Antibody that specifically binds to MUC-1 and use thereof

Abstract

Anti-MUC1 antibodies, anti-MUC1 / anti-CD3 bispecific antibodies, and pharmaceutical compositions comprising the same for preventing and / or treating cancer are provided.

Description

[Technical field]

[0001] Provided are anti-MUC1 antibodies, anti-MUC1 / anti-CD3 bispecific antibodies, and pharmaceutical compositions containing the same for preventing and / or treating cancer. [Background technology]

[0002] Mucin-1 (MUC1) is a type of mucin glycoprotein, a transmembrane glycoprotein that contains numerous glycosylated extracellular domains. The length of MUC1 is 200-500 nm at the cell surface, and MUC1 is located on the apical membrane of normal epithelial cells. MUC1 is expressed in glandular or luminal epithelial cells of many organs, such as the breast, stomach, esophagus, pancreas, urethra, lung, kidney, and gallbladder. Mucins are overexpressed in many carcinomas, including those of the pancreas, lung, breast, and ovary. Much research has been conducted on the pathological association of mucins with cancer, and in particular, several antibodies against MUC1 are known in the art.

[0003] However, their therapeutic efficacy can still be improved, and thus the present application provides anti-MUC1 antibodies or antigen-binding fragments thereof with improved characteristics. Summary of the Invention [Problem to be solved by the invention]

[0004] One embodiment provides an anti-MUC1 antibody or antigen-binding fragment thereof.

[0005] Another embodiment provides an anti-MUC1 / anti-CD3 bispecific antibody comprising an anti-MUC1 antibody, or antigen-binding fragment thereof, and an anti-CD3 antibody, or antigen-binding fragment thereof.

[0006] Yet another embodiment provides (i) an anti-MUC1 antibody or an antigen-binding fragment thereof, or (ii) a pharmaceutical composition for preventing or treating cancer, comprising the anti-MUC1 / anti-CD3 bispecific antibody as an active ingredient.

[0007] Yet another embodiment provides a method for preventing or treating cancer, comprising administering (i) an anti-MUC1 antibody, or antigen-binding fragment thereof, or (ii) said anti-MUC1 / anti-CD3 bispecific antibody to a subject in need thereof.

[0008] Yet another aspect provides the use of (i) an anti-MUC1 antibody, or antigen-binding fragment thereof, or (ii) said anti-MUC1 / anti-CD3 bispecific antibody for use in the prevention or treatment of cancer, or for use in the manufacture of an anti-cancer medicament.

[0009] Yet other aspects provide a polynucleotide encoding (i) an anti-MUC1 antibody or antigen-binding fragment thereof, or (ii) an anti-MUC1 / anti-CD3 bispecific antibody, a recombinant vector comprising said polynucleotide, and / or a recombinant cell comprising said recombinant vector.

[0010] Yet another embodiment provides a method for producing (i) an anti-MUC1 antibody, or antigen-binding fragment thereof, or (ii) an anti-MUC1 / anti-CD3 bispecific antibody, comprising expressing a polynucleotide encoding said (i) anti-MUC1 antibody, or antigen-binding fragment thereof, or (ii) said anti-MUC1 / anti-CD3 bispecific antibody. [Means for solving the problem]

[0011] Definition of Terms Animal-derived antibodies, which are produced by immunizing a target animal with a desired antigen, may generally cause immune rejection when administered to humans for therapeutic purposes. To prevent such immune rejection, chimeric antibodies have been developed. Chimeric antibodies are made by replacing the constant region of an animal-derived antibody, which causes anti-isotype reactions, with the constant region of a human antibody using genetic engineering methods. Although chimeric antibodies have improved the anti-isotype reaction to a large extent compared to animal-derived antibodies, they still contain animal-derived amino acids in the variable region, and thus have potential side effects against anti-idiotypic reactions. Humanized antibodies have been developed to improve such side effects. They are made by grafting the CDR (complementarity determining regions) site, which plays an important role in antigen binding, from the variable region of a chimeric antibody onto a human antibody framework.

[0012] In one embodiment, the anti-MUC1 antibody may be an animal-derived antibody (e.g., a mouse-derived antibody), a chimeric antibody (e.g., a mouse-human chimeric antibody), or a humanized antibody. The antibody or antigen-binding fragment may be isolated from a living organism or non-naturally occurring. The antibody or antigen-binding fragment may be recombinantly or synthetically produced. The antibody or antigen-binding fragment may be isolated from a living organism or non-naturally occurring. The antibody or antigen-binding fragment may be synthetically or recombinantly produced.

[0013] In other examples, the antibody may be derived (isolated) from any animal, including mammals, including humans, birds, etc. For example, the antibody may be a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken antibody. Here, a human antibody includes an antibody having a human immunoglobulin amino acid sequence and isolated from a human immunoglobulin library or an antibody isolated from an animal that has been transduced to one or more human immunoglobulins and does not express endogenous immunoglobulins.

[0014] The anti-MUC1 antibody may be a monoclonal antibody or a polyclonal antibody, for example, a monoclonal antibody. The monoclonal antibody may be produced according to methods well known in the art. For example, it may be produced using the phage display technique. Alternatively, the anti-MUC1 antibody may be a mouse-derived monoclonal antibody.

[0015] The heavy chain CDR and light chain CDR regions, or the remaining regions excluding the heavy chain variable region and the light chain variable region, as defined above in the anti-MUC1 antibody or antigen-binding fragment thereof may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.), e.g., from framework regions, and / or light chain constant regions and / or heavy chain constant regions of any of the above subtypes of immunoglobulin.

[0016] A complete antibody (e.g., IgG type) has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0017] The term "heavy chain" refers to a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three constant region domains C H1 , C H2 and C H3 The term "light chain" is intended to include a full-length heavy chain, including the heavy chain and hinge, and any fragment thereof. The term "light chain" is intended to include a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and constant region domain C L The term "light chain" is intended to mean any full-length light chain and any fragment thereof.

[0018] The term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin. Each of the heavy and light chains can contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide the main contact residues when an antibody binds to an antigen or epitope. Meanwhile, the terms "specifically bind" and "specifically recognize" as used herein have the same meaning as commonly known to those skilled in the art, and refer to the specific interaction between an antigen and an antibody to cause an immunological reaction.

[0019] The term "hinge region" refers to a region contained in an antibody heavy chain, which is located between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site in the antibody.

[0020] When an animal-derived antibody undergoes chimerization, the animal-derived IgG1 hinge is replaced with a human IgG1 hinge, but the animal-derived IgG1 hinge is shorter than the human IgG1 hinge, and the disulfide bonds between the two heavy chains are reduced from three to two, resulting in different hinge rigidity effects. Therefore, modification of the hinge region can increase the antigen-binding efficiency of the humanized antibody. Methods for deleting, adding, or substituting amino acids to modify the amino acid sequence of the hinge region are well known to those skilled in the art.

[0021] The "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure, and is a part of a polypeptide that contains a portion capable of binding to an antigen. For example, it may be, but is not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', or F(ab')2. The antigen-binding fragment of an antibody of the present invention may be an antibody fragment containing one or more of the above-mentioned complementarity determining regions, for example, one selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. The antigen-binding fragment can be obtained using a protease (for example, Fab can be obtained by limited cleavage of the entire antibody with papain, and F(ab')2 fragment can be obtained by cleavage with pepsin), or can be produced by genetic recombination technology.

[0022] Among the antigen-binding fragments, Fab is composed of the variable regions of the light chain and the heavy chain, the constant region of the light chain, and the first constant region of the heavy chain (C H1 ) and has one antigen-binding site.

[0023] Fab' is the heavy chain CH1 The F(ab')2 antibody is different from the Fab antibody in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain. The F(ab')2 antibody is generated by disulfide bonds formed by the cysteine ​​residues in the hinge region of Fab'.

[0024] Fv is the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv (single-chain Fv; scFv), the heavy chain variable region and the single chain variable region are generally linked by a covalent bond via a peptide linker or directly linked at the C-terminus, and can form a dimer-like structure like a two-chain Fv. The peptide linker is as described above and may be, for example, 1 to 100 amino acids long, for example, 2 to 50 amino acids long, or 5 to 25 amino acids long, and the type of amino acid contained therein is not limited.

[0025] The antigen-binding fragment can be obtained using proteolytic enzymes (for example, whole antibodies can be digested with papain to obtain Fab fragments, or with pepsin to obtain F(ab')2 fragments) or can be produced by recombinant gene techniques.

[0026] In an antigen-binding fragment, for example, scFV, the heavy chain variable region and the light chain variable region can be linked with or without a linker, for example, a peptide linker. The peptide linker may be a polypeptide consisting of any amino acid of 1 to 100 or 2 to 50, and the type of amino acid contained therein is not limited. The peptide linker may contain, for example, Gly, Asn and / or Ser residues, and also contains neutral amino acids such as Thr and / or Ala. The length of the linker can be determined in various ways within the limits that do not affect the function of the bispecific antibody. For example, the peptide linker may contain a total of 1 to 100, 2 to 50, or 5 to 25 of one or more amino acids selected from the group consisting of Gly, Asn, Ser, Thr and Ala. In one example, the peptide linker may be represented by (G4S)n (n is the number of (G4S) repeats), which is an integer of 1 to 10, for example, an integer of 2 to 5).

[0027] The term "antagonist" is intended to include any molecule that partially but completely blocks, inhibits, or neutralizes one or more biological activities of a target (e.g., MUC1). For example, an "antagonist" antibody refers to an antibody that inhibits or reduces the biological activity of an antigen (e.g., MUC1) to which the antibody binds. An antagonist can bind to a receptor for a ligand (target) and reduce receptor phosphorylation, or incapacitate or kill cells that have been activated by the ligand. An antagonist can also completely disrupt the receptor-ligand interaction, competitively bind to the receptor with the ligand, or substantially reduce the receptor-ligand interaction by changing the tertiary structure of the receptor or downregulating it.

[0028] Anti-MUC1 antibody or antigen-binding fragment thereof One example of the present invention provides a polypeptide capable of specifically recognizing and / or specifically binding to MUC1 (Mucin-1).

[0029] The MUC1 may be human MUC1, monkey, dog, rat, mouse, or the like MUC1. In one example, the human MUC1 is selected from the group consisting of MUC1 isoform M2 (identifier: A6ZID5; NCBI reference number: ABS01294.1), MUC1 isoform 1 (identifier: P15941-1), MUC1 isoform 2 (identifier: P15941-2), MUC1 isoform 3 (identifier: P15941-3), MUC1 isoform 4 (identifier: P15941-4), MUC1 isoform 5 (identifier: P15941-5), MUC1 isoform 6 (identifier: P15941-6), MUC1 isoform Y (identifier: P15941-7), MUC1 isoform 8 (identifier: P15941-8), MUC1 isoform 9 (identifier: P15941-9), MUC1 isoform The polypeptide may be, but is not limited to, MUC1 isoform F (identifier: P15941-10), MUC1 isoform Y-LSP (identifier: P15941-11), MUC1 isoform S2 (identifier: P15941-12), MUC1 isoform M6 (identifier: P15941-13), MUC1 isoform ZD (identifier: P15941-14), MUC1 isoform T10 (identifier: P15941-15), MUC1 isoform E2 (identifier: P15941-16), or MUC1 isoform J13 (identifier: P15941-17).

[0030] In one example, the human MUC1 is selected from the group consisting of NCBI Reference Sequences: NP_001018016.1, NP_001018017.1, NP_001037855.1, NP_001037856.1, NP_001037857.1, NP_001037858.1, NP_001191214.1, NP_001191215.1, NP_001191216.1, NP_001191217.1, NP_001191218.1, NP_0011 91219.1, NP_001191220.1, NP_001191221.1, NP_001191222.1, NP_001191223.1, NP_001191224.1, NP_001191225.1, NP_001191226.1, NP_002447.4, etc., but are not limited thereto, and each amino acid sequence can be found in NCBI Reference Sequence:NM_001018016.2, NM_001018017.2, NM_001044390.2, NM_001044391.2, NM_001044392.2, NM_001 044393.2, NM_001204285.1, NM_001204286.1, NM_001204287.1, NM_001204288.1, NM_001204289.1, NM_0012 NM_001204290.1, NM_001204291.1, NM_001204292.1, NM_001204293.1, NM_001204294.1, NM_001204295.1, NM_001204296.1, NM_001204297.1, NM_002456.5, etc.

[0031] As used herein, a protein, polypeptide, or polynucleotide having an amino acid sequence or a nucleotide sequence can all be used to mean that the protein, polypeptide, or polynucleotide comprises, essentially comprises, or consists of said sequence.

[0032] These polypeptides can be used as the complementarity determining region (CDR) of an anti-MUC1 antibody. In this case, the complementarity determining region can be determined by any conventional method, for example, but not limited to, the IMGT definition (http: / / www.imgt.org / IMGT_vquest / share / textes / ) or the Kabat definition (http: / / www.bioinf.org.uk / abs / ).

[0033] In another embodiment, a MUC1 target polypeptide molecule is provided that comprises one or more members selected from the group consisting of the above polypeptides.

[0034] The MUC1-targeting polypeptide molecule may comprise a heavy chain complementarity determining region, a light chain complementarity determining region, or a combination thereof, of an anti-MUC1 antibody described above; or a heavy chain variable region containing the heavy chain complementarity determining region, a light chain variable region containing the light chain complementarity determining region, or a combination thereof.

[0035] The MUC1-targeting polypeptide molecule can serve as, but is not limited to, an anti-MUC1 antibody, an antigen-binding fragment of the antibody, or an anti-MUC1 antibody analog (a structure having a similar backbone and function to an antibody; e.g., a peptibody, a nanobody, etc.), or a precursor or component part (e.g., a CDR) of a multimeric antibody, etc.

[0036] The term "peptibody" refers to a fusion protein in which a peptide is fused to all or part of an antibody constant region such as the Fc region, and has a structure and function similar to that of an antibody, in which one or more of the peptides described above can act as antigen-binding sites (heavy and / or light chain CDRs or variable regions).

[0037] The term "nanobody", also known as single-domain antibody, refers to an antibody fragment that contains a single variable domain of an antibody in a monomeric form and has the property of selectively binding to a specific antigen similar to an intact antibody. The molecular weight of nanobodies is generally about 12 kDa to about 15 kDa, which is very small compared to the typical molecular weight (about 150 kDa to about 160 kDa) of an intact antibody (containing two heavy chains and two light chains), and in some cases is smaller than a Fab fragment or scFv fragment.

[0038] Specifically, the present invention provides a polypeptide capable of specifically recognizing and / or binding to MUC1. The polypeptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:27, and SEQ ID NO:33, and more specifically, may have one or more amino acid sequences selected from the group consisting of SEQ ID NO:2 to 6, SEQ ID NO:8 to 15, SEQ ID NO:17 to 18, SEQ ID NO:20 to 26, SEQ ID NO:28 to 32, and SEQ ID NO:34 and SEQ ID NO:38.

[0039] The polypeptides and their applicable complementarity determining regions are summarized in Table 1 below. H -CDR1, V H -CDR2, and V H - CDR3 means heavy chain complementarity determining region, V L -CDR1, V L -CDR2, and V L - CDR3 means light chain complementarity determining region. [Table 1-1]

[0040] [Table 1-2]

[0041] In one example, an anti-MUC1 antibody or antigen-binding fragment thereof is provided that comprises one or more selected from the group consisting of the above polypeptides as a complementarity determining site.

[0042] The anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region, A polypeptide having the amino acid sequence of SEQ ID NO:1 (VH-CDR1), A polypeptide having the amino acid sequence of SEQ ID NO: 7 (VH-CDR2), and Polypeptide having the amino acid sequence of SEQ ID NO: 16 (VH-CDR3) The composition may include one or more selected from the group consisting of:

[0043] The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of the following general formula 1: [General Formula 1] (SEQ ID NO: 1) X1 -YWM- X2 X1 is D or S, X2 is N, S, or H.

[0044] In one example, the polypeptide of SEQ ID NO: 1 that can be used as the heavy chain CDR1 of the anti-MUC1 antibody or antigen-binding fragment thereof may have, for example, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 6, and the detailed sequences are set forth in Table 1 and the sequence listing, and in the amino acid sequences set forth, bold underlined portions represent modified amino acids.

[0045] The amino acid sequence of SEQ ID NO: 7 is the amino acid sequence of the following general formula 2: [General formula 2] (SEQ ID NO: 7) X3 -I- X4 - X5 -K- X6 -Y- X7 - X8 - X9 - X10 - X11 -Y- X12 -X13 -SVKG X3 is Q, F, or V, X4 is R or K, X5 is N, S, Q, or Y, X6 is P or A, X7 is N or G, X8 is Y, G, or S, X9 is E or T, X10 is T or K, X11 is Y or E, X12 is S, A, or V, X13 is N, Q, or A.

[0046] In one example, the polypeptide of SEQ ID NO: 7 that can be used as the heavy chain CDR2 of the anti-MUC1 antibody or antigen-binding fragment thereof may have, for example, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 8 to 15, and the detailed sequences are set forth in Table 1 and the sequence listing, and in the amino acid sequences set forth, bold underlined portions represent modified amino acids.

[0047] The amino acid sequence of SEQ ID NO: 16 is an amino acid sequence of the following general formula 3: [General Formula 3] (SEQ ID NO: 16) AMITSDGYA- X14 -DY X14 is M or F.

[0048] In one example, the polypeptide of SEQ ID NO: 16 that can be used as the heavy chain CDR3 of the anti-MUC1 antibody or antigen-binding fragment thereof may have, for example, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17-18, and the detailed sequences are set forth in Table 1 and the sequence listing, and in the amino acid sequences set forth, the bold underlined portions represent modified amino acids.

[0049] The anti-MUC1 antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region, A polypeptide having the amino acid sequence of SEQ ID NO: 19 (VL-CDR1); A polypeptide having the amino acid sequence of SEQ ID NO: 27 (VL-CDR2), and Polypeptide having the amino acid sequence of SEQ ID NO: 33 (VL-CDR3) The composition may include one or more selected from the group consisting of:

[0050] The amino acid sequence of SEQ ID NO: 19 is an amino acid sequence of the following general formula 4: [General Formula 4] (SEQ ID NO: 19) X15 - X16 -SQS- X17 -L- X18 -SS- X19 - X20 - X21 -NYLA X15 is K or R, X16 is S or A, X17 is L or V, X18 is L, Y, or H, X19 is N or S, X20 is Q or G, X21 is K or Y.

[0051] In one example, the polypeptide of SEQ ID NO: 19 that can be used as the light chain CDR1 of the anti-MUC1 antibody or antigen-binding fragment thereof may have, for example, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 20 to 26, and the detailed sequences are set forth in Table 1 and the sequence listing, and in the amino acid sequences set forth, bold underlined portions represent modified amino acids.

[0052] The amino acid sequence of SEQ ID NO: 27 is the amino acid sequence of the following general formula 5: [General Formula 5] (SEQ ID NO: 27) W- X22 -S- X23 -R- X24 - X25 X22 is A or G, X23 is T or S, X24 is E or A, X25 is S or T.

[0053] In one example, the polypeptide of SEQ ID NO: 27 that can be used as the light chain CDR2 of the anti-MUC1 antibody or antigen-binding fragment thereof may have, for example, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 28 to 32, and the detailed sequences are set forth in Table 1 and the sequence listing, and in the amino acid sequences set forth, bold underlined portions represent modified amino acids.

[0054] The amino acid sequence of SEQ ID NO: 33 is an amino acid sequence of the following general formula 6: [General formula 6] (SEQ ID NO: 33) QQ- X26 -F- X27 - X28 -P- X29 -T X26 is C or S, X27 is S or Q, X28 is Y, T, or S, X29 is L or Y.

[0055] In one example, the polypeptide of SEQ ID NO: 27 that can be used as the light chain CDR3 of the anti-MUC1 antibody or antigen-binding fragment thereof may have, for example, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 34 to 38, and the detailed sequences are set forth in Table 1 and the sequence listing, and in the amino acid sequences set forth, bold underlined portions represent modified amino acids.

[0056] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is

[0057] a heavy chain variable region comprising one or more heavy chain complementarity determining sites selected from the group consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 1 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 7 (VH-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO: 16 (VH-CDR3), or said one or more heavy chain complementarity determining sites;

[0058] a light chain variable region comprising one or more light chain complementarity determining sites selected from the group consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 19 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 27 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO: 33 (VL-CDR3), or said one or more light chain complementarity determining sites; a combination of said heavy chain complementarity determining regions and said light chain complementarity determining regions; or It may comprise a combination of the heavy and light chain variable regions.

[0059] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is a heavy chain variable region comprising one or more heavy chain complementarity determining sites selected from the group consisting of a polypeptide (VH-CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6, a polypeptide (VH-CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 16, and a polypeptide (VH-CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 18, or the one or more heavy chain complementarity determining sites; a light chain variable region comprising one or more light chain complementarity determining sites selected from the group consisting of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 26 (VL-CDR1), a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28 to 32 (VL-CDR2), and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 38 (VL-CDR3), or the one or more light chain complementarity determining sites; a combination of said heavy chain complementarity determining regions and said light chain complementarity determining regions; or It may comprise a combination of the heavy and light chain variable regions.

[0060] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is A heavy chain variable region comprising a polypeptide (VH-CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6, a polypeptide (VH-CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 16, and a polypeptide (VH-CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 18; and It may comprise a light chain variable region comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 26 (VL-CDR1), a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28 to 32 (VL-CDR2), and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 38 (VL-CDR3).

[0061] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof may comprise the heavy chain variable region CDR sequences and light chain variable region CDR sequences combined in Tables 2 and 3 below. [Table 2]

[0062] [Table 3]

[0063] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is a heavy chain complementarity determining region comprising a polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:14 (VH-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:17 (VH-CDR3), or a heavy chain variable region comprising said heavy chain complementarity determining regions; a light chain complementarity determining region comprising a polypeptide having the amino acid sequence of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:31 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36 (VL-CDR3), or a light chain variable region comprising said light chain complementarity determining regions; a combination of said heavy chain complementarity determining regions and said light chain complementarity determining regions; or It may comprise a combination of the heavy and light chain variable regions.

[0064] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is a heavy chain complementarity determining region comprising a polypeptide having the amino acid sequence of SEQ ID NO:2 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9 (VH-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:17 (VH-CDR3), or a heavy chain variable region comprising said heavy chain complementarity determining regions; a light chain complementarity determining region comprising a polypeptide having the amino acid sequence of SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:24 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:35 (VL-CDR3), or a light chain variable region comprising said light chain complementarity determining regions; a combination of said heavy chain complementarity determining regions and said light chain complementarity determining regions; or It may comprise a combination of the heavy and light chain variable regions.

[0065] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is 1) a polypeptide having the amino acid sequence of SEQ ID NO:2 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:9 (VH-CDR2), a polypeptide having the amino acid sequence of SEQ ID NO:17 (VH-CDR3), a polypeptide having the amino acid sequence of SEQ ID NO:21 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:28 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:35 (VL-CDR3); 2) a polypeptide having the amino acid sequence of SEQ ID NO:2 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:9 (VH-CDR2), a polypeptide having the amino acid sequence of SEQ ID NO:17 (VH-CDR3), a polypeptide having the amino acid sequence of SEQ ID NO:23 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:28 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:36 (VL-CDR3); 3) a polypeptide having the amino acid sequence of SEQ ID NO:2 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:9 (VH-CDR2), a polypeptide having the amino acid sequence of SEQ ID NO:17 (VH-CDR3), a polypeptide having the amino acid sequence of SEQ ID NO:24 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:29 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:35 (VL-CDR3); 4) a polypeptide having the amino acid sequence of SEQ ID NO:2 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:8 (VH-CDR2), a polypeptide having the amino acid sequence of SEQ ID NO:17 (VH-CDR3), a polypeptide having the amino acid sequence of SEQ ID NO:23 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:28 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:34 (VL-CDR3); or 5) a polypeptide having the amino acid sequence of SEQ ID NO:2 (VH-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:8 (VH-CDR2), a polypeptide having the amino acid sequence of SEQ ID NO:17 (VH-CDR3), a polypeptide having the amino acid sequence of SEQ ID NO:24 (VL-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO:29 (VL-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO:35 (VL-CDR3); It may include.

[0066] The anti-MUC1 antibody or antigen-binding fragment thereof can comprise one or more framework sequences of a heavy chain variable region, one or more framework sequences of a light chain variable region, or one or more framework sequences of a heavy chain variable region and one or more framework sequences of a light chain variable region, in addition to one or more types of complementarity determining sites selected from the group consisting of complementarity determining sites of general formulas 1 to 6. Examples of the framework sequences of the light chain variable regions and the framework sequences of the light chain variable regions are shown in Tables 4 to 7 below. [Table 4]

[0067] [Table 5]

[0068] [Table 6]

[0069] [Table 7]

[0070] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is The heavy chain variable region may be a heavy chain variable region containing the heavy chain complementarity determining sites (VH0 to VH9 in Table 2), a light chain variable region containing the light chain complementarity determining sites (VL0 to VL9 in Table 3), or a combination thereof. For example, the heavy chain variable region may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 86, and the light chain variable region may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 87 to 96.

[0071] Thus, the anti-MUC1 antibody or antigen-binding fragment thereof may: A heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 77 to 86; A light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 87 to 96; or It may include a combination of these.

[0072] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is a heavy chain variable region having the amino acid sequence of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, or SEQ ID NO:85; and It may comprise a light chain variable region having the amino acid sequence of SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:95.

[0073] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is a heavy chain variable region having the amino acid sequence of SEQ ID NO:78, SEQ ID NO:81, or SEQ ID NO:84; and It may comprise a light chain variable region having the amino acid sequence of SEQ ID NO:89, SEQ ID NO:91, or SEQ ID NO:92.

[0074] In one example, the anti-MUC1 antibody or antigen-binding fragment thereof is 1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 78 and a light chain variable region having the amino acid sequence of SEQ ID NO: 89; 2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 78 and a light chain variable region having the amino acid sequence of SEQ ID NO: 91; 3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 78 and a light chain variable region having the amino acid sequence of SEQ ID NO: 92; 4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 81 and a light chain variable region having the amino acid sequence of SEQ ID NO: 91; 5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 81 and a light chain variable region having the amino acid sequence of SEQ ID NO: 92; or 6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 84 and a light chain variable region having the amino acid sequence of SEQ ID NO: 91; It may include.

[0075] The term "epitope" is understood to mean an antigenic determinant, that part of an antigen that is recognized by an antibody. According to one embodiment, said epitope may be a site within the MUC1 protein, for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 109.

[0076] Thus, an anti-MUC1 antibody or antigen-binding fragment thereof may specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO:109.

[0077] multiple binding antibodies One example of the present invention relates to a multibinding or multispecific antibody comprising a polypeptide that specifically recognizes and / or can specifically bind to MUC1, and more particularly may be a bispecific antibody comprising a polypeptide that specifically recognizes and / or can specifically bind to MUC1 as well as a polypeptide that specifically recognizes and / or can specifically bind to a second target.

[0078] As used herein, the term "multimeric antibody" refers to an antibody that recognizes and / or binds to two or more different antigens or recognizes and / or binds to different sites of the same antigen, and one antigen-binding site of the multimeric antibody may comprise a polypeptide as described above.

[0079] Another example of the invention relates to a bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds MUC1 and a second antigen-binding domain that specifically binds CD3. In particular, one example of the invention relates to a bispecific antibody comprising a MUC1 antibody or antigen-binding fragment thereof according to the invention and one or more antigen-binding sites specific for a T cell activation antigen.

[0080] In the bispecific antibody, the T cell activation antigen can be a CD3 T cell coreceptor (CD3; cluster of differentiation 3) antigen (e.g., an anti-CD3 epsilon (ε) antigen), and the bispecific antibody can comprise an anti-CD3 antibody or an antigen-binding fragment thereof that includes an antigen-binding site for the CD3 antigen.

[0081] Specifically, the present invention relates to a bispecific antibody comprising an anti-MUC1 antibody or antigen-binding fragment thereof and an anti-CD3 antibody or antigen-binding fragment thereof (i.e., an anti-MUC1 / anti-CD3 bispecific antibody), which is capable of exerting an enhancing effect while maintaining the affinity and activity for the respective antigens, MUC1 and CD3.

[0082] The bispecific antibody specifically binds to the first antigen, MUC1, and can comprise an anti-MUC1 antibody or antigen-binding fragment thereof, which can be the same as those described above.

[0083] The bispecific antibody may also specifically bind to the second antigen, CD3, and may specifically comprise an anti-CD3 antibody or antigen-binding fragment thereof. The anti-CD3 antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof that binds to human, monkey, mouse, etc. CD3. The anti-CD3 antibody or antigen-binding fragment thereof is useful for targeting T cells expressing CD3 or stimulating T cell activation, for example, in circumstances where T cell-mediated cell killing is advantageous or preferred. The anti-CD3 antibody or antigen-binding fragment thereof of the present invention is included as a portion of a bispecific antibody that directs CD3-mediated T cell activation against a particular cell type, for example, a tumor cell or an infectious agent.

[0084] The anti-CD3 antibody or antigen-binding fragment thereof can be any antibody that specifically binds to CD3, including, but not limited to, OKT3 antibody or antigen-binding fragment thereof, UCHT-1 antibody or antigen-binding fragment thereof, SP34 antibody or antigen-binding fragment thereof, etc. The OKT3 antibody may be a mouse or humanized monoclonal antibody of immunoglobulin IgG2a isotype. The UCHT-1 antibody is known to react with the ~20 kDa CD3ε subunit of the human T cell receptor (TCR) / CD3 complex, and the SP34 antibody is known to bind to the CD3ε subunit. Methods for obtaining the OKT3 antibody, UCHT-1 antibody, or SP34 antibody are generally well known in the art.

[0085] In one embodiment, the anti-CD3 antibody or antigen-binding fragment thereof can comprise one or more CDR sequences, preferably six CDR sequences, selected from the group consisting of the following polypeptides (see Table 8 below): A polypeptide having the amino acid sequence of SEQ ID NO: 97 (VH-CDR1); A polypeptide having the amino acid sequence of SEQ ID NO: 98 (VH-CDR2); A polypeptide having the amino acid sequence of SEQ ID NO: 99 (VH-CDR3); A polypeptide having the amino acid sequence of SEQ ID NO: 100 (VL-CDR1); A polypeptide having the amino acid sequence of SEQ ID NO: 101 (VL-CDR2), and A polypeptide having the amino acid sequence of SEQ ID NO: 102 (VL-CDR3).

[0086] More particularly, the anti-CD3 antibody or antigen-binding fragment thereof comprises: It may comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO:103, and a light chain variable region having the amino acid sequence of SEQ ID NO:104 or SEQ ID NO:105. [Table 8]

[0087] The antigen-binding fragment of the anti-CD3 antibody may be selected from the group consisting of scFv, (scFv)2, Fab, Fab' and F(ab')2 of the anti-CD3 antibody.

[0088] An anti-MUC1 / anti-CD3 bispecific antibody according to the invention may be one in which the anti-CD3 antibody, or antigen-binding fragment thereof, is linked to the C-terminus or N-terminus of the anti-MUC1 antibody, or antigen-binding fragment thereof.

[0089] For example, the scFv may be an anti-CD3 antibody scFv bound to the N-terminus or C-terminus of the heavy chain of an anti-MUC1 antibody or an antigen-binding fragment thereof (anti-MUC1 heavy chain N-term-anti-CD3 scFv or anti-MUC1 heavy chain C-term-anti-CD3 scFv), or an anti-CD3 antibody scFv bound to the N-terminus or C-terminus of the light chain of anti-MUC1 (anti-MUC1 light chain N-term-anti-CD3 scFv or anti-MUC1 light chain C-term-anti-CD3 scFv). In addition, the scFv may be a bispecific antibody in which the anti-CD3 scFv is monovalently or divalently bound, so that there is one antigen-binding site of the anti-MUC1 antibody and one antigen-binding site of the anti-CD3 antibody (1+1), two and one, so that there are three antigen-binding sites in one bispecific antibody (1+2 or 2+1), or there are four antigen-binding sites, two by two (2+2), but is not limited thereto.

[0090] In one example, the anti-MUC1 / anti-CD3 bispecific antibody may comprise an anti-MUC1 antibody or antigen-binding fragment thereof, and an anti-CD3 antibody or antigen-binding fragment thereof linked to the C-terminus or N-terminus of the heavy or light chain of the anti-MUC1 antibody or antigen-binding fragment thereof, e.g., the N-terminus of the light chain. In one example, the anti-MUC1 / anti-CD3 bispecific antibody may comprise an intact antibody (e.g., an IgG type antibody) against MUC1, and an antigen-binding fragment of the anti-CD3 antibody (e.g., scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2) linked to the N-terminus of the light chain of the antibody.

[0091] In the anti-MUC1 / anti-CD3 bispecific antibody, the anti-MUC1 antibody or its antigen-binding fragment and the anti-CD3 antibody or its antigen-binding fragment can be linked with or without a linker, for example, a peptide linker. In addition, the heavy chain portion and the light chain portion in the antigen-binding fragment, for example, the heavy chain variable region and the light chain variable region in the scFv fragment, can also be linked with or without a peptide linker. The peptide linker linking the anti-MUC1 antibody or its antigen-binding fragment and the anti-CD3 antibody or its antigen-binding fragment and the peptide linker linking the heavy chain portion and the light chain portion in the antigen-binding fragment can be the same or different. The peptide linker can be a polypeptide consisting of any of 1 to 100 or 2 to 50 amino acids, and the type of amino acids contained therein is not limited. The peptide linker can contain, for example, Gly, Asn, and / or Ser residues, and also includes neutral amino acids such as Thr and / or Ala. Amino acid sequences suitable for peptide linkers are known in the art. Meanwhile, the length of the linker can be determined in various ways within the limits that do not affect the function of the bispecific antibody. For example, the peptide linker may comprise a total of 1 to 100, 2 to 50, or 5 to 25 of one or more amino acid sequences selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one example, the peptide linker may be represented by (G4S)n (n is the number of (GGGGS) repeats) and is an integer of 1 to 10, for example, an integer of 2 to 5).

[0092] In one example, the anti-MUC1 / anti-CD3 bispecific antibody may be in the form of an scFv of an anti-CD3 antibody bound to the N-terminus of the light chain located in the anti-MUC1 antibody or its antigen-binding fragment. As described above, an anti-MUC1 / anti-CD3 bispecific antibody in which an anti-CD3 antibody scFv is bound to the N-terminus of the light chain located in the anti-MUC1 antibody or its antigen-binding fragment may have better cytotoxicity against cancer cells, inhibitory effect on cancer cell proliferation, and / or therapeutic effect on cancer than an anti-MUC1 / anti-CD3 bispecific antibody in which an anti-CD3 antibody scFv is bound to the C-terminus of the light chain located in the anti-MUC1 antibody or its antigen-binding fragment.

[0093] Medical use Another embodiment provides a pharmaceutical composition for preventing or treating cancer, comprising said anti-MUC1 antibody or antigen-binding fragment thereof, or said anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof.

[0094] Another embodiment provides a method for preventing or treating cancer, comprising administering a pharma- ceutical effective amount of the anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof to a subject in need thereof. The method for preventing and / or treating cancer may additionally comprise, prior to the administering step, identifying a patient in need of cancer prevention and / or treatment.

[0095] The anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, can be usefully used in the prevention or treatment of cancer. The anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof provided herein functions as an agonist of MUC1, so that the anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, can be used to its full potential to exert its efficacy (e.g., anti-cancer efficacy such as cancer cell killing), and the cancer to be treated may advantageously have its cancer cells express MUC1, for example, it may be a solid cancer expressing MUC1. Specifically, one example of the present invention relates to a novel antibody that specifically binds to MUC-1 with improved targeting and treatment of solid cancers, and its use. In one example, the cancer includes, but is not limited to, one or more selected from the group consisting of blood cancer (e.g., B cell lymphoma, etc.), bone marrow cancer, thyroid cancer, uterine cancer (e.g., cervical cancer), skin cancer, melanoma, breast cancer, colon cancer, lung cancer, gastric cancer, prostate cancer, and pancreatic cancer, or a solid cancer in which an epitope of an antigen to which an anti-MUC1 antibody or antigen-binding fragment thereof specifically binds is expressed in 30% or more.

[0096] In the pharmaceutical compositions or methods, a pharma- ceutical effective amount of the anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, may be provided together with one or more additives selected from the group consisting of pharma- ceutically acceptable carriers, diluents, excipients, and the like.

[0097] The pharma- ceutically acceptable carrier is one that is commonly used in the formulation of antibodies, and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. The pharmaceutical composition may further contain, in addition to the above-mentioned components, one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., that are commonly used in the manufacture of pharmaceutical compositions.

[0098] The pharmaceutical composition, the anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof can be administered orally or parenterally. Parenteral administration can be by intravenous, subcutaneous, intramuscular, peritoneal, intradermal, topical, intranasal, pulmonary, and rectal administration. When administered orally, proteins or peptides are digested, so oral compositions must be formulated to coat or protect the active agent from digestion in the stomach. The composition may also be administered by any device that allows the active agent to be delivered to the target cells.

[0099] The appropriate dosage of the pharmaceutical composition, the anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof may be formulated in various ways depending on factors such as the formulation method, the administration method, the age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. For example, the daily dosage of the pharmaceutical composition, the anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof may be in the range of 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, and more specifically 0.1 to 50 mg / kg, but is not limited thereto. The daily dosage may be formulated in a single formulation in a unit dose form, or may be formulated in appropriate amounts, or may be prepared in a multi-dose container. The term "pharmacologically effective amount" means an amount of the active ingredient (i.e., the anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof) capable of exerting a desired effect, i.e., the effect of preventing and / or treating cancer, and can be formulated in a variety of ways depending on factors such as the formulation method, mode of administration, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity.

[0100] The pharmaceutical composition may be prepared in unit dose form or in multi-dose containers by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner easily understood by those skilled in the art, and may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

[0101] Additionally, the pharmaceutical compositions may be administered as individual therapeutic agents or in combination with other therapeutic agents, either sequentially or simultaneously with conventional therapeutic agents.

[0102] Alternatively, the pharmaceutical composition may be formulated into an immunoliposome since it contains an antibody or an antigen-binding fragment. The antibody-containing liposome may be prepared by methods well known in the art. The immunoliposome may be prepared by reverse phase evaporation as a lipid composition containing phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine. For example, the Fab' fragment of the antibody may be conjugated to the liposome by a disulfide-switching reaction. A chemotherapeutic agent such as doxorubicin may additionally be included in the liposome.

[0103] A patient to which the pharmaceutical composition or the preventive and / or therapeutic method is administered may be a mammal, for example, a primate such as a human or a monkey, or a rodent such as a rat or a mouse, but is not limited thereto, and may be a cancer patient who has developed resistance to an existing anticancer agent, for example, an antagonist against the target cell membrane protein (e.g., MUC1).

[0104] Methods for Producing Polypeptides, Recombinant Vectors, and Antibodies Another embodiment of the present invention provides a polynucleotide encoding the above-mentioned polypeptide. In one specific example, the polynucleotide may encode a polypeptide comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 6, 8 to 15, 17 to 18, 20 to 26, 28 to 32, 34, and 38.

[0105] In yet another specific example, the polynucleotide may encode a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 77-96.

[0106] Yet another embodiment provides a recombinant vector comprising the polynucleotide.

[0107] Yet another embodiment provides a recombinant cell transformed with the recombinant vector.

[0108] The term "vector" refers to a means for expressing a target gene in a host cell. For example, it includes a plasmid vector, a cosmid vector, and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The vector usable as the recombinant vector can be prepared by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.) or a virus (e.g., SV40, etc.) that is sometimes used in the art, but is not limited thereto.

[0109] In the recombinant vector, the polynucleotide can be operatively linked to a promoter. The term "operatively linked" refers to a functional connection between a nucleotide expression control sequence (e.g., a promoter sequence) and another nucleotide sequence. The control sequence can be "operatively linked" to control the transcription and / or reading of the other nucleotide sequence.

[0110] The recombinant vector is typically constructed as a vector for cloning or a vector for expression. The expression vector may be any vector commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed by a variety of methods known in the art.

[0111] The recombinant vector is constructed using a prokaryotic or eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, it generally contains a strong promoter (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) capable of proceeding transcription, a ribosome binding site for the initiation of decoding, and a transcription / decoding termination sequence. When a eukaryotic cell is used as a host, the replication origin contained in the vector that operates in a eukaryotic cell includes, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, and a BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, and an HSV tk promoter) may be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0112] The recombinant cell may be obtained by introducing the recombinant vector into a suitable host cell. The host cell may be any host cell known in the art as a cell capable of stably and continuously cloning or expressing the recombinant vector. Examples of prokaryotic cells include Bacillus strains such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. In the case of transformation into eukaryotic cells, examples of host cells include yeast (Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines and the like may be used, but are not limited to these.

[0113] The polynucleotide or a recombinant vector containing the same can be delivered (introduced) into a host cell by a delivery method well known in the art, for example, when the host cell is a prokaryotic cell, the CaCl2 method or electroporation method can be used, and when the host cell is a eukaryotic cell, the microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, gene bombardment method, etc. can be used, but are not limited to these.

[0114] The method of selecting the transformed host cell can be easily carried out by a method well known in the art using a phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0115] Yet another example provides a method for producing a polypeptide or an anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof, comprising expressing the polynucleotide in the recombinant cell. The expressing the polynucleotide may comprise culturing the recombinant cell under conditions that permit expression of the polynucleotide contained therein. The method for producing the polypeptide or anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof may additionally comprise, after the expressing or culturing step, isolating and / or purifying the polypeptide or anti-MUC1 antibody or antigen-binding fragment thereof, or the anti-MUC1 / anti-CD3 bispecific antibody or antigen-binding fragment thereof. Effect of the Invention

[0116] Anti-MUC1 antibody and anti-MUC1 / anti-CD3 bispecific antibody can effectively inhibit the growth of cancer cells, and therefore have excellent effects on the prevention or treatment of cancer. [Brief description of the drawings]

[0117] [Figure 1] Figure 1 shows the binding reaction of pancreatic cancer-specific MP1 monoclonal antibody to peripheral blood, normal pancreatic epithelial cells, and pancreatic cancer cells (granulocytes, lymphocytes, monocytes, normal pancreatic epithelial cells (H6C7), and five types of pancreatic cancer cell lines (AsPC1, BxPC3, Capan-1, Capan-2, PANC-1)). [Figure 2A] FIG. 2A shows the results of immunohistochemistry (IHC) in pancreatic cancer tissues, and FIG. 2B shows the results of IHC in breast cancer tissues, where T in the figure indicates the tumor site. [Figure 2B] This is a continuation of Figure 2A. [Diagram 3] FIG. 3 shows the results of western blot of the MP1 monoclonal antibody. [Figure 4] FIG. 4 shows the amino acid sequence of MUC1 identified by LC-MS / MS analysis. [Diagram 5] FIG. 5 shows the results of an ELISA assay of the reactivity of the MP1 monoclonal antibody against the MUC1 antigen. [Figure 6] FIG. 6 shows the results of a reactivity assay (IP-Western blot (IB)) of the MP1 monoclonal antibody against the MUC1 antigen. [Figure 7A] Figures 7A and 7B show the results of quantification of antigens on the surface of pancreatic cancer cell lines that react with MP1, and Figures 7C and 7D show the results of epitope mapping of MP1 monoclonal antibodies using a circular peptide array method. [Figure 7B] This is a continuation of Figure 7A. [Figure 7C] This is a continuation of Figure 7B. [Figure 7D] Continuation of Figure 7C. [Figure 8] FIG. 8 shows the pancreatic cancer cell binding reaction of the MP1 humanized antibody. The stained cell line was Capan-2, and the combination of heavy and light chain antibody genes and the cell binding positivity (%) are shown. [Figure 9] FIG. 9 shows the structure of an LCC-type anti-MUC1 / OKT3 antibody. [Figure 10A] 10A and 10B show the results showing cell surface binding of LCC-type anti-MUC1 / OKT3 antibodies to pancreatic cancer cells AsPC1. [Figure 10B] This is a continuation of Figure 10A. [Figure 11] FIG. 11 shows the results showing the cytotoxicity of LCC-type anti-MUC1 / OKT3 antibodies against pancreatic cancer cells Capan-2 and AsPC1. [Figure 12] FIG. 12 shows the structure of an LCN-type anti-MUC1 / OKT3 antibody. [Figure 13] FIG. 13 shows the results showing the cytotoxicity of LCN-type anti-MUC1 / OKT3 antibodies against pancreatic cancer cells, Capan-1. [Figure 14] FIG. 14 shows the results showing the cytotoxicity of LCN-type anti-MUC1 / OKT3 antibodies against pancreatic cancer cells AsPC1. [Figure 15] FIG. 15 shows the results showing the cytotoxicity of LCN-type anti-MUC1 / OKT3 antibodies against pancreatic cancer cells Capan-2 and AsPC1, gastric cancer cells SNU-601, and breast cancer cells T47D. [Figure 16] FIG. 16 shows the results of administering anti-MUC1 / OKT3 antibody to a NOG mouse animal model in which tumors were induced by injection of pancreatic cancer cells AsPC-1, and confirming the therapeutic effect on cancer cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0118] The present invention will now be described in more detail with reference to the following examples, which are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0119] Example 1. Discovery of MP1 monoclonal antibody In order to develop a new antibody specific to pancreatic cancer, the following experiments were carried out. The antibody developed was expressed not only in pancreatic cancer, but also in some breast and gastric cancers. As a solid cancer-specific antibody, it was named MP1.

[0120] Example 1-1. Method for designing and selecting target sites for producing MP monoclonal antibodies To develop monoclonal antibodies against specific antigens expressed in pancreatic cancer, we immunized mice with various pancreatic cancer cell lines AsPC1, BxPC3, Capan-1, Capan-2, and PANC-1, and established monoclonal antibodies against them by cell fusion method. After that, in the hybridoma selection process, we selected antibodies that were positive for pancreatic cancer cell lines and negative for normal pancreatic cell line H6C7 and peripheral blood.

[0121] Example 1-2. Selection of hybridoma-produced antibodies To develop pancreatic cancer-specific monoclonal antibodies, five pancreatic cancer cell lines (AsPC1, BxPC3, Capan-1, Capan-2, and PANC-1) were mixed in equal numbers and incubated at 1 × 10 per 6-week-old Balb / c female mouse. 7 The mice were immunized by injecting the spleen cells into the intraperitoneal cavity (IP) three times at three-week intervals. The spleens of the immunized mice were excised to obtain single cell suspensions, which were washed twice with RPMI1640 and mixed with 0.4% trypan blue (sigma) at 1:1 (v / v), and the number of cells was counted by trypan blue staining, which measures cells that are not stained under a microscope. X63 mouse myeloma cell line (ATCC CRL-1580) was used as the cell fusion partner cell, and the number of cells was counted after washing in the same manner as the spleen cells. The bone marrow (myeloma) cells and spleen cells were mixed, and PEG (polyethylene glycol) 1500 was added to fuse the cells, and the cells were centrifuged. The fused hybridoma cells were suspended in RPMI (20% FBS, hypoxanthine-aminopterin-thymidine) containing 1xHAT, dispensed in 150ul portions into 96-well plates, and then cultured in an incubator at 37°C and 5% CO2. After the fusion, the selection process was carried out in a medium containing HAT for a certain period of time, and when wells that formed colonies were observed, the medium was switched to HT medium and cultured in an incubator at 37°C and 5% CO2 for 48 hours. The normal pancreatic cell line H6C7 and the five pancreatic cancer cell lines used for immunization, AsPC1, BxPC3, Capan-1, Capan-2, and PANC-1, were each subjected to fluorescent staining and flow cytometry analysis as follows. 100 ul of hybridoma culture supernatant was added to the prepared cells and reacted at 4°C for 30 minutes, then 3 ml of PBS was added and centrifuged at 1700 rpm for 3 minutes to wash off unbound antibodies, and to confirm bound antibodies, a secondary antibody, goat anti-Mouse Ig-FITC (Jackson ImmunoResearch, USA), was diluted 200-fold and added, reacted at 4°C for 15 minutes, washed with 3 ml of PBS in the same manner as above, and then measured by flow cytometry.

[0122] Example 1-3. To confirm the degree of binding to peripheral blood, 100 ul of hybridoma supernatant was added to PBMC (peripheral blood mononuclear cells, Zenbio) and reacted at 4°C for 30 minutes, then 3 ml of PBS was added and centrifuged at 1700 rpm for 3 minutes to wash off unbound antibodies, and to confirm bound antibodies, a secondary antibody, goat anti-Mouse Ig-FITC (Jackson ImmunoResearch, USA), was added at a 200-fold dilution and reacted at 4°C for 15 minutes, then washed with 3 ml of PBS in the same manner as above, and the results were measured by flow cytometry (Figure 1). Figure 1 shows the results of a test of the reactivity of pancreatic cancer-specific MP1 monoclonal antibody with peripheral blood, normal pancreatic epithelial cells (H6C7), and various types of pancreatic cancer cells.

[0123] As shown in Figure 1, using the above method, a monoclonal antibody (hereinafter referred to as MP1) that was positive for various pancreatic cancer cell lines and negative for normal pancreatic epithelial cells H6C7, peripheral blood granulocytes, lymphocytes, and monocytes was selected, and finally, a single colony of MP1 hybridoma cells was obtained by limiting dilution.

[0124] Example 2. Antibody expression analysis in various cell lines Example 2-1. Antibody expression in various cell lines The binding of MP1 monoclonal antibody to various cancer cell lines obtained from KCLB (Korean Cell Line Bank) and ATCC (American Type Culture Collection) was confirmed by flow cytometry. Specifically, cancer cell lines were cultured in RPMI1640 medium or DMEM medium supplemented with 10% heat inactivated FBS in an incubator at 37℃ under 5% CO2 according to the culture medium and method provided by KCLB (Korean Cell Line Bank) and ATCC (American Type Culture Collection). Human pancreatic epithelial cell line H6C7 was purchased from Kerafast and cultured in Keratinocyte SFM (Invitrogen Cat#:17005042) in an incubator at 37℃ under 5% CO2.

[0125] The MP1 monoclonal antibodies of the present invention were added to the various cancer cells cultured above and reacted at 4° C. for 30 minutes, then washed with PBS, and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, USA) was added and cultured at 4° C. for 15 minutes. After washing again with PBS, the cells were analyzed with FACS caliber (Becton Dickinson, USA) to confirm the reactivity of the MP1 antibody in various solid cancer cell lines (Table 9).

[0126] As shown in Tables 9 and 10 below, it was confirmed that the MP1 monoclonal antibody binds strongly to pancreatic cancer, gastric cancer, breast cancer, some of the lung cancer cell lines, skin cancer, and prostate cancer cell lines, but does not bind or binds weakly to T cells, B cells, brain cancer, biliary tract cancer, and colon cancer cell lines, including the normal pancreatic epithelial cell line H6C7. PBMC showed negative binding to normal blood cells.

[0127] MP1 showed a positive reaction in various pancreatic cancer cells and a negative reaction in normal pancreatic epithelial cells, indicating that it can be used as a therapeutic agent for pancreatic cancer. [Table 9-1]

[0128] [Table 9-2]

[0129] [Table 10]

[0130] Example 2-2. IHC (Immunohistochemistry) Analysis The distribution of the MP1 monoclonal antibody-bound antigen in human pancreatic and breast cancer tissues was confirmed by immunohistochemistry (IHC). Human pancreatic and breast cancer tissues were obtained from Chungbuk National University Hospital, Korea, and paraffin sections were prepared at the Department of Pathology, Chungbuk National University Hospital.

[0131] Human breast cancer tissue to be stained was fixed in formalin using the usual method, embedded in paraffin, and microarray sections were prepared. Then, the tissue was placed on a slide to prepare a single tissue slide for staining reading. Next, immunohistochemical staining was performed using MP1 antibody in the following manner. The tissue was treated with xylene for 10 minutes three times, ethanol was treated with 100%, 80%, and 50% in sequence, and finally washed with distilled water to proceed with the deparaffinization process. After washing with 1XTBS, the slide was placed in a 10mM citric acid (pH 6.0) antigen regeneration solution and the antigen was regenerated in a microwave oven for 20 minutes. The slide temperature was gradually lowered using tap water, and the slide was stored in 1XTBS for 10 minutes. The tissue was treated with mouse MP1 antibody as the primary antibody and reacted at room temperature for 1 hour, then washed three times with 1XTBS, and reacted with anti-mouse antibody-HRP conjugate as the secondary antibody at room temperature for 30 minutes. The slides were washed three times with 1XTBS, stained with DAB for 1 minute and 30 seconds, washed three times with 1XTBS, counter stained for 45 seconds, and washed in running tap water for 10 minutes. The dehydration process was carried out in the reverse order of the deparaffinization process, and the slides were mounted and read under a microscope or photographed.

[0132] As shown in Figures 2A and 2B, it was confirmed that the antigen recognized by the MP1 monoclonal antibody was not distributed in normal tissues, but was specifically expressed in cancer tissues.

[0133] Example 3. Antigen Analysis for MP1 Monoclonal Antibody Example 3-1. Confirmation of antigen size of MP1 monoclonal antibody Western blot was performed to examine the protein size of the antigen recognized by the MP1 monoclonal antibody of the present invention. The pancreatic cancer cell line Capan-2, which has high expression of the antigen against MP1, was dissolved in a cell lysis solution (NP40 cell lysis buffer, Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma, Cat.#:1136170001), and then electrophoresed on an 8% SDS polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore Corporation, Billerica, MA, USA). The purified MP1 monoclonal antibody was then treated with a membrane and colored with an ECL system (GE Healthcare, Buckinghamshire, UK) to confirm the band (Figure 3). As can be seen from Figure 3, the measurement results confirmed that the size of the antigen recognized by the MP1 monoclonal antibody was approximately 300 kDa.

[0134] Example 3-2. Isolation and analysis of MP1 monoclonal antigen To identify the antigen recognized by the MP1 monoclonal antibody of the present invention, immunoprecipitation was performed. First, Capan-2 cell line, which is a cell line highly reactive to the MP1 monoclonal antibody, was cultured at 1 × 10 7The cells were lysed in a cell lysis buffer (NP40 cell lysis buffer, Thermo Fisher Scientific) at 1000 cells / ml, and the cell residues were removed by centrifugation. The supernatant was collected and used as an antigen separation material recognized by the MP1 antibody. The MP1 monoclonal antibody was added to Protein-G Sepharose, mixed at 4°C for 4 hours, washed twice with 1XPBS, and then the previously prepared Capan-2 lysate was added, mixed at 4°C for 4 hours, and washed twice with 1XPBS. The immune complex obtained from this was subjected to SDS-polyacrylamide gel electrophoresis, and the gel was stained with Coomassie Brilliant Blue G-250. The band estimated to be an antigen for MP1 at around 300 kDa was cut out and identified by ProteomeTech for LC-MS / MS analysis. Identification was performed by LC-MS / MS analysis and the results obtained were processed by MASCOT. 100 protein hits obtained from this series of analyses were expressed on the cell surface, and the analysis range was limited to antigens with a protein size of approximately 300 kDa. As a result, MUC1 was selected as the antigen for the MP1 monoclonal antibody (Figure 4). The peptide sequence isolated from MP1 is QGGFLGLSNIK (sequence number 107).

[0135] Example 3-3. Verification of antigen identification results of MP1 monoclonal antibody by ELISA To verify the antigen identification results obtained by LC-MS / MS, the reactivity of MUC1 monoclonal antibody against recombinant protein MUC1 (Sino Biological, Cat.#:12123-HCCH) was confirmed by ELISA and Western blotting assay.

[0136] 100ng of recombinant protein MUC1 per well was added to a Maxisorp ELISA plate and reacted at 37℃ for 1 hour to coat the antigen, then 200ul of 1x blocking solution (Sigma) was added per well and reacted at 37℃ for 1 hour to block. MP1 monoclonal antibody, control antibody, and 100ul of PBS were added to the prepared plate and reacted at 37℃ for 1 hour, then washed with PBS to remove unbound antibodies. Goat anti-Mouse IgG-HRP (Jackson ImmunoResearch, USA) was diluted and added and reacted for 30 minutes, then washed with PBS, and 50ul of TMB solution was added per well and reacted for 10 minutes, then 50ul of sulfuric acid was added to stop the reaction, and the absorbance was measured at 450nm. MP1 monoclonal antibody showed reactivity to recombinant protein MUC1 as shown in Figure 5. Figure 5 shows the results of ELISA verifying that the antigen for MP1 monoclonal antibody is MUC1.

[0137] Example 3-4. Verification of antigen identification results of MP1 monoclonal antibody by IP and IB In the above experiment, the recognition of MUC1 as an antigen by MP1 monoclonal antibody was again examined by Western blot. As described in Example 3-2, various pancreatic cancer cell lines BXPC3, Capan-1, Capan-2, and PANC1 lysates were prepared, and a commercial MUC1 antibody (BioXCell, Cat. #C595 (NCRC48)) and MP1 monoclonal antibody were mixed with Protein-G Sepharose at 4°C for 4 hours, washed twice with 1XPBS, and then the previously prepared lysate was added, mixed at 4°C for 4 hours, and washed twice with 1XPBS. The immune complex obtained from this was boiled for 10 minutes, electrophoresed on an 8% SDS polyacrylamide gel, and transferred to an Immobilon P membrane (Millipore Corporation, Billerica, MA, USA). The purified MP1 monoclonal antibody was then applied to a membrane and stained with an ECL system (GE Healthcare, Buckinghamshire, UK) to confirm the bands. Western blotting of the immune complex of a commercial MUC1 antibody reacted with a pancreatic cancer cell line using the MP1 antibody confirmed a band at the same position of 300 kDa as in the immune complex samples of Capan-2 lysate alone and MP1 and pancreatic cancer cell line lysate. Figure 6 shows the results of a western blot verifying that the antigen for the MP1 monoclonal antibody is MUC1.

[0138] Example 4. Quantification of cell surface antigens The expression levels of antigens that bind to MP1 monoclonal antibody on the surface of pancreatic cancer cells were measured using QIFIKIT (Dako, Cat.#:K0078), a quantitative flow cytometry kit for measuring cell surface antigens. The antibody binding affinity (ABC) of the cell line can be calculated by comparing the mean fluorescence intensity (MFI) of the beads using a calculation graph. Figure 7A shows the expression levels of antigens that react with MP1 detected on the cell surface of various pancreatic cancer cell lines (AsPC1, BxPC3, Capan-1, Capan-2, PANC1) using MP1 monoclonal antibody and QIFIKIT.

[0139] Example 5. Epitope mapping Next, a partial sequence of the MUC-1 antigen used for epitope mapping (amino acid sequence from amino acid 895 to 1264) is shown (Table 11). A site containing the amino acid sequence of the recombinant MUC-1 antigen used for antigen identification verification was selected, and epitope mapping was carried out using a circular peptide array.

[0140] As can be seen from FIG. 7B, the MP1 antibody bound to a peptide containing the PAHGVTS sequence in a dose-dependent manner, thus confirming that the antigen-binding site, or epitope, of MP1 is PAHGVTS (SEQ ID NO: 109). [Table 11]

[0141] Example 6. Sequence and isotype analysis of the MP1 antibody variable regions Example 6-1. Determination of isotype To determine the isotype of the MP1 monoclonal antibody prepared in Example 1, it was analyzed using a mouse immunoglobulin isotyping ELISA kit (BD Biosciences, USA), and the MP1 monoclonal antibody was confirmed to be mouse IgG2a / kappa light chain.

[0142] Example 6-2. MP1 antibody variable region sequence analysis The MP1 antibody gene was cloned using Mouse Ig-Primer Set (Merck Millipore, Cat.#:69831). PCR was performed using Mouse Ig-Primer Set from RNA isolated from MP1 hybridoma, and the PCR product was inserted into pGEM-T vector (Promega, Cat.#:A3600). The DNA base sequence was confirmed by sequencing, and the mouse antibody gene was confirmed through the IMGT site (www.imgt.org). Table 12 shows the sequences in the heavy and light chain variable regions of the analyzed MP1 antibody. In the amino acid sequences described below, the bold underlined parts indicate the complementarity determining sites. In Table 12 below, the sequence positions are prepared according to the standards of the Kabat numbering system. [Table 12]

[0143] Example 7. Development of MP1 humanized antibody Prior to producing the double antibody, in order to reduce the immunogenicity caused by the human anti-mouse antibody (HAMA) reaction when an antibody of mouse origin is administered to the human body and to secure an antibody with high druggability, a humanized antibody was produced in which the MP1 variable region and the remaining part were replaced with human Fc. It was confirmed that the antigen specificity and affinity of the selected humanized antibody are similar to those of the original mouse-derived MP1 antibody.

[0144] Example 7.1 Recombinant antibody sequence selection by in silico humanization The CDR sequences of the heavy and light chains of mouse antibody MP1 (heavy chain amino acid sequence: SEQ ID NO:77; light chain amino acid sequence: SEQ ID NO:87) are as follows: VH-CDR1: SEQ ID NO:2 (DYWMN); VH-CDR2: SEQ ID NO:8 (QIRNKPYNYETYYSNSVKG); VH-CDR3: SEQ ID NO:17 (AMITSDGYAMDY); VL-CDR1: SEQ ID NO:20 (KSSQSLLLSSNQKNYLA); VL-CDR2: SEQ ID NO:28 (WASTRES); VL-CDR3: SEQ ID NO:34 (QQCFSYPLT). Humanized antibodies with the same or superior antigen binding ability while maintaining maximum similarity between the heavy and light chain amino acid sequences of mouse antibody MP1 were selected by in silico recombination of sequence sites for framework regions based on the germline sequences encoding human antibody genes. The human antibody germline genes that are most similar to the heavy and light chain sequences of mouse antibody MP1 and were used as the backbone of the humanized recombinant antibody sequence are shown in Table 13 below.

[0145] The amino acid sequences of the heavy chain variable region and the light chain variable region of the selected humanized antibody are shown in Table 14. In the amino acid sequences shown below, the bold underlined parts indicate the complementarity determining regions. [Table 13]

[0146] [Table 14-1]

[0147] [Table 14-2]

[0148] Example 7.2 Expression and Analysis of Humanized Recombinant Antibodies The amino acid sequences corresponding to the expression genes of the nine heavy chains (including VH1 to VH9) and nine light chains (including VL1 to VL9) in Table 14 were codon-optimized to synthesize DNA sequences, which were then linked to human Fc region genes (human IgG1 Fc gene for the heavy chain and human Ig kappa constant gene for the light chain) by overlap PCR and cloned into the pcDNA3.4 vector included in the ExpiCHO-S Expression system kit (Gibco, USA). The light and heavy chain expression plasmids of the humanized MP1 antibody thus prepared were transfected into the ExpiCHO-S cell line using the ExpiCHO-S expression system kit (Invitrogen / Life technologies) and temporarily expressed and produced at a 48-well scale.

[0149] Using the culture medium of the expressed humanized antibody, 1) the presence or absence of cell surface binding was confirmed by staining the pancreatic cancer cell line Capan-2, and 2) the presence or absence of expression of the humanized antibody was confirmed by anti-human Ig antibody sandwich ELISA. From a total of 81 combinations of heavy chains and light chains (i.e., combinations of antibodies including a heavy chain containing any one of the nine heavy chain variable regions listed in Table 14 and a light chain containing any one of the nine light chain variable regions listed in Table 14), combinations of humanized antibodies showing a binding pattern similar to that of the original mouse antibody or chimeric antibody on the cell surface were confirmed, and based on the results of sandwich ELISA confirming the expression level of the temporarily expressed humanized antibody, it was confirmed that the antibody was generally expressed (Table 15). [Table 15]

[0150] In Table 15, cell surface binding (%) is the positive rate of antibody binding to the cell surface compared to the negative control group, and expression (sandwich ELISA) is the 450 nm OD value of sandwich ELISA.

[0151] Table 15 shows 20 antibodies with high cell surface binding (%) to Capan-2 out of the 81 antibodies prepared. As can be seen from Table 12, VH8 / VL3 (antibody with a combination of heavy chain number 8 / light chain number 3; i.e., an antibody containing a heavy chain containing the heavy chain variable region VH8 in Table 14 and a light chain containing the light chain variable region VL3 in Table 14; hereinafter the same), VH1 / VL5, VH4 / VL4, VH4 / VL5, VH1 / VL4, VH8 / VL4, VH1 / VL2, VH7 / VL4, VH7 / VL3, VH8 / VL2, VH4 / VL8, and VH4 / VL7 antibodies had excellent cell surface binding to Capan-2.

[0152] After expanding the antibody genes of the heavy and light chain combinations of humanized antibodies that were highly positive in the primary transient expression from a 48-well to a 6-well scale and performing transient expression again, the expressed antibodies reconfirmed their binding ability to the Capan-2 pancreatic cancer cell line (Figure 8).

[0153] As shown in Figure 8, VH1 / VL5, VH4 / VL4, VH4 / VL5, VH1 / VL4, VH1 / VL2, and VH7 / VL4 antibodies were superior in binding to the cell surface of Capan-2.

[0154] Example 8. In vitro efficacy: T-cell engaging bispecific Ab / format optimization Example 8-1. Expression of T cell engaging biantibody using humanized MP1 antibody Among various bispecific antibody forms, a tetravalent bispecific antibody was developed that exhibits T cell activity and high anti-cancer function, and at the same time retains an Fc site and maintains a half-life close to that of the original antibody. The humanized antibody variable region of the OKT3 antibody exhibiting T cell activity was used as the anti-CD3 antibody for T cell activity. The sequence of the humanized OKT3 antibody was quoted from US20090202532A1 patent and corresponds to SEQ ID NO: 103, SEQ ID NO: 105, and SEQ ID NO: 106 in Table 13 below. SEQ ID NO: 103 is the amino acid sequence of the heavy chain variable region of the humanized OKT3 antibody, SEQ ID NO: 104 is the amino acid sequence of the light chain variable region of the humanized OKT3 antibody, and SEQ ID NO: 105 shows the amino acid sequence of the scFv antibody of the humanized OKT3 antibody. The humanized OKT3 antibody is composed of a single chain Fv (scFv) linked by a [GGGS]n=5 linker between the heavy chain variable region sequence and the light chain variable region sequence, and is linked to the N-terminus (LCN version) or C-terminus (LCC version) of the light chain of the humanized MP1 antibody by a [GGGS]n=3 linker.

[0155] In the humanized OKT3 scFv, the 44th glycine in the heavy chain variable region and the 100th glycine in the light chain variable region were changed to cysteine ​​to induce interdomain disulfide bonds and increase stability (Protein Engineering, Design & Selection vol. 25 no. 7 pp. 321-329, 2012).

[0156] To express the double antibody, a humanized MP1 heavy chain gene (a gene encoding any one of the heavy chains VH1 to VH9 in Table 11) plasmid and an LCN or LCC type humanized MP1 light chain-OKT3scFv gene (a gene encoding a light chain sequence including a light chain complementarity determining site of VL4 or VL5 in Table 11-OKT3scFv gene) plasmid were transfected into ExpiCHO-S cells at a ratio of 1:1.7. After 7 or 10 days, antibody expression (sandwich ELISA using anti-human Ig), cell line binding (cell surface staining and flow cytometer analysis), and cytotoxicity assay were performed using the transfected double antibody culture solution, and the double antibody culture solution that had been confirmed to have activity was purified by Protein A affinity chromatography.

[0157] The sequences of the anti-MUC1 / anti-CD3 bispecific antibodies prepared as described above (anti-MUC1 antibody light chain sequence and anti-CD3 antibody scFv sequence) are shown in Table 13 below. The form in which the anti-CD3 antibody (OKT3 scFv) is bound to the C-terminus of the light chain of the anti-MUC1 antibody is designated as LCC, and the form in which the anti-CD3 antibody is bound to the N-terminus of the light chain is designated as LCN.

[0158] In Table 16 below, as an LCC-type anti-MUC1 / anti-CD3 bispecific antibody (see FIG. 9), LCC version humanized MP1 Light chain #4-OKT3scFv shows the light chain sequence (SEQ ID NO: 112) of an anti-MUC1 / anti-CD3 bispecific antibody in which a humanized OKT3 scFv antibody (SEQ ID NO: 106) is bound to the C-terminus of a light chain sequence (SEQ ID NO: 110) containing the light chain variable region of light chain number 4 (VL4) (SEQ ID NO: 91) in Table 14. LCC version humanized MP1 Light chain #5-OKT3scFv shows the light chain sequence (SEQ ID NO: 113) of an anti-MUC1 / anti-CD3 bispecific antibody in which a humanized OKT3 scFv antibody (SEQ ID NO: 106) is bound to the C-terminus of a light chain sequence (SEQ ID NO: 111) containing the light chain variable region of light chain number 5 (VL5) (SEQ ID NO: 92) in Table 14.

[0159] In Table 16 below, as an LCN-type anti-MUC1 / anti-CD3 bispecific antibody (see FIG. 12), LCN version humanized MP1 Light chain #4-OKT3scFv shows the light chain sequence (SEQ ID NO: 114) of an anti-MUC1 / anti-CD3 bispecific antibody in which a humanized OKT3 scFv antibody (SEQ ID NO: 106) is bound to the N-terminus of a light chain sequence (SEQ ID NO: 110) containing the light chain variable region of light chain number 4 (VL4) (SEQ ID NO: 91) in Table 14. LCN version humanized MP1 Light chain #4-OKT3scFv shows the light chain sequence (SEQ ID NO: 115) of an anti-MUC1 / anti-CD3 bispecific antibody in which a humanized OKT3 scFv antibody (SEQ ID NO: 106) is bound to the C-terminus of a light chain sequence (SEQ ID NO: 111) containing the light chain variable region of light chain number 5 (VL5) (SEQ ID NO: 92) in Table 14.

[0160] In the light chain sequences of the four types of anti-MUC1 / anti-CD3 bispecific antibodies, the light chain of the anti-MUC1 antibody was linked to the humanized OKT3 scFv antibody via a [GGGS]n=3 linker. The heavy chain of the anti-MUC1 / anti-CD3 bispecific antibody used had a heavy chain sequence including the heavy chain variable regions (SEQ ID NO: 78 to SEQ ID NO: 86) of heavy chain numbers 1 to 9 (VH1 to VH9) in Table 14. [Table 16-1]

[0161] [Table 16-2]

[0162] Example 8-2. Functional analysis of LCC-type humanized MP1 bibody The LCC-type anti-MUC1 / anti-CD3 bispecific antibodies prepared in Example 8-1 were subjected to cell surface binding and cytotoxicity assays for AsPC1 pancreatic cancer cells positive for MUC1 antigen. The expression of the anti-MUC1 / anti-CD3 bispecific antibodies at the protein level was confirmed by Sandwich ELISA using anti-human Ig / anti-human Ig-HRP (Table 17), and the binding of these bispecific antibodies to AsPC1 of MUC1 antigen-positive pancreatic cancer cells was confirmed by cell surface staining and flow cytometry (Figure 10).

[0163] As a control, anti-mesothelin / OKTscFv humanized bispecific antibody (MSTN-OKT3) was used. MSTN-OKT3 is an LCC-type bispecific antibody in which the humanized OKT3 scFv sequence (SEQ ID NO: 106) is linked to the C-terminus of the light chain of the h7D9.v3 antibody using the sequence of the h7D9.v3 antibody described in International Publication WO2012 / 087962. [Table 17]

[0164] In Table 17, expression (sandwich ELISA) is the 450 nm OD value of sandwich ELISA, and in one example, the antibody "VH1 / VL5" means an anti-MUC1 / anti-CD3 bispecific antibody comprising a heavy chain containing the heavy chain variable region of heavy chain number 1 (VH1) in Table 14 (sequence number 78) and a light chain (sequence number 113) in which humanized OKT3 scFv antibody (sequence number 106) is bound to the C-terminus of the light chain variable region of light chain number 5 (VL5) in Table 14 (sequence number 92).

[0165] Although the antibodies shown in Table 17 were expressed, the flow cytometer showed negative results in Figure 10 because the affinity of the original antibodies was lost during the process of combining with the humanized antibodies. Also, as can be seen from Figure 10, all of the bispecific antibody combinations expressed together with light chain #4-OKT3 scFv antibody (i.e., bispecific antibodies including a heavy chain variable region of any one of heavy chains #1 to #9 in Table 14 and a light chain in which humanized OKT3 scFv antibody is bound to the C-terminus of the light chain variable region of light chain #4) showed cell surface binding, but only the combinations of heavy chain #1 / light chain #5, heavy chain #2 / light chain #5, and heavy chain #7 / light chain #5 showed cell surface binding among the bispecific antibody combinations expressed together with light chain #5-OKT3 scFv antibody.

[0166] The expressed bispecific antibodies were subjected to a cytotoxicity assay using Capan-2 and AsPC-1 cell lines as target cells and human T cells as effector cells. The ratio of target cells to effector cells was 1:10, and the amount of bispecific antibodies added was 1ug / ml. After 48 hours, the death of target cells was measured using EZ-Cytox (DogenBio, Cat.#: EZ-1000), and the %Cytotoxicity was calculated using the following formula, which is shown in Figure 11.

[0167] %Cytotoxicity=(1-(measurement value of antibody-treated wells / measurement value of target and efficacy cell wells))X100

[0168] As can be seen from FIG. 11, the LCC-type anti-MUC1 / anti-CD3 bispecific antibody prepared in Example 8-2 was less effective than the control antibody (MSTN-OKT3), with a cytotoxicity of less than 30% and poor efficacy.

[0169] Example 8-3. Functional analysis of LCN-type humanized MP1 bibody (1) The LCN-type anti-MUC1 / anti-CD3 bispecific antibody prepared in Example 8-1 was subjected to cell surface binding and cytotoxicity assay for MUC1 antigen-positive Capan-1 and AsPC1 pancreatic cancer cells.

[0170] The expression of the LCN-type anti-MUC1 / anti-CD3 bispecific antibodies expressed as shown in Table 16 above at the protein level was confirmed by Sandwich ELISA using anti-human Ig / anti-human Ig-HRP, and the binding of these bispecific antibodies to Capan-1 and AsPC1 in MUC1 antigen-positive pancreatic cancer cells was confirmed by cell surface staining and flow cytometry.

[0171] The results are shown in Figures 13 and 14. As a control, an anti-MUC1 / anti-CD3 bispecific antibody (chi / 4) was used, which comprises a heavy chain containing the heavy chain variable region of the mouse MUC1 antibody in Table 12 (SEQ ID NO: 77) and a light chain in which the humanized OKT3 scFv antibody was bound to the N-terminus of a light chain sequence (SEQ ID NO: 110) containing the light chain variable region of light chain number 4 (VL4) in Table 14 (SEQ ID NO: 91).

[0172] As shown in Figures 13 and 14, it was confirmed that anti-MUC1 / anti-CD3 bispecific antibodies with combinations of heavy chain #1 / light chain #4 ("1 / 4", i.e., an anti-MUC1 / anti-CD3 bispecific antibody comprising a heavy chain containing the heavy chain variable region (SEQ ID NO: 78) of heavy chain #1 (VH1) in Table 14, and a light chain (SEQ ID NO: 114) in which a humanized OKT3 scFv antibody (SEQ ID NO: 106) is bound to the N-terminus of the light chain variable region (SEQ ID NO: 91) of light chain #4 (VL4) in Table 14; hereinafter the same), heavy chain #4 / light chain #4 ("4 / 4"), and heavy chain #7 / light chain #4 ("7 / 4") had superior toxic effects against cancer cells compared to the control group.

[0173] These results indicate that the LCN type bispecific antibody (Example 8-3) is able to bind to T cells while maintaining a closer microscopic distance between the cancer cells and T cells, which is more likely to cause cytotoxicity, than the LCC type bispecific antibody (Example 8-2), and thus is able to exhibit higher cytotoxicity.

[0174] Example 8-4. Functional analysis of LCN-type humanized MP1 bibody (2) The LCN-type anti-MUC1 / anti-CD3 bispecific antibody prepared in Example 8-1 was subjected to a cytotoxicity assay on AsPC-1 and Capan-2 (pancreatic cancer cell line), SNU-601 (gastric cancer cell line), and T47D (breast cancer cell line) cell lines positive for MUC1 antigen to confirm the in vitro cytotoxicity effect, and the results are shown in FIG. 15.

[0175] As the anti-MUC1 / anti-CD3 bispecific antibody, an anti-MUC1 / anti-CD3 bispecific antibody with a combination of heavy chain 1 / light chain 5 with a higher humanization rate was used, i.e., an anti-MUC1 / anti-CD3 bispecific antibody containing a heavy chain including the heavy chain variable region of heavy chain 1 (VH1) in Table 14 (SEQ ID NO: 78) and a light chain (SEQ ID NO: 115) in which the humanized OKT3 scFv antibody (SEQ ID NO: 106) is bound to the N-terminus of the light chain variable region of light chain 5 (VL5) in Table 14 (SEQ ID NO: 92).

[0176] As shown in FIG. 15, it was confirmed that the LCN-type anti-MUC1 / anti-CD3 bispecific antibody showed a cytotoxic effect on cancer cells in proportion to the concentration of the administered antibody.

[0177] Example 9. Evaluation of the therapeutic effect of humanized double antibodies using a pancreatic cancer mouse model To confirm the in vivo immune anti-cancer effect of the anti-MUC1 / anti-CD3 bispecific antibody, a NOG mouse animal model transplanted with the human pancreatic cancer cell line AsPC-1 was used to evaluate whether or not tumor size was reduced by administration of the anti-MUC1 / anti-CD3 bispecific antibody. The anti-MUC1 / anti-CD3 bispecific antibody used was the same antibody as that used in Example 8-4 (anti-MUC1 / anti-CD3 bispecific antibody with a combination of LCN type heavy chain #1 / light chain #5).

[0178] Preparation of mouse model To evaluate the anti-cancer effect of antibodies with substantial human immune cells, NOD / Shi-scid mice (IL-2RγKO mice, KOATECH) were used, which are deficient in the functions of T cells, B cells, NK cells, macrophages, and dendritic cells. 6 Pancreatic cancer cell line AsPC-1 was subcutaneously injected (IP, intraperitoneal injection) into the flank of 8-week-old NOG mice, and 1-2 weeks later, 100-150 mm 3 Mice with tumors of this volume were selected for the experiment, with 5 mice per group.

[0179] Preparation of Human PBMCs Human cryopreserved peripheral blood mononuclear cells (PBMCs) purchased from Zenbio were thawed in RPMI1640 (GeneDireX, Cat#: CC112-0500) containing 10% FBS 10 days before antibody injection, treated with IFN-r (jw Creagen, Cat#: HIFNG-100) 250ng / ml on day 1, added with IL-2 (jw Creagen, JW-H031-0100) 30ng / ml and OKT3 antibody (Miltenyi Biotec, Cat#: 130-093-387) 50ng / ml on day 2, and left for 5 days. After that, from day 6 to day 16 of culture, IL-2 30ng / ml and IFN-r 50ng / ml were cultured in RPMI1640 medium supplemented with 10% FBS to expand the number of PBMCs.

[0180] Anti-cancer efficacy evaluation of humanized MP1 dual antibody The NOG mice with tumors were divided into 6 groups. On day 0, 1 group was injected intraperitoneally with 200 μl of PBS, and 5 groups were injected with 5 × 10 6 The cell number of PBMCs was injected into the peritoneal cavity (i.e., 5 × 10 in 1X PBS). 6 The suspension was prepared so that the concentration of cells / 0.2 mL was injected into the abdominal cavity.) After this, the anti-MUC1 / anti-CD3 bispecific antibody was injected into PBMCs of four groups of mice at 1 mg / kg and 3 mg / kg, respectively, and then the antibodies were intravenously injected four times on the 3rd, 7th, 10th, and 14th days, and the size of the subcutaneously injected cancer cells was observed and measured.

[0181] Figure 16 shows the in vivo anti-cancer efficacy of anti-MUC1 / anti-CD3 bispecific antibodies in a humanized mouse pancreatic cancer animal model injected with human PBMCs. The antibody-administered groups (MP1-1 mg / kg and MP1-3 mg / kg) showed a higher anti-cancer effect (Tumor growth inhibition; TGI) than the group administered PBMCs only (Control-PBMC).

[0182] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below, rather than the above detailed description, and the equivalent concepts thereof.

Claims

1. A polypeptide having the amino acid sequence of SEQ ID NO:1 (VH-CDR1), A polypeptide having the amino acid sequence of SEQ ID NO: 7 (VH-CDR2), A polypeptide having the amino acid sequence of SEQ ID NO: 16 (VH-CDR3), A polypeptide having the amino acid sequence of SEQ ID NO: 19 (VL-CDR1), A polypeptide having the amino acid sequence of SEQ ID NO:27 (VL-CDR2), and A polypeptide having the amino acid sequence of SEQ ID NO:33 (VL-CDR3) An anti-MUC1 (Mucin-1) antibody or an antigen-binding fragment thereof, comprising:

2. A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6 (VH-CDR1); A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 15 (VH-CDR2); A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-18 (VH-CDR3); A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 26 (VL-CDR1); A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-32 (VL-CDR2), and A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-38 (VL-CDR3) The anti-MUC1 antibody or antigen-binding fragment thereof of claim 1,

3. A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 77-86; and A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-96. The anti-MUC1 antibody or antigen-binding fragment thereof of claim 1,

4. The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 1, which has an activity of inducing apoptosis of cancer cells expressing MUC1.

5. The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-MUC1 antibody is a monoclonal antibody.

6. The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 1, which specifically recognizes a peptide comprising the amino acid sequence of SEQ ID NO:

109.

7. The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv)2, Fab, Fab' and F(ab')2 of the anti-MUC1 antibody.

8. The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-MUC1 antibody is a mouse-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

9. A bispecific antibody comprising the MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and one or more antigen-binding sites specific for a T cell activation antigen.

10. 10. The bispecific antibody of claim 9, wherein the T cell activation antigen is the CD3 T cell coreceptor (CD3) antigen.

11. the bispecific antibody comprises an anti-CD3 antibody or an antigen-binding fragment thereof comprising an antigen-binding site for the CD3 antigen; The bispecific antibody of claim 10, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an OKT3 antibody or antigen-binding fragment thereof, an UCHT-1 antibody or binding fragment thereof, or an SP34 antibody or antigen-binding fragment thereof.

12. The bispecific antibody of claim 11 , wherein the anti-CD3 antibody or antigen-binding fragment thereof is linked to the C-terminus or N-terminus of the anti-MUC1 antibody or antigen-binding fragment thereof.

13. The bispecific antibody of claim 11, wherein the antigen-binding fragment of the anti-CD3 antibody is selected from the group consisting of scFv, (scFv)2, Fab, Fab' and F(ab')2 of the anti-CD3 antibody.

14. The anti-CD3 antibody or antigen-binding fragment thereof comprises: A polypeptide having the amino acid sequence of SEQ ID NO:97 (VH-CDR1), A polypeptide having the amino acid sequence of SEQ ID NO:98 (VH-CDR2); A polypeptide having the amino acid sequence of SEQ ID NO:99 (VH-CDR3), A polypeptide having the amino acid sequence of SEQ ID NO: 100 (VL-CDR1), A polypeptide having the amino acid sequence of SEQ ID NO: 101 (VL-CDR2), and Polypeptide having the amino acid sequence of SEQ ID NO: 102 (VL-CDR3) 12. The bispecific antibody of claim 11 , comprising:

15. A pharmaceutical composition for preventing or treating cancer, comprising an anti-MUC1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or a bispecific antibody according to any one of claims 9 to 14.

16. The pharmaceutical composition for preventing or treating cancer according to claim 15, wherein the cancer is one or more cancers selected from the group consisting of blood cancer, bone marrow cancer, thyroid cancer, uterine cancer, skin cancer, melanoma, breast cancer, colon cancer, lung cancer, gastric cancer, prostate cancer, and pancreatic cancer.

17. A polynucleotide encoding an anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a bispecific antibody according to any one of claims 9 to 14.

18. A recombinant vector comprising the polynucleotide of claim 17.

19. A recombinant cell comprising the recombinant vector of claim 18.

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