Immuno-oncology composition comprising Anti-MUC1 antibody

Abstract

The present invention relates to: an immuno-oncology composition comprising an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide comprising the C-terminal extracellular domain of Mucin 1 (MUC1); and a use for inducing an immuno-oncology response in a subject by using same. The anti-MUC1 antibody or antigen-binding fragment thereof that specifically binds to a polypeptide comprising the C-terminal extracellular domain of MUC1 according to the present invention does not exhibit direct cytotoxicity to cancer cells, induces an increase in the activity of immune cells, and exhibits antibody-dependent cellular cytotoxicity (ADCC) by inhibiting immune evasion of cancer. Therefore, the anti-MUC1 antibody or antigen-binding fragment thereof can be used as various types of immuno-oncology agents, immuno-oncology adjuvants, or combination therapeutic agents for inducing an immune response in a subject and enhancing an anticancer effect.

Description

Immuno-anticancer composition containing anti-MUC1 antibody

[0001] The present invention relates to an immuno-anticancer composition comprising an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of Mucin 1 (MUC1); and to a use of inducing an immuno-anticancer response in an individual using the same.

[0002]

[0003] MUC1 is a highly glycosylated monotransit type I transmembrane protein in which the N-terminal domain (MUC1-N) and the C-terminal extracellular domain form a stable heteromeric complex. MUC1 is highly polymorphic, and it is known that over 90 isoforms exist in the N-terminal domain's VNTR (variable number tandem repeat) region, differing in the number of tandem repeats. MUC1 is expressed in glandular or luminal epithelial cells of many organs, such as the breast, stomach, esophagus, pancreas, urethra, lungs, kidneys, and gallbladder. In normal tissues, the negatively charged carbohydrate of MUC1 forms a physical barrier that protects the basal epithelium from dehydration, pH changes, pollen, and microorganisms.

[0004] As such, MUC1 is expressed in all epithelial cells, but in cancer cells, a truncated form called MUC1-C is produced, and massive expression of MUC1-C has been confirmed particularly in breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, and multiple myeloma. These are known to play an important role in the growth of cancer tumors, and specifically, they are known to play a crucial role in promoting the migration, invasion, proliferation, and metastasis of cancer cells by playing an important role in interactions with integrins that are important for cancer cell growth; therefore, they are becoming important targets for the development of anticancer drugs.

[0005] Meanwhile, methods used to treat cancer include chemotherapy, which involves administering chemical anticancer drugs; cytotoxic anticancer therapy, which induces a direct apoptotic effect on cancer cells; targeted anticancer therapy, which eliminates cancer cells by delivering drugs that target specific genes, proteins, or signaling pathways known to play a key role in cancer development; and immunotherapy, which activates the function of immune cells. In particular, since conventional cytotoxic treatments that directly attack cancer cells can cause side effects such as toxicity to normal cells, immunotherapy, which treats cancer by maximizing the function of immune cells present in the body, has recently been gaining popularity.

[0006] However, there are not many types of approved immunotherapies to date, and there is a need to develop new immunotherapies targeting various targets.

[0007]

[0008] While researching novel immunotherapeutic agents, the inventors confirmed that an anti-MUC1 antibody or its antigen-binding fragment, which specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 of the present invention, can be utilized as an immunotherapeutic agent by inducing increased activity of immune cells and inhibiting immune evasion by cancer, thereby exhibiting antibody-dependent cellular cytotoxicity (ADCC).

[0009] Accordingly, the object of the present invention is to provide an immuno-anticancer composition comprising an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1, and the use thereof.

[0010]

[0011] To achieve the above objective, the present invention provides a composition for inducing increased immune cell activity, comprising as an active ingredient an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

[0012] In addition, the present invention provides a composition for inducing the internalization of the C-terminal extracellular domain of MUC1, comprising as an active ingredient an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

[0013] The present invention also provides a composition for inducing antibody-dependent cytotoxicity (ADCC), comprising as an active ingredient an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

[0014] In addition, the present invention provides an immuno-anticancer composition comprising, as an active ingredient, an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

[0015] In addition, the present invention provides an immuno-anticancer composition comprising an anti-MUC1 antibody that specifically binds to a polypeptide comprising the C-terminal extracellular domain of MUC1, or an antibody-conjugate comprising an antigen-binding fragment thereof.

[0016] In addition, the present invention provides an immuno-anticancer composition comprising an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1, or a bispecific antibody comprising an antigen-binding fragment thereof.

[0017] The present invention also provides an immuno-anticancer composition comprising an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1, or a chimeric antigen receptor (CAR) comprising an antigen-binding fragment thereof.

[0018] In addition, the present invention provides an immuno-anticancer composition comprising: an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide comprising the C-terminal extracellular domain of MUC1; a chimeric antigen receptor (CAR); and an immune cell comprising the chimeric antigen receptor.

[0019] In addition, the present invention provides an immuno-anticancer treatment method comprising the step of administering an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 to an individual in need thereof.

[0020]

[0021] An anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 according to the present invention exhibits antibody-dependent cellular cytotoxicity (ADCC) by inducing increased activity of immune cells and suppressing immune evasion by cancer without exhibiting direct cytotoxicity to cancer cells; therefore, it can be used as various forms of immunotherapeutic agents, immunotherapeutic adjuvants, or combination therapies to enhance anticancer effects by inducing an immune response in an individual.

[0022]

[0023] Figure 1 shows the results of confirming the binding of the anti-MUC1 antibody of the present invention to the full length of MUC1 and the C-terminal extracellular domain (MUC1-C) of MUC1 in various cancer cell lines.

[0024] Figure 2 shows the results of confirming the cytotoxicity of the anti-MUC1 antibody of the present invention against cancer cells in DU145, PC-3, T47D, MDA-MB-231, and CAPAN-2 cell lines.

[0025] Figure 3 is a figure showing the results of confirming the toxicity of the anti-MUC1 antibody in normal rats and mice according to the present invention. Figure 3a shows the experimental schedule, and Figure 3b shows the change in body weight of each experimental group.

[0026] Figure 4 shows the results of confirming the ADCC effect of the anti-MUC1 antibody by performing mixed culture or single culture with PBMC in various cancer cell lines.

[0027] Figure 5 shows the results of confirming the CAPAN-2 cell death effect in PBMC mixed culture or single culture experimental groups through fluorescence imaging analysis.

[0028]

[0029] The present invention relates to a composition for inducing increased immune cell activity, inducing the internalization of the C-terminal extracellular domain of MUC1, or inducing antibody-dependent cytotoxicity (ADCC), comprising as an active ingredient an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

[0030] An anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 according to the present invention exhibits antibody-dependent cellular cytotoxicity (ADCC) by inducing increased activity of immune cells and suppressing immune evasion by cancer without exhibiting direct cytotoxicity to cancer cells; therefore, it can be used as various forms of immunotherapeutic agents, immunotherapeutic adjuvants, or combination therapies to enhance anticancer effects by inducing an immune response in an individual.

[0031]

[0032] The present invention will be described in detail below.

[0033] The present invention is characterized in that an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide comprising a C-terminal extracellular domain of MUC1 can recognize an antigen that is not glycosylated, and the C-terminal extracellular domain of MUC1 recognized by the antibody of the present invention may be composed of an amino acid sequence represented by SEQ ID NO. 9 (SVV VQLTLAFREG TINVHDVETQ FNQYKTEAASRYNLTISDVS VSDVPFPFSA QS), and may be used interchangeably with 'MUC1-C'.

[0034] In the present invention, "antibody" is a general term for a substance produced within the immune system by stimulation of an antigen, and its type is not particularly limited. The antibody refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen and acts as a receptor that specifically recognizes the antigen; it may include polyclonal antibodies, monoclonal antibodies, total antibodies, and antibody fragments. The antibody may be unnaturally generated, for example, recombinant or synthetically generated. The antibody may be an animal antibody (e.g., mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody, and preferably a humanized or human antibody. The antibody may be a monoclonal antibody. Furthermore, unless otherwise noted, the antibody may also include an antigen-binding fragment of an antibody possessing antigen-binding ability, but is not limited thereto.

[0035] In the present invention, the term "antigen-binding fragment" refers to any fragment of the antibody of the present invention that possesses the antigen-binding function of the antibody. The antigen-binding fragment may be referred to interchangeably with terms such as "fragment" or "antibody fragment," and the antigen-binding fragment may be Fab, Fab', F(ab')2, Fv, etc., but is not limited thereto.

[0036] In the present invention, 'anti-MUC1 antibody' means an antibody that binds to MUC1, or preferably to the C-terminal extracellular domain (MUC1-C) of MUC1.

[0037] The anti-MUC1 antibody or its antigen-binding fragment of the present invention can specifically act on cancer or tumor cells in which the terminal extracellular domain of MUC1-C is expressed at a high level compared to normal cells and glycosylation is low, by specifically recognizing MUC1-C, and can recognize / bind MUC1 proteins expressed on the entire surface of the cell as well as on one surface. In addition, the anti-MUC1 antibody or its antigen-binding fragment can bind to MUC1 proteins, particularly MUC1-C, to induce various immune anticancer responses.

[0038] In the present invention, the immune anticancer response may mean that rather than the anti-MUC1 antibody or its antigen-binding fragment binding to MUC1 or MUC1-C on the surface of cancer cells to exhibit direct cytotoxicity against cancer cells, it strengthens the immune system of an individual requiring treatment and promotes the elimination of cancer cells by immune cells by suppressing the immune evasion mechanisms of cancer cells. That is, the anti-MUC1 antibody or its antigen-binding fragment of the present invention exhibits antibody-dependent cytotoxicity (ADCC) and can suppress the immune evasion of cancer cells.

[0039] In the present invention, 'induction of increased immune cell activity' may mean activating various immune cells involved in the immune system, such as macrophages, neutrophils, monocytes, dendritic cells, NK cells, B cells, or T cells, such as helper T cells or cytotoxic T cells (SY Gun et al., 2019), by means of the anti-MUC1 antibody of the present invention or its antigen-binding fragment, and may mean promoting an immune attack against cancer cells in vivo through mechanisms such as promoting tumor suppression in the tumor microenvironment following macrophage activation, stimulating effector T cells following NK cell activation, promoting antibody formation following B cell activation, or secreting perforin and granzyme cytotoxic factors and promoting cancer cell death through T cell activation (H Ahmed et al., 2023).

[0040] In the present invention, 'immune evasion inhibition' may mean improving the immunosuppressive environment by inhibiting the expression of immune checkpoint receptor ligands of cancer cells or by removing regulatory T cells within the tumor microenvironment. The anti-MUC1 antibody of the present invention or its antigen-binding fragment may bind to the CTLA-4 receptor to block cytotoxic inhibitory signaling, thereby enabling T cells to destroy cancer cells, or bind to the PD-1 receptor to block the interaction between PD-L1 and PD-1 of cancer cells, thereby activating immune cells. Alternatively, the anti-MUC1 antibody of the present invention or its antigen-binding fragment may inhibit the expression of PD-L1 on the surface of cancer cells, thereby enabling immune cells to better recognize cancer cells and effectively attack the cancer.

[0041] In the present invention, 'induction of cell internalization of the C-terminal extracellular domain of MUC1 (MUC1-C)' means that the anti-MUC1 antibody of the present invention or its antigen-binding fragment binds to MUC1-C and induces removal by internalizing MUC1-C into the cancer cell. MUC1-C on the surface of cancer cells promotes the migration, invasion, proliferation, and metastasis of cancer cells through interactions with integrins important for cancer cell growth, and since the overexpression of MUC1-C is sufficient to induce anchorage-independent growth and tumorigenicity (Huang et al., 2003; Li et al., 2003b; Ren et al., 2002; Schroeder et al., 2004), if the anti-MUC1 antibody of the present invention or its antigen-binding fragment induces the removal of MUC1-C by internalizing it, the anticancer effect by immune cells can be maximized.

[0042] That is, the anti-MUC1 antibody of the present invention exhibits cytotoxicity by the body's immune system rather than direct cancer cell death activity, so it can be used for inducing 'antibody-dependent cytotoxicity (ADCC)' and / or for immuno-anticancer purposes.

[0043] Accordingly, the present invention provides a composition for inducing antibody-dependent cytotoxicity (ADCC) comprising, as an active ingredient, an anti-MUC1 antibody that specifically binds to a polypeptide comprising the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof; or an immuno-anticancer composition comprising, as an active ingredient, an anti-MUC1 antibody that specifically binds to a polypeptide comprising the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

[0044] More specifically, the immuno-anticancer composition of the present invention may be used to induce increased immune cell activity, inhibit immune evasion of cancer, or induce the internalization of MUC1-C into cells.

[0045] The immuno-anticancer composition of the present invention may be used alone, but may also be used as an immuno-anticancer adjuvant or combination therapy to aid in anticancer therapeutic activity. Accordingly, the present invention provides an immuno-anticancer adjuvant comprising, as an active ingredient, an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

[0046] The above anticancer treatment includes, without limitation, treatments for cancer that inhibit the proliferation of cancer-related cells, suppress metastasis and invasion, and induce apoptosis, such as radiation therapy, surgical procedures, and chemotherapy. That is, the anti-MUC1 antibody of the present invention or its antigen-binding fragment promotes or assists the elimination of cancer cells by immune cells by strengthening the patient's immune system and suppressing the immune evasion mechanisms of cancer cells, and can be used as an adjunct to various existing anticancer agents or treatment methods.

[0047] Meanwhile, the anti-MUC1 antibody or its antigen-binding fragment of the present invention may, without limitation, include an anti-MUC1 antibody or its antigen-binding fragment that recognizes a polypeptide containing the C-terminal extracellular domain (MUC1-C) of MUC1, preferably the amino acid sequence represented by SEQ ID NO. 9 (SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQS), but may be an antibody produced from hMUC1-1H7 (KCLRF-BP-00395), and may include an antibody characterized by including a complementation determining region (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) or a heavy chain variable region and a light chain variable region of the antibody produced therefrom.

[0048] In one embodiment, the anti-MUC1 antibody of the present invention or the antigen-binding fragment thereof may comprise a heavy chain CDR1 represented by SEQ ID NO. 1, a heavy chain CDR2 represented by SEQ ID NO. 2, and a heavy chain CDR3 represented by SEQ ID NO. 3; and a light chain CDR1 represented by SEQ ID NO. 4, a light chain CDR2 represented by SEQ ID NO. 5, and a light chain CDR3 represented by SEQ ID NO. 6.

[0049] In addition, in one embodiment, the anti-MUC1 antibody of the present invention or its antigen-binding fragment may include a heavy chain variable region represented by SEQ ID NO. 7 and a light chain variable region represented by SEQ ID NO. 8, and may include a heavy chain variable region or a light chain variable region having sequence homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with said sequence, as long as the function and activity thereof are maintained.

[0050] In addition, in the present invention, one or more residues of the lysine residues of the light chain CDR1 (KASQDIKSYLS) represented by SEQ ID NO. 4 may be substituted with other amino acids, for example, arginine (R), histidine (H), aspartic acid (D) or glutamic acid (G), glycine (G), alanine (A), valine (V), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W), leucine (L), and isoleucine (I). In a more preferred embodiment, lysine may be substituted with arginine or glycine, for example, the antibody may be one in which both the first and second lysines are substituted with arginine, or the first lysine is substituted with arginine and the second lysine is substituted with glycine. When a mutation is induced in the lysine of the light chain sequence in this way, glycosylation of the corresponding portion is reduced, and a high-purity antibody or antigen-binding fragment may be produced.

[0051] In addition, the anti-MUC1 antibody of the present invention or its antigen-binding fragment may be included in an immuno-anticancer composition in the form of an antibody-drug conjugate (ADC), a bispecific antibody, a chimeric antigen receptor (CAR), or an immune cell comprising said chimeric antigen receptor.

[0052] The above antibody-drug conjugate is in the form in which a drug is conjugated to an anti-MUC1 antibody or its antigen-binding fragment, so that an anticancer drug can be stably bound to the anti-MUC1 antibody or its antigen-binding fragment and delivered to target cancer cells. The binding of the drug and the anti-MUC1 antibody of the present invention can be achieved in a manner known in the art, for example, through a linker.

[0053] The above-mentioned dual-specific antibody means a form in which, among the two arms of the antibody, one arm comprises an anti-MUC1 antibody according to the present invention or an antigen-binding fragment thereof, or an antigen-binding fragment thereof, and the other arm comprises an antibody specific to an antigen other than MUC1, preferably an antibody specific to a cancer-related antigen or an immune checkpoint protein antigen, or an antibody specifically binding to an immunogenic cell-related antigen or an antigen thereof. The antigen to which the antibody other than the anti-MUC1 antibody included in the dual antibody according to the present invention binds is preferably selected from Her2, EGFR, VEGF, VEGF-R, CD-20, MUC16, CD30, CD33, CD52, PD-1, PD-L1, CTLA4, BTLA4, EphB2, E-selectin, EpCam, CEA, PSMA, PSA, ERB3, c-MET, etc. as cancer-related antigens or immune checkpoint protein antigens, and may be selected from TCR / CD3, CD16(FcγRIIIa), CD44, CD56, CD69, CD64(FcγRI), CD89 and CD11b / CD18(CR3), etc., but is not limited thereto.

[0054] The chimeric antigen receptor (CAR) described above may have a general CAR structure having an anti-MUC1 antibody or an antigen-binding fragment thereof as a binding (target) domain according to the present invention, and, for example, includes an extracellular domain comprising an anti-MUC1 antibody or an antigen-binding fragment thereof that binds to MUC1 or MUC1-C; a transmembrane domain, one or more intracellular co-stimulation signaling domains, and a primary signaling domain.

[0055] In addition, the immune cells containing the chimeric antigen receptor may be CAR-T, CAR-NK (natural killer cell) and / or CAR-MA (Macrophage) cells.

[0056] The immuno-anticancer composition or immuno-anticancer adjuvant of the present invention may be used without limitation in MUC1-positive cancers, and the term 'MUC1-positive cancer' broadly refers to any cancer or tumor associated with or related to one or more pathological processes in which the expression of MUC1 or MUC1-C is characteristic of the disease or disorder. Such cancers include, but are not limited to, solid tumors and hematological malignancies. For example, it may include cancers of epithelial origin, namely adenocarcinoma and squamous cell carcinoma, and non-limiting examples include breast cancer, non-cellular lung cancer, uterine cancer, prostate cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, acute myeloid leukemia (AML), multiple myeloma, skin cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma, acute lymphoblastic leukemia (ALL), adult T-cell leukemia, chronic lymphoblastic leukemia (CLL), hair cell leukemia, myelodysplasia, It may include one or more cancers selected from the group consisting of myeloproliferative disorders, chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), human leukemia virus-type 1 (HTLV-1) leukemia, mastocytosis, acute lymphoblastic leukemia, lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, and solitary myeloma, and preferably may be breast cancer, non-cellular lung cancer, uterine cancer, prostate cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, acute myeloid leukemia (AML), or multiple myeloma.

[0057] In addition, the present invention relates to an immuno-anticancer treatment method comprising the step of administering an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 to an individual in need thereof.

[0058] In addition, the present invention relates to a method for inducing an immune anticancer response in an individual, comprising the step of administering an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 to an individual in need thereof.

[0059] The subject of the present invention is a subject being evaluated for or receiving treatment, and may be a mammal and preferably a human.

[0060] The anti-MUC1 antibody of the present invention or its antigen-binding fragment may be administered in a therapeutically effective amount capable of inducing an immune anticancer response in an individual, which refers to the amount of activator necessary to confer therapeutic benefits. For example, the 'therapeutically effective amount' is an amount that induces, improves, or otherwise causes improvement in pathological symptoms associated with the disease, disease progression, or physiological status, or enhances tolerance to the disorder.

[0061] In the treatment method of the present invention, an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide comprising the C-terminal extracellular domain of MUC1 may be administered in the form of a bispecific antibody comprising said anti-MUC1 antibody or an antigen-binding fragment thereof, a chimeric antigen receptor, or an immune cell comprising said chimeric antigen receptor.

[0062] In the present invention, the induction of an immune anticancer response may be an increase in immune cell activity in the individual; inhibition of immune evasion by cancer cells; internalization of MUC1-C into cells; or antibody-dependent cytotoxicity (ADCC).

[0063]

[0064] The definitions and numerical values ​​described in this specification shall be interpreted to include equivalents unless otherwise specified.

[0065] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by these embodiments.

[0066]

[0067] Experimental Example. Preparation of Anti-MUC1 (Mucin 1) Antibody

[0068] An anti-MUC1 antibody (PAb001) was prepared, and information on the heavy and light chain CDRs of the antibody and the heavy and light chain variable sequences of the humanized antibody is shown in Tables 1 and 2. The CDR sequences within the heavy and light chain variable sequences of the humanized antibody are each underlined. The anti-MUC1 antibody was used in the following experiments.

[0069]

[0070] Heavy chain amino acid sequence Light chain amino acid sequence CDR1(Sequence No. 1)GYTFTSYWMHCDR1(Sequence No. 4)KASQDIKSYLSCDR2(Sequence No. 2)YINPGTGYIEYNQKFKDCDR2(Sequence No. 5)YATRLADCDR3(Sequence No. 3)STAPFDYCDR3(Sequence No. 6)LQYDESPYT

[0071] Sequence Information Heavy chain variable sequence (Sequence No. 7) EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMHWVQQAPGKGLEWIGYINPGTGYIEYNQKFKDRVTITADKSTDTAYMELSSLRSEDTAVYYCASSTAPFDYWGQGTLVTVSS Light chain variable sequence (Sequence No. 8) EIVLTQSPGTLSLSPGERATLSCKASQDIKSYLSWYQQKPGQAPRLLIYYATRLADGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCLQYDESPYTFGQGTKLEIKR

[0072]

[0073]

[0074] Example 1. Confirmation of MUC1 binding of anti-MUC1 antibody

[0075] To determine whether the anti-MUC1 antibody of the present invention can bind to endogenous MUC1 protein in cancer cells, Western blotting was performed using lysates of acute myeloid leukemia cell lines THP-1 and MV4-11, pancreatic cancer cell line CAPAN-2, prostate cancer cell lines DU145 and PC-3, triple-negative breast cancer cell line MM-231 (MDA-MB-231), and breast cancer cell line T47D. Cell lysates of each cell were immunoprecipitated (IP) with the anti-MUC1 antibody, and immunoblotting was performed using a commercially available anti-MUC1-N antibody (MUC1-NT Ab) or anti-MUC1-C antibody (MUC1-CT Ab), and the results are shown in Figure 1.

[0076] In Figure 1 (A), binding of the anti-MUC1 antibody to MUC1-C was observed at approximately 25 kDa, and in (B), binding of the anti-MUC1 antibody to MUC1-N was observed at approximately 150 kDa, confirming binding to the full-length protein of MUC1 or MUC1-C in DU154, T47D, and CAPAN-2 cell lines.

[0077]

[0078] Example 2 Confirmation of the anti-tumor inhibitory effect of the anti-MUC1 antibody

[0079] Experiments were conducted to confirm the tumor-suppressing effect of the anti-MUC1 antibody in a cancer mouse model prepared by inoculating the pancreatic cancer CAPAN-2 cell line. For xenograft analysis, 5 x 10⁶ in 50% Matrigel 6 CAPAN-2 cells were subcutaneously inoculated into the dorsal right flank of 16 BALB / cAnCrj-nu / nu mice. The tumor volume was 75 mm² 3When this occurred, the mice were randomly divided into two treatment groups of eight mice each (eight mice / group: PBS treatment group and anti-MUC1 antibody treatment group). The antibody was administered intravenously twice a week at a dose of 10 mg / kg, and tumor diameters were measured at 7-day intervals using calipers, and changes in the mice's body weight were measured. Changes in tumor size and body weight following anti-MUC1 antibody treatment are shown in Tables 3 and 4.

[0080]

[0081]

[0082]

[0083]

[0084] As shown in Table 3, the tumor size decreased significantly compared to the PBS-treated control group starting from day 35 following the administration of the anti-MUC1 antibody, and as shown in Table 4, it was confirmed that there was no change in body weight. These results demonstrate that the anti-MUC1 antibody can effectively achieve tumor suppression without exhibiting toxicity.

[0085]

[0086] Example 3. Cancer cell death effect of anti-MUC1 antibody

[0087] To confirm whether the anti-MUC1 antibody of the present invention exhibits a direct killing effect on cancer cells, the anti-MUC1 antibody was treated to prostate cancer cell lines PC-3, DU145, breast cancer cell lines T47D, MDA-MB-231 (MM-231), and pancreatic cancer cell line CAPAN-2 at a concentration of 0.1 to 10 μg / ml, and the inhibitory effect on cell proliferation was confirmed. The results of confirming the cancer cell killing effect according to the treatment concentration for each cell type are shown in Figure 2.

[0088] As shown in Figure 2, even when the anti-MUC1 antibody was increased to 10 μg / ml, no significant cytotoxicity or apoptotic effect was observed in any of the cells. This result surprisingly demonstrates that the tumor suppression effect observed in Example 2 was not due to the direct cancer cell apoptotic effect of the anti-MUC1 antibody.

[0089]

[0090] Example 4. Confirmation of toxicity of anti-MUC1 antibody in normal rats and mice

[0091] Since it was confirmed through Example 3 that the anti-MUC1 antibody does not have a direct cancer cell death effect, additional experiments were conducted to determine whether the anti-MUC1 antibody exhibits self-toxicity regardless of the presence of cancer cells. Specifically, the antibody of the present invention was intravenously injected at 10, 30, and 60 mg / kg into 7-week-old normal SD rats (n=4) and C57BL6 mice (n=4), and changes in body weight of each mouse were observed. A schematic diagram of the experimental method and changes in body weight of each experimental group are shown in Figure 3.

[0092] As shown in Figure 3b, no significant changes in body weight were observed in both mouse and rat models, and no adverse reactions or deaths to the drug occurred in any of the individuals. This means that the anti-MUC1 antibody does not exhibit inherent toxicity.

[0093]

[0094] Example 5. Confirmation of ADCC (Antibody-Dependent Cellular Cytotoxicity) Effect of Anti-MUC1 Antibody

[0095] Experiments were conducted to confirm whether the anti-tumor effect of the anti-MUC1 antibody shown in Example 2 was due to ADCC. Specifically, the anti-MUC1 antibody was applied to cancer cell lines CAPAN-2, DU145, PC3, and MM-231, and PBMCs were cultured either in a mixed culture or alone to confirm the apoptotic effect in each cancer cell. The apoptotic effect confirmed in each cancer cell is shown in Figure 4.

[0096] As shown in Figure 4, no distinct apoptotic effect was observed in the group of cancer cells not co-cultured with PBMC, whereas an apoptotic effect caused by treatment with the anti-MUC1 antibody of the present invention was observed in all the groups of cancer cells co-cultured with PBMC. In particular, an increase in apoptosis of approximately 60% and 40% was confirmed in pancreatic cancer cells CAPAN2 and breast cancer cells DU145, respectively. These results indicate that the anti-tumor effect of the anti-MUC1 antibody is due to ADCC.

[0097] Additionally, experimental groups in which CAPAN-2 cell lines treated with anti-MUC1 antibodies were cultured in combination with PBMC or CAPAN-2 cells cultured alone with PBMC were stained and fluorescence imaging analysis was performed to confirm cell death, and the results are shown in Figure 5.

[0098] As shown in Figure 5, in the control group that was not treated with the antibody of the present invention, no PI (Propidium Iodide) reaction indicating cell death was observed, but in treatment groups 1 and 2, in which PBMC co-culture was performed with antibody treatment, cell death (yellow) was observed in both.

[0099]

[0100] Example 6. Confirmation of the ADCC effect mechanism of the anti-MUC1 antibody

[0101] Experiments were conducted to determine whether the ADCC effect exhibited by the anti-MUC1 antibody is achieved by inducing increased immune cell activity or the internalization of MUC1-C into cells.

[0102]

[0103] 6.1 Confirmation of Apoptosis Signaling Activation

[0104] Experiments were conducted to determine whether the anti-MUC1 antibody activates Fcγ signaling using reporter cell lines expressing FcγRIIIa. These target cells were co-cultured with effector cell lines (Jurkat) engineered to overexpress human CTLA-4 on the cell surface and to express Fcγa upstream of the NFAT response element (RE) that induces the expression of firefly luciferase. The cells were treated with either the anti-MUC1 antibody or the control antibody (IgG1) and cultured. Luminescence measurements showed that the anti-MUC1 antibody activated Fcγa signaling in the effector cells.

[0105] In addition, Jurkat tumor cells were treated with an anti-MUC1 antibody, and antibody-mediated NK cell activation by CD16 was measured. As a result, the degranulation fraction of NK cells increased following treatment with the anti-MUC1 antibody, and it was confirmed that the anti-MUC1 antibody can induce NK cell-mediated ADCC.

[0106]

[0107] 6.2 Confirmation of the Internalization-Promoting Effect of MUC1-C

[0108] Since MUC1-C on the surface of cancer cells evades and suppresses immune anticancer responses by interfering with the interaction between receptors on the cancer cell surface and immune cells, the internalization and clearance of MUC1-C can enhance the immune anticancer effect. Experiments were conducted to confirm whether the anti-MUC1 antibody induces clearance by promoting its internalization into cancer cells after binding to MUC1-C. Pancreatic and colorectal cancer cells were treated with the anti-MUC1 antibody of the present invention and cultured; subsequently, to confirm cell behavior, the cells were immobilized and stained using a secondary antibody. Afterward, the intracellular migration of MUC1-C bound to the anti-MUC1 antibody was confirmed using a confocal laser microscope. In addition, changes in the expression of MUC1-C present on the cell surface and the total expression of MUC1-C following treatment with the anti-MUC1 antibody were confirmed through quantitative protein analysis.

[0109] As a result, it was confirmed that the anti-MUC1 antibody is involved in the internalization of MUC1-C present on the cell surface.

[0110]

[0111] Based on the above results, it was confirmed that the anti-MUC1 antibody of the present invention can be utilized as an immuno-anticancer agent that increases the activity of immune cells and induces the internalization of MUC1-C, thereby exhibiting antibody-dependent cell-mediated cytotoxicity.

Claims

1. A composition for inducing increased immune cell activity, comprising as an active ingredient an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

2. A composition for inducing the internalization of the C-terminal extracellular domain of MUC1, comprising as an active ingredient an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

3. A composition for inducing antibody-dependent cytotoxicity (ADCC) comprising, as an active ingredient, an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

4. In any one of paragraphs 1 through 3, The above antibody or its antigen-binding fragment comprises a heavy chain CDR1 represented by SEQ ID NO. 1, a heavy chain CDR2 represented by SEQ ID NO. 2, and a heavy chain CDR3 represented by SEQ ID NO. 3; and A composition comprising a light chain CDR1 represented by SEQ ID NO. 4, a light chain CDR2 represented by SEQ ID NO. 5, and a light chain CDR3 represented by SEQ ID NO.

6.

5. A composition according to claim 4, characterized in that one or more of the lysine residues of the light chain CDR1 are substituted with other amino acids.

6. A composition according to claim 5, characterized in that the lysine residue is substituted with an amino acid selected from the group consisting of arginine (R), histidine (H), aspartic acid (D) or glutamic acid (G), glycine (G), alanine (A), valine (V), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W), leucine (L) and isoleucine (I).

7. An immuno-anticancer composition comprising, as an active ingredient, an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 or an antigen-binding fragment thereof.

8. In claim 7, the above composition is an immuno-anticancer composition for inducing increased immune cell activity, inhibiting immune evasion of cancer, or inducing the internalization of the C-terminal extracellular domain of MUC1.

9. An immuno-anticancer composition comprising an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1, or an antibody-conjugate comprising an antigen-binding fragment thereof.

10. An immuno-anticancer composition comprising an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1, or a bispecific antibody comprising an antigen-binding fragment thereof.

11. An immuno-anticancer composition comprising an anti-MUC1 antibody that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1, or a chimeric antigen receptor (CAR) comprising an antigen-binding fragment thereof.

12. An immuno-anticancer composition comprising: an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1; and an immune cell comprising the chimeric antigen receptor.

13. In any one of claims 7 to 12, the composition is used for breast cancer, non-cellular lung cancer, uterine cancer, prostate cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, acute myeloid leukemia (AML), multiple myeloma, skin cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma, acute lymphoblastic leukemia (ALL), adult T-cell leukemia, chronic lymphoblastic leukemia (CLL), hair cell leukemia, myelodysplasia, myeloproliferative disorder, chronic myeloid An immuno-anticancer composition for treating one or more cancers selected from the group consisting of leukemia (CML), myelodysplastic syndrome (MDS), human leukemia virus-type 1 (HTLV-1) leukemia, mastocytosis, acute lymphoblastic leukemia, lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, and solitary myeloma.

14. A step of administering an anti-MUC1 antibody or an antigen-binding fragment thereof that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 to an individual in need thereof; an immuno-anticancer treatment method comprising:

15. An immuno-anticancer treatment method according to claim 14, wherein the anti-MUC1 antibody or its antigen-binding fragment that specifically binds to a polypeptide containing the C-terminal extracellular domain of MUC1 is administered in the form of a bispecific antibody containing the anti-MUC1 antibody or its antigen-binding fragment, a chimeric antigen receptor, or an immune cell containing the chimeric antigen receptor.

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