3M4 pzfv cells (Donor 1, Figures 13A-13L). The legend is the same for each of Figures 13A-13X. [Figure 13-3] This figure shows the upregulation of CD25 and CD69 on CD4 T cells and CD8 T cells in the presence of PBMCs derived from two healthy donors, as well as the induction of tumor cell lethality and T cell activation, as measured by the release of IL6, IL8, IL10, IFNγ, TNFα, and granzyme B by GO2 TCBs on T3M4 pzfv (Donor 2, Figures 13M-13X). The legend is the same for each of Figures 13A-13X. [Figure 13-4]This figure shows the upregulation of CD25 and CD69 on CD4 T cells and CD8 T cells in the presence of PBMCs derived from two healthy donors, as well as the induction of tumor cell lethality and T cell activation, as measured by the release of IL6, IL8, IL10, IFNγ, TNFα, and granzyme B by GO2 TCBs on T3M4 pzfv (Donor 2, Figures 13M-13X). The legend is the same for each of Figures 13A-13X. [Figure 14] These figures show the tumor cell lethality (Figures 14A-14B) and the induction of T cell activation, as measured by the upregulation of CD25 and CD69 on CD8 T cells and CD4 T cells using GO2 TCBs on MCF7 cs in the presence of PBMCs (Figures 14C-14F, respectively). The legend is the same for each of Figures 14A-14F. [Figure 15] This figure shows the binding of GO2 TCB and HMFG1 TCB to MCF10A (human non-tumor-forming mammary epithelial cell line) (Figure 15A) and HBEpiC (human bronchial epithelial cells) (Figure 15B). [Figure 16] This figure shows tumor cell lethality (Figure 16A) and induction of T cell activation, as measured by the upregulation of CD25 in CD4 T cells (Figure 16B) and CD8 T cells (Figure 16C) by GO2 TCBs and HMFG1 TCBs on MCF10A cells in the presence of PBMCs. [Figure 17] This is an illustration of GO2 and GO2 TCB flowing through a flow cell to which glycopeptides are coupled. [Figure 18A] This is a sensorgram showing the binding of GO2 to glycopeptides in humans and cynomolgus monkeys. [Figure 18B] This is a sensorgram showing the binding of GO2 TCB to glycopeptides in humans and cynomolgus monkeys. [Figure 19-1] Figures 19A-B show the binding (binding activity) of GO2 antibodies to human and cynomolgus monkey glycopeptides, and estimated "apparent" KD values. [Figure 19-2]Figures 19C-D show the binding (binding activity) of GO2 TCB to glycopeptides in humans and cynomolgus monkeys, and estimated "apparent" KD values. [Modes for carrying out the invention]

[0026] 5. Detailed explanation 5.1 Antibodies The inventors have developed a novel anti- The body was developed. These are exemplified by the antibody 5F7, which is referred to herein as "GO2". To mimic the glycosylation pattern of MUC1 presented on tumor cells, purified... GalNAc-T2 is a recombinant human glycosyltransferase polypeptide. Glycosylated with GalNAc-T4 and GalNAc-T1, in MUC1 One of the existing tandem repeats is VTSAPDTRPAPGSTAPPAHG (sequence number) Regarding antibodies that bind to three representative copies of glycosylated 60-mer of product 50): GO2 was identified through the screening process.

[0027] The anti-glycoMUC1 antibody of this disclosure is exemplified by antibody GO2 and is used in the diagnosis and treatment of cancer. It is a useful tool in law.

[0028] Therefore, in certain embodiments, this disclosure relates to the glycation of MUC1 presented on tumor cells. The coform (referred to herein as "GlycoMUC1") preferably in the United States GalNAc-T2, GalNAc-T4, as described in Specification No. 6,465,220 and a 60-mer peptide glycosylated with GalNAc-T1 (VTSAPD Antibodies and antigen-binding fungibles that bind to TRPAPGSTAPPAHG)3 (SEQ ID NO: 47) Provide incentives.

[0029] The anti-glycoMUC1 antibodies of this disclosure are polyclonal, monoclonal, and genetically engineered. Furthermore, the properties may be modified in any other way, for example, These include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, primate-derived antibodies, single-chain antibodies, Examples include bispecific antibodies and bivariable domain antibodies. In various embodiments, the anti The body includes all or part of the constant region of the antibody. In some embodiments, the constant region is IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG 1. IgM (IgG2, IgG3 or IgG4), and an isotype selected from IgM Yes. In a specific embodiment, the anti-glycoMUC1 antibody of this disclosure is used in the constant region of IgG1. Includes isotypes within the region.

[0030] The term "monoclonal antibody," as used herein, refers to antibodies produced via hybridoma technology. Not limited to antibodies produced by monoclonal antibodies, monoclonal antibodies can be produced by any eukaryote, prokaryote, or This includes phage clones, which are any available or known in the industry. Derived from a single clone by means of the present invention. A monoclon useful in the disclosure of the present invention Naru antibodies utilize hybridoma, recombinant, and phage display technologies, or those related to them. It can be prepared using various techniques known in the industry, including the use of combinations of these. Many uses of the disclosure of this invention include the in vivo use of anti-glycoMUC1 antibodies in humans. In this application, chimeric antibodies, primate-derived antibodies, humanized antibodies, or human antibodies are preferably used. It is possible.

[0031] The term "chimeric" antibody, as used herein, refers to non-human immunoglobulins, for example, Variable sequences derived from a mouse antibody or similar, and typically human immunoglobulin templates. This refers to an antibody that has a selected human immunoglobulin constant region. The methods are publicly known in this industry. For example, by this reference, they are all included in this specification. Incorporated into the book, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, B ioTechniques 4:214-221;Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent No. 5,807,715; Patent No. 4,816,567; and No. 4, Please refer to Specification No. 816397.

[0032] The "humanized" form of non-human (e.g., mouse) antibodies is derived from non-human immunoglobulins. It is a chimeric immunoglobulin containing the minimum sequence of the CDR region. Generally, humanized antibodies are CDR regions. The entire or substantially entire region corresponds to the CDR region of non-human immunoglobulins, and the FR region At least one that is entirely or substantially entirely the FR region of a human immunoglobulin sequence Typically, it is expected to contain virtually all of the two variable domains. Humanized antibodies are immunodeficiency antibodies. At least a portion of the constant region (Fc) of epidemic globulins, typically human immunoglobulin concepts It may contain at least a portion of the nucleotide sequence. The method of antibody humanization is in the industry It is publicly known. For example, all of them are incorporated herein by reference, Riec hmann et al., 1988, Nature 332:323-7; Queen et al., U.S. 5,530,101 Specification No. 5,585,089; Specification No. 5,693,761; No. 5,69 Patent No. 3,762; and Patent No. 6,180,370; European Patent No. 239400 Specification; PCT International Publication No. 91 / 09967 Pamphlet; U.S. 5,225,5 Specification No. 39; European Patent No. 592106; European Patent No. 519596; Pa dlan, 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 the United States Please refer to Specification No. 5,565,332.

[0033] "Human antibodies" include antibodies that have the amino acid sequence of human immunoglobulins, and furthermore, Antibodies isolated from an immunoglobulin library, or one or more human immunoglobulins Are these animals transgenic in terms of immunoglobulin and do not express endogenous immunoglobulin? It includes isolated antibodies. Human antibodies are from a library of antibodies derived from human immunoglobulin sequences. This is produced by various methods known in the industry, such as phage display methods that use These can be manufactured. Each of these is incorporated herein by reference in whole. U.S. Patent No. 4,444,887 and No. 4,716,111; PCT International Publication No. 98 / 46645; International Publication No. 98 / 50433 Pamphlet; International Publication No. 98 / 24893 Pamphlet; International Publication No. 98 / 1665 Pamphlet No. 4; International Publication No. 96 / 34096; International Publication No. 96 / 33 See Pamphlet No. 735 and International Publication No. 91 / 10741. Human antibodies also cannot express functional endogenous immunoglobulins, but human immunoglobulins It can also be produced by using transgenic mice that can express the phosphorus gene. Yes, it is possible. For example, the PCT International Publication, which is incorporated herein in its entirety by reference. Pamphlet No. 98 / 24893; International Publication Pamphlet No. 92 / 01047; International Public release pamphlet No. 96 / 34096; International release pamphlet No. 96 / 33735; U.S. Patent No. 5,413,923; Patent No. 5,625,126; Patent No. 5,633 ,425 specification;No. 5,569,825 specification;No. 5,661,016 specification;No. Specification No. 5,545,806; Specification No. 5,814,318; Specification No. 5,885,793 See the specifications; Specification No. 5,916,771; and Specification No. 5,939,598. I want to. Fully human antibodies that recognize selected epitopes are called "guided selection." It can be generated using the technology described. In this approach, selected non-human Monoclonal antibodies, such as mouse antibodies, are a selection of fully human antibodies that recognize the same epitope. Used to guide the selection (refer to Jespers et al., 1988, Biotechnology 12:899-903). see).

[0034] "Primate-like antibodies" contain both a monkey variable region and a human constant region. The methods for doing so are publicly known in this industry. For example, by reference, they are all included in this specification. Incorporated in this document are U.S. Patent No. 5,658,570; No. 5,681,722. See the specification; and Specification No. 5,693,780.

[0035] The anti-glycoMUC1 antibody disclosed herein consists of a full-length (intact) antibody molecule and a molecule that binds to glycoMUC1. It includes both antigen-binding fragments that can be used. Examples of antigen-binding fragments include For example, though not limited to these, Fab, Fab', F(ab')2, FvF Examples include ligments, single-chain Fv fragments, and single-domain fragments.

[0036] The Fab fragment consists of a constant domain in the light chain (CL) and a first constant domain in the heavy chain. It contains (CH1). The Fab' fragment is one or from the hinge region of the antibody. The attachment of several residues at the carboxyl terminus of the heavy chain CH1 domain containing multiple cysteines. The addition makes it different from the Fab fragment. The F(ab') fragment is F(ab')2 It is produced by the cleavage of disulfide bonds in the pepsin digestion product, hinge cysteine. The additional chemical coupling of antibody fragments is known to those skilled in the art. The F(ab')1 fragment lacks the Fc fragment of the intact antibody and is faster than the intact antibody. They are eliminated from the animal's circulation and may have less nonspecific tissue binding than intact antibodies. (See, for example, Wahl et al., 1983, J. Nucl. Med. 24:316).

[0037] The "Fv" fragment is the smallest flag of an antibody that contains the complete target recognition and binding site. This is a ment. This region is a single heavy chain and in association by a firm non-covalent bond. Dimer of one light chain variable domain (V H -V L It consists of a dimer. In this stereoconfiguration, each possible The three CDRs in the variant domain interact, V H -VL The target binding site on the dimer surface is defined. Often the six CDRs confer target binding specificity to the antibody. However , in some cases, a single variable domain (or half of the Fv containing only the three CDRs specific for the target) may, although with a lower affinity than the entire binding site, have the ability to recognize and bind to the target.

[0038] A "single-chain Fv" or "scFv" antigen-binding fragment contains the V H and V L domains , in which case these domains are present in a single polypeptide chain. Generally , the Fv polypeptide further contains a polypeptide linker between the V H domain and the V L domain, which enables the scFv to form a structure desirable for target binding.

[0039] A "single-domain antibody" is composed of a single V H or V L domain that exhibits sufficient affinity for glycoMUC1. In a specific embodiment, the single-domain antibody is a camelized antibody (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0040] Also, the anti-glycoMUC1 antibodies of the present disclosure may be bispecific and other multispecific antibodies. Bispecific antibodies are monoclonal antibodies having binding specificity for two different epitopes on the same or different antigens, often human or humanized antibodies. In the disclosure of the present invention, one of the binding specificities may be directed toward glycoMUC1. On the other hand, any other antigen, such as cell surface proteins, receptors, receptor subunits Tissue-specific antigens, virus-derived proteins, virus-encoded envelopes Directed towards rope proteins, bacterial proteins, or bacterial surface proteins, etc. It may be. In certain preferred embodiments, it may have two specificities and other multiple specificities. The anti-glycoMUC1 antibody and antigen-binding fragment target the second MUC1 epitope, Epitopes on other proteins co-expressed with MUC1 on rhinocytes, or activated T cells It specifically binds to epitopes on other proteins present on different cells. The bispecific antibodies shown are IgG-type bispecific antibodies and single-chain-based bispecific antibodies. Includes.

[0041] The IgG-like bispecific antibodies of this disclosure are various types of IgG-like antibodies known in the industry. One of the bispecific antibodies in the formula, for example, quadroma bispecific antibody, "nob-in- "Hole (knobs-in-hole)" bispecific antibody, CrossMab bispecific antibody, charge pair Bispecific antibodies with a specific property, general light chain bispecific antibodies, single-arm Fab-immunoglobulin Robulin gamma bispecific antibody, disulfide-stabilized Fv bispecific antibody, Du etMab, controlled Fab arm exchange bispecific antibody, chain exchange operated domain D's bispecific antibody, 2-arm leucine zipper heterodimer monoclonal bispecific Heterogender antibodies, κλ body bispecific antibodies, bivariable domain bispecific antibodies, and cross-reactivity. They may also be bivariable domain bispecific antibodies. For example, this reference will show their whole Kohler and Milstein, 1975, Nature 256:495-497; Milstein, which is incorporated into this specification ein and Cuello, 1983, Nature 305:537-40; Ridgway et al., 1996, Protein Eng. 9:61 7-621; Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92; Gunasekaran e t 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 En g 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 23:195-20 2; 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.

[0042] In some embodiments, the bispecific antibody of this disclosure is CrossMab. The ossMab technologies are incorporated herein in their entirety by this reference, and are internationally publicly available. Pamphlet No. 2009 / 080251, International Publication No. 2009 / 080252 Fret, International Publication No. 2009 / 080253 Pamphlet, International Publication No. 2009 / 0 Pamphlet No. 80254, International Publication No. 2013 / 026833, International Publication Brochure No. 2016 / 020309, and Schaefer et al., 2011, Proc Natl It is described in detail in Acad Sci USA 108:11187-92. In short, CrossMab The technology facilitates correct chain association between the heavy and light chains within one Fab arm of bispecific IgG. Based on the crossover of domains between and . The CrossMab bispecific antibodies of this disclosure are bispecific "Cro" is a type of antibody in which the heavy and light chains of the Fab portion of one arm of an heterozygous IgG antibody are exchanged. ssMab FAB "Antibodies may also be used. In other embodiments, the CrossM of the Disclosure ab bispecific antibodies are the heavy chain of the Fab portion of one arm of a bispecific IgG antibody and "CrossMab" is a system where only the variable domains of the light chain are exchanged. VH-VL "An antibody" It is also good. In further embodiments, the CrossMab bispecific antibody of the present disclosure is two Only the constant domains of the heavy and light chains of the Fab portion of one arm of the hemispecific IgG antibody The "CrossMab" that has been replaced CH1-CL "Antibodies may also be used." CrossMa b CH1-CL The antibody is CrossMab FAB and ClawsMab VH-VL What is In contrast, it does not have the expected byproducts, and therefore, in some embodiments, Cross sMab CH1-CL Bispecific antibodies are preferred. Klein et al., 2016, mAbs, 8(6):101 See 0-1020. Further embodiments of the CrossMab of this disclosure are shown in Section 5. This will be discussed later in section 2.

[0043] In some embodiments, the bispecific antibodies of this disclosure are used in controlled Fab arm exchanges. This is a bispecific antibody. For a method to produce Fab-arm exchange bispecific antibodies, see the reference. The entirety of these is incorporated herein by PCT International Publication No. 2011 / 13174. As described in Pamphlet No. 6 and Labrijn et al., 2014 Nat Protoc. 9(10):2450-63 In short, controlled Fab arm exchange bispecific antibodies contain the CH3 domain. By separately expressing two parental IgG1 cells containing a single matching point mutation, In vitro, parent IgG1 is mixed under redox conditions to allow recombination of half the molecules. And, by removing the reduced form and re-oxidizing the interchain disulfide bond, thereby achieving bispecificity. It can be produced by forming antibodies.

[0044] The bispecific antibody of this disclosure comprises an Fc domain composed of a first and a second subunit. It may also contain. In one embodiment, the Fc domain is an IgG Fc domain. In certain embodiments, the Fc domain is the IgG1Fc domain. In the implementation form, the Fc domain is the IgG4Fc domain. More specific implementation form In this state, the Fc domain has an amino acid substitution at position S228, particularly the amino acid substitution S2 The IgG4Fc domain includes 28P (Kabat EU index numbering). This amino acid substitution reduces Fab arm exchange of IgG4 antibody 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 certain embodiments, the Fc domain is the human IgG1Fc domain. An exemplary sequence of the IgG1Fc region is shown in Sequence ID No. 42.

[0045] In certain embodiments, the Fc domain is a first and second subunit of the Fc domain. Includes modifications that facilitate the association of two subunits of the human IgG Fc domain. The site of the largest protein-protein interaction is located within the CH3 domain. Therefore, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

[0046] In a specific embodiment, the association of the first and second subunits of the Fc domain Modifications that facilitate this are the so-called "knob-into-hole" modifications. This involves a "knob" modification to one of the two subunits of the Fc domain, and the Fc domain The other of the two main subunits includes a modification of "Whole". Knob-Into The Woo-Hall technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,6 Specification No. 95,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, J. This method is described in Immunol Meth 248:7-15, 2001. Generally, this method is used for heterodimers. The projections are designed to enter the cavity in order to promote formation and inhibit homodimer formation. The first polypeptide has a projection ("knob") at its boundary, and the second polypeptide has a boundary This includes introducing a corresponding cavity ("hole") of the first polypeptide. Smaller amino acid side chains from the boundary to larger side chains (e.g., tyrosine or tryptophan) It is constructed by replacing a larger amino acid side chain with a smaller amino acid side chain. By replacing it with (for example, alanine or threonine), the second polypeptide A cavity of the same or similar size as the protrusion is created at the boundary.

[0047] Therefore, in some embodiments, the CH3 domain of the first subunit of the Fc domain By replacing the amino acid residues in the main chain with amino acid residues that have a larger side chain volume. As a result, within the CH3 domain of the first subunit, the CH3 domain of the second subunit A protrusion is created that can enter the cavity within the main, and the second subunit of the Fc domain Replace the amino acid residues in the CH3 domain with amino acid residues that have a smaller side chain volume. By replacing it, the CH3 domain of the second subunit contains the first subunit's A cavity is created in which a projection within the CH3 domain can be inserted. Preferably, a larger The amino acid residue having the volume of the side chain is arginine (R), phenylalanine (F), Selected from the group consisting of tyrosine (Y) and tryptophan (W). Preferably, The amino acid residues having a smaller side chain volume are alanine (A), serine (S), and s Selected from the group consisting of rheonine (T) and valine (V). The protrusions and cavities are For example, site-directed mutagenesis can be used to change the nucleic acid that codes for polypeptides. It can be prepared by or by peptide synthesis. An exemplary substitution is shown in Y470T. be.

[0048] In certain such embodiments, in the first subunit of the Fc domain, 366 The threonine residue at position 1 is replaced by a tryptophan residue (T366W), Fc In the second subunit of the domain, the tyrosine residue at position 407 is a valine residue. Replaced by (Y407V), the threonine residue at position 366 is optionally serine Substituted with a residue (T366S), the leucine residue at position 368 is replaced by an alanine residue. It has been replaced (L368A) (numbered by the Kabat EU index). In a further embodiment, in the first subunit of the Fc domain, in addition, position 354 The serine residue in is replaced by a cysteine ​​residue (S354C), or at position 356. The glutamic acid residue in (E356C) (especially 35) is replaced by a cysteine ​​residue. The serine residue at position 4 is replaced by a cysteine ​​residue), the fourth of the Fc domain In subunit 2, in addition, the tyrosine residue at position 349 is a cysteine ​​residue. Replaced by (Y349C) (number by Kabat EU index) (Attached). In certain embodiments, the first subunit of the Fc domain is an amino acid substitution. Including S354C and T366W, the second subunit of the Fc domain is an amino acid Includes replacements Y349C, T366S, L368A and Y407V (Kabat EU In (Numbering by DEX)

[0049] In some embodiments, fixed steering (e.g., Gunasekaran et al., 2010, As described in J Biol Chem 285(25):19637-46, the first and This can facilitate the meeting of the second subunit.

[0050] In some embodiments, the Fc domain is an Fc receptor and / or effector. Contains one or more amino acid substitutions that reduce binding to the enzyme.

[0051] 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 activated Fc It is a receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor. More specifically, human FcγRIIIa, FcγRI, or FcγRIIa, Most specifically, it is human FcγRIIIa. In one embodiment, effector function complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody One or more selected from the group consisting of dependent cell phagocytosis (ADCP) and cytokine secretion. It is a number. In a particular embodiment, the effector function is ADCC.

[0052] Typically, the same one or more amino acid substitutions affect two subunits of the Fc domain. It is present in each of the two. In one embodiment, one or more amino acid substitutions are Fc Reduces the binding affinity of the domain to the Fc receptor. In one embodiment, one or more The amino acid substitution reduces the binding affinity of the Fc domain to the Fc receptor by at least half. Reduce it to at least one-fifth, or at least one-tenth.

[0053] In one embodiment, the Fc domains are E233, L234, L235, N297, P Includes amino acid substitutions at positions selected from the groups 331 and P329 (Kabat EU (Numbering by index). In a more specific embodiment, the Fc domain is L2 34, containing amino acid substitutions at positions selected from the group L235 and P329 (Kaba (Numbering by EU index). In some embodiments, the Fc domain is Includes mino acid substitutions L234A and L235A (numbered by Kabat EU index) (Numbering). In one such embodiment, the Fc domain is the IgG1Fc domain. In particular, the human IgG1Fc domain. In one embodiment, the Fc domain is P The amino acid substitution is at position 329. In a more specific embodiment, the amino acid substitution is P3 29A or P329G, specifically P329G (according to the Kabat EU index) (Numbering). In one embodiment, the Fc domain includes an amino acid substitution at position P329. Further locations can be selected from E233, L234, L235, N297 and P331. Includes amino acid substitutions (numbered according to the Kabat EU index). More specific examples In the application form, further amino acid substitutions include E233P, L234A, L235A, L2 These are 35E, N297A, N297D, or P331S. In certain embodiments, F The c domain contains amino acid substitutions at positions P329, L234, and L235 (Kab (numbered by the EU index). In a more specific embodiment, the Fc domain This includes amino acid mutations L234A, L235A and P329G ("P329G" LALA, PGLALA, or LALAPG). Specifically, in a particular embodiment. In this, each subunit of the Fc domain has amino acid substitutions L234A, L235A and This includes P329G (Kabat EU index numbering), i.e., Fc domain In each of the first and second subunits of yin, the leucine residue at position 234 The base is replaced by an alanine residue (L234A), and the leucine residue at position 235 is, The alanine residue is replaced (L235A), and the proline residue at position 329 is replaced by glycine It is replaced by a residue (P329G) (according to the Kabat EU index) (Numbering). In one such embodiment, the Fc domain is IgG1Fc domain The main domain, particularly the human IgG1Fc domain, is the primary one.

[0054] The single-chain-based bispecific antibodies of this disclosure are similar to various types of single-chain-based antibodies known in the industry. Bispecific antibodies, such as bispecific T cell derivatives (BiTEs) (bispecific T cell inducers) BiTE antibody, diabody, tandem diabody ), dual-affinity retargeting molecules (DART), and It may be either a bispecific killer cell derivative or a whole of them by reference. This is incorporated herein by reference, Loffler et al., 2000, Blood 95:2098-103; Holliger et al. 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, Cli n Cancer Res 19:3844-55; Liu et al., 2017, Front. Immunol. 8:38; and Yang et a See l., 2017, Int. J. Mol. Sci. 18:48.

[0055] In some embodiments, the bispecific antibody of this disclosure is a bispecific T cell derivative (Bi BiTE is a single polypeptide chain molecule having two antigen-binding domains. In this configuration, one antigen-binding domain binds to the T cell antigen, and the second antigen-binding domain targets the target. Binds to antigens presented on the surface (the whole of these is incorporated herein by reference) PCT International Publication No. 05 / 061547; Baeuerle et al., 2008, D See Rugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321: 974-977. Therefore, the BiTE of this disclosure has an antigen-binding domain that binds to T cell antigens, and It has a second antigen-binding domain directed towards GlycoMUC1.

[0056] In some embodiments, the bispecific antibodies of this disclosure are biaffinity retargeting molecules (DA RT) is. DART associates (especially through covalent interactions) and It also includes at least two polypeptide chains that form two epitope binding sites, and these The epitope binding site may recognize the same epitope, or it may recognize a different epitope. It may recognize the p. Each of the polypeptide chains of DART is a variable immunoglobulin light chain. It includes the region and the immunoglobulin heavy chain variable region, but these regions interact and epithet It does not form a link-binding site. Rather, one of the DART polypeptide chains (for example, The first) immunoglobulin heavy chain variable region is different (for example, the second) DART(trademark) It interacts with the immunoglobulin light chain variable region of the lipeptide chain to form an epitope binding site. Similarly, the immunoglobulin of one of the DART polypeptide chains (e.g., the first one) The chain variable region is a different (e.g., a second) immunoglobulin heavy chain of the DART polypeptide chain. It interacts with the variable region to form an epitope binding site. DART is monospecific, bi It may be multiple specific, triple specific, etc., and therefore 1, 2, 3 or more. At the same time as different epitopes (which may be the same or different antigens) They can be combined. DART can also be monovalent, divalent, trivalent, tetravalent, pentavalent, hexavalent, etc. This may also be the case, and therefore, one, two, three, four, five, six or more molecules are the same These two properties of DART (i.e., the degree of singularity and By combining valencies, for example, a tetravalent (i.e., a set of four epitopes) can be bonded. A bispecific antibody (i.e., capable of binding to two epitopes) It can produce things like ( ). DART molecules are, by reference, the whole of them specified herein. The PCT International Publication No. 2006 / 113665 pamphlet, which will be incorporated into the international publication, will be included in the international publication. Pamphlet No. 2008 / 157379, and International Publication No. 2010 / 080538 It is disclosed in the brochure.

[0057] In some embodiments of the bispecific antibodies of this disclosure, one of the binding specificities is GlycoMU. One signal is directed towards C1, while the other is directed towards antigens expressed on immune effector cells. When used herein, "immune effector cells" or "effector cells" means, Cellular cellular spheroids in the mammalian immune system that can be activated and affect the viability of target cells. It refers to cells within the natural repertoire. Immune effector cells include lymphocytes, for example. Examples include natural killer (NK) cells, T cells including cytotoxic T cells, or B cells. In addition to being able to do so, monocytes or macrophages, dendritic cells and neutrophils, etc. Myeloid cells can also be considered immune effector cells. Therefore, the effector T cells are preferably NK cells, T cells, B cells, monocytes, macrophages, and dendritic cells. Or neutrophils. Replenishing abnormal cells with effector cells is done by replacing them with effector cells. The cells either directly kill the abnormal cells they replace, or indirectly open up that lethality. To initiate this process, immune effector cells approach and take over abnormal target cells. This means that it is incorporated. To avoid nonspecific interactions, the bispecific anti- The body is at least over-affected by these immune effector cells compared to other cells in the body. It is preferable to specifically recognize antigens on overexpressed immune effector cells. Target antigens presented on fector cells include CD3, CD8, CD16, and CD25. Examples include CD28, CD64, CD89, NKG2D, and NKp46. Preferably, the antigen on the immune effector cells is CD3, which is expressed on T cells.

[0058] When used herein, "CD3" refers to primates (e.g., hi) unless otherwise specified. (t) non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats) This term refers to all natural CD3 derived from any vertebrate source, including mammals. In addition to unprocessed "full-length" CD3, processing occurs within cells. This term encompasses all forms of CD3. This term also refers to naturally occurring variants of CD3. Ants, for example, splice variants or allele variants, are also included. The most preferred antigen on Fector cells is the CD3 epsilon chain. This antigen is abnormal. It has been shown to be very effective in supplementing T cells to other cells. Therefore, The disclosed bispecific antibody preferably specifically recognizes CD3 epsilon. The amino acid sequence of 3-epsilon is UniProt (www.uniprot.org) accession number P077. 66 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq This is shown in NP_000724.1. Crab-eating macaque [Macaca fasciculari] The amino acid sequence of CD3 epsilon is NCBI GenBank number BAB718. As shown in 49.1. In the case of therapeutic use in humans, the CD3 binding domain is human CD3 (for example) In this case, a bispecific antibody that specifically binds to the human CD3 epsilon chain is used. In the case of preclinical trials in animals or cell lines, the species used in the preclinical trial (for example, primates) In the test, the CD3-binding domain in cynomolgus monkeys specifically bound to CD3. A bispecific antibody can be used.

[0059] As used herein, "specifically binds" to a target antigen of a particular species or similar species. The binding domain that "specifically recognizes" this binds to or recognizes antigens from other species. Antibodies that do not exclude and therefore have one or more binding domains that exhibit cross-reactivity between species. This includes, for example, "specifically binding" to human CD3 or "specifically recognizing" it. The CD3-binding domain also binds to cynomolgus CD3, or It is possible to recognize this, and vice versa.

[0060] In some embodiments, the bispecific antibodies of this disclosure bind to the CD3 epitope. Regarding this, monoclonal antibody H2C (PCT International Publication No. 2008 / 119567 pamphlet) It may compete with (as described in the lett). In other embodiments, the dual features of this disclosure Regarding the binding of heterozygous antibodies to the CD3 epitope, monoclonal antibody V9 (Rodrigue s et al., 1992, Int J Cancer Suppl 7:45-50 and U.S. Patent No. 6,054,297 It can compete with (as described in the details). Furthermore, in other embodiments, two of the disclosures The heavily specific antibody, in relation to binding to the CD3 epitope, is the monoclonal antibody FN18 ( This can compete with the work described in Nooij et al., 1986, Eur J Immunol 19:981-984. In further embodiments, the bispecific antibodies of this disclosure bind to the CD3 epitope. Regarding the combination, the monoclonal antibody SP34 (Pessano et al., 1985, EMBO J 4:337-340) It can compete with (what is listed).

[0061] The anti-glycoMUC1 antibodies of this disclosure include derivatized antibodies. For example, however limited However, derivatized antibodies are typically glycosylated, acetylated, pegylated, and lycosylated. By oxidation, amidation, derivatization with known protecting / blocking groups, and proteolysis Modified by cleavage, linkage to cell ligands or other proteins, and various chemical modifications. None of these are limited to, but include specific chemical cleavage, acetylation, formylation, This can be done by known techniques such as the metabolic synthesis of tunicamycin. In addition, derivatives For example, using ambrx technology, it contains one or more non-natural amino acids. This is also acceptable (see, for example, Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0062] The anti-glycoMUC1 antibody or binding fragment has at least one constant region mediated Antibodies or facultatives whose sequences have been modified to alter their biological effector function. It may be a lagment. For example, in some embodiments, the anti-glycoMUC1 antibody is modified Compared to unmodified antibodies, at least one constant region mediates biological effects. It may be modified to reduce its receptor function, for example, its binding to the Fc receptor (FcγR). It is modified to reduce the binding to FcγR, which is a specific region required for FcγR interaction. This is reduced by mutating the constant region segment of the antibody's immunoglobulin in that region. This is possible (for example, Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; (see also Lund et al., 1991, J. Immunol. 147:2657-2662). The ability of the antibody to bind FcγR reduction can also reduce other effector functions that rely on FcγR interactions, such as opsonization, phagocytosis and antigen-dependent cell cytotoxicity ("ADCC").

[0063] The anti-glycoMUC1 antibodies or binding fragments described herein acquire or modify at least one biologically mediated effector function of the constant region, or are improved compared to unmodified antibodies, e.g., antibodies modified to enhance FcγR interactions and / or binding fragments (see, e.g., U.S. Patent Application Publication No. 2006 / 013 No. 4709). For example, the anti-glycoMUC1 antibodies of the present disclosure have a higher affinity for FcγRIIA, FcγRIIB, and / or FcγRI than the corresponding wild-type constant region and may have a constant region that binds to IIA.

[0064] Thus, the antibodies of the present disclosure may have alterations in biological activity that cause an increase or decrease in opsonization, phagocytosis, or ADCC. Such alterations are known in the art. For example, modifications in antibodies that reduce ADCC activity are described in U.S. Patent No. 5,834,597. An exemplary variant that reduces ADCC corresponds to "mutant 3" in which residue 236 is deleted and residues 234, 235, and 237 (using EU numbering) are substituted with alanine (shown in Figure 4 of U.S. Patent No. 5,834,597).

[0065] In some embodiments, the anti-glycoMUC1 antibodies of the present disclosure have a low level of fucose either have or lack fucose. Antibodies lacking fucose have been shown to correlate with enhanced ADC C activity, especially at low antibody doses. See Shields et al., 2002, J. Biol. Chem. 277:267 33-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. Methods for preparing antibodies with less fucose include growth in rat myeloma YB2 / 0 cells (ATCC C RL1662). YB2 / 0 cells express FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme required for polypeptide fucosylation, at low levels.

[0066] In yet another aspect, the anti-glycoMUC1 antibody or binding fragment may, for example, be mutated in the immunoglobulin constant region segment at specific regions involved in FcRn interaction to increase or decrease its binding affinity to the neonatal Fc receptor, FcRn (see, for example, WO 2005 / 123780 pamphlet). In certain embodiments, an IgG class anti-glycoMUC1 antibody is mutated such that at least one of the amino acid residues 250, 314, and 428 in the heavy chain constant region is substituted, for example, at positions 250 and 428, or 250 and 314, or 314 and 428, or 250, 314, and 428, either alone or in any combination thereof, with a specific combination being positions 250 and 428. In the case of position 250, the amino acid residue to be substituted can be any amino acid residue other than threonine, for example, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine Nylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, Asparagine, proline, glutamine, arginine, serine, valine, tryptophan, Alternatively, it may be tyrosine, etc. In the case of position 314, the amino acid residue to be substituted is leuk. It may be any amino acid residue other than syn, for example, but not limited to these, a Lanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, hi Stidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, Arginine, serine, threonine, valine, tryptophan, or tyrosine, etc. It is also acceptable. In the case of position 428, the amino acid residue to be substituted is any amino acid other than methionine. It may also be an acid residue, for example, but is not limited to these, alanine, cysteine, a Spartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine Lysine, leucine, asparagine, proline, glutamine, arginine, serine, sle Onine, valine, tryptophan, or tyrosine may also be suitable. Specific combinations of substitutions are incorporated herein by reference in U.S. Article 7,217, These mutations are identified in Table 1 of Specification No. 797. Such mutations increase binding to FcRn. This is done to protect the antibody from degradation and increase its half-life.

[0067] In yet another embodiment, the anti-glycoMUC1 antibody of the antigen-binding fragment of this disclosure is For example, Jung and Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki et al. , 2004, Protein Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Patent Application As described in Specification No. 2007 / 0280931, one of its highly variable regions or It has one or more inserted amino acids.

[0068] In yet another embodiment particularly useful for diagnostic applications, the antigen-binding fragment of the present disclosure is an anti- The lyco-MUC1 antibody is attached to the detectable region. The detectable region is: Radioactive portion, colorimetric molecule, fluorescent portion, chemiluminescent portion, antigen, enzyme, detectable bead (for example) If magnetic or high electron density (e.g., gold beads), or a molecule that bonds to another molecule (for example) Examples include biotin or streptavidin.

[0069] Radioactive isotopes or radionuclides are, 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc , 111 In, 125 I, 131 I can be cited.

[0070] Fluorescent labels include rhodamine, lanthanide fluorescence, fluorescein and its derivatives. Fluorescent dye, GFP (GFP stands for "Green Fluorescent Protein") Green fluorescent protein (represented by), dansyl, umbelliferone, phycoerythrin, f Phycocyanin, allophycocyanin, o-phthalaldehyde, and phthaldehyde Luoresamine can be mentioned as an example.

[0071] Enzyme labels include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase (「G6PDH」), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase.

[0072] Chemiluminescent labels or chemiluminescers, such as isoluminol, luminol, and dioxetane

[0073] Other detectable moieties include molecules such as biotin, digoxigenin, or 5-bromodeoxyuridine

[0074] In certain embodiments, the anti-glycoMUC1 antibodies or antigen-binding fragments of the present disclosure compete with an antibody or antigen-binding fragment that includes GO2 or the heavy and light chain variable regions of GO2 (SEQ ID NOs: 3 and 4, respectively).

[0075] The competition can be assayed in cells that express the glycoMUC1 epitope that binds GO2, or in a glycosylated MUC1 peptide that contains the epitope that binds GO2, such as the 60-mer peptide glycosylated with GalNAc-T2, GalNAc-T4, and GalNAc-T1 described in U.S. Patent No. 6,465,220, (VTSAPDT RPAPGSTAPPAHG)3. As a control, cells that do not express the epitope or non-glycosylated peptides can be used.

[0076] ​​​​​​​​Cells that can be used in competitive assays include, but are not limited to, breast cancer cells. The strains MCF7 or T47D, and the glycoMUC1 epitope, are engineered to express these. Recombinant cells are one example. In one non-specific example, CHO IdID cells are U Lacking DP-Gal / GalNAc epimerase, and possessing GalNAc and Gal respectively In the absence of exogenous additions, O-glycosylation and galactosylation of GalNAc are insufficient. This is a condition in which MUC1 is manipulated to express and GalNAc is absent. Either it proliferates in the presence of GO2, and in the latter case, it produces a Tn glycoform of MUC1 to which GO2 binds. Cells that express the unglycosylated form of MUC1 are negative. It can be used as a comparison.

[0077] Assays concerning competition include, but are not limited to, those involving radioactive substance labeling. Munoassay (RIA), Enzyme-linked immunosorbent assay (ELISA), Sandwich ELI SA, fluorescence-activated cell sorting (FACS) assays, and Biacore assays These are some examples.

[0078] Antibody competition assay between a reference antibody and a test antibody (regardless of species or isotype) In performing this, first, detectable markers, e.g., so that subsequent identification is possible. For example, label the reference with a fluorophore, biotin, or enzyme (or radioactive label). It is also possible. In this case, cells expressing glycoMUC1 are treated with an unlabeled test antibody. An incubated and labeled reference antibody is added, and the intensity of the bound label is measured. The test antibody is labeled by binding to overlapping epitopes. When competing with the test antibody, the intensity is expected to decrease compared to the control reaction performed without the test antibody. It can be done.

[0079] In a specific embodiment of this assay, assay conditions (e.g., specified cell density) The concentration of the labeled reference antibody that yields 80% of the maximum binding at (degrees) is "conc8 0% First, the ) is determined, and the competitive assay is performed using 10 × concrete unlabeled test antibodies. 80% and the conc of the labeled reference antibody 80% It will continue.

[0080] Inhibition is the inhibition constant, or K i It can be expressed as follows: K i =IC 50 / (1+[reference Ab concentration] / K d ) It is calculated according to the formula, in which IC 50 This test antibody results in a 50% reduction in the binding of the reference antibody. It is the concentration of K d This is the dissociation constant of the reference antibody, which is the dissociation constant of the glycoMUC1. This is a measure of affinity. Antibodies that compete with the anti-glycoMUC1 antibodies disclosed herein are: Under the assay conditions described herein, 10 pM to 10 nM K i It may have.

[0081] In various embodiments, 80% of the maximum binding occurs under the specific assay conditions used. At the reference antibody concentration and at a test antibody concentration 10 times higher than the reference antibody concentration, at least approximately 20% Or more, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, or any of the aforementioned values. If the test antibody reduces the binding of the reference antibody within a certain percentage range, then the test antibody The body is considered to be competing with the reference antibody.

[0082] In one example of a competitive assay, a 60-mer peptide, which is a glycosylated MUC1, was used. By bringing the rate into contact with a peptide solution (for example, a concentration of 1 μg / mL in PBS) Leave at 4°C overnight to adhere to a solid surface, such as a microwell plate. Wash (e.g., 0.1% Tween 20 in PBS), block (e.g., S upperblock, Thermo Scientific, Rockford, IL. ). ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in PBS) ) a subsaturated amount of biotinylated GO2 (e.g., at a concentration of 80 ng / mL) and Further diluted (e.g., 2.8 μg / mL, 8.3 μg / mL, or 25 μg / mL concentration) Unlabeled GO2 ("reference" antibody) or competing anti-glycoMUC1 antibody (at a certain degree) Add the antibody mixture ("test" antibody) to the wells and gently shake the plate for 1 hour. Incubate for a period of time. Wash the plate and add 1 μg of ELISA buffer to each well. Add streptavidin conjugated with g / mL of HRP, and set the plate for 1 hour. Incubate. Wash the plate and add the substrate (e.g., TMB, Biofx Labo). The compound is formed by the addition of ratories Inc., Owings Mills, MD. Antibodies were detected. Stop buffer (e.g., Bio FX Stop reagent, Biofx L) was used. By adding aboratories Inc., Owings Mills, MD) Stop the reaction and use a microplate reader (e.g., VERSAmax, Molecula) Absorbance was measured at 650 nm using r Devices (Sunnyvale, CA). To determine.

[0083] In this competition assay, to test the competition between GO2 and another anti-glycoMUC1 antibody... Other variations can also be used. For example, in a certain embodiment, antiglyceride MU The C1 antibody is used as the reference antibody, and GO2 is used as the test antibody. In addition, G Instead of the 60-mer peptide which is cosylated MUC1, (e.g., on the cell surface during culture) For example, a membrane-bound glyco-MUC1 expressed on the surface of one of the aforementioned cell types can be used. Yes. Generally, about 10 4 from 10 6 individual transformants, for example, about 10 5 individual transformants It is used. Other forms of competing assays are publicly known in the industry and can be adopted. ru.

[0084] In various embodiments, the anti-glycoMUC1 antibody of the present disclosure is an anti-glycoMUC1 antibody , 0.08μg / mL, 0.4μg / mL, 2μg / mL, 10μg / mL, 50μg / In mL, at a concentration of 100 μg / mL, or in a concentration within the range of any of the aforementioned values ​​(for example) If used at concentrations ranging from 2 μg / mL to 10 μg / mL, labeled GO The bond of 2 is at least 40%, at least 50%, at least 60%, at least 70% %, at least 80%, at least 90%, or a percentage in the range of any of the aforementioned values Reduced by the scent (for example, the anti-glycoMUC1 antibody of this disclosure is labeled GO2 (Reduces the bonding by 50% to 70%).

[0085] In other embodiments, GO2 is 0.4 μg / mL, 2 μg / mL, 10 μg / mL At concentrations of 50 μg / mL, 250 μg / mL, or within a range of any of the aforementioned values. When used at a specific concentration (for example, in a range of concentrations from 2 μg / mL to 10 μg / mL), G O2 binds at least 40% of the labeled anti-glycoMUC1 antibody of this disclosure, and less 50%, at least 60%, at least 70%, at least 80%, at least 90% Reduce by a percentage, or a percentage within the range of any of the aforementioned values ​​(e.g., GO 2 reduces the binding of the labeled anti-glycoMUC1 antibody of this disclosure by 50% to 70%. ).

[0086] In the assay described above, the GO2 antibody detects the CDR or heavy and light chain variable regions of GO2. Any antibody or antigen-binding fragment containing the region, such as a humanized or chimeric GO2, e.g., GO2. It can be replaced with a counterpart.

[0087] In certain embodiments, the anti-glycoMUC1 antibody or antigen-binding fragment of the Disclosure is Table 1 includes heavy and / or light chain variable sequences (or nucleotide sequences) (Coded) In other embodiments, the anti-glycoMUC1 antibody or antigen-binding fungiblet of the Disclosure The nucleoin contains the heavy and / or light chain CDR sequences listed in Table 1 (or nucleotide). (Encoded by the sequence). Such anti-glycoMUC1 antibodies and antigen-binding flags. Even if the ment framework sequence is the natural mouse framework sequence listed in Table 1, It may be a good, or non-natural (e.g., humanized or human) framework sequence. .

[0088] In other embodiments, the present disclosure relates to Sequence IDs 3 and 4, respectively, and at least 95% , possessing heavy chain and light chain variable regions with 98%, 99%, or 99.5% sequence identity. The present invention provides an anti-MUC1 antibody or antigen-binding fragment.

[0089] In yet another embodiment, the anti-glycoMUC1 antibody or antigen-binding fragment of the present disclosure. This is a single-chain variable fragment (scFv). An example scFv is a light-chain variable fragment. The N-terminal side of the nt contains a heavy chain variable fragment. In some embodiments, the heavy chain of scFv Chain-variable fragments and light-chain-variable fragments have a linker sequence of 4-15 amino acids. They are covalently bonded. scFv may be in the form of a bispecific T cell derivative, or This may also be within a chimeric antigen receptor (CAR).

[0090] 5.2 Anti-glycoMUC1 and anti-CD3 bispecific antibodies In some embodiments, the bispecific antibodies of this disclosure are CD3 (e.g., CD3 as listed in Table 4). A first antigen-binding domain that specifically binds to R or VH and VL, and It may also contain a second antigen-binding domain that specifically binds to biglycoMUC1. The antigen-binding domain exhibits the characteristics described above for the glycoMUC1 antibody, independently of the other, Alternatively, they may be included in combination (for example, combinations of CDRs identified in Tables 1-3, For example, any of the CDR combinations described in the numbered embodiments 3 to 17 below CDR combinations containing no acid sequences, or containing VH and VL sequences as identified in Table 1.

[0091] [Table 4-1]

[0092] [Table 4-2]

[0093] In some embodiments, the first antigen-binding domain is the heavy chain CDR-H of SEQ ID NO: 34. 1. Heavy chain variable regions including CDR-H2 of SEQ ID NO: 35 and CDR-H3 of SEQ ID NO: 36 Domain; as well as light chain CDR-L1 of SEQ ID NO: 37, CDR-L2 of SEQ ID NO: 38 and sequence Includes a light chain variable region, including CDR-L3, number 39.

[0094] In some embodiments, the second antigen-binding domain is, for example, in the numbered embodiments. A CDR containing any of the amino acid sequences of the CDR combinations described in 3 to 17, for example (i) Heavy chain CDR-H1 (SEQ ID NO: 5), CDR-H2 (SEQ ID NO: 6), and CDR (SEQ ID NO: 7) -Heavy chain variable region including H3; and light chain CDR(CDR-L)1 of Sequence ID No. 8, sequence number It includes a light chain variable region, including CDR-L2 of sequence number 9 and CDR-L3 of sequence number 10.

[0095] In a particular embodiment, the bispecific antibody is (i) CDR-H1, which specifically binds to CD3 and contains the amino acid sequence of SEQ ID NO: 34, CDR-H2 containing the amino acid sequence of column number 35, and containing the amino acid sequence of sequence number 36. A heavy chain variable region containing CDR-H3; and a CDR containing the amino acid sequence of SEQ ID NO: 37. -L1, CDR-L2 containing the amino acid sequence of SEQ ID NO: 38, and the amino acid sequence of SEQ ID NO: 39 A first antigen-binding domain containing a light chain variable region including CDR-L3 containing an acid sequence; and to (ii) Specifically binds to glycoMUC1, and (i) contains the amino acid sequence of SEQ ID NO: 33 CDR-H1, more preferably CDR-H1 containing the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: CDR-H2 containing 29 amino acid sequences, more preferably containing the amino acid sequence of SEQ ID NO: 6 CDR-H1 and CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, more preferably CDR-H3. This is a heavy chain variable region containing CDR-H3, which includes the amino acid sequence of SEQ ID NO: 7; and SEQ ID NO: CDR-L1 contains the amino acid sequence of 8, and CDR-L2 contains the amino acid sequence of SEQ ID NO: 9. CDR-L3 containing the amino acid sequence of SEQ ID NO: 31, more preferably the amino acid sequence of SEQ ID NO: 10 A second antigen-binding domain containing a light chain variable region including CDR-L3 containing a minoic acid sequence. Includes.

[0096] In some embodiments, the first antigen-binding domain has the amino acid sequence of SEQ ID NO: 40 and At least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain variable regions Regional sequence, and amino acid sequence of SEQ ID NO: 41 and at least approximately 95%, 96%, 97%, 9 It contains 8%, 99%, or 100% identical light chain variable region sequences.

[0097] In some embodiments, the first antigen-binding domain is the heavy chain variable region of SEQ ID NO: 40 Includes the column and the light chain variable region sequence of sequence number 41.

[0098] In some embodiments, the second antigen-binding domain has a small amino acid sequence compared to SEQ ID NO: 3. At least 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain variable region is identical. The sequence, and the amino acid sequence of SEQ ID NO: 4, and at least approximately 95%, 96%, 97%, 98% , containing 99% or 100% identical light chain variable region sequences.

[0099] In some embodiments, the second antigen-binding domain is the heavy chain variable region sequence of SEQ ID NO: 3. and includes the light chain variable region sequence of sequence number 4.

[0100] In some embodiments, the first and / or second antigen-binding domains are located within the Fab molecule. In some embodiments, the first antigen-binding domain is the Fab light chain and the Fab This is a cross-Fab molecule in which either the variable or constant region of the heavy chain is exchanged. In one embodiment, the second antigen-binding domain is preferably a conventional Fab molecule. .

[0101] In some embodiments, both the first and second antigen-binding domains of the bispecific antibody are Both are Fab molecules, and one of the antigen-binding domains (especially the first antigen-binding domain) Then, the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other. Occasionally, i) In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 It is substituted with a positively charged amino acid (numbered by Kabat), and the first antibody In the constant domain CH1 of the primordial domain, the amino acid at position 147 or 213 The amino acid at the position is substituted with an amino acid that has a negative charge (Kabat EU Numbering by index; or ii) In the constant domain CL of the second antigen-binding domain, at position 124, amino The acid is substituted with a positively charged amino acid (numbered by Kabat), and the second In the constant domain CH1 of the antigen-binding domain, the amino acid at position 147 or 21 The amino acid at position 3 is substituted with a negatively charged amino acid (Kabat EU (Numbering by index).

[0102] The bispecific antibody does not contain either of the modifications described in i) and ii). VH / VL cross The constant domains CL and CH1 of the antigen-binding domain, which have substitution, are substituted for each other. They are not there (i.e., they remain unexchanged).

[0103] In a more specific embodiment, i) In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 It is independently substituted with lysine (K), arginine (R), or histidine (H). (Kabat numbering), in the constant domain CH1 of the first antigen-binding domain The amino acid at position 147 or 213 is glutamic acid (E), Alternatively, it is independently substituted with aspartic acid (D) (Kabat EU Index Numbering by; or ii) In the constant domain CL of the second antigen-binding domain, at position 124, amino The acid is independently substituted with lysine (K), arginine (R), or histidine (H). (Kabat numbering), in the constant domain CH1 of the second antigen-binding domain Therefore, the amino acid at position 147 or position 213 is glutamic acid (E). , or independently substituted with aspartic acid (D) (Kabat EU Index) (Numbering by S)

[0104] In one such embodiment, the constant domain CL of the second antigen-binding domain The amino acid at position 124 is lysine (K), arginine (R), or histidine. (H) is independently substituted (numbered by Kabat), and the second antigen-binding domain In the constant domain CH1 of the protein, the amino acid at position 147 or position 213 The amino acids are independently substituted with glutamic acid (E) or aspartic acid (D). (Numbered according to the Kabat EU index).

[0105] In a further embodiment, in the constant domain CL of the second antigen-binding domain, 1 The amino acid at position 24 is lysine (K), arginine (R), or histidine (H). They are substituted independently (numbered by Kabat), and the second antigen-binding domain is constant. In domain CH1, the amino acid at position 147 is glutamic acid (E), or It is independently substituted with spartic acid (D) (number according to the Kabat EU index) (Numbering).

[0106] In a particular embodiment, in the constant domain CL of the second antigen-binding domain, 12 The amino acid at position 4 is either lysine (K), arginine (R), or histidine (H). It is substituted in a fixed position (numbered by Kabat), and the amino acid at position 123 is ri It is independently substituted with syn (K), arginine (R), or histidine (H) (Ka (Numbering by bat), in the constant domain CH1 of the second antigen-binding domain, 14 The amino acid at position 7 is either glutamic acid (E) or aspartic acid (D), independently. It has been replaced (numbered by the Kabat EU index), and is ranked 213th. The amino acids are independently substituted with glutamic acid (E) or aspartic acid (D). (Numbered according to the Kabat EU index).

[0107] Furthermore, in a specific embodiment, in the constant domain CL of the second antigen-binding domain The amino acid at position 124 is substituted with lysine (K) (number by Kabat). (Note) The amino acid at position 123 is substituted with lysine (K) (according to Kabat) (Numbering), in the constant domain CH1 of the second antigen-binding domain, at position 147 The amino acid is substituted with glutamic acid (E) (Kabat EU Index) (Numbering by), the amino acid at position 213 is substituted with glutamic acid (E). (Numbering by Kabat EU Index)

[0108] In a more specific embodiment, the constant domain CL of the second antigen-binding domain Furthermore, the amino acid at position 124 is substituted with lysine (K) (according to Kabat). (Numbering), the amino acid at position 123 is substituted with arginine (R) (Kab (numbering by at), in the constant domain CH1 of the second antigen-binding domain, 147 The amino acid at the position is substituted with glutamic acid (E) (Kabat EU index (Numbering by X), the amino acid at position 213 is substituted with glutamic acid (E). (Numbered according to the Kabat EU Index).

[0109] In a particular embodiment, the amino acid substitution according to the above embodiment is used for the second antigen-binding domain. When this occurs in the constant domain CL and constant domain CH1 of the gene, the second antigenic linkage The constant domain CL of the synthetic domain is the constant domain CL of the kappa isotype.

[0110] In some embodiments, the first and second antigen-binding domains are optionally peptides. They are fused together via a linker.

[0111] In some embodiments, the first and second antigen-binding domains are, respectively, Fab molecules. (i) The second antigen-binding domain is at the C-terminus of the Fab heavy chain, and the first antigen-binding domain (ii) The first antigen-binding domain is fused to the N-terminus of the Fab heavy chain of (ii) The Fab heavy chain is fused at the C-terminus, and the second antigen-binding domain is fused at the N-terminus of the Fab heavy chain. It is one of the following.

[0112] In some embodiments, the bispecific antibody provides monovalent binding to CD3.

[0113] In certain embodiments, a bispecific antibody is a single antigen antibody that specifically binds to CD3. It contains a binding domain and two antigen-binding domains that specifically bind to glycoMUC1. Therefore, in some embodiments, the bispecific antibody specifically binds to glycoMUC1. It contains a third antigen-binding domain. In some embodiments, the third antigenic portion is It is identical to the antigen-binding domain in 1 (for example, the Fab molecule also has the same amino acid sequence). include).

[0114] In certain embodiments, the bispecific antibody is composed of a first and a second subunit. It further includes an Fc domain. In one embodiment, the Fc domain is an IgG Fc domain. This is the main part. In certain embodiments, the Fc domain is the IgG1Fc domain. In another embodiment, the Fc domain is the IgG4Fc domain. More specifically In a typical embodiment, the Fc domain has an amino acid substitution at position S228, particularly an amino acid substitution. This is an IgG4Fc domain containing acid-substituted S228P (Kabat EU Index) (Numbering). In a further specific embodiment, the Fc domain is a human Fc domain. Yes. In a more specific embodiment, the Fc domain is the human IgG1Fc domain. An exemplary sequence of the human IgG1Fc region is shown in Sequence ID No. 42.

[0115] In some embodiments, there are first, second, and, if present, third antigen-binding domains. These are Fab molecules, and (a)(i) the second antigen-binding domain is the Fab heavy chain At the C-terminus, it is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding The fusion domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain. (ii) The first antigen-binding domain is at the C-terminus of the Fab heavy chain, and the second The antigen-binding domain is fused to the N-terminus of the Fab heavy chain, and the second antigen-binding domain is The C-terminus of the Fab heavy chain is fused to the N-terminus of the first subunit of the Fc domain. (b) The third antigen-binding domain, if present, is at the C-terminus of the Fab heavy chain. It is fused to the N-terminus of the second subunit of the Fc domain.

[0116] In certain embodiments, the Fc domain is, for example, as described in Section 5.1. This includes modifications that facilitate the association of the first and second subunits of the Fc domain.

[0117] In some embodiments, the Fc domain is as described in Section 5.1, for example. Furthermore, one or more agents that reduce binding to Fc receptors and / or effector functions. Includes amino acid substitution.

[0118] In a particular embodiment, the bispecific antibody is (i) A first antigen-binding domain that specifically binds to CD3, and the first antigen-binding domain The main component is the crossed Fab molecule, and the variable or constant regions of the Fab light chain and Fab heavy chain. Either, or especially the variable region, is the first antigen-binding domain that has been replaced; (ii) Second and third antigen-binding domains that specifically bind to glycoMUC1 Therefore, the heavy chain CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and C of SEQ ID NO: 7 Heavy chain variable region including DR-H3; as well as light chain CDR-L1 of SEQ ID NO: 8, SEQ ID NO: 9 Including the light chain variable region including CDR-L2 and CDR-L3 of Sequence ID No. 10, the second and The third antigen-binding domain is a Fab molecule, and in particular, a conventional Fab molecule. Second and third antigen-binding domains; (iii) Fc dome consisting of first and second subunits capable of stable assembly in Includes, The second antigen-binding domain is at the C-terminus of the Fab heavy chain, and is the Fab of the first antigen-binding domain. It is fused to the N-terminus of the heavy chain, and the first antigen-binding domain is at the C-terminus of the Fab heavy chain, Fc It is fused to the N-terminus of the first subunit of the domain, and the third antigen-binding domain is F It is fused at the C-terminus of the ab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0119] In one embodiment, the first antigen-binding domain is the heavy chain CDR-H1 of SEQ ID NO: 34. Heavy chain variable regions including CDR-H2 of SEQ ID NO: 35 and CDR-H3 of SEQ ID NO: 36; Also, the light chain CDR-L1 of SEQ ID NO: 37, CDR-L2 of SEQ ID NO: 38 and SEQ ID NO: Includes a light chain variable region containing 39 CDR-L3.

[0120] In one embodiment, the first antigen-binding domain has the amino acid sequence of SEQ ID NO: 40 and a small number of others. At least 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain variable region is the same. The amino acid sequence of sequence number 41 and at least approximately 95%, 96%, 97%, 98% , containing 99% or 100% identical light chain variable region sequences.

[0121] In one embodiment, the first antigen-binding domain is the heavy chain variable region sequence of SEQ ID NO: 40 It includes the light chain variable region sequence of sequence number 41.

[0122] In one embodiment, the second and third antigen-binding domains are the amino acid combination of SEQ ID NO: 3 Heavy chains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the column are possible. The variant region sequence, and the amino acid sequence of SEQ ID NO: 4, are at least approximately 95%, 96%, and 97% of the original sequence. It contains 98%, 99%, or 100% identical light chain variable region sequences. Preferably, antigen-binding The main component is an amino acid from any of the CDR combinations described in the numbered embodiments 3 to 17. It contains a CDR containing an acid sequence. In one embodiment, the second and third antigen-binding domains are This includes the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4.

[0123] The Fc domain according to the above embodiment has all of the features described above with respect to the Fc domain. These may be included individually or in combination.

[0124] In some embodiments, the antigen-binding domain and the Fc region are linked by a peptide linker. For example, by peptide linkers such as those in SEQ ID NOs. 45 and 46. They are fused together.

[0125] In one embodiment, the constant domains CL of the second and third Fab molecules of (ii) Furthermore, the amino acid at position 124 is substituted with lysine (K) (according to Kabat). (Numbering), the amino acid at position 123 is either lysine (K) or arginine (R), It is replaced with arginine (R) (numbered by Kabat), and the second of (ii) And in the constant domain CH1 of the third Fab molecule, the amino acid at position 147 is G It is substituted with glutamic acid (E) (numbered according to the Kabat EU index), The amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU (Numbering by index).

[0126] In one embodiment, the bispecific antibody contains at least 80% of the sequence of SEQ ID NO: 43, and 8 Polypepto containing 5%, 90%, 95%, 96%, 97%, 98%, or 99% identical sequences Butide (preferably CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, and the amino acid sequence of SEQ ID NO: 9) CDR-L2 containing the no-acid sequence, and CDR-L3 containing the amino acid sequence of SEQ ID NO 31 (including), the sequence of sequence number 44 and at least 80%, 85%, 90%, 95%, 96%, 9 Polypeptides containing 7%, 98%, or 99% identical sequences (preferably those listed in Table 4) (Including CD3 heavy chain and light chain CDR sequences), sequence of SEQ ID NO: 45 and at least 80%, 8 Polypepto containing 5%, 90%, 95%, 96%, 97%, 98%, or 99% identical sequences Butide (preferably CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 29) Contains CDR-H2 containing the amino acid sequence, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 25. (m), and the sequence of sequence number 46 and at least 80%, 85%, 90%, 95%, 96% , polypeptides containing 97%, 98%, or 99% identical sequences (preferably, sequence number CDR-H1 containing 33 amino acid sequences, CDR-H containing the amino acid sequence of SEQ ID NO: 29 2. CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, and containing the amino acid sequence of SEQ ID NO: 37 CDR-L1, CDR-L2 containing the amino acid sequence of SEQ ID NO: 38, and SEQ ID NO: 39 It contains CDR-L3, which includes the amino acid sequence of the following.

[0127] In one embodiment, the bispecific antibody is a polypeptide containing the sequence of SEQ ID NO: 43 ( (2 polypeptides), polypeptide containing the sequence of sequence number 44, sequence of sequence number 45 The polypeptide includes a polypeptide containing the sequence of sequence number 46.

[0128] 5.3 Antibody-drug conjugates Another aspect of this disclosure includes the anti-glycoMUC1 antibody and antigen-binding fragment of this disclosure. This concerns antibody-drug conjugates (ADCs). An ADC is generally one or more One or more cytotoxic substances and / or cell proliferation inhibitors are transmitted via the linker. Linked thereto, the anti-glycoMUC1 antibody and / or conjugated filament described herein. It contains a component. In a specific embodiment, the ADC has structural formula (I): [DL-XY] n -Ab A compound or salt thereof, in which each "D" is independently a cytotoxic substance. Represents a substance that inhibits the quality and / or cell growth ("drug"); each "L" is independent of the others. "Ab" represents the linker; "Ab" is the antiglyco-MUC1 antigen-binding domain, for example, as specified herein. This represents the anti-glycoMUC1 antibody or binding fragment described; each "XY" is a phosphorus Functional group R on the car x and the "complementary" functional group R on the antibody y This represents the connection formed between and n This represents the number of drugs linked to the ADC, or the drug-to-antibody ratio (DAR) of the ADC.

[0129] Specific embodiments of various antibodies (Ab) that may include ADCs are shown above in relation to antiglyco-MUC This includes various embodiments of 1 antibody and / or conjugated fragments.

[0130] In specific embodiments of some of the ADCs and / or salts of structural formula (I), each D is the same They are the same, and / or each L is the same.

[0131] Cytotoxic substances and / or cell proliferation substances that may constitute the antiglyco-MUC1 ADCs of this disclosure In addition to the growth inhibitory substance (D) and linker (L), numerous cell injuries linked to the ADC Specific embodiments of the inhibitory substance and / or cell proliferation inhibitory substance are described in more detail below. do.

[0132] 5.3.1. Cytotoxic substances and / or cell proliferation inhibitors Cytotoxic substances and / or cell proliferation inhibitors affect cells, especially cancer and / or To inhibit the proliferation and / or replication of tumor cells, and / or to kill such cells It could be any drug known to have cytotoxic and / or cell proliferation inhibitory effects. Numerous drugs with inhibitory properties are known from the literature. Cytotoxic substances and / or cell proliferation inhibitors. Non-limiting examples of the class of controllable substances include, but are not limited to, radiation. Sexual radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DN A Insertion agent (e.g., sub-groove binder, e.g., sub-groove binder), RNA / DNA metabolite antagonist Cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase Examples include lactose inhibitors, mitochondrial inhibitors, and antimitotic agents.

[0133] The following are specific, non-limiting examples of drugs within a particular range of these various classes. This will be shown.

[0134] Alkylating agent: asaley ((L-leucine, N-[N-acetyl-4-[bi S-(2-chloroethyl)amino]-DL-phenylalanyl]-,ethyl ester;N SC167780;CAS Registry Number 3577897));AZQ((1,4-cyclohex Sadiene-1,4-dicarbamate, 2,5-bis(1-aziridinyl)-3,6-diode Xo-, diethyl ester; NSC182986; CAS Registry Number 57998682)) ;BCNU((N,N'-bis(2-chloroethyl)-N-nitrosourea;NSC40 9962; CAS Registry Number 154938); Busulfan (1,4-butanediol di Methanesulfonate; NSC750; CAS Registry Number 55981; (Carboxyphthalate) (T) Platinum (NSC27164; CAS Registry Number 65296813); CBDCA ((ci s-(1,1-cyclobutanedicarboxylate)diammineplatinum(II)); NSC24 1240;CAS Registry Number 41575944);CCNU((N-(2-chloroethyl )-N'-cyclohexyl-N-nitrosourea; NSC79037; CAS Registry Number 1 3010474)); CHIP (Iproplatin; NSC256927); Chlorambucus Ru (NSC3088; CAS Registry Number 305033); Chlorozotocin ((2-[[[( 2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-gluco Pyranose; NSC178248; CAS Registry Number 54749905); Cisplatin (Cisplatin; NSC119875; CAS Registry Number 15663271); Clomeson (clomesone) (NSC338947; CAS Registry Number 88343720); cyanomol Horinodoxorubicin (NCS357704; CAS Registry Number 88254073); Shik Logison (cyclodisone) (NSC348948; CAS Registry Number 99591738); Dianhydrogalactitol (5,6-diepoxydulcitol; NSC132313; CAS Registry Number 23261203); Fluorodopan ((5-[(2- Chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil; NSC73 754;CAS Registry Number 834913); Hepsulfame (NSC329680;CAS Registration number 96892578); Hicanton (NSC142982; CAS Registry Number 232) 55938); Melphalan (NSC8806; CAS Registry Number 3223072); Meth CCNU((1-(2-chloroethyl)-3-(trans-4-methylcyclohexa n)-1-nitrosourea; NSC95441; 13909096); Mitomycin C (NSC26980; CAS Registry Number 50077); Mitozolomide (N SC353451; CAS Registry Number 85622953); Nitrogen Mustard (( Bis(2-chloroethyl)methylamine hydrochloride; NSC762; CAS Registry Number 55867);PCNU((1-(2-chloroethyl)-3-(2,6-dioxo-3- Piperidyl)-1-nitrosourea; NSC95466; CAS Registry Number 1390902 9)); Piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl Pill)-piperazine dihydrochloride; NSC344007); piperazinedione (N SC135758; CAS Registry Number 41109802); Pipobroman ((N,N-bis (3-bromopropionyl)piperazine; NSC25154; CAS Registry Number 54911 )); Porphyromycin (N-methylmitomycin C; NSC56410; CAS registration Registration number 801525); Spirohydanto in mustard (NSC172112; CAS registration number Record number 56605164); Teroxirone (Triglycidyl isocyanure Tetraplatin (NSC296934;CAS Registry Number 2451629);NSC 363812; CAS Registry Number 62816982); Thiotepa (N,N',N''-tri -1,2-Ethanediylthiophosphoramide; NSC6396; CAS Registry Number 5224 4); Triethylene melamine (NSC9706; CAS Registry Number 51183); Uracil Nitrogen mustard (desmethyldopan); NSC34462; CAS Registry Number 66751); Yoshi-864((Bis(3-methyloxypropyl (Amine hydrochloride; NSC102627; CAS Registry Number 3458228).

[0135] Topoisomerase I inhibitor: Camptothecin (NSC94600; CAS Registry Number 76) 89-03-4); Various camptothecin derivatives and analogs (e.g., NSC1008) 80, NSC603071, NSC107124, NSC643833, NSC6299 71, NSC295500, NSC249910, NSC606985, NSC7402 8, NSC176323, NSC295501, NSC606172, NSC60617 3, NSC610458, NSC618939, NSC610457, NSC61045 9, NSC606499, NSC610456, NSC364830, and NSC60 6497); Morpholine isoxorubicin (NSC3546) 46;CAS Registry Number 89196043);SN-38(NSC673596;CAS Registry Number (Record number 86639-52-3).

[0136] Topoisomerase II inhibitor: Doxorubicin (NSC123127; CAS Registry Number) 25316409); Amonafide (benzoisoquinoline dione; NSC308847; CAS Registry Number 69408817); m-AMSA((4'-(9-Acridinylamino )-3'-Methoxymethanesulfon anilide; NSC249992; CAS Registry Number 51 264143); Anthrapyrazole derivative ((NSC355644); Etoposide ( VP-16; NSC141540; CAS Registry Number 33419420); Pyrazoroacryl Zin((pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine,9- Methoxy-N,N-dimethyl-5-nitro-,monomethanesulfonate; NSC3661 40;CAS Registry Number 99009219); Bisanthren Hydrochloride (NSC337 766;CAS Registry Number 71439684);Daunorubicin (NSC821151;C AS registration number 23541506); Deoxydoxorubicin (NSC267469; CA S Registration Number 63950061); Mitoxantrone (NSC301739; CAS Registration Number Item No. 70476823); Menogalil (NSC269148; CAS Registry Number 716289) 61); N,N-Dibenzyldaunomycin (NSC268242; CAS Registry Number 70 878512); Oxanthrazole (NSC349174; CAS registration number) Record number 105118125); rubidazone (NSC164011; CAS Registration number 36508711); Teniposid (VM-26; NSC122819; CAS registration (Record number 29767202).

[0137] DNA insertion agents: Anthramycin (CAS registry number 4803274); Thicamycin A (CAS Registry Number 89675376); Tomimycin (CAS Registry Number 3505055 6); DC-81 (CAS Registry Number 81307246); Cibilomycin (CAS Registry Number (Category 12684332); Pyrrolobenzodiazepine derivative (CAS Registry Number 9454900) 95);SGD-1882((S)-2-(4-aminophenyl)-7-methoxy-8- (3-4(S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11 α-Dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-8-i (Lu)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2a][1,4]diazepam Pin-5 (11aH)-on); 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]di Azepine-5(11aH)-one; NSC694501; CAS Registry Number 232931 576).

[0138] RNA / DNA antimetabolite: L-alanosine (NSC153353; CAS registry number) 59163416); 5-Azacitidine (NSC102816; CAS Registry Number 3206) 72); 5-Fluorouracil (NSC19893; CAS Registry Number 51218); ac Bicin (NSC163501; CAS Registry Number 42228922); aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl [Cylamino]benzoyl-]L-aspartic acid (NSC132483); Aminopter Phosphorus derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl [Amino]benzoyl]L-aspartic acid (NSC184692); aminopterin derivative Conductor N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amine [N]benzoyl]L-aspartic acid monohydrate (NSC134033); folic acid antagonist (antifo)((N α -(4-amino-4-deoxypterol)-N 7 - Hemiphthaloyl -L-ornithine; NSC623017); Baker's soluble folate antagonist Drug (antifol) (NSC139105; CAS registry number 41191042); dichloroa Lillawsone ((2-(3,3-dichloroallyl)-3-hydroxy-1, 4-Naphthoquinone; NSC126771; CAS Registry Number 36417160); Brekina Ru (NSC368390; CAS Registry Number 96201886); Futraful ((Prodra 5-Fluoro-1-(tetrahydro-2-furyl)-uracil; NSC14895 8;CAS Registry Number 37076689); 5,6-Dihydro-5-Azacitidine (NSC 264880; CAS Registry Number 62402317); Methotrexate (NSC740; CAS Registry Number 59052); Methotrexate derivative (N-[[4-[[(2,4-di [amino-6-pteridinyl]methyl]methylamino]-1-naphthalenyl]carbonyl] L-glutamic acid; NSC174121); PALA((N-(phosphonoacetyl)-L -Aspartate; NSC224131; CAS Registry Number 603425565); Pyrazó Flynn (NSC143095; CAS Registry Number 30868305); Trimethrexate (NSC352122; CAS Registry Number 82952645).

[0139] DNA metabolite antagonist: 3-HP (NSC95678; CAS registry number 3814797) ;2'-Deoxy-5-Fluorouridine (NSC27640; CAS Registry No. 5091) 9); 5-HP (NSC107392; CAS Registry Number 19494894); α-TGD R(α-2'-deoxy-6-thioguanosine; NSC71851 CAS Registry Number 21) 33815); Aphydicolinglycinate (NSC303812; CAS Registry Number 92 802822);ara C (Cytosine Arabinoside; NSC63878; CAS Registry Number) (Registered No. 69749); 5-aza-2'-deoxycytidine (NSC127716; CAS registration number) Number 2353335); β-TGDR (β-2'-deoxy-6-thioguanosine; NS) C71261; CAS Registry Number 789617); Cyclocitidine (NSC145668; CAS Registry Number 10212256); Guanazole (NSC1895; CAS Registry Number 1 455772); Hydroxyurea (NSC32065; CAS Registry Number 127071); Inosingricodial aldehyde (NSC118994; CAS registry number 23590990) ; Macbethine II (NSC330500; CAS Registry Number 73341738); Pirazoro Imidazole (NSC51143; CAS registry number 6714290); Thioguanine (N SC752; CAS Registry Number 154427); Thioprine (NSC755; CAS Registry Number 154427) No. 50442).

[0140] Cell cycle modulator: silibinin (CAS registry number 22888-70-6); galling Epigallocatechin glyceride (EGCG; CAS registry number 989515); procyanidin derivative (For example, procyanidin A1 [CAS registry number 103883030], procyanidin B1 [CAS Registry Number 20315257], Procyanidin B4 [CAS Registry Number 29 106512], arecatannin B1 [CAS Registry Number 79763283 ]); Isoflavones (for example, genistein [4% 5,7-trihydroxyisoflavones ;CAS Registry Number 446720], Daidzein [4',7-Dihydroxyisoflavones, CAS Registry Number 486668; Indole-3-carbinol (CAS Registry Number 700 061); Quercetin (NSC9219; CAS Registry Number 117395); Estramus Chin (NSC89201; CAS Registry Number 2998574); Nocodazole (CAS Registry Number 2998574) Number 31430189); Podophyllotoxin (CAS Registry Number 518285); Tartaric acid Vinorelbine (NSC608210; CAS Registry Number 125317397); Cryptof Ishin (NSC667642; CAS Registry Number 124689652).

[0141] Kinase inhibitors: Afatinib (CAS registry number 850140726); Axitinib (CAS Registry Number 319460850); ARRY-438162 (Binimethinib) (C AS Registry Number 606143899; Bosutinib (CAS Registry Number 380843754) Cabozantinib (CAS Registry Number 1140909483); Ceritinib (CAS Registry Number (Certificate No. 1032900256); Crizotinib (CAS Registry Number 877399525); Dub Rafenib (CAS Registry Number 1195765457); Dasatinib (NSC732517) ;CAS Registry Number 302962498); Erlotinib (NSC718781;CAS Registry Number Registration number 183319699); Everolimus (NSC733504; CAS registry number 15 9351696); Fostamatinib (NSC745942; CAS Registry Number 90111) 9355); Gefitinib (NSC715055; CAS Registry Number 184475352) Ibrutinib (CAS Registry Number 936563961); Imatinib (NSC71605) 1; CAS Registry Number 220127571); Lapatinib (CAS Registry Number 3880827 88); Lenvatinib (CAS Registry Number 857890392); Mbritinib (CAS 3 66017096); Nilotinib (CAS Registry Number 923288953); Nintedanib (CAS Registry Number 656247175); palbociclib (CAS Registry Number 571190 302); Pazopanib (NSC737754; CAS Registry Number 635702646); Pe Gaptanib (CAS Registry Number 222716861); Ponatinib (CAS Registry Number 111 4544318); Rapamycin (NSC226080; CAS Registry Number 5312388) 9); Regorafenib (CAS Registry Number 755037037); AP23573 (Lidaf) (Orolimus) (CAS Registry Number 572924540); INCB018424 (Lukisoly) Tinib (CAS Registry Number 1092939177); ARRY-142886 (Selmeth Nib) (NSC741078; CAS Registry Number 606143-52-6); Sirolimus ( NSC226080; CAS Registry Number 53123889); Sorafenib (NSC724) 772;CAS Registry Number 475207591); Sunitinib (NSC736511;CA S registration number 341031547); Tofacitinib (CAS registration number 477600752) ); Temsirolimus (NSC683864; CAS Registry Number 163635043); Tiger Metinib (CAS Registry Number 871700173); Vandetanib (CAS Registry Number 443) 913733); Vemurafenib (CAS Registry Number 918504651); SU6656 (CAS Registry Number 330161870); CEP-701 (lesaurtinib) nib))(CAS Registry Number 111358884);XL019(CAS Registry Number 9457 55566); PD-325901 (CAS Registry Number 391210109); PD-98 059 (CAS Registry Number 167869218); ATP-competitive TORC1 / TORC2 inhibition Agents, for example, PI-103 (CAS registry number 371935749), PP242 (CAS registry number 371935749) (Registration number 1092351671), PP30 (CAS Registry Number 1092788094), T orin 1 (CAS Registry Number 1222998368), LY294002 (CAS Registry Number 1222998368), Number 154447366), XL-147 (CAS Registry Number 934526893), CA L-120 (CAS Registry Number 870281348), ETP-45658 (CAS Registry Number (Category 1198357797), PX866 (CAS Registry Number 502632668), GDC -0941 (CAS Registry Number 957054307), BGT226 (CAS Registry Number 12 45537681), BEZ235 (CAS registration number 915019657), XL-76 5 (CAS Registry Number 934493762), etc.

[0142] Protein synthesis inhibitor: Acriflavin (CAS registry number 65589700); Amica Syn (NSC177001; CAS Registry Number 39831555); Albeca Syn (CAS Registration number 51025855); Astromycin (CAS Registry Number 55779061); Azithromycin (NSC643732; CAS Registry Number 83905015); Bekanama Icin (CAS Registry Number 4696768); Chlortetracycline (NSC13252) ;CAS Registry Number 64722); Clarithromycin (NSC643733; CAS Registry Number 81103119); Clindamycin (CAS Registry Number 18323449); Clo Mocyclin (CAS Registry No. 1181540); Cycloheximide (CAS Registry No. 6 6819); Dactinomycin (NSC3053; CAS Registry Number 50760); Darf Opristin (CAS Registry Number 112362502); Demeclocycline (CAS Registry Number 112362502) Number 127333); Dibekacin (CAS Registry Number 34493986); Dihydrostre Ptomycin (CAS Registry No. 128461); Dilithromycin (CAS Registry No. 62 013041); Doxycycline (CAS Registry Number 17086281); Emetine (N SC33669; CAS Registry Number 483181); Erythromycin (NSC55929) ;CAS Registry Number 114078); Flurithromycin (CAS Registry Number 8366420 8); Furamycetin (Neomycin B; CAS Registry Number 119040); Gentamycin (NSC82261; CAS Registry Number 1403663); Glycylcycline, for example, Gecycline (CAS Registry Number 220620097); Hygromycin B (CAS Registry Number 220620097) Number 31282049); Isepamycin (CAS Registry Number 67814760); Josa Mycin (NSC122223; CAS Registry Number 16846245); Kanamycin (C AS Registry Number 8063078); Ketolides, e.g., telithromycin (CAS Registry Number 191114484), cesromycin (CAS registry number 205110481), and Solithromycin (CAS Registry No. 760981837); Lincomycin (CAS Registry No. Number 154212); Limecycline (CAS Registry Number 992212); Mechrocycline Phosphorus (NSC78502; CAS Registry Number 2013583); Metacycline (Rondoma) Ishin (rondomycin); NSC356463; CAS Registry Number 914001); Midekama Icin (CAS Registry Number 35457808); Minocycline (NSC141993; C AS Registry Number 10118908; Myocamycin (CAS Registry Number 55881077) Neomycin (CAS Registry Number 119040); Netylmycin (CAS Registry Number 56 391561); Oleandmycin (CAS Registry Number 3922905); Oxazolidi Non-, for example, eperezolid (CAS registry number 165800044), linezolid (CAS Registration number 165800033), posizolid (CAS Registry Number 25226) 0029), ladezolid (CAS Registry Number 869884786), lambezolid (ranbez olid (CAS Registry Number 392659380), stezolid (CAS Registry Number 16882 8588), tedizolide (CAS Registry Number 856867555); oxytetracycline N (NSC9169; CAS Registry Number 2058460); Paromomycin (CAS Registry Number (Certificate No. 7542372); Penimepicycline (CAS Registry Number 4599604); Peptidyl Transferase inhibitors, such as chloramphenicol (NSC3069;CAS Registration number 56757) and Azidam phenicol (CAS registration number 13838089) ), florfenicol (CAS registry number 73231342), and thianfenicol Lu (CAS Registry Number 15318453), and proiromucilin, for example, letapamulin (CAS Registry Number 224452668), Chiamlin (CAS Registry Number 55297955 ), derivatives such as barnemurin (CAS registry number 101312929); pirurimycin (CAS Registry Number 79548735); Puromycin (NSC3055; CAS Registry Number 53792); Quinupristin (CAS Registry Number 120138503); Ribostama Icin (CAS Registry Number 53797356); Rokitamycin (CAS Registry Number 7401) 4510); Lolitetracycline (CAS Registry Number 751973); Roxithromycetin n (CAS Registry Number 80214831); shisomycin (CAS Registry Number 3238511) 8); Spectinomycin (CAS Registry Number 1695778); Spiramycin (CAS Registry Number 1695778) Registration number 8025818); streptogramin, e.g., pristinamycin (CAS registration number 8025818) (Registration number 270076603), quinupristin / dalfopristin (CAS Registry Number 12 6602899), and Virginiamycin (CAS Registry Number 11006761); Treptomycin (CAS Registry Number 57921); Tetracycline (NSC10857) 9; CAS Registry Number 60548; Tobramycin (CAS Registry Number 32986564) ;Troleandmycin (CAS Registry Number 2751099);Tylosin (CAS Registry Number Item No. 1401690; Verdamicin (CAS Registry Number 49863481) ).

[0143] Histone deacetylase inhibitor: Abexinostat (CAS Registry Number 7833556) 02); Bellinostat (NSC726630; CAS Registry Number 414864009); Chidamide (CAS Registry Number 743420022); Entinostat (C AS Registry Number 209783802); Givinostat (CAS Registry Number 7 32302997); Mosetinostat (CAS Registry Number 726169739); Panobi Nostat (CAS Registry Number 404950807); Kyxinostat (CAS Registry Number 8 75320299); Resminostat (CAS Registry Number 864814880); Romid Psine (CAS Registry No. 128517077); Sulforaphane (CAS Registry No. 447) 8937); Thiouraidobutyronitrile (Kevetrin(trademark); CAS Registry Number 6659890); Valproic acid (NSC93819; CAS registry number 99661); Boli Nostat (NSC701852; CAS Registry Number 149647789); ACY-12 15 (Rocilinostat; CAS Registry Number 1316214524); C UDC-101 (CAS Registry Number 1012054599); CHR-2845 (Tefino Tefinostat; CAS Registry Number 914382608; CHR-3996(C AS registration number 1235859138); 4SC-202 (CAS registration number 9104624) 30);CG200745(CAS Registry Number 936221339);SB939(Plastic Nostat (CAS Registry Number 929016966).

[0144] Mitochondrial inhibitor: Pancratistatin (NSC349156; CAS Registry No. 9 6281311); Rhodamine-123 (CAS Registry Number 63669709); Edelf Edelfosine (NSC324368; CAS Registry Number 70641519); d- Tocopherol alpha-succinate (NSC173849; CAS Registry Number 434503) 3); Compound 11β (CAS Registry Number 865070377); Aspirin (NSC406) 186; CAS Registry Number 50782); Ellipticine (CAS Registry Number 519233); Berberine (CAS Registry Number 633658); Cerurenin (CAS Registry Number 173978) 96); GX015-070 (Obatoclax(registered trademark); 1H-indole, 2 -(2-((3,5-dimethyl-1H-pyrrole-2-yl)methylene)-3-methoxy -2H-pyrrole-5-yl)-;NSC729280;CAS Registry Number 8037126 76); Cerastrol (tripterin; CAS registry number 34157830); Metfor Min (NSC91485; CAS Registry Number 1115704); Brilliant Green (N SC5011; CAS Registry Number 633034); ME-344 (CAS Registry Number 1374) 524556).

[0145] Antimitotic agent: allocolchicine (NSC406042); Auris Tatin, for example MMAE (monomethyl auristatin E; CAS registry number 474645- 27-7) and MMAF (Monomethyl Auristatin F; CAS Registry No. 745017) -94-1; Halichondrin B (NSC609395); Colchicine (NSC757; C AS registration number 64868); Colchicine derivative (N-benzoyl-dea Cetylbenzamide; NSC33410; CAS Registry Number 63989753); Drasta Chin 10 (NSC376128; CAS Registry Number 110417-88-4); Meitanshi (NSC153858; CAS Registry Number 35846-53-8); Rhizoxin in) (NSC332598; CAS Registry Number 90996546); Taxol (NSC1 25973; CAS Registry Number 33069624); Taxol derivative ((2'-N-[3 -(dimethylamino)propyl]glutaramate taxol; NSC60 8832); Thiocorchicine (3-Demethylthiocolchicine; NSC361792); Lithylcysteine ​​(NSC49842; CAS Registry Number 2799077); Vimbala sulfate Stine (NSC49842; CAS Registry Number 143679); Vincristine Sulfate (NS C67574; CAS Registry Number 2068782).

[0146] Of these drugs, those that include an attachment site for antibodies, or those that include an attachment site for antibodies Anything that can be modified to include may be included in the ADC disclosed herein. ru.

[0147] In specific embodiments, cytotoxic substances and / or cell proliferation inhibitory substances are anti It is a mitotic agent.

[0148] In another specific embodiment, a cytotoxic substance and / or a cell proliferation inhibitory substance is auristatin, for example, monomethyl auristatin E ("MMAE") or monomethyl auristatin E It is Chil-Olistin F ("MMAF").

[0149] 5.3.2. Linker In the antiglycoMUC1 ADC of this disclosure, cytotoxic substances and / or cell proliferation The inhibitory substance is linked to the antibody by a linker. The antibody of the ADC contains a cytotoxic substance. and / or linkers that link cell growth inhibitors are short, long, hydrophobic, hydrophilic The linker may be flexible or rigid, or the linker may have different properties. Each segment may independently possess one or more of the above characteristics, including segments that have the above characteristics. They may consist of segments that resist more than one drug. They may be polyvalent so as to be covalently linked to a single site on the body, or they may be single It may be monovalent, so that one drug is covalently linked to a single site on the antibody.

[0150] As those skilled in the art would expect to understand, the linker contains cytotoxic substances at one position. and / or form a covalent linkage to a cell proliferation inhibitor, with the antibody at the other position. By forming covalent bonds to cytotoxic substances and / or cell proliferation, The growth-inhibiting substance is linked to the antibody. Covalent linkage occurs between the functional group on the linker and the drug. It is formed by reaction with functional groups on antibodies. When used herein, "linker" The expression "(i) linker to cytotoxic and / or cell proliferation inhibitory substances" Functional groups that can be linked by covalent bonds, and linkers that can be covalently linked to antibodies. (ii) a linker in an unconjugated form containing possible functional groups; (ii) a linker It can be covalently linked to antibodies, inhibiting cytotoxic substances and / or cell proliferation. Linkers containing functional groups covalently linked to the control substance, or vice versa. (iii) partially conjugated forms; and (iii) cytotoxic substances and / or fine The linker is covalently linked to both the cell proliferation inhibitor and the antibody, and is completely controlled. This disclosure is intended to include a jugated form. Linker and antiglycoMUC1 In addition to ADCs, there are also synths, which are used to conjugate linkers—drugs—to antibodies. In some specific embodiments, a functional group on the linker and between the linker and the antibody The parts containing the covalent bonds that have been formed are each R x And as a specific example, XY It will be shown.

[0151] Linkers are preferably, but not necessarily, adapted to extracellular conditions. In contrast, it is chemically stable and is cleaved, destroyed, and / or otherwise destroyed within the cell. It may be designed to be specifically degraded by other means. Alternatively, inside the cell A linker not designed to be specifically cleaved or broken down may be used. The choice between a stable linker and an unstable linker affects the inhibition of cytotoxic substances and / or cell proliferation. It can be determined by the toxicity of the substance. In the case of drugs toxic to normal cells, stable cells Ink is preferred. It is selective for normal cells or targets them to reduce toxicity. The use of drugs may be permitted, and the chemical stability of the linker in the extracellular environment is not that important. No. Various linkers useful for linking drugs to antibodies in the ADC state are available in this industry. It is publicly known that any of these linkers, and other linkers, are cytotoxic substances. The substance and / or cell proliferation inhibitory substance is linked to the antibody of the antiglycoMUC1 ADC of this disclosure. It can be used for tying knots.

[0152] Linking many cytotoxic and / or cell proliferation inhibitory substances to a single antibody molecule. An example of a multivalent linker that can be used is, for example, one whose entire contents are referenced in this book. The International Publication No. 2009 / 073445 pamphlet, which is incorporated into the specification; International Publication Pamphlet No. 2010 / 068795; International Publication Pamphlet No. 2010 / 138719 Lett; International Publication No. 2011 / 120053 Pamphlet; International Publication No. 2011 / 17 Brochure No. 1020; International Publication No. 2013 / 096901; International Publication Pamphlet No. 2014 / 008375; International Publication Pamphlet No. 2014 / 093379 Lett; International Publication No. 2014 / 093394 Pamphlet; International Publication No. 2014 / 09 It is described in pamphlet No. 3640. For example, developed by Mersana et al. Fleximer linker technology provides high-DAR ADCs with superior physicochemical properties. It has the ability to make this possible. As shown below, Mersana technology enables the arrangement of ester bonds. This is based on incorporating drug molecules into a soluble polyacetal backbone via a series of columns. This method involves a highly loaded ADC (most) while maintaining excellent physicochemical properties. It brings about a large 20 DAR.

[0153] Further examples of dendritic linkers are incorporated herein by reference, U.S. Patent Application Publication No. 2006 / 116422; U.S. Patent Application Publication No. 2005 / 2 Specification No. 71615; de Groot et al. (2003) Angew. Chem. Int. Ed. 42:4490-4494;A mir 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 Let ters 12:2213-2215;Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-176 8; This can be found in King et al. (2002) Tetrahedron Letters 43:1987-1990.

[0154] Examples of monovalent linkers that can be used are, for example, each incorporated herein by reference. 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. Me d. Chem. 54:3606-3623; U.S. Patent No. 7,223,837; U.S. Patent No. 8,56 U.S. Patent No. 8,728; U.S. Patent No. 8,535,678; and International Publication No. 200 It is listed in pamphlet number 4010957.

[0155] For example, but not limited to, the antiglycoMUC1 ADCs of this disclosure include Some of the severable and non-severable linkers obtained will be described later.

[0156] 5.3.3. Cuttable Linker In certain embodiments, the selected linker is detachable in vivo. A capable linker may contain chemically or enzymatically unstable or degradable links. Good. A cleavable linker is generally a cell-internal process that releases a drug, for example. Reduction in the cytoplasm, exposure to acidic conditions in lysosomes, or specific proteins within cells It relies on cleavage by enzymes or other enzymes. The cleavable linker is generally chemical or It incorporates one or more chemical bonds that can be cleaved enzymatically, and the same Sometimes, the remaining part of the linker is impossible to cut. In certain embodiments, the linker is chemical Contains chemically unstable groups, such as hydrazones and / or disulfide groups. Linkers containing unstable groups exhibit different properties between plasma and certain intracytoplasmic compartments. To utilize. In the case of a linker containing hydrazone, intracellular conditions that facilitate drug release are: The acidic environment of endosomes and lysosomes, on the other hand, contains disulfide-containing linkers. - is reduced in a cytosol containing a high thiol concentration, such as glutathione. In certain embodiments, the plasma stability of a linker containing a chemically unstable group is chemically This can be increased by introducing steric hindrance using substituents near the unstable group. can.

[0157] Acid-instability compounds, such as hydrazones, are in the neutral pH environment of blood (pH 7.3-7.5). During systemic circulation, it remains intact, but ADCs are affected by the cell's weakly acidic endosomes (pH 5.0 When internalized in the compartments of the lysosome (pH 4.5-5.0) and 6.5°C, hydrolysis occurs. Upon receiving the signal, the drug is released. This pH-dependent release mechanism is related to the nonspecific release of the drug. They are linked together. To increase the stability of the hydrazone group of the linker, the linker is, To achieve more efficient release in lysosomes while minimizing loss in circulation. This can be modified by chemical modifications that can be adjusted, such as substitution.

[0158] Linkers containing hydrazones have additional cleavage sites, such as additional acid instability cleavage sites. It may contain cleavage sites and / or enzyme-instability sites. Exemplary hydrazones The ADC containing the linker has the following structure:

[0159] [ka] In the formula, D and Ab are cytotoxic substances and / or cell proliferation inhibitory substances, respectively. The term "drug" represents the drug and "Ab," where n represents the number of drug-linkers linked to the antibody. In a linker, for example, in a linker (Ig), the linker has two cleavable groups, namely It includes a disulfide moiety and a hydrazone moiety. In the case of such a linker, it is modified Effective release of free drugs does not require acidic pH or disulfide reduction and acidic pH. The key is that linkers such as (Ih) and (Ii) are effective for a single hydrazone cleavage site. It has been shown that...

[0160] While intact during systemic circulation, ADCs become internalized within acidic cellular compartments, leading to hydrolysis. Carbonates are examples of additional linkers that release drugs upon receiving a reaction. Una linker allows cytotoxic substances and / or cell proliferation inhibitors to co-transmit via oxygen. It may be useful if it can be attached via a coupling mechanism.

[0161] Other acid-unstable groups that may be present in the linker include cis-aconityl. Linkers are one example. The cis-aconityl chemistry promotes amide hydrolysis under acidic conditions. To advance the process, a carboxylic acid is used in conjunction with the amide bond.

[0162] The cleavable linker may also contain a disulfide group. Disulfides are physiological It is designed to be thermodynamically stable at pH and to release the drug once it is internalized within the cell. In this case, the cytosol provides a significantly more reducing environment compared to the extracellular environment. The separation of disulfide bonds is generally achieved when a linker containing disulfides is circulating. It is moderately stable and selectively releases the drug in the cytosol, and the cytoplasmic thio It requires the presence of cofactors, such as (reduced) glutathione (GSH). Disulfide isomerase, an intracellular enzyme protein, or the cleavage of disulfide bonds Possible similar enzymes may also contribute to the preferential cleavage of disulfide bonds within cells. It is possible. In approximately 5 tumor cells, GSH or cysteine ​​is present in circulation (this is Compared to GS, which has a significantly lower concentration (as it is the most abundant low molecular weight thiol), H has been reported to be present in cells at concentrations ranging from 0.5 to 10 mM, in this case Irregular blood flow causes a state of hypoxia, which enhances the activity of reducing enzymes, and therefore G The lutathione concentration becomes even higher. In certain embodiments, a disulfide is contained. The in vivo stability of the linker depends on its chemical modification, for example, disulfide The bond can be strengthened by using steric hindrances adjacent to the bond.

[0163] An example ADC containing a disulfide-containing linker has the following structure:

[0164] [ka] In the formula, D and Ab represent the drug and antibody, respectively, and n is the drug linked to the antibody. - Represents the number of linkers, where R is selected independently of hydrogen or alkyl for each occurrence. Selected. In certain embodiments, the steric hindrance adjacent to the disulfide bond is increased. This increases the stability of the linker. Structures such as (Ij) and (Il) are one Alternatively, if multiple R groups are selected from lower alkyl groups, for example methyl groups, in vivo It shows increased stability.

[0165] Another type of cleavable linker that can be used is one that is specifically cleaved by enzymes. This is a linker. Such linkers are typically peptide-based or It contains a peptide region that acts as a substrate for the enzyme. The peptide-based linker is plasma Furthermore, in the extracellular environment, it tends to be more stable than chemically unstable linkers. The proteolytic enzymes in the lysosome are inconveniently high compared to endogenous inhibitors and lysosomes. Because of the H value in blood, peptide bonds generally have very low activity in the blood. It exhibits excellent stability in serum. Drug release from antibodies is specifically facilitated by lysosomes. This is caused by the action of theases, such as cathepsin and plasmin. Aase may be present at high levels in certain tumor cells.

[0166] In an exemplary embodiment, the cleavable peptide is Gly-Phe-Leu-Gly Tetra (SEQ ID NO: 128), Ala-Leu-Ala-Leu (SEQ ID NO: 129), etc. Peptides, or Val-Cit, Val-Ala, Met-(D)Lys, Asn-( D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val , His-Val, NorVal-(D)Asp, Ala-(D)Asp5, Met-L ys, Asn-Lys, Ile-Pro, Me3Lys-Pro, PhenylGly-(D )Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Me t-(D)Lys, Asn-(D)Lys, AM Met-(D)Lys, Asn-(D Dipeptides such as )Lys, AW Met-(D)Lys, and Asn-(D)Lys Selected from. In certain embodiments, longer peptides are hydrophobic, therefore dip Butyrospermocytes are preferred over longer polypeptides.

[0167] Doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepine, talyso Tallysomycin and auristatin / auristatin family members This document describes various dipeptide-based cleavable linkers useful for linking drugs to antibodies. The following are included herein by reference (each incorporated herein by reference, Dubowchik et al., 19 98, 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;Walk er et al., 2004, Bioorg. Med. Chem. Lett. 14:4323-4327;Sutherland et al., 2013, (See Blood 122: 1455-1463; and Francisco et al., 2003, Blood 102: 1458-1465) These dipeptide linkers, or modified versions of these dipeptide linkers All of the above can be used in the antiglycerid MUC1 ADC of this disclosure. Other dipeptide linkers include Seattle Genetics' Brentuxima Buvedotin SGN-35 (Adcetris®), Seattle Genetics' SGN-75 (anti-CD-70, Val-Cit-monomethylauris) Tatin F (MMAF), Seattle Genetics' SGN-CD33A (anti-C D-33, Val-Ala-(SGD-1882)), Celldex Therape Glenbatumumab (CDX-011) from utics (anti-NMB, Val-Cit-monomegaly) Chil-Olistatin E (MMAE) and Cytogen PSMA-ADC AD (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE) Examples of what can be found in C include...

[0168] As an enzymatically cleavable linker, to spatially separate the drug from the enzymatic cleavage site One example is a self-destructing spacer. Direct attachment of drugs to the peptide linker. This can cause the release of amino acid adducts of drugs through protein degradation, Its activity is impaired by this. The use of self-destructing spacers is after the hydrolysis of the amide bond. This allows for the removal of sufficiently active, chemically unmodified drugs.

[0169] One self-destructing spacer is a bifunctional para-aminobenzyl alcohol group, and this It is linked to the peptide via an amino group to form an amide bond, but contains an amine. The drug is activated via carbamic acid functionalization to the hydroxyl group of benzyl in the linker (PABC) It may be attached in this manner. The resulting prodrug is activated by protease-mediated cleavage. The drug is modified, undergoing a 1,6 elimination reaction, and the unmodified drug, carbon dioxide, and phosphorus are removed. The remainder of the Kerr group is released. The following scheme is for the fragmentation of p-amide benzyl ether. Describes transformation and drug release.

[0170] [ka] In the formula, XD represents the unmodified drug.

[0171] Heterocyclic variants of this self-destructing group are also described. For example, those incorporated herein by reference. See U.S. Patent No. 7,989,434, which can be included.

[0172] In some embodiments, the enzymatically cleavable linker is β-glucuronic acid-based It is a linker. The easy release of drugs is facilitated by the lysosomal enzyme β-glucuronidase. This can be achieved through the cleavage of the β-glucuronide glycosidic bond. This enzyme, It is abundant within lysosomes and is overexpressed in some tumor types, but outside the cell. Enzyme activity is low in this case. The β-glucuronide-based linker is hydrophilic to β-glucuronide. It can be used to avoid the tendency of ADCs to aggregate due to their properties. In one embodiment, a β-glucuronic acid-based linker is linked to a hydrophobic drug. Preferred as a linker for C. The following scheme is a β-glucuronic acid-based phosphorus This describes the release of drugs from an ADC containing CAR.

[0173] [ka]

[0174] Auristatin, camptothecin and doxorubicin analogs, CBI secondary groove binder , and various cuts useful for linking drugs such as psymberin to antibodies Interruptible β-glucuronic acid-based linkers are described (each by reference in this book). The specification includes Nolting, Chapter 5 “Linker Technology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1 045, pp. 71-100, Laurent Ducry (Ed.), Springer Science & Business Medica, LLC, 2 013;Jeffrey et al., 2006, Bioconjug. Chem. 17:831-840;Jeffrey et al., 2007, Bi oorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. See 127:11254-11255). All of these β-glucuronic acid-based linkers are described herein. It can be used in antiglycerid MUC1 ADCs.

[0175] In addition, cytotoxic substances and / or cell proliferation inhibitors containing phenol groups are This allows the phenol to be covalently bonded to the linker via the oxygen. Such linkers are described in the International Publication No. 2007 / 089149 pamphlet. Diaminoethane "SpaceLink" is used to deliver phenol, unlike conventional "P The method relies on using an ABO-based self-destructing group. Linker cleavage is schematically shown below. Described in the formula, where D is a cytotoxic substance having a hydroxyl group of phenol and / Alternatively, it represents a substance that inhibits cell proliferation.

[0176] [ka]

[0177] A severable linker may include an inseverable portion or segment, A segment or portion that can be cut is, in order to make it cut, It may be contained within an external, inseparable linker. As just one example, polyethylene gum Coal (PEG) and related polymers have cleavable groups in the polymer backbone. It may contain. For example, polyethylene glycol or polymer linker may be one or comprising multiple cleavable groups, such as disulfide, hydrazone, or dipeptide That's fine.

[0178] Other degradable links that may be present in the linker include PEG carboxylic acids or active Formed by the reaction of the carboxylic acid of modified PEG with the alcohol group on a bioactive drug. An example is ester linkage, in which case such an ester group is generally under physiological conditions It hydrolyzes and releases bioactive drugs. The links that can be decomposed by hydrolysis are these Not limited to, but include carbonate linkage; imine linkage resulting from the reaction of amines and aldehydes; and alcohol linkage. Phosphate ester linkage formed by reacting aldehyde with a phosphate group; aldehyde and Acetal linkage is a reaction product of alcohols; it is a reaction product of formate and alcohols. Orthoester linkage; and, but not limited to, phosphoamide at the ends of polymers. The oligonucleotide formed by the 5' hydroxyl group of the oligonucleotide and the 5' hydroxyl group of the oligonucleotide. One example is the Ochido linkage.

[0179] In certain embodiments, the linker is an enzymatically cleavable peptide moiety, for example, Formation (IVa) or (IVb):

[0180] [ka] The formula includes a linker or a salt thereof, wherein the peptide is cleavable by a lysosomal enzyme. Possible peptides (exemplified by C→N, carboxyl and amino "terminuses" are not shown) This represents; T is one or more ethylene glycol units or alkylene chains, R represents polymers containing those combinations; a These are hydrogen, alkyl, sulfonate and me Selected from chlorosulfonates; p is an integer in the range of 0 to 5; q is 0 or 1 x is 0 or 1; y is 0 or 1;

[0181] [ka] This represents the attachment point of the linker to cytotoxic and / or cell proliferation inhibitory substances. ; * This indicates the attachment point to the rest of the linker.

[0182] 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- Selected from Cit; Ile-Cit; Phe-Arg; and Trp-Cit. In the embodiment, the dipeptide is selected from Cit-Val and Ala-Val. It can be done.

[0183] Linker by structural formula (IVa) that may be included in the antiglycoMUC1 ADC of this disclosure Specific exemplary embodiments include the linker exemplified below (exemplified The linker contains a group suitable for covalently linking the linker to the antibody.

[0184] [ka]

[0185] [ka]

[0186] The linker according to structural formula (IVb) that may be included in the antiglycoMUC1 ADC of this disclosure Specific exemplary embodiments include the linker exemplified below (exemplified The linker contains a group suitable for covalently linking the linker to the antibody.

[0187] [ka]

[0188] [ka]

[0189] [ka]

[0190] [ka]

[0191] In certain embodiments, the linker is an enzymatically cleavable peptide moiety, for example, Formation (IVc) or (IVd):

[0192] [ka] The formula includes a linker or a salt thereof, wherein the peptide is cleavable by a lysosomal enzyme. Possible peptides (exemplified by C→N, carboxyl and amino "terminuses" are not shown) This represents; T is one or more ethylene glycol units or alkylene chains, R represents polymers containing those combinations; a These are hydrogen, alkyl, sulfonate and me Selected from chlorosulfonates; p is an integer in the range of 0 to 5; q is 0 or 1 x is 0 or 1; y is 0 or 1;

[0193] [ka] This represents the attachment point of the linker to cytotoxic and / or cell proliferation inhibitory substances. ; * This indicates the attachment point to the rest of the linker.

[0194] Linker by structural formula (IVc) that may be included in the antiglycoMUC1 ADC of this disclosure Specific exemplary embodiments include the linker exemplified below (exemplified The linker contains a group suitable for covalently linking the linker to the antibody.

[0195] [ka]

[0196] Linker by structural formula (IVd) that may be included in the antiglycoMUC1 ADC of this disclosure Specific exemplary embodiments include the linker exemplified below (exemplified The linker contains a group suitable for covalently linking the linker to the antibody.

[0197] [ka]

[0198] [ka]

[0199] In certain embodiments, structural formulas (IVa), (IVb), (IVc), or (IV The linker containing d) further has a carbonate portion that can be cleaved by exposure to an acidic medium. Includes. In certain embodiments, the linker transmits cytotoxic substances and / or via oxygen. It is attached to a cell proliferation inhibitory substance.

[0200] 5.3.4. Linkers that cannot be cut While a severable linker may offer certain advantages, the antiglyco-MUC1 ADC of this disclosure Linkers containing the drug do not necessarily have to be cleavable. In the case of a non-cleavable linker, The release of substances does not depend on the different properties between the plasma and certain intracytoplasmic compartments. Release involves the internalization of ADCs via antigen-mediated endocytosis and delivery to the lysosomal compartment. This occurs afterward, where the antibody is broken down to the amino acid level via intracellular protein degradation. It is assumed that this process involves a drug, a linker, and the linker being covalently bonded. It releases drug derivatives formed by attached amino acid residues. Uncleavable phosphorus Amino acid drug metabolites from conjugates with a linker are conjugated with a cleavable linker. Compared to gypsum, it is more hydrophilic and generally has lower membrane permeability, and therefore This results in less bystander action and less nonspecific toxicity. The ADC with an unbreakable linker is greater than the ADC with a breakable linker in circulation. It has high stability. The unbreakable linker may be an alkylene chain or a natural Polymers, such as polyalkylene glycol polymers and amide polymer-based polymers. It may be present, or alkylene chains, polyalkylene glycol (glocol) and / or Alternatively, it may contain segments of an amide polymer.

[0201] Various non-cleavable linkers used to link drugs to antibodies are described. Each of the following is incorporated herein by reference: Jeffrey et al., 2006, Bioconjug. C hem. 17;831-840;Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280;a See nd Jiang et al., 2005, J. Am. Chem. Soc. 127:11254-11255. These Any of the linkers may be included in the antiglycoMUC1 ADC of this disclosure.

[0202] In certain embodiments, the linker is inseparable in vivo, for example, in a structural formula A linker by (VIa), (VIb), (VIc), or (VId) (as exemplified) As described above, the linker is a suitable base for covalently linking the linker to the antibody:

[0203] [ka] or a salt thereof, in the formula, R a These are hydrogen, alkyl, sulfonate and methyl sulfonate. Selected from Nate; R x This refers to a functional group that can covalently link the linker to the antibody. This is the part that includes;

[0204] [ka] This represents the linker's attachment point to cytotoxic and / or cell proliferation inhibitory substances. .

[0205] The structural formulas (VIa) to (VId) that may be included in the antiglycoMUC1 ADC of this disclosure are as follows: Specific exemplary embodiments of the linker include the linker exemplified below. (As illustrated, the linker has a suitable base for covalently linking the linker to the antibody.) Including,

[0206] [ka] This indicates an attachment point to a cytotoxic substance and / or a cell proliferation inhibitor.

[0207] [ka]

[0208] 5.3.5. Bases used to attach the linker to the antibody Various groups are used to obtain ADCs by attaching a linker-drug synth to an antibody. It is possible. The attachment group may be intrinsically electrophilic, for example, Mallet Imide groups, activated disulfides, activated esters, such as NHS esters and HOBt esters. Steryl, formic acid halides, acid halides, alkyl halides and benzyl, for example Haloacetamides are one example. As discussed below, they can be used in accordance with this disclosure. Emerging technologies for "self-stabilizing" maleimides and "cross-linking disulfides" that enable this There are also techniques available. The specific substrate used is expected to depend in part on the attachment site to the antibody. .

[0209] ADC species that undergo spontaneous hydrolysis under antibody conjugation conditions to improve stability An example of the resulting "self-stabilizing" maleimide group is illustrated in the following schematic diagram. U.S. Patent Application Publication No. Specification No. 20130309256; further, Lyon et al., Nature Biotech published on See also line, doi:10.1038 / nbt.2968.

[0210] [ka]

[0211] [ka]

[0212] Polytherics are derived from the reduction of natural hinged disulfide bonds. A method for bridging pairs of hydryl groups is disclosed. Badescu et al., 2014, Bioconjugate C See hem. 25:1124-1136. This reaction is illustrated in the following diagram. Advantages of this method. This involves the complete reduction of IgG (producing four pairs of sulfhydryls) followed by four equivalents of This refers to the ability to synthesize DAR4 ADC enriched by reaction with a lycylating agent. ADCs containing "disulfide" have also been reported to have increased stability.

[0213] [ka]

[0214] Similarly, as described below, males that can bridge pairs of sulfhydryl groups Imide derivatives (1, below) have been developed. International Publication No. 2013 / 085925 Please refer to the frets.

[0215] [ka]

[0216] 5.3.6. Considerations for Linker Selection As is well known to those skilled in the art, the selected linkers for a particular ADC are not limited to these. However, there is no attachment site to the antibody (e.g., lys, cys, or other amino acid residues), The effects are influenced by various factors, including the structural constraints of the drug's pharmacological action groups and the drug's lipophilicity. It is possible to receive. The selected linker for a specific ADC is a specific antibody / multiple A balance of these various factors is required for combination drugs. Regarding the general overview of factors affected by linker selection, see Nolting, Chapter 5. “Linker Technology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates : Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Spri See nger Science & Business Medica, LLC, 2013.

[0217] For example, ADCs can detect bystander antigen-negative cells located near antigen-positive tumor cells. It has been observed that it causes lethality. The mechanism of bystander cell lethality by ADCs is This demonstrated that metabolites formed during the intracellular processing of ADCs can play a role. The neutral cytotoxic metabolites produced by the metabolism of ADCs in antigen-positive cells are Although it appears to play a role in the lethality of Istandale cells, the charged metabolites are membrane This could potentially prevent the spread of the virus through other media, thus affecting bystander lethality. It is not possible to provide. In certain embodiments, the linker is a cellular metabolite of ADC Selected to mitigate the bystander lethality caused by specific implementations. Morphologically, the linker is selected to increase bystander lethality.

[0218] Furthermore, the properties of the linker also affect the aggregation of ADCs under usage and / or storage conditions. It is possible. Typically, ADCs reported in the literature contain 3-4 or more per antibody molecule. It contains the following drug molecules (see, for example, Chari, 2008, Acc Chem Res 41:98-107). Attempts to obtain a higher drug-to-antibody ratio ("DAR") are particularly challenging when considering both the drug and the linker. If the material is hydrophobic, the ADC often aggregates, leading to failure (King et al., 2002, JM ed Chem 45:4336-4343;Hollander et al., 2008, Bioconjugate Chem 19:358-361;Burk e et al., 2009 Bioconjugate Chem 20:1242-1250). In many cases, higher than 3-4. DAR may be beneficial as a means of increasing potency. Cytotoxic substances and / or cells In cases where the growth inhibitor is inherently hydrophobic, a DAR greater than 3-4 is particularly desirable. In a typical example, a relatively hydrophilic linker is selected as a means to reduce ADC aggregation. It may be desirable to do so. Therefore, in certain embodiments, the linker stores It incorporates chemical components that reduce ADC aggregation during storage and / or use. (Linker) To reduce the aggregation of ADCs, polar or hydrophilic groups, such as charged groups or It may incorporate a group that becomes charged at a physiological pH. For example, a linker. This involves, for example, deprotonating a carboxylic acid at a physiological pH, or, for example, purging an amine. The rotonized charged group, such as a salt or other group, may be incorporated.

[0219] Numerous cytotoxic substances and / or have been reported to result in high DAR levels of 20. An exemplary polyvalent linker that can be used to link cell proliferation inhibitors to antibodies is, among The entirety of these works is incorporated herein by reference, International Publication No. 2009 / 073. Pamphlet No. 445; International Publication No. 2010 / 068795; International Publication No. Pamphlet No. 2010 / 138719; International Publication No. 2011 / 120053 Pamphlet International Publication No. 2011 / 171020 Pamphlet; International Publication No. 2013 / 096 Pamphlet No. 901; International Publication No. 2014 / 008375; International Publication No. Pamphlet No. 2014 / 093379; International Publication No. 2014 / 093394 This information is included in the international publication brochure No. 2014 / 093640.

[0220] In certain embodiments, aggregation of ADCs during storage or use is detected by size exclusion chromatography. According to Raffie (SEC), it is less than approximately 10%. In certain embodiments Aggregation of ADCs during storage or use can be detected by size exclusion chromatography (SEC). If that is decided, then it will be less than 10%, for example, less than 5%, less than 4%, less than 3%, or about 2%. Less than, approximately less than 1%, approximately less than 0.5%, approximately less than 0.1%, or even lower than that. stomach.

[0221] 5.3.7. Method for fabricating antiglyco-MUC1 ADCs The antiglyco-MUC1 ADCs of this disclosure can be synthesized using well-known chemical methods. The selected chemical method involves, among many others, cytotoxic substances and / or cell proliferation. The inhibitory substance depends on the properties of the linker and the base used to attach the linker to the antibody. It is expected that... Generally, the ADC according to formula (I) is prepared according to the following scheme... It is possible: DLR x +Ab-R y →[DL-XY] n -Ab (I) In the formula, D, L, Ab, XY, and n are as previously defined, and R x and R y As discussed above, complementary elements are capable of forming covalent bonds with each other. It represents a base.

[0222] R x and R y The properties of the base are, Synton DLR x Chemicals used to link to antibodies It is expected to depend on the chemical method. Generally, the chemical method used is to ensure the integrity of the antibody. For example, it should not alter its ability to bind to its target. Preferably, The binding properties of a conjugated antibody are extremely different from those of an unconjugated antibody. They are expected to be similar. Various methods for conjugating molecules into biomolecules such as antibodies. Such chemical methods and techniques are publicly known in the industry, and in particular for conjugating antibodies. The following is well known. For example, Amon et al., “Monoclonal Antibodies For Immunotar getting Of Drugs In Cancer Therapy,” in: Monoclonal Antibodies And Cancer Therap y, Reisfeld et al. Eds., Alan R. Liss, Inc., 1985;Hellstrom et al., “Antibodie s For Drug Delivery,” in: Controlled Drug Delivery, Robinson et al. Eds., Marce l Dekker, Inc., 2nd Ed. 1987;Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in: Monoclonal Antibodies '84: Biological And Clin ical 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;PCT countries Please refer to International Publication No. 89 / 12624. Any of these chemical methods Synthon can also be used to link antibodies.

[0223] Functional group R is useful for linking synthons to accessible lysine residues. x The number and chemical formula The law is publicly known, and examples include, but are not limited to, NHS esters and iso Thiocyanates are one example.

[0224] Useful for linking synthons to accessible free sulfhydryl groups of cysteine ​​residues. Functional group R x The number and chemical methods are well known, and are not limited to these, but include, for example: Examples include haloacetyl and maleimide.

[0225] However, the chemical properties of the conjugation are not limited to the available side chain groups. The side chains of amines, etc., can be linked to other useful groups by attaching appropriate small molecules to the amine. For example, it can be converted to hydroxyl. This strategy allows for the antibody to access the amino acid residue. By conjugating polyfunctional small molecules to the side chain of the base molecule, usable linkage on antibodies is achieved. It can be used to increase the number of parts. Then these "converted" parts Functional group R is suitable for covalently linking synthons to the active group. x However, it is included in Shinton.

[0226] Antibodies can also be manipulated to include amino acid residues for conjugation. It can. Non-genetically encoded amino acid residues are useful for conjugating drugs in the ADC state. An approach to manipulating antibodies to include the group involves synthons attached to non-coding amino acids. Along with useful chemical methods and functional groups for bonding, Axupet al., 2012, Proc Natl Acad S This is described by ci USA. 109(40):16101-16106.

[0227] Typically, synthons are located on the side chains of amino acid residues of antibodies, for example, accessible lysine residues. Linked to the primary amino group of the base or the sulfhydryl group of an accessible cysteine ​​residue. Free sulfhydryl groups are obtained by reducing the interchain disulfide bonds. It is possible.

[0228] R y If R is a sulfhydryl group (for example, x (When maleimide is used in concatenation) Generally, antibodies first completely or partially reduce the cysteine ​​residues between the chains. Destroy the Rufid Bridge.

[0229] Mutations in one or more codons can cause cysts that do not participate in disulfide crosslinking. The residue can be manipulated to enter the antibody. Reduction of cysteine ​​without these correspondences. This generates sulfhydryl groups suitable for conjugation. Manipulated cysteine Preferred locations for incorporation include, but are not limited to, human Ig On the G1 heavy chain, positions S112C, S113C, A114C, S115C, and A176C 5180C, S252C, V286C, V292C, S357C, A35 9C, S398C, S428C (Kabat numbering), and human Ig kappa On the light chain, V110C, S114C, S121C, S127C, S168C, V205C (Kabat numbering) is one example (for example, US 7,521, U.S. Patent No. 541, U.S. Patent No. 7,855,275 and U.S. Patent No. 8,455, (See Specification No. 622).

[0230] As expected to be understood by those skilled in the art, numerous cell wounds linked to antibody molecules Harmful substances and / or cell proliferation inhibitors; some antibodies contain one linked drug. Some antibodies contain two linked drugs, while others contain three linked drugs. Some antibodies contain drugs (although some antibodies do not contain linked drugs), and the aggregate of ADCs is this It is possible to modify it so that it can be qualitatively heterogeneous. Among the many degrees of heterogeneity, However, the chemicals used to link cytotoxic substances and / or cell proliferation inhibitors It is expected to depend on the method. For example, the antibody is reduced and the sulfhydr for attachment When a lyl group is formed, each molecule has 0, 2, 4, 6, or 8 linked drug molecules. Often, a heterogeneous mixture of antibodies is produced. Furthermore, controlling the molar ratio of the attachment compounds... By limiting the number of molecules, 0, 1, 2, 3, 4, 5, 6, 7, or 8 links can be formed per molecule. Antibodies containing the drug are often produced. Therefore, depending on the context, It is expected that the DAR can be the average of the antibody aggregate. For example, "DAR4" has not been purified to isolate specific DAR peaks, and per antibody The number of cell proliferation inhibitors and / or cytotoxic substances attached differs from one another. For example, an antibody may contain a heterogeneous mixture of ADC molecules (0, 2, 4, 6, or 8 drugs per antibody). It may appear that this refers to an ADC preparation in which the average drug-to-antibody ratio is 4. In some embodiments, "DAR2" is heterogeneous, where the average drug-to-antibody ratio is 2. This refers to an ADC preparation.

[0231] If an enriched preparation is desired, a predetermined number of linked cytotoxic substances and / or Antibodies containing cell proliferation inhibitory substances are obtained through the purification of heterogeneous mixtures, for example, by scalculating This can be obtained via chromatography, for example, hydrophobic interaction chromatography. ru.

[0232] Purity can be evaluated by various methods, as is well known in this industry. As a concrete example, ADC preparations are analyzed via HPLC or other chromatography. This may be done, and the purity may be evaluated by analyzing the area under the curve of the obtained peak. stomach.

[0233] 5.4 Chimeric Antigen Receptors The disclosure of the present invention relates to the anti-glycoMUC1 antibody or antigen-binding fragment described herein. The present invention provides a chimeric antigen receptor (CAR) containing an antigen.

[0234] The CARs of this disclosure typically have an extracellular domain operably linked to a transmembrane domain. It includes, which are sequentially operably linked to intracellular domains for signal transduction.

[0235] The extracellular domain of the CAR disclosed herein is an anti-glycoMUC1 antibody or antigen-binding fragment. Includes an array of (for example, as described in Section 5.1 or Embodiments 1 to 90) nothing.

[0236] Exemplary transmembrane domain sequences and intracellular domain sequences are shown in Section 5.4, respectively. This is described in sections 1 and 5.4.2.

[0237] Numerous fusion proteins described herein (e.g., Examples 92 and 94-96) ) is a CAR, and disclosures related to CARs apply to such fusion proteins. .

[0238] 5.4.1. Transmembrane domain Regarding the transmembrane domain, the CAR is operably linked to the extracellular domain of the CAR. It can be designed to include (for example, fused) transmembrane domains.

[0239] The transmembrane domain may be derived from either a natural or synthetic source. In some cases, the domain may originate from any membrane-bound or transmembrane protein. The transmembrane region for specific applications shown is the T cell receptor, CD28, CD3 epsilon, C D45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 Alpha, Bass It may be derived from the ta or zeta chain (i.e., including at least its transmembrane region). (This may be done.) In some cases, various human Ig (immunoglobulin) hinges may be used. Hinges can also be used in a similar manner.

[0240] In one embodiment, the transmembrane domain is synthetic (i.e., not naturally occurring). Examples of synthetic transmembrane domains include those containing primarily hydrophobic residues such as leucine and valine. It is a peptide. Preferably, each terminus of the synthetic transmembrane domain contains phenylalanine and triglycerides. A triplicate of putophan and valine is expected to be found. Optionally, a short oligonucleotide may be found. Alternatively, a polypeptide linker, preferably 2 to 10 amino acids in length, is used as a transmembrane ligand. A linkage can be formed between the cytoplasmic signaling domain of the CAR. The lysine-serine duo provides a particularly suitable linker.

[0241] In one embodiment, the transmembrane domain in the CAR of this disclosure is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain is the amino acid sequence YLHLGAL Includes GRDLWGPSPVTGYHPLL.

[0242] In one embodiment, the transmembrane domain in the CAR of this disclosure is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain is the amino acid sequence FWVLV Includes VVGGVLACYSLLVTVAFIIFWV

[0243] In some cases, the transmembrane domain of the CAR of this disclosure is the CD8a hinge domain Includes. In one embodiment, the CD8a hinge domain has the amino acid sequence TTTPAPRP Includes PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

[0244] 5.4.2. Intracellular Domains The intracellular signaling domain of CAR in this disclosure is normal for immune cells that express CAR. It is involved in the activation of at least one effect function. The term "effect function" is This refers to the specialized function of a cell. For example, the effector function of a T cell is related to cell lysis activity and This can be helper activity, including cytokine secretion. Therefore, the term "intracellular signaling" The "transduction domain" converts effector function signals, allowing cells to perform specialized functions. This refers to the portion of a protein that directs a signaling pathway. Typically, it encompasses the entire intracellular signaling domain. While this can be employed, in many cases it is not necessarily required to use the entire chain. Insofar as the shortened portion of the intracellular signaling domain is used, The shortened part is that as long as it converts the effect function signal, the chain remains intact. It can be used instead. Therefore, the term intracellular signaling domain is All intracellular signaling domains sufficient to convert effector function signals It means that it includes the shortened part.

[0245] Preferred examples of intracellular signaling domains for use in the CARs of this disclosure include: T cell receptors (T) act cooperatively after binding to antigen receptors to initiate signal transduction. The cytoplasmic sequences of CR and co-receptors, in addition to any derivatives of these sequences or This includes variants and any synthetic sequences having the same functional capabilities.

[0246] The signal generated by the TCR alone may be insufficient for complete activation of T cells. Secondary or co-stimulatory signals are also required. Therefore, T cell activation requires two distinct signals. A class of cytoplasmic signaling sequences, namely, antigen-dependent primary activity via TCRs. The signaling sequence that initiates the reaction (primary cytoplasmic signaling sequence), and antigen-independent Signaling sequences that act in a sexual manner and deliver secondary or co-stimulatory signals (secondary cells) It can be said that this is mediated by intraplasmic signaling sequences.

[0247] Primary cytoplasmic signaling sequences can be either stimulated or inhibited by T It regulates the primary activation of the CR complex. Primary cytoplasmic signaling pathway acts in a stimulus-mediated manner. The sequence is a signal known as an immune receptor tyrosine-based activation motif or ITAM. It may contain a transmission motif.

[0248] Primary cytoplasmic signaling containing ITAM with specific applications in the CAR of this disclosure Examples of signal transduction sequences include TCR zeta, FcR gamma, FcR beta, CD3 gamma, and C. D3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and C Examples include those derived from D66d. It is particularly preferable that the sequence includes a cytoplasmic signaling sequence derived from CD3-zeta.

[0249] In a preferred embodiment, the cytoplasmic domain of CAR contains a primary molecule containing ITAM. Including the intracytoplasmic signaling sequence domain itself (for example, that of CD3-zeta), or in combination with any other desirable intracellular domain useful in the context of the CAR of this disclosure It is designed to include the CD3 zeta chain portion. For example, the cytoplasmic domain of CAR is designed to include the CD3 zeta chain portion. It may also include a co-stimulus signaling region.

[0250] The co-stimulus signaling region refers to the CAR portion of the co-stimulus molecule that includes the intracellular domain. Co-stimulatory molecules are antigen receptors or their lignament molecules necessary for the efficient response of lymphocytes to antigens. These are cell surface molecules other than 4-. Examples of such molecules include CD27, CD28, 4- 1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytes Function-related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H Examples include ligands that specifically bind to CD83, and others.

[0251] The cytoplasmic signaling sequence within the cytoplasmic signaling portion of the CAR disclosed herein is Randa They may be linked together in any or specified order. Optionally, short oligos may be used. Alternatively, polypeptide linkers, preferably with a length of 2 to 10 amino acids, form the linkage. It is possible. The glycine-serine duo provides a particularly suitable linker.

[0252] In one embodiment, the cytoplasmic domain is the signaling domain of CD3-zeta and includes the signaling domain of CD28. In another embodiment, the intracellular domain This involves the signaling domain of CD3-zeta and the signaling domain of 4-1BB. include.

[0253] 5.5 Nucleic acids, recombinant vectors, and host cells The disclosure of the present invention relates to the immunoglobulin light chain and heavy chain genes of anti-glycoMUC1 antibodies. Nucleic acid molecules, vectors containing such nucleic acids, and the anti-glycoMUC1 antibody of the present disclosure. This includes host cells capable of producing [the substance]. In certain embodiments, nucleic acid molecules are [the substance]. AntiglycoMUC1 antibodies and antigen-binding fragments (e.g., Section 5.1 and As described in Embodiments 1 to 90), in addition, a fusion protein containing them (for example (For example, as described in Embodiments 91-96) and chimeric antigen receptors (e.g., section Coded as described in Section 5.4 and Embodiments 97-98, the host cells are It is possible to express the following. Exemplary vectors of this disclosure are shown in Embodiments 111-113. Exemplary host cells are described in embodiments 114-117.

[0254] The anti-glycoMUC1 antibody disclosed herein enhances immunoglobulin light chain and heavy chain inheritance in host cells. It can be prepared by recombinant expression of the offspring. To recombinantly express the antibody, the host cells It has one DNA fragment that encodes the light and heavy chains of the antibody immunoglobulin. Alternatively, multiple recombinant expression vectors are transfected, thereby transfecting the light and heavy chains. These are expressed in host cells, and optionally they are secreted into the culture medium in which the host cells are cultured. Antibodies can be recovered from the culture medium. Standard recombinant DNA methods involve antibody heavy chains and Light chain genes are obtained, these genes are incorporated into a recombinant expression vector, and the vector is then used in a host. For example, Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook) , Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocol s in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associate To introduce into the documents described in U.S. Patent No. 4,816,397 (s, 1989) and U.S. Patent No. 4,816,397 It will be used.

[0255] To generate nucleic acids encoding such anti-glycoMUC1 antibodies, first, the light chain and We obtain DNA fragments that encode heavy chain variable regions. These DNAs are, for example, poly Using melase chain reaction (PCR), germ cells encode variable light and heavy chain sequences. It can be obtained by amplifying and modifying cellular DNA or cDNA. Human heavy-stranded DNA The germline DNA sequences of the light chain variable region genes are publicly known in the industry (for example, See 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., 19 92, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol. 24:827-836 See also; the contents of each of these are incorporated herein by reference).

[0256] V related to anti-glycoMUC1 antibodies H and V L DNA fragments that code for segments Once obtained, these DNA fragments can be further processed using standard recombinant DNA techniques. By manipulating it in this way, for example, the variable region gene can be converted into a full-length antibody chain gene or a Fab fragment gene. It can be converted into a child or scFv gene. In these operations, V H or V L The DNA fragment encoding another protein, such as an antibody constant region or fragment It is operably ligated to another DNA fragment that encodes a flexible linker. The term "operably linked" when used in this context refers to two DNA fragments. The amino acid sequence encoded by the DNA remains within the frame, as shown by the two DNA fragments. The intention is that it means "to combine".

[0257] V H The isolated DNA encoding the region is V HThe DNA encoding the heavy chain constant region Create another DNA molecule encoding (CH1, CH2, CH3, and optionally CH4) By mobilizing the linkage, it can be converted into a full-length heavy chain gene. Human heavy chain constant The sequences of regional genes are publicly known in this industry (e.g., Kabat et al., 1991, Sequen ces of Proteins of Immunological Interest, Fifth Edition, USDepartment of Heal (See Human Services, NIH Publication No. 91-3242), encompassing these areas. The DNA fragment can be obtained by standard PCR amplification. The heavy chain constant region is The determination of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD It may be a constant region, but in certain embodiments, it may be a constant region of IgG1 or IgG4 In the case of Fab fragment heavy chain genes, V H The DNA that codes for this is the heavy chain CH1 It may be operably ligated to another DNA molecule that encodes only a constant region.

[0258] V L The isolated DNA encoding the region is V L The DNA encoding the light chain constant region By operably linking to another DNA molecule encoding CL, the full-length light chain gene (In addition, it can be converted to the Fab light chain gene.) The sequence of the human light chain constant region gene is , which is publicly known in the industry (for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Servi See ces, NIH Publication No. 91-3242), DNA fragments encompassing these regions The light chain constant region can be obtained by standard PCR amplification. It may be a steady-state region, but in certain embodiments, it is a steady-state region.

[0259] In order to create the scFv gene, V H and V L The DNA fragment that codes for , V H and V L The array is V H and V L The domains merged by a flexible linker. To enable expression as a continuous single-chain protein, it encodes a flexible linker. Another fragment, for example, another fragment encoding the amino acid sequence (Gly4~Ser)3 It may be operably coupled to the mechanism (e.g., Bird et al., 1988, Science 242) :423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;McCaffer See ty et al., 1990, Nature 348:552-554.

[0260] The portion obtained as described above in order to express the anti-glycoMUC1 antibody of this disclosure DNA encoding light and heavy chains, either in its entirety or full length, is controlled by the transcription and translational regulatory mechanisms of genes. It is inserted into the expression vector so that it can be operably linked to the column. In this context, the term " "Operationally linked" means that the transcription and translation control sequences within the vector are linked to the transcription of the antibody gene. And in order to perform their intended function of regulating translation, antibody genes are transferred to the vector. This is intended to mean that it will be ignited. The expression vector and expression control sequence are Selected to match the host cells used for expression. Antibody light chain gene and antibody heavy chain gene. The offspring may be inserted into separate vectors, or more typically, both genes may be inserted into separate vectors. These are inserted into the same expression vector.

[0261] The antibody gene is processed using a standard method (e.g., antibody gene fragment and vector phase). Complementary restriction site ligation, or, in the absence of a restriction site, blunt end ligation Inserted into the expression vector by () . Light or heavy chain related to the anti-glycoMUC1 antibody Prior to sequence insertion, the expression vector may already contain the antibody constant region sequence. For example. V related to anti-glycoMUC1 monoclonal antibodies H and V L The sequence is incorporated into the full-length antibody gene. One approach to the conversion is V H The segment is created in the CH segment within the vector. Movable connection, V L The segment is operably linked to the CL segment in the vector. To that end, the heavy chain constant and light chain constant regions are pre-encoded in the expression vector. This involves inserting a recombinant expression vector into the host cell. In addition, or alternatively, the recombinant expression vector can be used in the host cell. The antibody chain gene may also encode a signal peptide that facilitates the secretion of the antibody chain. This is so that the signal peptide is linked to the amino terminus of the antibody chain gene within the frame. It can be cloned into a processor. The signal peptide is an immunoglobulin signal Lupeptides or heterologous signal peptides (i.e., derived from non-immunoglobulin proteins) It may also be a signal peptide.

[0262] In addition to the antibody chain gene, the recombinant expression vector of the present disclosure expresses the antibody chain gene in host cells. It has regulatory elements that control expression. The term "regulatory elements" refers to promoters, enhancers, etc. and other expression regulatory elements that control the transcription or translation of antibody chain genes (e.g., poly It is intended to include an adenylation signal. Such regulatory sequences include, for example, Gode l, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Di It is described in ego, Calif., 1990. Those skilled in the art will know that the expression vector, including the selection of the regulatory sequence, The design of the transformer involves selecting host cells to be transformed and determining the level of expression of the desired protein. It is expected that they understand that it may be determined by factors such as [mention specific factors here]. Suitable regulatory sequences for host-like cell expression include those that produce high levels of protein in mammalian cells. The viral element that directs the current virus, for example, a pro-cytomegalovirus (CMV) derived from the virus. Motor and / or enhancer (e.g., CMV promoter / enhancer), Mianvirus 40 (SV40)-derived promoters and / or enhancers (e.g.) (SV40 promoter / enhancer), adenovirus-derived promoter and / Or enhancers (e.g., adenovirus major late promoter (AdMLP)), Also included are promoters and / or enhancers derived from polyomas. For further explanation of the Luss adjustment elements and their arrangement, see, for example, Stinski. U.S. Patent No. 5,168,062 by Bell et al., U.S. Patent No. 4,510, U.S. Patent No. 245, and U.S. Patent No. 4,968,615 by Schaffner et al. Please refer to the detailed document.

[0263] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of this disclosure are expressed in host cells. Sequences that regulate the replication of the vector (e.g., origin of replication) and selectable marker genes It may have any additional sequence. Selectable marker genes are introduced into the vector. Facilitates the selection of host cells (e.g., all by Axel et al., U.S. 4,399, Specification No. 216, Specification No. 4,634,665 and Specification No. 5,179,017 (See reference). For example, typically, the selectable marker gene is G418, hygromycin. Alternatively, resistance to drugs such as methotrexate can be introduced into host cells into which the vector has been introduced. It contributes. A suitable selectable marker gene is dihydrofolate reductase (DHFR ) gene (DHFR - (For use with methotrexate selection / amplification in host cells) The neo gene (for G418 selection) is an example. For the expression of the light and heavy chains, Expression vectors encoding heavy and light chains are translated into host cells using standard techniques. To be affected. Various forms of the term "transfection" include, for example, electroporation. Ration, lipofection, calcium phosphate precipitate, DEAE-dextran Common for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as through sfection. It is intended to encompass a variety of technologies used in [the project].

[0264] The antibodies described herein may be expressed in either prokaryotic or eukaryotic host cells. In this embodiment, antibody expression, or properly folded and immunologically active Optimal antibody secretion occurs in eukaryotic cells, such as mammalian host cells. Recombination of the recombinant cells of this disclosure Exemplary mammalian host cells for antibody expression include the Chinese hamster ovary. CHO cells) (e.g., Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:421) DHFR as described in 6-4220 - These include CHO cells, for example, Kaufman and Sh. Along with DHFR-selectable markers as described in arp, 1982, Mol. Biol. 159:601-621 Examples include NSO myeloma cells, COS cells, and SP2 cells (used in [treatment]). Antibody residue When a recombinant expression vector encoding a gene is introduced into a mammalian host cell, the antibody will... This enables antibody expression within cells or the secretion of antibodies into the culture medium in which host cells proliferate. Antibodies are produced by culturing host cells for a sufficient period of time. It can be recovered from the culture medium using protein purification methods. Host cells also retain intact antibodies. To be used to produce a part of the body, such as a Fab fragment or scFv molecule. This is possible. Variations of the above procedure are understood to be within the scope of the disclosure of this invention. For example, either the light chain or the heavy chain of the anti-glycoMUC1 antibody of this disclosure (but not both). In some cases, it is desirable to transfect host cells with DNA that codes for (not present). .

[0265] Regarding the expression of CAR in this disclosure, see, for example, Section 5.4, and further, Embodiment 97 And as described in 98, preferably the host cell is a T cell, preferably a human T cell. These are cells. In some embodiments, the host cells are cross-linked with MUC1 on tumor cells. When this occurs, it exhibits anti-tumor immunity. Detailed methods for producing T cells of this disclosure are described in section It is described in section 5.5.1.

[0266] Recombinant DNA technology also allows for the use of light chains and other components that are not necessarily required for binding to glycoMUC1. Used to remove part or all of the DNA that codes for one or both of the heavy chains. It is also possible that molecules expressed from such shortened DNA molecules are also antibodies of this disclosure. It is included in.

[0267] For recombinant expression of the anti-glycoMUC1 antibody of this disclosure, host cells undergo two processes of this disclosure. The current vector, i.e., the first vector encoding a heavy chain-derived polypeptide and the light chain A second vector encoding the derived polypeptide may be cotransfected. The two vectors may contain the same selectable marker, or they may contain the same selectable marker. They may each contain different selectable markers. Alternatively, both heavy and light chains may be included. A single vector encoding a lipeptide may be used.

[0268] If nucleic acids begin to encode one or more parts of an anti-glycoMUC1 antibody, even if For example, antibodies with different CDR sequences, antibodies with reduced affinity to the Fc receptor, or different Further modifications or mutations are required to generate nucleic acids encoding a subclass of antibody. It can be introduced into a code array.

[0269] The anti-glycoMUC1 antibody of this disclosure can also be synthesized (e.g., Solid Phase Peptide) Described in *ide Synthesis*, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill. They can also be produced (depending on the method). Variant antibodies can also be produced using a cell-free platform. It can be produced using (for example, Chu et al., Biochemia No.2, 2001 (Roche Mole) (Cyclical Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology (See 17:420-426).

[0270] Once the anti-glycoMUC1 antibody of this disclosure is produced by recombinant expression, immunoglobulins By any method known in the art for the purification of the fruit, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), far By heart separation, fractional lysis, or any other standard protein purification technique This can be purified. Furthermore, the anti-glycoMUC1 antibody and / or the disclosure of the present invention The bound fragments are, in order to facilitate purification, as described herein or otherwise. It can be fused with heterologous polypeptide sequences that are known in this industry.

[0271] Once isolated, the anti-glycoMUC1 antibody can be analyzed, for example, using high-performance liquid chromatography (HCM). According to Fisher (Laboratory Techniques in Biochemistry and Molecular Biochemistry) (See ogy, Work and Burdon, eds., Elsevier, 1980), or Superdex (trademark) Gel filtration chromatography using 75 columns (Pharmacia Biotech AB, Uppsala, Sweden) It may be further refined by [a specific method].

[0272] 5.5.1. Recombination production of CARs in T cells In some embodiments, the nucleic acid encoding the antiglycoMUC1 CAR of this disclosure is It is delivered to cells using a trovirus or lentiviral vector. CAR expression Retroviruses and lentiviral vectors are used as carriers to transfect cells. Use or encapsulated, conjugated, or naked vectors in a cell-free local or It uses systemic delivery to deliver to different types of eukaryotic cells, and even to tissues and the entire organism. It can be achieved. The method used requires stable expression or sufficient all It could be a method for that purpose.

[0273] In other embodiments, the CAR sequence is derived using mRNA transcribed in vitro. It is delivered to the cell. mRNA CAR transcribed in vitro is transferred as a carrier. Using encapsulated cells, or encapsulated, bound, or naked mRNA Using cell-free local or systemic delivery, different types of eukaryotic cells, and even tissues, It can also be delivered to the entire organism. The method used requires transient expression. It can be a sufficient method for any purpose.

[0274] In another embodiment, the desired CAR is expressed in the cell by a transposon. It is possible.

[0275] One advantage of the RNA transfection method disclosed herein is that RNA transfection RNA is essentially transient and does not involve a vector; that is, RNA transduction is a form of genetic modification. The child is delivered to lymphocytes, and after short-term in vitro cell activation, any additional These are expressed there as a minimal expression cassette without requiring the viral sequence. Under these conditions, the likelihood of the introduced gene being incorporated into the host cell genome is low. The process does not necessarily improve the efficiency of RNA transfection or uniformly modify the entire lymphocyte population. It's not because of that ability to change.

[0276] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) is possible in both cases. Both utilize two different strategies that have been continuously tested in various animal models. RNs transcribed in vitro by lipofection or electroporation Transfect with A. Preferably, achieve sustained expression of the transferred IVT-RNA. To achieve this, it is desirable to stabilize IVT-RNA using various modifications.

[0277] Some IVT vectors have been standardized as templates for in vitro transcription. It is used in a manner in which the genes are modified to produce stabilized RNA transcripts. This is publicly known from the literature. Currently, the protocols used in this industry are as follows: Structure: A 5' RNA polymerase promoter that enables RNA transcription, followed by a 3' The target gene adjacent to the untranslated region (UTR) at either the and / or 5', and Plasmid having a 3' polyadenyl cassette containing 50-70 A nucleotides Vector-based. Before in vitro transcription, the circular plasmid is transferred to a polyadenylic cassette. Downstream, it is linearized by type II restriction enzymes (the recognition sequence corresponds to the cleavage site). Therefore, the polyadenylic cassette corresponds to the late poly(A) sequence in the transfer. As a result, some nucleotides remain as part of the enzymatic cleavage site after linearization, with their 3' ends remaining. The poly(A) sequence at the edge is extended or masked. This non-physiological operation Barhang affects the amount of protein produced intracellularly from such constructs. It is unclear whether or not it will be granted.

[0278] RNA offers numerous advantages over more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and the protein product is trans. It is produced immediately after infection. Furthermore, RNA enters the cytoplasm, not the nucleus. Because only this is required, a typical transfection method is extremely high transfection This leads to a higher success rate. In addition, plasmid-based approaches allow for the target gene to be extracted from the cells under study. This requires activating the promoter that drives the expression of [the substance].

[0279] In another embodiment, RNA constructs are converted to cells by electroporation. It can be delivered. For example, U.S. Patent Application Publication No. 2004 / 0014645. , U.S. Patent Application Publication No. 2005 / 0052630, U.S. Patent Application Publication No. 2005 Specification No. / 0070841, U.S. Patent Application Publication No. 2004 / 0059285, U.S. To mammalian cells, as taught in National Patent Application Publication No. 2004 / 0092907 See the formulations and methods for electroporation of nucleic acid constructs. Various parameters such as electric field strength required for electroporation of any known cell type - Generally, this is publicly known in numerous patents and applications, in addition to relevant research literature in this field. It is knowledge. For example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,2 See Patent No. 64 and U.S. Patent No. 7,173,116. Devices for the therapeutic application of poration, such as MedPulser(trademark) DNA E Rectroporation treatment system (Inovio / Genetronics, San Diego, Calif.) is commercially available and has a U.S. license of 6,567,694. U.S. Patent No. 6,516,223, U.S. Patent No. 5,993,434 Detailed Statement, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701 , and as described in patents such as U.S. Patent No. 6,233,482, elect Roporation is described, for example, in U.S. Patent Application Publication No. 20070128708. Therefore, it can also be used for in vitro cell transfection. Electroporation is also used to deliver nucleic acids to cells in vitro. Therefore, many available devices and electronics known to those skilled in the art can be used. Using one of the electroporation systems, transfer nucleic acids containing expression constructs into cells. Electroporation-mediated administration is an excitation method for delivering target RNA to target cells. It provides a new and innovative method.

[0280] 5.5.1.1 T cell sources The T cell source is obtained from a subject before it is increased and genetically modified. The term "subject" refers to: It is intended to include organisms capable of eliciting an immune response (e.g., mammals). Examples of subjects include Examples include humans, dogs, cats, mice, rats, and their transgenic species. It is possible. Preferably, the subject is a human.

[0281] T cells originate from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, and the site of infection. It can be obtained from numerous sources, including tissue, ascites, pleural exudate, splenic tissue, and tumors. In certain embodiments of the disclosure of the present invention, various T cell lines available in the industry are used. It can be used. In certain embodiments of the disclosure of the present invention, T cells are Ficoll (Trademark) From blood units collected from subjects using various techniques known to those skilled in the art, such as separation. It can be obtained. In a preferred embodiment, cells from the circulating blood of an individual are obtained. Obtained by LASIS. Apheresis products are typically lymphocytes, including T cells. It contains monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Morphologically, cells collected by apheresis are washed to remove the plasma fraction. For subsequent processing steps, the cells can be placed in a suitable buffer or medium. In one embodiment of this disclosure, the cells are washed with phosphate-buffered saline (PBS). Alternative In this embodiment, the washing solution lacks calcium and magnesium. It may also be good, or it may lack many divalent cations, even partially. Here too, What should be done is that the initial activation step in the absence of calcium has further activation. It is expected that those skilled in the art can easily understand that the washing process is known to those skilled in the art by methods, for example, semi - automated "flow - through" centrifuges according to the manufacturer's instructions ( for example, Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) can be achieved by using. After washing, the cells are suspended in various biocompatible buffers, such as Ca - free, Mg - free PBS, PlasmaLyte A, or other salt solutions with or without buffers etc. Alternatively, the unnecessary components of the apheresis sample can be removed and the cells can be directly resuspended in the culture medium.

[0282] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL (trademark) gradient or counter - flow centrifugal elution method. Specific subsets of T cells, such as CD3 CD28’, CD + 4 + CD8 + CD45RA + and CD45RO + T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubating with anti - C D3 / anti - CD28 (i.e., 3×28) conjugate beads, such as DYNAB EADS (registered trademark) M - 450 CD3 / CD28T for a period sufficient for positive selection of the desired T cells In one embodiment, the period is about 30 minutes. In a further embodiment, the period ranges from 30 minutes to 36 hours or a longer time range, and the range of all integer values ​​within that time. Further embodiments In this case, the duration is at least 1, 2, 3, 4, 5, or 6 hours. Furthermore, another preferred In a preferred embodiment, the duration is 10 to 24 hours. The incubation period is 24 hours. Isolation of T cells from patients with leukemia. In this case, using a longer incubation time, for example 24 hours, will increase cell yield. It can be done. Longer incubation times are suitable for tumor tissue or immunodeficient individuals. When isolating tumor-infiltrating lymphocytes (TILs), T cells are different from other cell types. It can be used to isolate T cells in a very small number of situations. Furthermore, Using longer incubation times can increase the capture efficiency of CD8+ T cells. Therefore, simply shortening the time it takes for T cells to bind to CD3 / CD28 beads is not enough. Or, by lengthening and / or increasing or decreasing the ratio of beads to T cells By reducing (as further described herein), at the start of culture, or At other time points in the process, a subset of T cells is either present or absent. It can be selected preferentially without being limited. In addition, anti-CD3 and on beads or other surfaces / or by increasing or decreasing the ratio of anti-CD28 antibody at the start of culture or at other desired time points, a subset of T cells, whether present or absent. It can still be preferred. A person skilled in the art would, in the circumstances of this disclosure, multiple times It is expected that you will be aware that the option of can also be used. In certain embodiments, Perform the selection procedure and use the cells that were "not selected" in the activation and growth process. This may be desirable. "Unselected" cells are also subjected to further selection rounds. It is also possible.

[0283] The enrichment of T cell populations through negative selection is related to surface markers specific to negatively selected cells. This can be achieved by using antibodies in combination. One method is to present antibodies on negatively selected cells. Negative magnetic immunoassay using a cocktail of monoclonal antibodies against cell surface markers Cell sorting and / or selection via cytometry or flow cytometry. Example For example, by negative selection, CD4 + To enrich the cells, a monoclonal antibody cocktail is used. Typically, CD14, CD20, CD11b, CD16, HLA-DR, and CD Contains an antibody against 8. In certain embodiments, typically CD4 + CD25 + , C D62L hi GITR + , and FoxP3 + Enriching regulatory T cells that express this, Alternatively, it may be desirable to select them as positive. Alternatively, in certain embodiments Furthermore, regulatory T cells are activated by anti-C25 conjugate beads or other similar selective methods. And then it becomes depleted.

[0284] For isolation of a desired cell population by positive or negative selection, cells and surfaces (e.g.) The concentration of particles (such as beads) may vary. In certain embodiments, beads and significantly reduce the volume of cells mixed together (i.e., increase the cell concentration) ), it may be desirable to ensure maximum contact between cells and beads. For example, In an embodiment, a concentration of 2 billion cells per milliliter is used. In one embodiment , a concentration of 1 billion cells per milliliter is used. In a further embodiment, more than 100 million cells per milliliter are used. In a further embodiment, 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million , 45 million, or 50 million cell concentrations per milliliter are used. In yet another embodiment , cell concentrations from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per milliliter are used. In a further embodiment, a concentration of 125 million or 150 million cells per milliliter may be used. The use of high concentrations can result in an increase in cell yield, activation of cells, and an increase in cells. Further, the use of high cell concentrations enables more efficient capture of cells in which the expression of the target antigen of interest may be weak, such as CD28-negative T cells, or capture from samples in which many tumor cells are present (i.e., blood of leukemia, tumor tissue, etc.). Such cell populations may have therapeutic value and are expected to be desirable to obtain. For example, the use of high concentrations of cells enables more efficient selection of CD8 <​​​​​​​​​​​​​​​​​​​​It expresses CD28 and has a dilute concentration of CD8 + It is captured more efficiently than T cells. One embodiment In this case, the cell concentration used is 5 × 10 6 In other embodiments, use The concentration used is approximately 1 × 10⁻⁶ 5 From / ml to 1 x 10 6 / ml, and any adjustments in between A range of numbers is also acceptable.

[0286] In other embodiments, cells are rotated in a rotating device for various lengths of time. The incubation can be performed at a rate of 2°C to 10°C or at room temperature.

[0287] The T cells for stimulation may also be frozen after the washing process. I don't intend to be bound by theory. However, the freezing and subsequent thawing processes remove granulocytes and some monocytes from the cell population. This provides a more homogeneous product after the washing step to remove plasma and platelets. The cells may be suspended in a freezing solution. Many freezing solutions and parameters are available in the industry. It is publicly known and is expected to be useful in this situation, but one method is 20% PBS containing DMSO and 8% human serum albumin, or 10% dextran 4 0 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, if 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.4 5% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum alcohol A culture medium containing bumin and 7.5% DMSO, or, for example, Hespan and This includes using other suitable cell freezing media containing PlasmaLyte A, and then The cells are frozen to -80°C at a rate of 1° / min and stored in the gas phase of a liquid nitrogen storage tank. Other controlled refrigeration methods include immediate refrigeration at -20°C or uncontrolled refrigeration in liquid nitrogen. It can be used in the same way as frozen food.

[0288] In certain embodiments, before activation using the method disclosed in the present invention, the cells are stored at low temperatures. The thawed cells are thawed, washed as described herein, and left to stand at room temperature for 1 hour.

[0289] Furthermore, in the period prior to when the increased cells described herein are deemed necessary, the subject It is also expected in the circumstances of this disclosure that blood samples or apheresis products may be collected. Therefore, the source of the cells that you want to increase will be at every necessary time point. Desired cells, such as T cells, can be collected and are expected to benefit from T cell therapy. T-cell therapy for various diseases or conditions, such as those described herein. It can be isolated and frozen for later use. In one embodiment, blood samples The pull or apheresis is taken from an overall healthy subject. In certain embodiments... Furthermore, blood samples or apheresis carry a risk of causing disease, but the disease... The target cells are taken from generally healthy subjects who have not developed the disease, and are used for subsequent use. They are isolated and frozen. In certain embodiments, T cells increase over time. It can be frozen and used. In certain embodiments, the sample is... Immediately after the diagnosis of a specific disease described in the specification, but before any treatment, from the patient Collected. In further embodiments, various related treatment methods, for example, are not limited to these. Although not used, natalizumab, efalizumab, antiviral agents, chemotherapy agents, radiation, and immunotherapy are used. Inhibitors, such as cyclosporine, azathioprine, methotrexate, and mycophenolate. Salt, and FK506, antibodies, or other immunosuppressants, such as CAMPATH, anti-CD3 Antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, Mykov Before drug treatments such as enolates, steroids, FR901228, and radiation therapy. The cells are isolated from a blood sample or apheresis from the subject. These drugs The substance inhibits calcineurin, a calcium-dependent phosphatase (cyclos). Porins and FK506) are important for signaling induced by growth factors. The key is to inhibit the p70S6 kinase (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 further embodiments, cells are chemically Therapeutic agents, such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. Bone marrow or stem cell transplantation or T-cell depletion therapy using (for example, before For subsequent use (at the same time or thereafter), each patient is isolated and frozen. .

[0290] In a further embodiment of the disclosure of the present invention, T cells are obtained from the patient immediately after treatment. In this regard, after certain cancer treatments, especially after treatment with drugs that damage the immune system, patients The quality of the T cells obtained immediately after treatment during the period when the patient is expected to recover from the usual treatment is With regard to the ability to increase in ex vivo, it may be optimal or improved. It has been observed that in ex vivo using the methods described herein, After the procedure, these cells are in a favorable condition for enhanced engraftment and in vivo growth. There is a possibility that, therefore, in the context of the disclosure of the present invention, during this recovery period, T cells It is expected that blood cells, including dendritic cells or other hematopoietic lineage cells, will be collected. Furthermore, in certain embodiments, mobilization (e.g., mobilization in GM-CSF) and conditioning The conditioning regimen is particularly important for the re-establishment and recirculation of specific cell types within a defined time frame after therapy. To produce a state in the object that is favorable for ringing, regeneration, and / or increase It can be used. Examples of cell types include T cells, B cells, dendritic cells, and others. These include immune system cells.

[0291] 5.5.1.2 Activation and Increase of T Cells T cells are generally, for example, U.S. Patent No. 6,352,694; No. 6,534 ,055 specification;No. 6,905,680 specification;No. 6,692,964 specification;No. Specification No. 5,858,358; Specification No. 6,887,466; No. 6,905,681 Specification; No. 7,144,575; Specification No. 7,067,318; No. 7,172 ,869 specification;No. 7,232,566 specification;No. 7,175,843 specification;No. Specification No. 5,883,223; Specification No. 6,905,874; Specification No. 6,797,514 Specification; Specification No. 6,867,041; and U.S. Patent Publication No. 200601210 It is activated and increased using the method described in Specification No. 05.

[0292] Generally, the T cells described herein are stimulated by drugs that stimulate CD3 / TCR complex-related signaling. and contact the surface to which ligands that stimulate co-stimulatory molecules on the T cell surface are attached. It is increased by, for example, anti-CD3 antibodies or their antigen binding. In particular, the T cell population is increased by, for example, anti-CD3 antibodies or their antigen binding. By contacting the fragment, or the anti-CD2 antibody immobilized on the surface, This is a calcium ionophore along with a protein kinase C activator (e.g., briostat). By bringing it into contact with (n), it can be stimulated as described herein. For co-stimulation of accessory molecules on the cell surface, ligans bind to accessory molecules. For example, a population of T cells is used under conditions suitable for stimulating T cell proliferation. It can be brought into contact with anti-CD3 antibodies and anti-CD28 antibodies. CD4 + T cells or CD8 + To stimulate the proliferation of either T cells, anti-CD3 antibodies and anti-CD28 antibodies Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone). Examples include Besancon and France, and are generally the same as other methods known in the industry. It can be used in the following way (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al. l., J. Exp. Med. 190(9):13191328, 1999;Garland et al., J. Immunol Meth. 227(1-2 (53-63, 1999).

[0293] In certain embodiments, the primary stimulation signal and co-stimulation signal related to T cells are, This can be provided through various protocols. For example, drugs that provide each signal. It may be in the form of a solution, or it may be coupled to a surface. When coupling, the drugs are either on the same surface (i.e., in a "cis" configuration) or on a different surface. It may be coupled to the surface (i.e., in a "trans" configuration). Alternatively Alternatively, one drug may be coupled to the surface, while the other drug is in the form of a solution. In one embodiment, the drug providing the co-stimulatory signal is bound to the cell surface and is primary active The drug that provides the sexualization signal is in the form of a solution or coupled to a surface. In certain embodiments, both agents may be in the form of a solution. In this, the drug may be in a soluble form, and then cells expressing the Fc receptor Regardless of which surface it is crosslinked to, or to the antibody or other binding agent that is expected to bind to the drug Good. In this regard, for example, the use in the activation and increase of T cells in the disclosure of the present invention Regarding the artificial antigen-presenting cells (aAPCs) expected in relation to the United States Patent Application Publication No. 2 Please refer to Specification No. 0040101519 and Specification No. 20060034810. .

[0294] In one embodiment, the two drugs are placed on the same bead, i.e., "cis", or on separate beads. It is fixed on the bead, that is, in one of the "trans" configurations. For example, The drug that provides the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment. Yes, drugs that provide co-stimulatory signals include anti-CD28 antibodies or their antigen-binding fragments. In one embodiment, both drugs are immobilized together with the same beads in equivalent molecular amounts. CD4 +To increase T cells and promote T cell proliferation, beads bound in a 1:1 ratio were used. Each antibody is used. In a particular aspect of the disclosure of the present invention, observations are made using a 1:1 ratio. Compared to the increase observed, an increase in T cell increase is observed when beads are bound to anti A CD3:CD28 antibody ratio is used. In one particular embodiment, a 1:1 ratio. Compared to the increase observed using the rate, an increase of approximately 1 to 3 times is observed. In this state, the ratio of CD3:CD28 antibody bound to the beads changes from 100:1 to 1:1 The range is 00 and the range of all integer values ​​in between. In one embodiment of the disclosure of the present invention More anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., CD3:C The ratio of D28 is less than 1. In certain embodiments of this disclosure, the antibody bonded to the beads The ratio of CD28 antibody to anti-CD3 antibody is greater than 2:1. (One specific embodiment) In this study, antibodies conjugated to beads with a CD3:CD28 ratio of 1:100 are used. In another embodiment, antibodies conjugated to beads with a CD3:CD28 ratio of 1:75 are used. It is used. In a further embodiment, it is used to bind beads in a CD3:CD28 ratio of 1:50. Combined antibodies are used. In another embodiment, a CD3:CD28 ratio of 1:30 is used. Antibodies conjugated to beads are used. In one preferred embodiment, a 1:10 ratio of CD3: Antibodies conjugated to CD28-containing beads are used. In another embodiment, 1:3 Antibodies conjugated to beads in a CD3:CD28 ratio are used. In yet another embodiment... Antibodies bound to beads with a CD3:CD28 ratio of 3:1 are used.

[0295] The ratio of particles to cells is 1:500 to 500:1, and any integer values ​​in between. However, it can be used to stimulate T cells or other target cells. To make it easy to understand, the ratio of particles to cells is determined by the particle size relative to the target cell. There is a possibility that... For example, small beads can only bind to a few cells, but... Larger beads can bind to many cells. In certain embodiments, relative to the particles of cells The ratio is in the range of 1:100 to 100:1, and any integer value in between, and further In one embodiment, the ratio is in the range of 1:9 to 9:1 and any integer in between. These include values, and these can also be used to stimulate T cells. Anti-T cell stimulation occurs As mentioned above, the ratio of CD3 and anti-CD28 coupled particles to T cells varies. This is also acceptable, but specific preferred values ​​include 1:100, 1:50, 1:40, 1: 30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 , 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 Examples include 10:1 and 15:1, with one preferred ratio being at least 1:1 grains. This is the ratio to the child's T cells. In one embodiment, the particle ratio is 1:1 or lower. A ratio relative to the cells is used. In one particular embodiment, preferred particles: cells The ratio is 1:5. In a further embodiment, the ratio of particles to cells is the stimulation It can be changed depending on the day. For example, in one embodiment, the ratio of particles to cells The ratio is 1:1 to 10:1 on day 1, and then additional particles are added at a 1:1 ratio until day 10. Add cells daily or every other day in a final ratio of 1:10 (based on the number of cells added on the day of addition). It is added to. In one particular embodiment, the ratio of particles to cells is the daily dose of stimulation. The ratio is 1:1 to the eye and is adjusted to 1:5 on the 3rd and 5th days of stimulation. In another embodiment... The particles are added daily or every other day, reaching a final 1:1 ratio on day 1, stimulating the irritation. On the 3rd and 5th days, the ratio is 1:5. In another embodiment, the ratio of particles to cells The ratio is 2:1 on day 1 of stimulation and is adjusted to 1:10 on days 3 and 5 of stimulation. In another embodiment, the particles are added daily or every other day, with a final 1:1 ratio on day 1. The ratio is 1:10 on the third and fifth days of stimulation. Those skilled in the art will know that various other ratios are It is expected that you understand that the disclosure of this invention may be suitable for use. In particular, the ratio is expected to vary depending on particle size and cell size and type. It can be done.

[0296] In a further embodiment of the disclosure of the present invention, cells, such as T cells, are coated with a drug. The beads are combined with the cells, then the beads and cells are separated, and the cells are then cultured. In an alternative embodiment, the drug-coated beads and cells are separated before culturing. They are not separated, but are cultured together. In a further embodiment, first, by applying a force such as magnetism This concentrates the beads and cells, and as a result increases the ligation of cell surface markers. This is done to induce cellular stimulation.

[0297] For example, cell surface proteins are fitted with anti-CD3 and anti-CD28 receptors. By bringing paramagnetic beads (3 x 28 beads) into contact with T cells, ligation occurs. This is possible. In one embodiment, cells (for example, 10 4from 10 9 (individual T cells) Yobi beads (for example, DYNABEADS® M-450CD3 in a 1:1 ratio) (CD28T paramagnetic beads) are placed in a buffer, preferably PBS (calcium and magnesium). Combine in a container that does not contain divalent cations such as nesium. Here again, those skilled in the art will know all the details. It is easy to understand that cell concentration can be used. For example, target cells are sample It may be extremely rare, or it may constitute only 0.01% of the sample, The entire sample (i.e., 100%) may consist of the target cells of interest. Therefore, any number of cells is included in the disclosure of the present invention. In a particular embodiment, By significantly reducing the volume in which particles and cells are mixed together (i.e., increasing the concentration of cells) In addition, it may be desirable to ensure maximum contact between cells and particles. For example. In one embodiment, a concentration of approximately 2 billion cells per 1 ml is used. Another embodiment In this, more than 100 million cells are used per 1 ml. In a further embodiment, Per 1 ml: 10 million, 15 million, 20 million, 25 million, 300 Cell concentrations of 0,000, 35 million, 40 million, 45 million, or 50 million It is used. In yet another embodiment, 75 million cells and 8000 cells per 1 ml. Cell concentrations of 10,000,000, 85 million, 90 million, 95 million, or 100 million cells are used. In a further embodiment, 125 million or 150 million cells per 1 ml A concentration of 10,000 can be used. The use of high concentrations increases cell yield, activates cells, and This can lead to an increase in the number of cells. Furthermore, the use of high cell concentrations can target the desired antigen. This enables more efficient capture of cells that may have weak expression, such as CD28-negative T cells. Such cell populations may have therapeutic value, and in certain embodiments... It is expected that obtaining it will be desirable. For example, the use of high concentrations of cells is usually compared to CD28 expression. This enables more efficient selection of relatively weak CD8+ T cells.

[0298] In one embodiment of the disclosure of the present invention, the mixture is prepared over several hours (about 3 hours) to about 14 days. It can be cultured for any integer time between those two times. In another embodiment... The mixture can be cultured 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. The culture period for T cells is 60 days or longer. To achieve this, several cycles of stimulation may be desirable. For example, serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), Insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12 Factors necessary for proliferation and survival, such as IL-15, TGFβ, and TNF-α, or A suitable culture medium which may contain any other additives for cell proliferation known to those skilled in the art (e.g.) For example, minimal essential medium or RPMI medium 1640 or X-vivo15 (Lonza Other additives for cell proliferation include, but are not limited to, those containing the same active ingredient as the sulfate. Surfactants, plasmamates, and N-acetylcysteine ​​and 2-mercaptoyl Reducing agents such as tanol are examples. Culture media include amino acids, sodium pyruvate, and serum-free or with an appropriate amount of serum (or plasma) or pre-treated serum with added vitamins. A defined set of hormones, and / or sufficient amounts of sites for T cell proliferation and increase. Cain has been replenished with either RPMI1640, AIM-V, DMEM, or MEM. , α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optim Examples include antibiotics, such as penicillin and streptomycin. The substance is present only in experimental cultures and not in the culture of cells intended for injection into the target. Cells thrive under conditions necessary to support proliferation, such as a suitable temperature (e.g., 37°C). It is maintained under a specific atmosphere (for example, air plus 5% CO2).

[0299] T cells exposed to various stimulation times may exhibit different characteristics. For example, typical blood Alternatively, peripheral blood mononuclear cell products that have undergone apheresis are cytotoxic or suppressive. T cell population (T C CD8 + A larger population of helper T cells (T H CD4 + ) has The increase in T cells by stimulating CD3 and CD28 receptors ex vivo is About 8-9 days before, mainly T H It produces a population of T cells, which are composed of cells, but this process takes about 8-9 days. After that, the T cell population becomes even larger T C It contains a population of cells. Therefore, the purpose of the treatment Depending on the circumstances, primarily T H Injecting into a T-cell population that includes cells can be advantageous in some cases. Similarly, T C If an antigen-specific subset of cells is isolated, this subset can be used In some cases, it may be beneficial to increase it to a higher degree.

[0300] Furthermore, in addition to CD4 and CD8 markers, other expressions during the cell proliferation process. Type markers change significantly, but mostly reproducibly. Therefore, such re This expressiveness allows for the ability to tailor activated T cell products to specific purposes.

[0301] 5.6 Composition The anti-glycoMUC1 antibody and / or anti-glycoMUC1 ADC of this disclosure is anti-glyco MUC1 antibody and / or ADC, as well as one or more carriers, excipients and / or Alternatively, the composition may be in the form of a composition containing a diluent. The composition may be used for specific purposes, for example, It may be formulated for veterinary use or for medicinal use in humans. Compositions (e.g., dry powders, liquid formulations, etc.) as well as excipients, diluents and / or carriers. The morphology of the body, through the intended use of antibodies and / or ADCs, further therapeutic use, It is expected to be determined by the method of administration.

[0302] For therapeutic use, the composition is a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. It may be supplied as part of a larger quantity. This composition may be in any preferred form (for the patient). Depending on the preferred method of administration, the pharmaceutical composition may be administered orally, transdermally, subcutaneously, intranasally, or intravenously. It is administered to patients via various routes, including internally, intramuscularly, intratumorally, subarachnoidally, externally, or locally. It is possible. In any given case, the most suitable route of administration is the specific antibody and and / or ADC, subject, and the nature and severity of the subject's disease and physical condition It is expected that this will be determined accordingly. Typically, when a pharmaceutical composition is administered intravenously or subcutaneously... It is expected.

[0303] The pharmaceutical composition contains a predetermined amount of the anti-glycoMUC1 antibody of this disclosure per dose and / or it can be conveniently provided in a single dosage form containing antiglyco-MUC1 ADC. The amount of antibody and / or ADC contained in a unit dose depends on the disease being treated, in addition to the amount of antibody and / or ADC present in the unit dose. It is expected to be determined by other factors well known in the industry. Such unit dosing is a single dose. In the form of a lyophilized dry powder containing an amount of antibody and / or ADC suitable for administration. It may be present or in liquid form. The unit dosage form of dry powder is included in the kit. Packaged with a syringe, a suitable amount of diluent, and / or other components useful for administration. It may be done. A unit dose in liquid form contains an amount of antibody and / or suitable for a single dose. It can be conveniently supplied in the form of a syringe pre-filled with ADC.

[0304] The pharmaceutical composition is also supplied in bulk form containing an amount of ADC suitable for multiple doses. That's fine.

[0305] The pharmaceutical composition shall have the desired purity for storage as a lyophilized preparation or aqueous solution. Antibodies and / or ADCs are used in optional pharmaceutical applications typically employed in the industry. Permitted carriers, excipients, or stabilizers (all of which are referred to herein as “carriers”) ), that is, buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, acid fasteners It may also be prepared by mixing with a chemical agent and other compound additives. Remington's P See harmaceutical Sciences, 16th edition (Osol, ed. 1980). Additives should be non-toxic to recipients at the dosage and concentration adopted.

[0306] Buffers help maintain pH within a range close to physiological conditions. Buffers are various It may be present at any concentration, but typically exists in concentrations ranging from approximately 2 mM to approximately 50 mM. It is expected that... Suitable buffering agents for use in the disclosure of the present invention include, for example, citric acid. Acid buffer (e.g., monosodium citrate-disodium citrate mixture, citrate-c Trisodium cereal mixture, citrate-monosodium citrate mixture, etc., succinic acid Comboliary (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture) Compounds, succinic acid-disodium succinate mixture, etc.), tartaric acid buffer (e.g., tartaric acid- Sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture (compounds, etc.), fumarate buffer (e.g., fumarate-monosodium fumarate mixture, fumarate) Acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture (etc.), gluconate buffer (for example, gluconate-sodium gluconate (sodium gluconate) nate) mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate (potassium gluconate) mixture, etc., oxalate buffer (e.g., oxalate-oxalate) Sodium mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture (compounds, etc.), lactate buffer (e.g., lactate-sodium lactate mixture, lactate-sodium hydroxide mixture) (Mixed lactic acid, potassium lactate mixture, etc.) and acetate buffer (e.g., acetate-sodium acetate) Both organic and inorganic acids, such as lium mixtures and acetic acid-sodium hydroxide mixtures. These include salts. In addition, phosphate buffer, histidine buffer and trimethyl Amine salts, such as Tris, can be used.

[0307] Preservatives may be added to inhibit microbial growth, approximately 0.2% to 1%. It can be added in amounts within the range of w / v. Suitable storage for use in the disclosure of the present invention Examples of agents include phenol, benzyl alcohol, metacresol, methylparaben, pro Pyrparaben, octadecyldimethylbenzylammonium chloride, benzalkonium Halides (e.g., chlorides, bromides, and iodides), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol Examples include chlorohexanol, 3-pentanol, and 3-pentanol. Isotonic agents are sometimes used. It is also known as a "stabilizer," and in order to ensure the isotonicity of the liquid composition disclosed in the present invention It can be added to, for example, polyhydric sugar alcohols, such as trihydroxyl groups or Sugar alcohols with more hydroxyl groups, such as glycerin, erythritol, and arabic Examples include ethanol, xylitol, sorbitol, and mannitol. Stabilizers are excipients. This refers to a broad category, ranging from volume expanders to those that solubilize or denature therapeutic agents. These additives can have a variety of functional properties, ranging from those that help prevent adhesion to the container walls to other additives. Typical stabilizers include polyhydric sugar alcohols (as listed above); for example, arginine, lysine. Glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucothoe amino acids such as 2-phenylalanine, glutamic acid, threonine, etc. Calcium, trehalose, stachyose, mannitol, sorbitol, xylitol, rib Examples include chol, myoinositol, galactitol, glycerol, etc. Organic sugars or sugar alcohols such as cyclitol, for example, inositol; polyethylene Recall; amino acid polymers; e.g., urea, glutathione, thioctic acid, thioglycol Sodium thioglycerol, α-monothioglycerol, and sodium thiosulfate Sulfur-containing reducing agents such as; low molecular weight polypeptides (e.g., 10 residues or more) Peptides with few residues; for example, human serum albumin, bovine serum albumin, gelatin or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, for example. For example, monosaccharides such as xylose, mannose, fructose, and glucose; for example, lactose , disaccharides such as maltose, sucrose, and trehalose; and, for example, raffino This includes trisaccharides such as sucrose, as well as polysaccharides such as dextran. The stabilizer may be present in an amount ranging from 0.5 to 10 wt% relative to the weight of the ADC. good.

[0308] Nonionic surfactants or detergents (also known as "humectants") are glycoproteins. In addition to aiding in the solubilization of proteins, it protects glycoproteins from aggregation induced by stirring. It may be added to so as to cause the formulation to be able to process proteins without causing denaturation. It is also acceptable to be exposed to stressed shear surfaces. Suitable nonionic surfactants include: Polysorbate (20, 80, etc.), poloxamer (184, 188, etc.) Examples include (and), and pluronic polyols. Nonionic surfactants are approximately 0.05 The range is from mg / mL to approximately 1.0 mg / mL, for example, from approximately 0.07 mg / mL to approximately 0.2 mg / mL. It may be present in the range of g / mL.

[0309] Additional excipients for compound formulations include fillers (e.g., starch) and chelating agents (e.g., E). DTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and encapsulated substances. A mediator was mentioned.

[0310] 5.7 How to use The anti-glycoMUC1 antibodies or conjugated fragments described herein are used in various diagnostic applications. It can be used in immunoassay. For example, antibodies and binding fragments can be used in immunoassay. (i) For example, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation. Assays, e.g., immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), fluorescence activity It can be used in chemical cell sorting (FACS) and Western blotting, among other applications. ru.

[0311] The anti-glycoMUC1 antibodies or conjugated fragments described herein are also in vivo It is also useful for radiographic imaging, in which case radiopaque agents or radioisotopes are used. Antibodies labeled in a detectable part of the body are administered to a target, preferably into the bloodstream, and labeled. The presence and location of the antibodies in the host are assayed. This imaging technique is used for malignant tumors. It is useful in disease staging and treatment.

[0312] AntiglycoMUC1 antibodies or conjugated fragments, ADCs and C as described herein. AR is a cancer that expresses glycoMUC1, particularly epithelial cancers, such as breast cancer, ovarian cancer, and pancreatic cancer. It is useful in treating cancer, and lung cancer.

[0313] When using the CAR of this disclosure in therapy, the therapeutic method of this disclosure expresses glycoMUC1 For subjects having tumors, see, for example, section 5.4 or embodiment 97 or embodiment 98 As described herein, the effective use of genetically modified cells engineered to express the CAR of this disclosure This includes administering a certain amount. Methods for modifying cells, particularly T cells, to express CARs are, This is described in Section 5.5.1. [Examples]

[0314] [Example 1] Identification of anti-glycoMUC1 antibodies 6.1.1. Overview Different O-glycan densities and Tn(GalNAcα1-O-Ser / Thr) glyco Chemoenzymatic synthesis of multiple repeat MUC1 glycopeptides having a recombinant glycosylation Developed using a different polypeptide, GalNAc- Lansferase isoforms were used to assign the occupancy site of O-glycans (Be (nnett et al., 1998). The optimal vaccine design is Tnglycerin with a high O-glycan density. It was found to be a coform, and the glycopeptide conjugated to KLH was found to be resistant. It was found that this could be overcome. In wild-type Balb / c mice, complete O-glycan The glycopeptide with occupation reacts most strongly with MUC1 expressed in breast cancer cell lines. Because it elicited an antibody response, this represents the most effective vaccine design. The humoral immune response showed remarkable specificity against cancer cells.

[0315] 6.1.2. Materials and Methods 6.1.2.1 Chemoenzymatic synthesis of the polymer Tn MUC1 glycopeptide MUC1 60-mer(VTSAPDTRPAPGSTAPPAHG)n=3 (array) Peptide number 47) Combine as first reported by Fontenot et al., 1993 Success. The control peptide used was MUC2(PTTTPISTTTMVTPTPTP TC) (Sequence ID 51) and MUC4 (CPLPVTDTSSASTGHATPLPV Obtained from the tandem repeat (TR) of (Sequence No. 52). U.S. Patent No. 6,465,22 As described in Specification No. 0, purified recombinant human glycosyltransferase The polypeptides GalNAc-T2, GalNAc-T4, and GalNAc-T1 (Bennett et al., 1998; Schwientek et al., 2002) were used to in vitro study the peptide. The peptide was glycosylated with 25 mM cacodylic acid. Buffer solution (pH 7.4), 10 mM MnCl2, 0.25% Triton X-100, and in a reaction mixture containing 2 mM UDP-GalNAc (peptide 1 mg / mL) The procedure was performed using a 60-mer peptide containing 2 GalNAcs per 1 mg TR. Glycosylation of (MUC160Tn6) was achieved using GalNAc-T1. Three GalNAcs were incorporated per TR (MUC160Tn9), -Achieved using T2. All 5 in MUC1 TR(MUC160Tn15) Substitution of the presumed O-glycosylation site is used as a substrate in reaction with GalNAc-T4. The procedure was performed using C160Tn9. Glycosylation was performed using a nanoscale reversed-phase column (Po ros R3, PerSeptive Biosystems, Framingham; Glycopeptides were monitored using MA and MALDI-TOF mass spectrometry. A gradient of 0.1% trifluoroacetic acid (TFA) and 0–80% acetonitrile was used. , 1100 Hewlett Packard system (Avondale, PA) Zorbax 300SB-C3 column (9.4mm x 25cm) (Agilent) High-performance liquid chromatography at Technologies, Palo Alto, CA. Purified by HPLC. The yield of the glycosylation reaction was quantified and estimated using a standard method. Ratio of HPLC peaks measured by UV210 absorbance using a 10 μg peptide as a reference. The procedure was performed by comparison. The yield of GalNAc glycosylation of peptides was 80-90% overall. The recovery rate was 0%. The purified glycopeptide underwent delayed extraction. Equipped with Voyager DE or Voyager DE Pro MALDI-TOF MALDI-TOF quality in a mass spectrometer (PerSeptive Biosystems) Characterized by quantitative analysis. The MALDI matrix is ​​composed of 0.1% TFA and 30% A 10 g / L of 2,5-dihydroxyammonium compound dissolved in a 2:1 mixture with cetonitrile aqueous solution It was fuzzy acid (Aldrich, Milwaukee, WI). Approximately 0.1% TFA A sample dissolved at a concentration of 1 pmol / uL is placed in a 1 μL sample solution on a probe tip. The samples were prepared for analysis by adding 1 μL of matrix to the container. The mass spectrum was obtained in linear mode. Data processing was performed using GRAMS / 386 software. This was done using the following products (Galactic Industries, Salem, NH): .

[0316] 6.1.2.2 Immunization Protocol Glycopeptides are used with glutaraldehyde in KLH(Pierce, Rockf The conjugation efficiency was measured against the MUC1 fraction. The reaction was analyzed by size exclusion chromatography using a PD-10 column with ELISA. It was evaluated by analysis. Essentially, all reactivity was found in the excluded fraction. In fractions that were not excluded and were expected to contain peptides, non-significant reactivity was observed. Further evaluation involves ELISA with the corresponding glycopeptide of KLH condyl Comparative titration analysis of the conjugation was included. When the conjugation was nearly complete, 1:30 The ratio of 0 to the glycopeptide of KLH should result in both analyses being condyl The grafting was nearly perfect. Female Balb / c wild-type mice were given a total volume of 200. 10 or 15 μg of (sugar) peptide (Freund's adjuvant, Sigma) in uL The mice were subcutaneously injected with a 1:1 mixture. They received four immunizations at 14-day intervals. Blood sample obtained due to bleeding from the tail or eye one week after the third and fourth immunizations. I got the lure.

[0317] 6.1.2.3 Generation of mouse MAb anti-Tn-MUC1 60-mer fully galNAc glycosylated and coupled with KLH MAb was produced from wild-type Balb / c mice immunized with MUC1 glycopeptide. The screening involved ELISA for glycopeptides, followed by breast cancer cell lines (MCF7). Based on immunocytology using T47D and immunohistochemistry using cancer tissue. The selection was based on similar response patterns in the total serum of the same mice.

[0318] 6.1.2.4 ELISA ELISA in a 96-well MaxiSorp plate (Nunc, Denmark) The procedure was performed using the following method: Plates were placed in bicarbonate-carbonate buffer (pH 9.6) at a concentration of 1 μg / mL. Coated overnight at 4°C with the glycopeptide, then in 5% phosphate-buffered saline (PBS). Block with bovine serum albumin (BSA), followed by serum (diluted with PBS) or M The antibody was incubated with Ab at room temperature for 2 hours. The bound antibody was then used to treat peroxidase condyloma. Rabbit anti-mouse immunoglobulin (DakoCytomation, Glostr Peroxidase condyloma (up, Denmark) or isotype-specific antibodies Goat anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3(S) outhern Biotechnology Associates, Birming Detected using ham, AL). Plates were examined using O-phenylenediamine tablets (DakoC). The image was developed using ytomation and read at 492nm. The control antibody was an anti-MUC1 antibody. Certain HMFG2 and SM3 (Burchell et al., 1987) and anti-carbohydrate antibody 5F4 (Tn) and 3F1(STn) (Mandel et al., 1991) were included in the control serum. The study included mice immunized with MUC4 mucin peptide linked to KLH.

[0319] 6.1.3.Results A glycopeptide-specific mAb against GalNAc-MUC1 was used as an immunogen in KL We use a 60-mer glycopeptide of GalNAc-MUC1 conjugated to H. It was produced using ELISA assay. ) is a purified recombinant human glycosyltransferase called GalNAc-T1, In vitro glycosylation using GalNAc-T2 and GalNAc-T4 MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG) 3 (arrangement) The corresponding MUC1 peptide, which specifically reacts with (number 47) and does not contain a GalNAc- residue, reacts with (number 47) and does not contain a GalNAc- residue. Or it does not react with unrelated glycopeptides that have the same type of Tn glycoform. Figure 1 shows the results of the ELISA assay.

[0320] [Example 2] Characterization of anti-glycoMUC1 antibodies 6.2.1. Overview The monoclonal antibody GO2(5F7) was used to target the Tn glycosylation of MUC1, which is associated with cancer cells. It was characterized in terms of the specificity of its binding to the foam.

[0321] 6.2.2. Materials and Methods 6.2.2.1 Immunocytochemistry The cell line was fixed in ice-cold acetone or methanol:acetone for 10 minutes. Cells were incubated with mouse serum (1:200 / 1:400 / 1:800) or MAb at 5°C. Incubate overnight, followed by fluorescein isothiocyanate (FITC) conjugate. Gated rabbits with anti-mouse immunoglobulin (DakoCytomation) at room temperature for 4 The slides were incubated for 5 minutes in glycerol containing p-phenylenediamine. Mounted with a Zeiss fluorescence microscope (FluoresScience, Hallber). I was tested using GMOOS (Germany).

[0322] 6.2.2.2 Immunohistochemistry Formalin-fixed paraffin wax-embedded tissue samples were obtained from breast cancer. All cases were classified by histological type. It was conventionally classified by the following: Immunostaining was performed using the avidin-biotin peroxidase complex. The following method was used: wax was removed from the paraffin sections, rehydrated, and 0% methanol was used. The sections were treated with 0.5% H2O2 for 30 minutes. The sections were rinsed in TBS and mixed with non-immune rabbit serum for 20 minutes. The section was incubated for several minutes. The section was rinsed and incubated overnight with the primary antibody at 5°C. Rinse the hand and add rabbit anti-mouse serum (D) labeled with biotin diluted 1:200 with TBS. Incubate with akoCytomation for 30 minutes, rinse with TBS, and avidin - Biotin peroxidase complex (DakoCyto-mation) for 1 hour The tissue was incubated. The sections were rinsed with TBS and 0.05M TB containing 0.1% H2O2 was added. The newly prepared 0.05% 3,3'-diaminobenzidine tetrahydrochloride in S The tissue was developed using a specific method. Sections were stained with hematoxylin, dehydrated, and mounted.

[0323] 6.2.3.Results Immunohistochemistry was performed using GO2 for colorectal cancer, pancreatic cancer, and invasive breast cancer. Staining of colorectal cancer tissue (Figure 2) revealed intracellular and surface structures in the majority of cancer cells. Strong labeling was demonstrated. In contrast, no responsiveness was observed in healthy columnar epithelial cells. The level was extremely low. Labeling in healthy cells was limited to intracellular structures. This is because Large amounts of biosynthetic intermediates (GalNAc modified) are found in highly secretory cells such as columnar epithelium of the intestine. This is thought to be due to the presence of glycoproteins. A similar pattern is found in the pancreas (Figure 3). ) and breast cancer tissue (Figure 4) showed a strong reaction with cancer cells, but the surrounding healthy tissue Intracellular structures in healthy epithelial or connective tissue cells are non-reactive or have limited reactivity. I couldn't.

[0324] [Example 3] Sequence analysis of anti-glycoMUC1 antibodies Prepare mRNA from hybridomas that produce the monoclonal antibody GO2(5F7). Then, it was reverse-transcribed and sequenced.

[0325] The nucleotide sequences that encode the heavy chain and light chain variable regions along with their signal sequences are These are described in Sequence ID 11 and Sequence ID 12, respectively. Sequence ID 11 and Sequence ID The heavy and light chain variable regions coded by 12 are sequence numbers 1 and 12, respectively. It is described in issue 2. The predicted mature variable region sequence (after signal peptide cleavage) is as follows: These are described in SEQ ID NOs. 3 and 4, respectively, and SEQ ID NOs. 13 and 14, respectively. Encoded by: The predicted heavy chain CDR sequences (defined as IMGT) are, respectively, the sequences. The predicted light chain CDR sequences (defined as IMGT), listed in numbers 5-7, are, respectively, sequences. It will be listed under numbers 8-10.

[0326] [Example 4] Drug delivery to cancer cells using anti-glycoMUC1 antibodies 6.4.1. Overview The ability of the monoclonal antibody GO2(5F7) to deliver cytotoxic substances to target cells We conducted tests regarding force.

[0327] 6.4.2. Materials and Methods OVCar human ovarian cancer cells were placed in 24-well cell culture plates, 1,000 cells / well. It was added with a gel. Monoclonal antibody GO2 and the antitubulin agent monomethyl o - Listatin F (MMAF) (Anti-mFc-NC-MMAF) (Moradec catalog number) Add the secondary antibody conjugated to (AM-101-AF) to the plate and concentrate as follows: GO2 and ADC were obtained at a concentration of 1 / 2 (μg / ml).

[0328] [Table 5]

[0329] The plates were incubated at 37°C for 48 hours. After 48 hours of incubation, Add AlamarBlue® (Invitrogen) to each well, and 60 Fluorescence was measured at 0 nm.

[0330] 6.4.3.Results The results are shown in Figure 5. The results show that cytotoxicity is related to the concentration of the primary antibody (GO2) and the presence of the antibody. This indicates that it depends on the concentration and presence of the secondary ADC conjugate antibody. In other words, GO2 is coupled with a secondary antibody containing the cytotoxic substance MMAF. In combination, this induces cytotoxicity in this cancer cell line.

[0331] [Example 5] Quantification of circulating tumor cells using anti-Muc1 antibodies 6.5.1. Overview The monoclonal antibody GO2(5F7) is used to quantify circulating tumor cells. We tested their abilities.

[0332] 6.5.2. Materials and Methods GO2 was conjugated into magnetic separation beads and interacted with samples of tumor cells at different concentrations. The treatment was carried out. The cells and beads were removed from the solution using a magnet and washed several times to remove any remaining cells and beads. The material was removed. Then, it was conjugated with horseradish peroxidase. The GO2 was applied to magnetic separation beads containing bound cancer cells, and incubated. Next, the unbound conjugated GO2 was washed away. TNB was used as the substrate. A colorimetric reaction was carried out using [a specific method]. The reaction was terminated with sulfuric acid, and then the OD450 of the sample was [a specific method]. The reading value was taken.

[0333] 6.5.3.Results The results are shown in Figure 6. The assay results demonstrated binding of GO2 tumor cells.

[0334] [Example 6] Immunohistochemical staining of tumor tissue using anti-glycoMUC1 antibody 6.6.1. Materials and Methods Six formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMA) Sections were cut to a thickness of 2.5 μm. Breast cancer (BC), colorectal cancer (CRC) , ovarian cancer (OVC), non-small cell lung cancer (NSCLC), and prostate cancer (PrC) tumors TMAs were used in the study. Each TMA contained 25-47 tumor tissue cores for evaluation. It was available. Core sizes were 1mm, 2mm, or 3mm depending on the TMA. Each tissue core is a specimen from a single patient.

[0335] For staining, use Discovery XT Autostainer (Ventana Medica The procedure was performed using (Systems). Cell condition 1 (CC1) solution (Ventana Med After antigen retrieval using ical Systems, GO2 is used with Dako Green Medi. Apply at a concentration of 1 μg / mL in Um (green medium) antibody diluent at 37°C. The cells were incubated for 60 minutes. The binding of GO2 to tumor cells was visualized using DAB (brown precipitate). (Ventana Medica) Optiview DAB IHC Detection Kit Detected using (Systems)

[0336] 6.6.2.Results Figure 7 shows representative images of MUC1-positive TMA tumor cores. BC and OVC TM In area A, the majority of the spots (>90%) were GO2 to moderate or severe tumor cells. The coupling was shown. 70% and 51% of the cases of NSCLC and CRC were, respectively, It showed antibody binding to tumor cells to varying degrees and strength. In prostate cancer, antigen expression was more It seemed that the number was small. Only 28% of the spots in TMA1 had GO-2 applied. They showed moderate to strong staining intensity. The staining pattern was always intracytoplasmic, and many In some cases, it was membrane-bound. Apical membrane staining patterns were not observed in most cores. Ta.

[0337] [Example 7] Production and purification of anti-MUC1 antibodies in a bispecific (TCB) manner on T cells. 6.7.1. Materials and Methods 6.7.1.1 Production of expression vectors In the Fc region, knob-into-hole and P329G / L234A / L235A ( "PGLALA" mutation, and MUC1 CH1 (147E / 213E; "EE TC containing charged residues in the ) and CL(123R / 124K; "RK") region The GO2 antibody was converted to form B (see SEQ ID NOs. 43-46). Figure 8 shows the TCB antibody. To illustrate, simply put, we synthesize variable heavy and variable light chains of GO2 mAb (Gene (art, Regensburg, Germany), they are suitable for human steady-state heavy chain or The expression vectors in these vectors were inserted into suitable expression vectors that fused with human constant light chains. The set includes the 5'UTR and BGH polyadenylation sites, along with the CMV promoter, i It consists of nucleotide A. In addition, the plasmid contains HE, which contains the EBV nuclear antigen (EBNA). For stable maintenance in K293 cells, oriP derived from Epstein-Barr virus is used. It contains a region.

[0338] 6.7.1.2 Transient transfection and production HEK293EBNA cells are transfected using the PEI-mediated transfection procedure as follows: The cells transiently produced antibodies. HEK293EBNA cells responded to 6 mM L-glutamic acid. It is cultured in a serum-free suspension in Excell culture medium containing . 500 ml shake. For antibody production in the flask, 300 million HE cells were added 24 hours before transfection. Seed K293EBNA cells (therefore, for alternative scales, the total amount (Prepare). For transfection, centrifuge the cells at 210 × g for 10 minutes, and remove the supernatant. Replace with 20 ml of pre-warmed CD CHO medium. Replace the expression vector with 20 ml of CD CHO medium. Mix in CHO medium to achieve a final DNA volume of 200 μg. 540 μl PEI After adding the solution, vortex it for 15 seconds, then incubate at room temperature for 10 minutes. Then, mix the cells with the DNA / PEI solution and transfer them to a 500 ml shaking flask. Incubate in an incubator containing a 5% CO2 atmosphere at 37°C for 3 hours. After incubation, add 6 mM glutamine, 1.25 mM valproic acid, and 12.5% ​​P. 160 ml of Ex-cell® medium (Sigma-Al) containing epsoy Add drich and culture the cells for 24 hours. One day after transfection, 12 Add % feed 7 (48 mL) + 3 g / L glucose. After 7 days, 4 Collect the culture supernatant for purification by centrifugation for 5 minutes. Filter the solution through sterile filtration (0.22 μg). (Using an m filter), add sodium azide to a final concentration of 0.01% w / v. Then dissolve... Store the liquid at 4°C.

[0339] 6.7.1.3 Antibody purification The secreted protein was analyzed using affinity chromatography with protein A. Then, it was purified by size exclusion chromatography. Affinity chromatography For the imaging, the supernatant was mixed with 20 mM sodium phosphate and 20 mM sodium citrate. Protein A MabSelect SuRe column (G) equilibrated at pH 7.5 Loaded into E Healthcare. Washed with equilibrium buffer. The binding protein was removed. The binding protein was treated with 20 mM sodium citrate, 1 Stepwise (standard) prepared using 00 mM sodium chloride, 100 mM glycine, and pH 3.0 Elution was performed using either quasi-IgG or gradient (bispecific antibody) elution. The samples were collected and... To adjust the pH of the fraction, add 1 / 10 (v / v) 0.5M sodium phosphate pH 8.0. The protein was concentrated, filtered, and then mixed with 20 mM histidine. HiLoad Superdex 2 equilibrated at 40 mM sodium chloride, pH 6.0 Loaded into column 00 (GE Healthcare).

[0340] The contents of the aggregate of eluted fractions were analyzed by analytical size exclusion chromatography. Therefore, each 30 μl fraction was treated with 200 mM arginine and 25 mM K2PO4. 4. TSK equilibrated in 125 mM sodium chloride, 0.02% NaN3, pH 6.7. I applied to a G3000SWXL column (Tosoh Corporation, 7.8mm x 30cm). The fraction containing less than 2% oligomers is pooled and concentrated using a centrifugal concentrator (Millipore Using Amicon (registered trademark) ULTRA-15, 30k MWCO) 1-1. The solution was concentrated to a final concentration of 5 mg / ml. The purified protein was stored at -80°C.

[0341] 6.7.2.Results Table 5 shows the production yield and quality of GO2 TCB antibodies.

[0342] [Table 6]

[0343] [Example 8] For monitoring target expression in undigested patient-derived tumor samples ex vivo Jurkat-NFAT Reporter Assay 6.8.1. Overview Using the Jurkat NFAT reporter assay, undigested primary cells were analyzed ex vivo. Target expression (glycoMUC1) in human tumor samples was measured using GO2 TCB. I watched it.

[0344] 6.8.2. Materials and Methods 6.8.2.1 Materials • GO2 TCB (See Example 7) • DP47 TCB (non-targeted negative control) • Matrigel (item number 734-1101, Corning / VWR, Sw (Itzerland) Corning® Costar® Ultra-Low Attach Menthol multiwell plate (item number CLS7007-24EA, Sigma) • Cell culture microplate, 96 wells (item number 655098, Greine) (Bio-one, Switzerland) • RPMI1640 medium (item number 42401-018, FisherScien) (tific, Schweiz) Jurkat medium: 2 g / l D-glucose, 2 g / l NaHCO3, 10%F CS, 25 mM HEPES, 2 mM L-glutamine, 1 × NEAA, 1 × pyruvate RPMI1640 medium containing sodium and 200 μg / ml hygromycin B • Jurkat NFAT luciferase reporter cells (Promega) • Tumor samples received from Indivumed GmbH, Germany. Sun The pull was transported overnight in a transport medium. Approximately 24 hours after the surgery, the sample was finely calibrated. I did.

[0345] 6.8.2.2 Method Add 17 μl of cold Matrigel to a 96-well cell culture microplate. The preparation was carried out by incubating the plate at 37°C for 2 minutes, and then the tumor was dissected. A piece was added (3-pack). 33 μl of cold Matrigel was added to each well, and pre- The wells were incubated again at 37°C for 2 minutes. 100 μl (50 nM or TCB antibody dilution (5nM) (Hygromycin-free but 2× penicillin / strep) (diluted in Jurkat medium containing tomycin) was added. Jurkat-NFA T reporter cells were harvested and their viability was evaluated using ViCell. Cells were scanned at 350× Centrifuge at 0g for 7 minutes, then repackage in hygromycin-free Jurkat medium. The cells were suspended. 50 μl of cell suspension was added to each well (50,000 cells / well). ). Incubate the plates in a humidified incubator at 37°C for 4 to 5 hours. It was then removed for luminescence readout. 50 μl of ONE-Glo solution was added to each. The solution was added to the wells and incubated in the dark at room temperature for 10 minutes. WALLAC Victo r3 ELISA reader (PerkinElmer2030) at 5 seconds / well detection It was used in between, and the light emission was detected.

[0346] 6.8.3.Results Figures 9-11 show the results from tumor samples from three patients. Results shown in Figure 9. This refers to patients with malignant neoplasms of the bronchi and lungs: squamous cell carcinoma of the middle lobe, bronchi, or lung. These results are from the tumor samples obtained. The results shown in Figure 10 are from the bronchial and pulmonary malignancies. Sexual neoplasms: Tumor samples obtained from patients with non-keratinizing squamous cell carcinoma of the lower lobe, bronchi, or lung. This is from a sample. The results shown in Figure 11 are malignant neoplasms of the bronchi and lungs: upper lobe. From tumor samples obtained from patients with bronchial or lung, acinar-type adenocarcinoma. Therefore, each bar shown in Figures 9-11 represents the average of three consecutive values. The standard error is shown in the error bars. This is shown. The dotted line indicates incubation with the tumor sample without using TCB at all. The luminescence of Jurkat NFAT cells is shown. An unpaired two-sided t-test was performed for statistical analysis. Used. P-values ​​less than 0.05 were considered significant and are indicated with an asterisk. * P ≤ 0.05; * * P ≤ 0.001; *** P ≤ 0.001). In each of Figures 9-11, GO2 Tumor samples incubated with TCB were inked together with DP47-negative control TCB. It exhibited significantly higher luminescence than the tubulated sample.

[0347] [Example 9] In vitro characterization of GO2 TCB 6.9.1. Overview GO2 TCB recognizes tumor-specific abnormally glycosylated MUC1 (Example 7) ) was functionally characterized in tumor cells expressing MUC1.

[0348] 6.9.2. Materials and Methods 6.9.2.1 Cell lines and PBMCs Tumor cell lines manipulated with T3M4 pfzv and MCF7 cs were subjected to 10% FCS. MCF10A was cultured in DMEM containing 2 mM glutamine. This is a mammary epithelial cell line (ATCC® CRL-10317). HBEpiC is, These are human bronchial epithelial cells (Science II number 3210). All cells are collected from healthy volunteers. PBMCs were isolated by gradient centrifugation using blood.

[0349] 6.9.2.2 Target binding by flow cytometry The indicated target cells were collected using cell dissociation buffer, washed with PBS, and then treated with FACS buffer. The solution was resuspended. Antibody staining was performed in a 96-well round-bottom plate. Per well 200,000 cells were seeded. The plate was centrifuged at 400g for 4 minutes, and then... The solution was removed. The test antibody was diluted with FACS buffer, and 30 μl of the antibody solution was incubated at 4°C for 30 minutes. The antibody was added to the cells over a period of minutes. To remove unbound antibodies, the cells were rinsed twice with FACS buffer. Wash, and then dilute the secondary antibody (PE conjugate AffiniPure F(a b')2 Fragment specific to goat anti-human IgG Fcγ fragment, Jackson ImmunoResearch sample number 109-116-170 was added. The sample was incubated at 4°C for 30 minutes. After incubation, unbound secondary antibodies were washed off. Before measurement, cells were 20 Resuspend in 0 μl of FACS buffer and use BD Fortessa for flow cytometry. Analysis was performed by Tory. The assay was run in triplicate.

[0350] 6.9.2.3 T cell-mediated tumor cell death and T cell activation Target cells were harvested using trypsin / EDTA, counted, and their viability was checked. The cells were resuspended in their respective culture media at a final concentration of 300,000 cells / ml. Then, 100 μl of the target cell suspension was transferred to each well of a 96-flat-bottom plate. The plates were incubated overnight at 37°C in a tertoscope to allow the cells to adhere to the plates. The following day, PBMCs were isolated from whole blood. The blood was diluted 2:1 with PBS and Leucose 15 ml of Histopaque-1077 (number 10771, Sigma) in a p-tube It was placed on top of (a-Aldrich) and centrifuged at 450g for 30 minutes without applying the brakes. After centrifugation, the band containing cells was collected using a 10 ml pipette and then transferred to a 50 ml tube. Transferred to a tube. Fill the tube with PBS up to 50 ml and centrifuged (400 g, 10 (300g, room temperature). Remove the supernatant and resuspend the pellet in PBS. After centrifugation (300g, After 10 minutes (at room temperature), discard the supernatant, pool the two tubes, and repeat the washing process (this (350g, 10 minutes, centrifugation at room temperature). Then, resuspend the cells and adjust the cell count. The pellet was then pooled in 50 ml of PBS. After counting the cells, centrifugation was performed (350°C). g, 10 min, room temperature), 600 cells in RPMI containing 2% FCS and 2nM glutamine. The cells were resuspended at 10,000 cells / ml. The culture medium was removed from the plated target cells, and 2% FCS was added. Test antibodies diluted with RPMI containing 2nM glutamine were added to effector cells. 300,000 cells in the solution were transferred to each well, and an E:T ratio of 10:1 was obtained. To determine the release of the substance, target cells were lysed with Triton X-100 for 24 hours. And 48 hours later, cytotoxicity detection kit (1644793, Roche Applie LDH release was determined using (d Science). In T cells after tumor cell lethality... The upward adjustment of the activation marker was measured by flow cytometry. The PBMCs were collected, transferred to a 96-well round-bottom plate, and diluted with FACS buffer. D4APC (300514, BioLegend), CD8FITC (344704, B ioLegend), CD25BV421(302630, BioLegend), CD Stained with 69PE (310906, BioLegend) antibody. Incubated at 4°C for 30 minutes. After batting, the cells were washed twice with FACS buffer. BD Fortessa II Before measuring fluorescence, the cells were resuspended in 200 μl of FACS buffer. I performed the action "I" three times in a row.

[0351] 6.9.2.4 Cytokine / chemokine release by cytometry bead array Flow using a cytometry bead array (CBA) in accordance with the manufacturer's guidelines - Cytokine / chemokine secretion in the supernatant was measured by cytometry. T cells The supernatant from the vector lethal assay was collected and stored at -20°C. The supernatant was then thawed. The tests were conducted according to the manufacturer's instructions. The following CBA kit (BD Biosciences) was used. ) used: CBA Human Interferon Gamma (IFNγ) Flex Set (E7) CBA Human Granzyme B Flex Set (D7), CBA Human IL6 Flex Set Set (A7), CBA Human IL8 Flex Set (A9), CBA Human IL10 Flex ex set (B7) and CBA human tumor necrosis factor (TNF) Flex set (D9). Measure the sample using BD FACS Canto II and Diva software The analysis was performed using (BD Biosciences). The assay was run in triplicate. Ta.

[0352] 6.9.3.Results Both are breast cancer cell lines engineered to express abnormally glycosylated MUC1. Binding of GO2 TCB to MCF7 and the pancreatic cancer cell line T3M4 was confirmed (Figure 12). Subsequently, using newly isolated PBMCs, both tumor cell lines, MCF7 and T3 The activity of GO2 TCB in M4 was tested (Figures 13 and 14). After 24 hours, Lethal tumor cell death was detected in both cell lines, and it had become even stronger after 48 hours. At the same time, the two activation markers CD25 and CD69 were upregulated, as well as IL6 , by release into the supernatant of IL8, IL10, IFNγ, TNFα and granzyme B Strong activation of determined CD4 T cells and CD8 T cells occurred. As a negative control, Each of these contains a non-targeted TCB.

[0353] GO2 TCB does not bind to normally glycosylated MUC1 on epithelial cells. To prove this, we used MCF10A, a human non-tumor-forming mammary epithelial cell line, and primary he Binding to HBEpiC, a type of bronchial epithelial cell, was tested. Normal cells were used as a positive control. HMFG1 does not distinguish between MUC1 expressed on the surface and MUC1 expressed on tumor cells. TCBs were found to be present. HFMG1 TCBs were found to bind to both tested cells. Since it was released, MUC1 expression was confirmed, but GO2 TCBs do not bind to these cells. They could not be matched (Figure 15). In addition, the GO2 TCB was tested and it was found to be MU Whether it induces lethality or T cell activation in the presence of normal epithelial cells expressing C1 I observed this. When I tested this with MCF10A cells, lethal cells detectable by GO2 TCBs were found to be... There is no T cell activation, whereas HMFG1 TCB is lethal in addition to T cell activation. Activation was also induced (Figure 16).

[0354] [Example 10] Functional characterization of GO2 and GO2 TCB antibodies by surface plasmon resonance 6.10.1. Overview GO2 and GO2 TCB (Example 7) were characterized by surface plasmon resonance. .

[0355] 6.10.2. Materials and Methods 6.10.2.1 Binding of GO2 and GO2 TCB to immobilized glycopeptides Binding of GO2 antibodies and GO2 TCBs to human and cynomolgus monkey glycopeptides. (Table 6) was evaluated by surface plasmon resonance (SPR). All SPR experiments were B Using an iacore T200 at 25°C, HBS-EP (0.01) was used as the running buffer. M HEPES, pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% Performed using surfactant P20 (Biacore, Freiburg / Germany). did.

[0356] [Table 7]

[0357] Dissolve the biotinylated glycopeptide in PBS to a final concentration of 0.9 to 1.8 mg. The values ​​were adjusted between / ml (Table 6). Biotinylated glycopeptides were added to streptavidin (SA ) Directly coupled to the flow cell of the sensor chip. Up to 880 resonance units (RU) A fixed level was used. A 30 μL / min flow through a flow cell was used for 240 seconds. Alternatively, GO2 antibody or GO2 TCB was injected at a concentration of 1000 nM (Figure 17). Dissociation was monitored for 500 seconds. The refractive index difference of the bulk protein was fixed. The response was corrected by subtracting the response obtained from a non-reference flow cell.

[0358] 6.10.2.2 Binding activity of GO2 and GO2 TCB to immobilized glycopeptides sex The binding activity of GO2 and GO2 TCB was evaluated by surface plasmon resonance (SPR). It was worthwhile. All SPR experiments were conducted on a Biacore T200 at 25°C with running buffer. As a liquid, HBS-EP (0.01M HEPES, pH 7.4, 0.15M NaCl) 3 mM EDTA, 0.005% surfactant P20, Biacore, Freiburg The procedure was performed using (Germany). Biotinylated glycopeptides (Table 6) were strained The putavidin (SA) sensor chip was directly coupled to the flow cell. Maximum 200 A fixed level of resonance units (RUs) was used.

[0359] Using a 30 μL / min flow through a flow cell, over 120 seconds, 3.9 to 10 GO2 antibody or GO2 TCB was injected at a concentration in the 00 nM range (1:2 dilution). The separation was monitored for 400 seconds. The difference in refractive index of the bulk protein was measured against a reference plate where the protein was not fixed. The response obtained in Roesel was corrected by subtracting it, despite the bivalent nature of the interaction. Using Biaeval software (GE Healthcare), the curve was converted to a 1:1 ratio. By applying this to a Langmuir joint, we obtained KD. Therefore, the "apparent" K D can only be used for comparative purposes.

[0360] 6.10.3.Results As can be seen from the sensorgram in Figure 18, the GO2 antibody (Figure 18A) and GO2 TCB (Figure 18B) binds to both human and cynomolgus monkey glycopeptides. GO2 antibodies and GO2 TCBs have higher binding activity than human glycopeptides, and crab It binds to quisal glycopeptide.

[0361] As can be seen in Figure 19, the divalent GO2 binder to human glycopeptides The binding of (IgG, TCB) was at a three-digit nanomolar concentration (Figures 19A and 19C), In contrast, binding to cynomolgus glycopeptide is at a two-digit nanomolar concentration (Figure 19). B and 19D).

[0362] [Example 11] Pharmacokinetic and tolerable studies of an exploratory single dose of GO2 TCB in cynomolgus monkeys. 6.11.1. Overview The purpose of this study is to determine the effectiveness of administering a single intravenous injection to cynomolgus monkeys. This is to determine the pharmacokinetics and tolerability of the GO2 TCB described in Example 7.

[0363] 6.11.2. Materials and Methods 6.11.2.1 Preparation of GO2 TCB GO2 TCB (2.12 mg / mL) and combination buffer (20 mM histidine, 1 Frozen stock solution in 40 mM NaCl, 0.01% Tween 20 (pH 6.0) The liquid is melted overnight in a refrigerator set to maintain a temperature of 4°C. Dosage formulation of the test item. The material, under sterile conditions, is diluted with a compound buffer to meet the required dose level. Adjust to the desired concentration.

[0364] The dosage formulation is prepared within 2 hours prior to injection and stored at room temperature until use. Polypropylene containers are used for the preparation and storage of substances to prevent adsorption. The mixture should not be filtered, stirred, or shaken. Any mixing should be done gently. This is done either by quick pipetting or by gently shaking the container.

[0365] 6.11.2.2 Animals Crab-eating macaques (Macaca fascicularis) are 2-4 years old and weigh less than 4 kg. )) will be used in the study. The animals will be kept in the experimental facility for at least 6 weeks prior to the start of administration. Acclimatize them in a toxicology facility.

[0366] During the week prior to the start of administration, the animals undergo thorough veterinary examinations (performed immediately upon arrival) and clinical testing. Based on detailed observational records, body weight profiles, and clinical-pathological investigations, participation in the experiment was determined. Get approved.

[0367] The animals selected for the study were provided with group compatibility information and then provided with Animals are randomly assigned to cages based on the number of individual studies. Animals are grouped into groups of up to five. It will be assigned to a cage.

[0368] 6.11.2.3 Management If possible, the animals should be placed in enclosures on two levels, with a maximum of five groups separated by sex. They were isolated, with the lower floor measuring 1.61 x 1.66 x 2.5m and the upper floor measuring 1.61 x 1.66 x The measurement is 2.03m. The base material is wood shavings. The base contains dried wood for research purposes. There are no known contaminants that are expected to be present.

[0369] The targeting conditions for the animal room environment are as follows: Temperature: 18~24℃ Humidity: 40~70% Ventilation: A minimum of 10 air changes per hour. Light cycle: 12 hours of light and 12 hours of dark (interrupted by research procedures / activities) (Excluding the letter.)

[0370] Automatic temperature and humidity control is performed, and this is continuously monitored and recorded. The cycle is automatically controlled.

[0371] Special Diets Service (SDS) MP(E) Short SQC Meal (Short S QC Diet will be provided as a daily ration throughout the study. Approximately 2 per animal. A 00-gram feed is provided once a day. The feed contains ingredients that are expected to interfere with the research objectives. There are no known contaminants. Animals take water without restriction from a common source. Research is being conducted in the water. There are no known contaminants that are expected to interfere with the purpose.

[0372] Enrich the indoor environment of animals to promote social interaction, play, and exploration. The animals are kept in a safe place, and have plastic toys, balls, and climbing frames. It has materials such as glass and stainless steel mirrors. These are frequently used to reduce wear and tear. It will be replaced. Before replacement, all toy and climbing frame will be cleaned of cross-stains. They are thoroughly cleaned to avoid contamination. The animals are also usually cleaned daily with feed mixtures. In addition, a variety of other treats such as vegetables, nuts, biscuits, and fruits are also offered.

[0373] Veterinary care will be available throughout the course of the study, and clinical signs or other If the change is deemed normal, the animal will be examined by veterinary staff.

[0374] 6.11.2.4 Experimental Design GO2 TCB is administered to one male and one female animal.

[0375] [Table 8]

[0376] GO2 TCB is administered as a single intravenous injection in the saphenous vein or tail vein at least 8 days apart. It is administered to appropriate animals by a slow intravenous bolus injection (1-2 minutes). This ensures that only one male and one female receive the new dose level on any given day. This is done with a time lag. It is based on observations from previous dose levels (including clinical and pathological data). Then, increase or decrease the dose in the next dose group. Each dose level is used in naive animals. The dosage is administered using a syringe fitted with a Vygon needle. ru.

[0377] An autopsy of the animal will be performed approximately 72 hours after administration (after the last planned sample has been taken). Regarding all animals that must be terminated before the scheduled date due to the degree of clinical symptoms , complete clinical pathology (additional sampling before termination if possible) and histopathology Nell is analyzed.

[0378] Intravenous injection is a clinically applicable route of administration, therefore this route is suitable for this research. It has been meticulously selected. It covers the clinically important dose range and minimizes potential harm to animals. Low and high dose levels were selected to achieve similar efficacy. Selected based on experience in cynomolgus monkeys using T cell bispecific antibodies, high doses are This is equivalent to three times that amount.

[0379] 6.11.3.Results GO2 TCB is acceptable at the tested dose.

[0380] 7. Specific embodiments and citation of references While various specific embodiments have been described and illustrated, they do not deviate from the essence and scope of this disclosure. It is expected that various modifications can be made without further explanation. The disclosure of this invention is This is illustrated by the numbered embodiments described below.

[0381] 1.a. GlycoMUC1 epitopes that are overexpressed on cancer cells compared to normal cells. It preferentially binds to; b. Regarding binding to breast cancer cell lines MCF7 or T47D, the heavy chain variable of SEQ ID NO: 3 ( Antibodies or antigen-binding fragments containing the VH) sequence and the light chain variable (VL) sequence of SEQ ID NO: 4 An anti-glyco MUC1 antibody or antigen-binding fragment that competes with the target.

[0382] 2.a. GalNAc-T, a purified recombinant human glycosyltransferase. 1. In vitro glycosylation using GalNAc-T2 and GalNAc-T4 Modified MUC1 Tandem Repeat (VTSAPDTRPAPGSTAPPAHG) 3( (hereinafter referred to as the "first epitope") is connected to it; and, b. Regarding binding to breast cancer cell lines MCF7 or T47D, the heavy chain variable of SEQ ID NO: 3 ( Antibodies or antigen-binding fragments containing the VH) sequence and the light chain variable (VL) sequence of SEQ ID NO: 4 An anti-glyco MUC1 antibody or antigen-binding fragment that competes with the target.

[0383] 3. Complementarity-determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 29 CDR-H2 containing the amino acid sequence of SEQ ID NO: 25, CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, CDR containing the amino acid sequence of SEQ ID NO: 9 Embodiment 1 also includes CDR-L3 containing the amino acid sequence of -L2 and SEQ ID NO: 31. This is the anti-glycoMUC1 antibody or antigen-binding fragment described in Embodiment 2.

[0384] 4. CDR-H1 contains the amino acid sequence of SEQ ID NO: 5, as described in Embodiment 3. MUC1 antibody or antigen-binding fragment.

[0385] 5. CDR-H1 is an antiglyceridium according to Embodiment 3, comprising the amino acid sequence of SEQ ID NO: 23. CoMUC1 antibody or antigen-binding fragment.

[0386] 6. CDR-H1 is an antiglyceridium according to Embodiment 3, comprising the amino acid sequence of SEQ ID NO: 28. CoMUC1 antibody or antigen-binding fragment.

[0387] 7. CDR-H1 is an antiglyceridium according to Embodiment 3, comprising the amino acid sequence of SEQ ID NO: 32. CoMUC1 antibody or antigen-binding fragment.

[0388] 8. CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6, any of Embodiments 3 to 7. One of the anti-glycoMUC1 antibodies or antigen-binding fragments described below.

[0389] 9. CDR-H2 contains the amino acid sequence of SEQ ID NO: 24, as in any of Embodiments 3 to 7. The anti-glycoMUC1 antibody or antigen-binding fragment described in one of the following:

[0390] 10. CDR-H3 contains the amino acid sequence of SEQ ID NO: 7, in any of Embodiments 3 to 9. The anti-glycoMUC1 antibody or antigen-binding fragment described in one of the following:

[0391] 11. CDR-L1 contains the amino acid sequence of SEQ ID NO: 30, as in Embodiments 3 to 10. Either one of the anti-glycoMUC1 antibody or antigen-binding fragments described below.

[0392] 12. CDR-L1 contains the amino acid sequence of SEQ ID NO: 26, as in Embodiments 3 to 10. Either one of the anti-glycoMUC1 antibody or antigen-binding fragments described below.

[0393] 13. CDR-L2 contains the amino acid sequence of SEQ ID NO: 27, as in Embodiments 3 to 12. Either one of the anti-glycoMUC1 antibody or antigen-binding fragments described below.

[0394] 14. CDR-L3 contains the amino acid sequence of SEQ ID NO: 10, as described in Embodiments 3 to 13. Either one of the anti-glycoMUC1 antibody or antigen-binding fragments described below.

[0395] 15. VH contains the complementarity determination region (CDR) of sequence numbers 5-7, and VL contains sequence numbers AntiglycoMUC1 antibody according to Embodiment 1 or Embodiment 2, comprising 8-10 CDRs or antigen-binding fragment.

[0396] 16. VH contains the complementarity determination region (CDR) of sequence numbers 23-25, and VL is sequence Anti-gang according to Embodiment 1 or Embodiment 2, including CDRs numbered 26, 27, and 10. Lyco-MUC1 antibody or antigen-binding fragment.

[0397] 17. VH includes the complementarity determination regions (CDRs) of sequence numbers 28, 29, and 25. VL includes CDRs of sequence numbers 30, 9, and 31 in Embodiment 1 or Embodiment 2 The anti-glycoMUC1 antibody or antigen-binding fragment described above.

[0398] 18. A chimeric antibody or a humanized antibody, as described in any one of Embodiments 1 to 17. AntiglycoMUC1 antibody or antigen-binding fragment.

[0399] 19. VH contains an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 3. Furthermore, VL contains an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 4. AntiglycoMUC1 antibody or antigen-binding flag according to any one of Embodiments 1 to 18 Ment.

[0400] 20. VH contains an amino acid sequence that has at least 97% sequence identity with SEQ ID NO: 3. Furthermore, VL contains an amino acid sequence that has at least 97% sequence identity with SEQ ID NO: 4. AntiglycoMUC1 antibody or antigen-binding flag according to any one of Embodiments 1 to 18 Ment.

[0401] 21. VH contains an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 3. Furthermore, VL contains an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 4. AntiglycoMUC1 antibody or antigen-binding flag according to any one of Embodiments 1 to 18 Ment.

[0402] 22. VH contains the amino acid sequence of SEQ ID NO: 3, and VL contains the amino acid sequence of SEQ ID NO: 4. The anti-glycoMUC1 antibody or antigen-binding fungibler described in Embodiment 1 or Embodiment 2, including Gument.

[0403] 23. A polyvalent anti-glycoMUC1 antibody according to any one of Embodiments 1 to 22, or This is an antigen-binding fragment.

[0404] 24. Any of Embodiments 1 to 22, which are in the form of a single-chain variable fragment (scFv). The anti-glycoMUC1 antibody or antigen-binding fragment described above.

[0405] 25.scFv contains a heavy chain variable fragment at the N-terminus of the light chain variable fragment. The anti-glycoMUC1 antibody or antigen-binding fragment described in Embodiment 24.

[0406] The heavy-chain variable fragment and light-chain variable fragment of 26.scFv are 4-15 ami. The anti-glycoMUC1 antibody described in Embodiment 24 is covalently bound to the linker sequence of the ano acid. or antigen-binding fragment.

[0407] 27. Antiglycerides according to any one of Embodiments 1 to 22, which are in the form of multispecific antibodies. MUC1 antibody or antigen-binding fragment.

[0408] 28. A multispecific antibody binds to a second epitope that is different from the first epitope. The specific antibody is the anti-glycoMUC1 antibody or antigen-binding fragmentation antibody described in Embodiment 27. nt.

[0409] 29. Bispecific antibodies include CrossMab, Fab arm exchange antibody, and bispecific T-cell antibodies. Embodiment 2 is a cytoplasmic derivative (BiTE) or a biaffinity retargeting molecule (DART). The anti-glycoMUC1 antibody or antigen-binding fragment described in 8.

[0410] 30. The antiglycerid M according to Embodiment 29, wherein the bispecific antibody is CrossMab. UC1 antibody or antigen-binding fragment.

[0411] 31. Bispecific antibodies are CrossMab FAB The anti-g Lyco-MUC1 antibody or antigen-binding fragment.

[0412] 32. Bispecific antibodies are CrossMab VH-VL The embodiment described in Embodiment 30 is as follows: Anti-glycoMUC1 antibody or antigen-binding fragment.

[0413] 33. Bispecific antibodies are CrossMab CH1-CL This is described in Embodiment 30. AntiglycoMUC1 antibody or antigen-binding fragment.

[0414] 34. The antiglyceridium according to Embodiment 29, wherein the bispecific antibody is a Fab arm exchange antibody. CoMUC1 antibody or antigen-binding fragment.

[0415] 35. Embodiment 29, in which the bispecific antibody is a biaffinity retargeting molecule (DART). The anti-glycoMUC1 antibody or antigen-binding fragment described above.

[0416] 36. Embodiment 29, in which the bispecific antibody is a bispecific T cell derivative (BiTE). The anti-glycoMUC1 antibody or antigen-binding fragment described above.

[0417] 37. The second epitope is the MUC1 epitope, in any of embodiments 28 to 35 Any one of the anti-glycoMUC1 antibody or antigen-binding fragments described below.

[0418] 38. The second epitope is overexpressed on cancer cells compared to normal cells (MUC). One epitope is the antiglycoMUC1 anti-epitope described in any one of embodiments 28 to 35. Body or antigen-binding fragment.

[0419] 39. Any of embodiments 28 to 36, wherein the second epitope is a T cell epitope. The anti-glycoMUC1 antibody or antigen-binding fragment described in one of the following:

[0420] 40. T cell epitopes include CD3 epitope, CD8 epitope, CD16 epitope P, including CD25 epitope, CD28 epitope, or NKG2D epitope, The anti-glycoMUC1 antibody or antigen-binding fragment described in Form 39.

[0421] 41. The T cell epitope includes the CD3 epitope, and the CD3 epitope is optional. The anti-glycoMUC1 antibody described in Embodiment 40 is an epitope present in human CD3. Or an antigen-binding fragment.

[0422] 42. CD3 epitopes include CD3 gamma epitopes, CD3 delta epitopes, and CD3 epitopes. Embodiment 41, which includes a 3-epsilon epitope or a CD3 zeta epitope. Anti-glycoMUC1 antibody or antigen-binding fragment.

[0423] 43. Any one of Embodiments 1 to 42 is conjugated to a detectable portion. The anti-glycoMUC1 antibody or antigen-binding fragment described below.

[0424] 44. Embodiments in which the detectable marker is an enzyme, radioisotope, or fluorescent label. The anti-glycoMUC1 antibody or antigen-binding fragment described in 43.

[0425] 45. The first antigen-binding domain and glycan that bind to CD3 (optionally human CD3). A bispecific antibody containing a second antigen-binding domain that binds to MUC1, Sex antibodies, regarding binding to breast cancer cell lines MCF7 or T47D, are heavy chain antibodies of SEQ ID NO: 3. Antibodies or antigen-binding fluids containing a variable (VH) sequence and the light chain variable (VL) sequence of SEQ ID NO: 4 Competing with the ligment, the first antigen-binding domain is the heavy chain CDR-H1 of sequence number 34, sequence Heavy chain variable region including CDR-H2 (number 35) and CDR-H3 (sequence number 36); In addition, the light chain CDR-L1 of SEQ ID NO: 37, CDR-L2 of SEQ ID NO: 38, and SEQ ID NO: 39 A bispecific antibody containing a light chain variable region including CDR-L3.

[0426] 46. ​​The second antigen-binding domain is (i) CDR-H1 of SEQ ID NO: 5, C of SEQ ID NO: 6 Heavy chain variable regions including DR-H2 and CDR-H3 of SEQ ID NO: 7; and SEQ ID NO: 8 Includes light chain CDR-L1, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10. A bispecific antibody according to Embodiment 45, comprising a light chain variable region.

[0427] 47. The first antigen-binding domain is at least approximately 95% different from the amino acid sequence of SEQ ID NO: 40. , 96%, 97%, 98%, 99%, or 100% identical heavy chain variable region sequences, and sequence The amino acid sequence of number 41 and at least approximately 95%, 96%, 97%, 98%, 99% or Dual configurations as described in Embodiment 45 or Embodiment 46, including 100% identical light chain variable region arrangements. Specific antibodies.

[0428] 48. The first antigen-binding domain is the heavy chain variable region sequence of SEQ ID NO: 40 and SEQ ID NO: 4 A bispecific antibody according to Embodiment 47, comprising one light chain variable region sequence.

[0429] 49. The second antigen-binding domain is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 3. 96%, 97%, 98%, 99%, or 100% identical heavy chain variable region sequences, and sequence numbers The amino acid sequence of No. 4 is at least approximately 95%, 96%, 97%, 98%, 99%, or 10%. A dual configuration according to any one of embodiments 45 to 48, including 0% identical light chain variable region sequences. Specific antibodies.

[0430] 50. The second antigen-binding domain is the heavy chain variable region sequence of SEQ ID NO: 3 and SEQ ID NO: 4 A bispecific antibody according to Embodiment 49, comprising a light chain variable region sequence.

[0431] 51. Embodiment 4, in which the first and / or second antigen-binding domain is a Fab molecule. A bispecific antibody listed in any one of the numbers from 5 to 50.

[0432] 52. The first antigen-binding domain is located in the variable or constant region of the Fab light chain and Fab heavy chain. The bispecific antibody according to Embodiment 51 is a cross-Fab molecule in which either of the following is exchanged. body.

[0433] 53. Both the first and second antigen-binding domains of the bispecific antibody are Fab molecules. Furthermore, in one of the antigen-binding domains (especially the first antigen-binding domain), the Fab light chain and The variable domains VL and VH of the Fab heavy chain are substituted for each other. a. In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 It is substituted with a positively charged amino acid (numbered by Kabat), and the first antibody In the constant domain CH1 of the primordial domain, the amino acid at position 147 or 213 The amino acid at the position is substituted with an amino acid that has a negative charge (Kabat EU Numbering by index; or b. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 It is substituted with a positively charged amino acid (numbered by Kabat), and the second antibody In the constant domain CH1 of the primordial domain, the amino acid at position 147 or 213 The amino acid at the position is substituted with an amino acid that has a negative charge (Kabat EU (Numbering by index), The constant domains CL and CH1 of the antigen-binding domain, which have VH / VL exchange, are mutual. A bispecific antibody according to Embodiment 52, which is not replaced by the other antibody.

[0434] 54.a. In the constant domain CL of the first antigen-binding domain, at position 124, Mino acids are independently substituted with lysine (K), arginine (R), or histidine (H). (Kabat numbering) The constant domain CH1 of the first antigen-binding domain In this case, the amino acid at position 147 or position 213 is glutamic acid ( E) or independently substituted with aspartic acid (D) (Kabat EU index Numbering by X); or b. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 It is independently substituted with lysine (K), arginine (R), or histidine (H). (Kabat numbering), in the constant domain CH1 of the second antigen-binding domain The amino acid at position 147 or 213 is glutamic acid (E), Alternatively, it is independently substituted with aspartic acid (D) (Kabat EU Index) (Numbering by), the bispecific antibody described in Embodiment 53.

[0435] 55. In the constant domain CL of the second antigen-binding domain, at position 124, amino The acid is independently substituted with lysine (K), arginine (R), or histidine (H). (Kabat numbering), in the constant domain CH1 of the second antigen-binding domain Therefore, the amino acid at position 147 or position 213 is glutamic acid (E). , or independently substituted with aspartic acid (D) (Kabat EU Index) (Numbering by S), the bispecific antibody described in Embodiment 54.

[0436] 56. In the constant domain CL of the second antigen-binding domain, at position 124, amino The acid is independently substituted with lysine (K), arginine (R), or histidine (H). (Kabat numbering), in the constant domain CH1 of the second antigen-binding domain The amino acid at position 147 is either glutamic acid (E) or aspartic acid (D). Embodiment 5, independently replaced (numbered by Kabat EU index) The bispecific antibody described in 5.

[0437] 57. In the constant domain CL of the second antigen-binding domain, at position 124, amino The acid is independently substituted with lysine (K), arginine (R), or histidine (H). (Kabat numbering), the amino acid at position 123 is lysine (K), arginine They are independently substituted with nin (R) or histidine (H) (numbered by Kabat). (k) In the constant domain CH1 of the second antigen-binding domain, at position 147, amino The acid is independently substituted with glutamic acid (E) or aspartic acid (D) (K (as numbered by the abat EU index), the amino acid at position 213 is glutamic acid. It is independently substituted with nic acid (E) or aspartic acid (D) (Kabat EU (Numbering by index), the bispecific antibody according to Embodiment 55.

[0438] 58. In the constant domain CL of the second antigen-binding domain, at position 124, amino The acid is substituted with lysine (K) (numbered by Kabat), at position 123. The amino acids are substituted with lysine (K) (numbered by Kabat), and the second antigen In the constant domain CH1 of the binding domain, the amino acid at position 147 is glutamine. It is substituted with acid (E) (numbered according to the Kabat EU index), and is ranked 213th. The amino acid in this is substituted with glutamic acid (E) (Kabat EU index) (Numbering by Kus), the bispecific antibody described in Embodiment 57.

[0439] 59. In the constant domain CL of the second antigen-binding domain, at position 124, amino The acid is substituted with lysine (K) (numbered by Kabat), at position 123. The amino acids are substituted with arginine(R) (numbered by Kabat), and the second In the constant domain CH1 of the antigen-binding domain, the amino acid at position 147 is glutathione. It is substituted with minic acid (E) (numbered according to the Kabat EU index), 21 The amino acid at position 3 is substituted with glutamic acid (E) (Kabat EU-in (Numbering by DEX), the bispecific antibody described in Embodiment 57.

[0440] 60. The constant domain CL of the second antigen-binding domain is a kappa isotype constant domain A bispecific antibody according to any one of embodiments 53 to 59, which is in CL.

[0441] 61. The first and second antigen-binding domains, optionally via a peptide linker, A bispecific antibody according to any one of embodiments 45 to 60, which is fused with each other.

[0442] 62. The first and second antigen-binding domains are Fab molecules, respectively, (i) second The antigen-binding domain is at the C-terminus of the Fab heavy chain, and the first antigen-binding domain of the Fab heavy chain (ii) The first antigen-binding domain is fused to the N-terminus, or the C-terminus of the Fab heavy chain. Either it is fused at the end, or it is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain. , the bispecific antibody described in Embodiment 61.

[0443] 63. Bispecific antibodies provide monovalent binding to CD3, as in embodiments 45 to 62. A bispecific antibody as described in any one of the following.

[0444] 64. Embodiment 6, comprising two antigen-binding domains that specifically bind to glycoMUC1. The bispecific antibody described in 3.

[0445] 65. The two antigen-binding domains that specifically bind to glycoMUC1 have the same amino acid configuration. A bispecific antibody according to Embodiment 64, including a column.

[0446] 66. A bispecific antibody has an Fc domain composed of a first and second subunit. Furthermore, the bispecific antibody described in any one of embodiments 45 to 65.

[0447] 67. The dual specificity described in Embodiment 66, wherein the Fc domain is an IgG Fc domain. sexual antibodies.

[0448] 68. The dual feature according to Embodiment 67, wherein the Fc domain is an IgG1Fc domain. Heterogeneous antibodies.

[0449] 69. The dual feature according to Embodiment 67, wherein the Fc domain is an IgG4Fc domain. Heterogeneous antibodies.

[0450] 70. The IgG4Fc domain has an amino acid substitution at position S228 (Kabat E (U index numbering), preferably including amino acid substitution S228P, Embodiment 69 The bispecific antibody described above.

[0451] 71. The bispecific antimicrobial agent according to Embodiment 66, wherein the Fc domain is a human Fc domain. body.

[0452] 72. The Fc domain is the human IgG1Fc domain, and the human IgG1Fc domain The dual specificity described in Embodiment 71 may optionally include Sequence ID 42. sexual antibodies.

[0453] 73. The first, second, and, if present, third antigen-binding domains are, respectively, Fab It is a molecule, and (a)(i) the second antigen-binding domain is at the C-terminus of the Fab heavy chain, the first antigen The first antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the proto-binding domain, and the first antigen-binding domain is Fa Is the C-terminus of the β-heavy chain fused to the N-terminus of the first subunit of the Fc domain, or (ii) The first antigen-binding domain is at the C-terminus of the Fab heavy chain, and the second antigen-binding domain The second antigen-binding domain is fused to the N-terminus of the Fab heavy chain, and the second antigen-binding domain is fused to the C-terminus of the Fab heavy chain. And it is either fused to the N-terminus of the first subunit of the Fc domain;( b) If present, the third antigen-binding domain is at the C-terminus of the Fab heavy chain, and the Fc domain Fusing to the N-terminus of the second subunit, as described in any one of embodiments 66 to 72 The listed bispecific antibody.

[0454] 74. The Fc domain facilitates the meeting of the first and second subunits of the Fc domain. A bispecific antibody according to any one of embodiments 66 to 73, including the modification thereof.

[0455] 75. The Fc domain reduces binding to Fc receptors and / or effector functions. The embodiment according to any one of embodiments 66 to 74, comprising one or more amino acid substitutions. Bispecific antibodies.

[0456] 76.a. A first antigen-binding domain that specifically binds to CD3, and the first antigen-binding domain The combined domain is a crossed Fab molecule, and is a variable or constant domain of the Fab light chain and Fab heavy chain. Either region, preferably a variable region, is replaced with the first antigen-binding domain; b. Second and third antigen-binding domains that specifically bind to glycoMUC1, Heavy chain CDR-H1 (SEQ ID NO: 5), CDR-H2 (SEQ ID NO: 6), and CDR (SEQ ID NO: 7) - Heavy chain variable region containing H3; as well as light chain CDR-L1 of SEQ ID NO: 8, CD of SEQ ID NO: 9 Includes light chain variable regions including R-L2 and CDR-L3 of Sequence ID No. 10, and second and third The antigen-binding domains are the second and third antigen-binding domains, which are Fab molecules, respectively. ; c. Fc domains consisting of first and second subunits capable of stable association. Includes, The second antigen-binding domain is at the C-terminus of the Fab heavy chain, and is the Fab of the first antigen-binding domain. It is fused to the N-terminus of the heavy chain, and the first antigen-binding domain is at the C-terminus of the Fab heavy chain, Fc It is fused to the N-terminus of the first subunit of the domain, and the third antigen-binding domain is F The C-terminus of the ab heavy chain is fused to the N-terminus of the second subunit of the Fc domain. A bispecific antibody as described in Form 45.

[0457] 77. The first antigen-binding domain is the heavy chain CDR-H1 of SEQ ID NO: 34, and SEQ ID NO: 35 Heavy chain variable region including CDR-H2 and CDR-H3 of sequence number 36; and sequence number Light chain CDR-L1 of sequence number 37, CDR-L2 of sequence number 38, and CDR- of sequence number 39 A bispecific antibody according to Embodiment 77, comprising a light chain variable region including L3.

[0458] 78. The first antigen-binding domain is at least approximately 95% different from the amino acid sequence of SEQ ID NO: 40. , 96%, 97%, 98%, 99%, or 100% identical heavy chain variable region sequences, and sequence The amino acid sequence of number 41 and at least approximately 95%, 96%, 97%, 98%, 99% or A bispecific antibody according to Embodiment 77, comprising 100% identical light chain variable region sequences.

[0459] 79. The first antigen-binding domain is the heavy chain variable region sequence of SEQ ID NO: 40 and SEQ ID NO: 4 A bispecific antibody according to Embodiment 78, comprising a light chain variable region sequence 1.

[0460] 80. The second and third antigen-binding domains have at least the same amino acid sequence as SEQ ID NO: 3. Approximately 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain variable region sequences, And the amino acid sequence of SEQ ID NO: 4 is at least approximately 95%, 96%, 97%, 98%, and 99% Or any one of embodiments 76 to 79, which includes a 100% identical light chain variable region arrangement. The described bispecific antibody.

[0461] 81. The second and third antigen-binding domains are the heavy chain variable region and sequence number of SEQ ID NO: 3. A bispecific antibody according to Embodiment 80, comprising the light chain variable region of No. 4.

[0462] 82. Fc domains are described in sections 5.1 and 5.2 with respect to Fc domains. Embodiments 76 to 81 include any of the features described above, either individually or in combination. A bispecific antibody as described in one of the following.

[0463] 83. The antigen-binding domain and the Fc region are fused to each other by the peptide linker. A bispecific antibody according to any one of embodiments 76 to 82.

[0464] 84. The peptide linker is as shown in SEQ ID NO: 45 and / or SEQ ID NO: 46. A bispecific antibody according to Embodiment 83, comprising a peptide linker.

[0465] 85. In the constant domain CL of the second and third Fab molecules, at position 124 The amino acid is substituted with lysine (K) (numbered by Kabat), at position 123. The amino acid is lysine (K) or arginine (R), preferably arginine (R). It has been replaced (numbered by Kabat), and the second and third Fab parts of (ii) In the constant domain CH1 of this child, the amino acid at position 147 is glutamic acid (E). It has been replaced (numbered by the Kabat EU index), and is ranked 213th. Mino acids are substituted with glutamic acid (E) (according to the Kabat EU Index). A bispecific antibody according to any one of embodiments 76 to 84 (numbered).

[0466] 86. Sequence of sequence number 43 and at least 80%, 85%, 90%, 95%, 96%, 9 Polypeptides containing 7%, 98%, or 99% identical sequences, and the sequence of sequence number 44 and a small amount At least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% are identical. Polypeptides containing the sequence, the sequence of sequence number 45 and at least 80%, 85%, 90%, 9 Polypeptides containing 5%, 96%, 97%, 98%, or 99% identical sequences, and The array in column number 46 and at least 80%, 85%, 90%, 95%, 96%, 97%, 98% Any of embodiments 76 to 85, comprising polypeptides containing % or 99% identical sequences. A bispecific antibody as described in one of the following.

[0467] 87. The bispecific antibody contains a polypeptide containing the sequence of SEQ ID NO: 43, and a polypeptide containing the sequence of SEQ ID NO: 44. A polypeptide containing the column, a polypeptide containing the sequence of sequence number 45, and sequence number 46 A bispecific antibody according to Embodiment 86, comprising a polypeptide containing a sequence.

[0468] 88. The two polypeptides according to Embodiment 87, comprisin...

Claims

1. (a) (i) and (ii) below: (i) (1) Complementarity-determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 23, (2) CDR-H2 containing the amino acid sequence of SEQ ID NO: 24, and (3) CDR-H3 containing the amino acid sequence of SEQ ID NO: 25 Heavy chain variable region (VH) including, and (ii) Fc region; Heavy chains containing; and (b) (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10 Light chain containing a variable light chain region (VL) An antiglyco-MUC1 antibody comprising, wherein VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence encoded in SEQ ID NO: 13, and VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence encoded in SEQ ID NO:

14.

2. (a) (i) and (ii) below: (i) (1) Complementarity-determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 28, (2) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, and (3) CDR-H3 containing the amino acid sequence of SEQ ID NO: 25 Heavy chain variable region (VH) including, and (ii) Fc region; Heavy chains containing; and (b) (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 31 Light chain containing a variable light chain region (VL) An antiglyco-MUC1 antibody comprising, wherein VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence encoded in SEQ ID NO: 13, and VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence encoded in SEQ ID NO:

14.

3. (a) (i) and (ii) below: (i) (1) Complementarity-determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 5, (2) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6, and (3) CDR-H3 containing the amino acid sequence of SEQ ID NO: 7 Heavy chain variable region (VH) including, and (ii) Fc region; Heavy chains containing; and (b) (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 10 Light chain containing a variable light chain region (VL) An antiglyco-MUC1 antibody comprising, wherein VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence encoded in SEQ ID NO: 13, and VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence encoded in SEQ ID NO:

14.

4. An anti-glyco-MUC1 antibody according to any one of claims 1 to 3, which is a chimeric antibody or a humanized antibody.

5. An antiglyco-MUC1 antibody according to any one of claims 1 to 4, comprising VH and VL.

6. Including VH and VL, (a) The VH includes an amino acid sequence having at least 97% sequence identity with the amino acid sequence encoded in SEQ ID NO: 13, and the VL includes an amino acid sequence having at least 97% sequence identity with the amino acid sequence encoded in SEQ ID NO: 14, (b) The VH includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence encoded in SEQ ID NO: 13, and the VL includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence encoded in SEQ ID NO: 14, (c) The VH comprises an amino acid sequence having 100% sequence identity with the amino acid sequence encoded in SEQ ID NO: 13, and the VL comprises an amino acid sequence having 100% sequence identity with the amino acid sequence encoded in SEQ ID NO:

14. The anti-glyco-MUC1 antibody according to any one of claims 1 to 5.

7. (a) Polyvalent, or (b) The form of a multispecific antibody, The anti-glyco-MUC1 antibody according to any one of claims 1 to 6.

8. The anti-glyco-MUC1 antibody according to claim 7, which is a bispecific antibody that binds to a glyco-MUC1 epitope (hereinafter referred to as the "first epitope") and a second epitope different from the first epitope.

9. The anti-glyco-MUC1 antibody according to claim 8, wherein the bispecific antibody is CrossMab.

10. The aforementioned CrossMab ＦＡＢ CrossMab ＶＨ－ＶＬ Or CrossMab ＣＨ１－ＣＬ The anti-glyco-MUC1 antibody according to claim 9.

11. The anti-glyco-MUC1 antibody according to claim 8, wherein the second epitope is a MUC1 epitope that is overexpressed on cancer cells compared to normal cells.

12. The anti-glyco-MUC1 antibody according to claim 8, wherein the second epitope is a T cell epitope.

13. The anti-glyco-MUC1 antibody according to claim 12, wherein the T cell epitope is a human CD3 epitope.

14. The anti-glyco-MUC1 antibody according to claim 13, wherein the human CD3 epitope comprises a CD3 gamma epitope, a CD3 delta epitope, a CD3 epsilon epitope, or a CD3 zeta epitope.

15. A fusion protein comprising an anti-glyco-MUC1 antibody according to any one of claims 1 to 14, operably linked to at least a second amino acid sequence.

16. An antibody-drug conjugate comprising an anti-glyco-MUC1 antibody according to any one of claims 1 to 14, conjugated to a cytotoxic substance.

17. A nucleic acid comprising the coding region of an anti-glyco-MUC1 antibody according to any one of claims 1 to 14.

18. A vector comprising the nucleic acid described in claim 17.

19. A host cell manipulated to express a nucleic acid containing the coding region of an anti-glyco-MUC1 antibody according to any one of claims 1 to 14.

20. A pharmaceutical composition for treating cancer, comprising an anti-glyco-MUC1 antibody according to any one of claims 1 to 14, a fusion protein according to claim 15, an antibody-drug conjugate according to claim 16, a nucleic acid according to claim 17, a vector according to claim 18, or a host cell according to claim 19.

21. A pharmaceutical composition for use in the manufacture of a pharmacopoeia for treating cancer, comprising an anti-glyco-MUC1 antibody according to any one of claims 1 to 14, a fusion protein according to claim 15, an antibody-drug conjugate according to claim 16, a nucleic acid according to claim 17, a vector according to claim 18, or a host cell according to claim 19.

22. The pharmaceutical composition according to claim 20 or 21, wherein the cancer is breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, or colorectal cancer.

23. A method for detecting cancer in a biological sample, (a) Contacting a biological sample with the anti-glycoMUC1 antibody described in any one of claims 1 to 14, and (b) Detection of the binding of the anti-glycoMUC1 antibody. A method that includes this.

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