Her2 targeting agent

Abstract

To provide an antibody or antigen-binding fragment thereof that binds to HER2 expressed on a cancer cell or a fragment thereof.SOLUTION: The present invention provides an isolated antibody or antigen-binding fragment of the antibody having a stronger binding reactivity to HER2 expressed on a cancer cell than to HER2 expressed on a noncancer cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to HER2 targeting agents. [Background technology]

[0002] HER2 is a receptor tyrosine kinase with a structure similar to that of the epidermal growth factor receptor (EGFR), and is also called EGFR2, ERBB2, CD340, or NEU. The HER2 protein is also expressed in normal cells and is involved in regulating cell proliferation and differentiation, but amplification and / or mutation of the HER2 gene can cause uncontrollable cell proliferation, making the cells malignant, and it is known that HER2 is expressed in many cancers, including salivary gland cancer, gastric cancer, breast cancer, and ovarian cancer. Trastuzumab is an antibody drug that specifically binds to HER2 expressed on the surface of cancer cells. By binding to HER2 on cancer cells, trastuzumab can suppress signals that stimulate the proliferation of cancer cells or induce immune activity to destroy cancer cells.

[0003] In order to exert such a cell proliferation inhibitory effect or cytotoxic effect specifically on cancer cells, an antibody that specifically binds only to cancer cells is required. The CasMab method has been developed as a technique for producing antibodies specific to cancer cells (Non-Patent Documents 1 to 3). In the CasMab method, an antigen protein expressed in LN229 cells, a glioblastoma cell line with a specific glycan profile, is used as an immunogen to produce an antibody. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kato and Kaneko, 2014, Sci. Rep., 4: 5924 [Non-Patent Document 2] Yamada et al., 2017, Monoclon. Antib. Immunodiagn. Immunother., 36(2): 72 [Non-Patent Document 3] Kaneko et al., 2020, Biochem. Biophys. Rep., 24: 100826 Summary of the Invention

[0005] The present invention provides an antibody or antigen-binding fragment thereof that binds to HER2. The present invention preferably provides an antibody or antigen-binding fragment thereof that specifically binds to HER2. The present invention provides an antibody or antigen-binding fragment thereof that has binding affinity to HER2 expressed on cancer cells. More preferably, the present invention provides an antibody or antigen-binding fragment thereof that may have stronger binding affinity to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells. More preferably, the present invention provides an antibody or antigen-binding fragment thereof that does not significantly react to HER2 expressed on non-cancer cells and may specifically react to HER2 expressed on cancer cells. The present invention also provides a method for treating cancer in a patient using the antibody or antigen-binding fragment thereof that binds to HER2. Furthermore, the present invention provides a method for detecting HER2-positive cancer cells, comprising a patient contacting the antibody or antigen-binding fragment thereof that binds to HER2 with a cancer sample obtained from the patient, the method comprising the step of: detecting HER2-positive cancer cells; and determining whether a patient is responsive to HER2-targeted therapy using the antibody or antigen-binding fragment thereof that binds to HER2.

[0006] The present inventors have produced an antibody that specifically recognizes HER2 expressed in cancer cells. The present inventors have also found that HER2 expressed in normal cells and HER2 expressed in cancer cells can be distinguished by an antibody, and have produced an antibody that can distinguish between them. The present invention is based on such findings.

[0007] According to the present invention, the following aspects are provided. [0] An isolated antibody or antigen-binding fragment thereof that has binding reactivity with HER2 expressed on cancer cells. [1] An isolated antibody or antigen-binding fragment thereof that has stronger binding reactivity to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells. [2] (i) The antibody or antigen-binding fragment thereof according to [0] or [1] above, which binds to a peptide comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36). [3] (ii) The antibody or antigen-binding fragment thereof according to [0], [1] or [2] above, which has a stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A in a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622), for example, which has a stronger reactivity to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) having a point mutation of W614A, or a peptide having a point mutation of K615A or F616A {wherein the portion of the extracellular domain of HER2 may be the region of amino acid numbers 613 to 622 in the amino acid sequence set forth in SEQ ID NO: 2}. [4] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 18; A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20, and A light chain CDR1 having the amino acid sequence of SEQ ID NO: 21; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 22; and having a light chain variable region having a light chain CDR3 having the amino acid sequence of SEQ ID NO:23; (iv) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 24; A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 25, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 26, and A light chain CDR1 having the amino acid sequence of SEQ ID NO: 27; A light chain CDR2 having the amino acid sequence of SEQ ID NO: 28, and having a light chain variable region having a light chain CDR3 having the amino acid sequence of SEQ ID NO:29; or The antibody or antigen-binding fragment thereof according to any one of [0] and [1] to [3] above, which has at least one substitution, addition or deletion in one or more CDRs of (iii) or (iv) above. [5] (vii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14; and having a light chain variable region having the amino acid sequence of SEQ ID NO: 15; (viii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16; and having a light chain variable region having the amino acid sequence of SEQ ID NO:17; or The antibody or antigen-binding fragment thereof according to any one of [0] and [1] to [4] above, which has at least one substitution, addition or deletion in each of the variable regions (vii) or (viii) above. [6] The antibody or antigen-binding fragment thereof described in any one of [0] and [1] to [5] above, which binds to a peptide produced by LN229 cells, a glioblastoma cell line, and which consists of the extracellular domain of HER2 described in SEQ ID NO: 3. [7] The antibody or antigen-binding fragment thereof described in any one of [0] and [1] to [6] above, wherein the cancer cells are SK-BR-3 cells, a breast cancer cell line, and the non-cancerous cells are HaCaT cells, a normal human epidermal keratinocyte cell line. [8] A pharmaceutical composition comprising, as an active ingredient, a HER2 targeting agent having the antibody or antigen-binding fragment thereof described in any one of [0] and [1] to [7] above. [9] The antibody or antigen-binding fragment thereof according to any one of [0] and [1] to [7] above, which is conjugated with a cytotoxic agent.

[10] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in [9] above as an active ingredient.

[11] The pharmaceutical composition according to [8] or

[10] above for use in treating cancer.

[12] A cancer treatment agent comprising, as an active ingredient, the antibody or antigen-binding fragment thereof according to any one of [0] and [1] to [7] above.

[13] A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of [2] to [6] above.

[0008]

[14] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 18; A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20, and A light chain CDR1 having the amino acid sequence of SEQ ID NO: 21; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 22; and having a light chain variable region having a light chain CDR3 having the amino acid sequence of SEQ ID NO:23; (iv) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 24; A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 25, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 26, and A light chain CDR1 having the amino acid sequence of SEQ ID NO: 27; A light chain CDR2 having the amino acid sequence of SEQ ID NO: 28, and having a light chain variable region having a light chain CDR3 having the amino acid sequence of SEQ ID NO:29; or An antibody or antigen-binding fragment thereof that specifically binds to an epitope in the extracellular domain of HER2, which has at least one substitution, addition or deletion in one or more CDRs of (iii) or (iv) above.

[15] An antibody or antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells but can specifically react with HER2 expressed on cancer cells.

[0009]

[16] A method for producing, selecting, or identifying an antibody or an antigen-binding fragment of an antibody, comprising the steps of: A method comprising producing, selecting or identifying an antibody or an antigen-binding fragment of an antibody that does not significantly react with HER2 expressed on non-cancer cells but can specifically react with HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments of antibodies that bind to HER2 or a fragment thereof.

[17] A method for producing, selecting, or identifying an antibody or an antigen-binding fragment of an antibody, comprising the steps of: A method comprising producing, selecting or identifying an antibody or an antigen-binding fragment of an antibody that satisfies at least one of the following (i) to (iii) from a group of antibodies or antigen-binding fragments of the antibodies that bind to HER2 or a fragment thereof: (i) binds to a peptide comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36); and (ii) has a stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A in a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622), for example, has a stronger reactivity to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a peptide having a point mutation of W614A in a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622), or a peptide having a point mutation of K615A or F616A; and (iii) It has stronger binding reactivity to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells.

[18] A method for producing, selecting, or identifying an antibody or an antigen-binding fragment of an antibody, comprising the steps of: A method comprising producing, selecting or identifying an antibody or antigen-binding fragment thereof that binds to HER2 or a fragment thereof from a group of antibodies or antigen-binding fragments of antibodies that satisfy at least one selected from the group consisting of (i) to (ii) below. (i) binds to a peptide comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36); and (ii) It has stronger reactivity to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a peptide having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A in a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622).

[19] The method according to

[17] or

[18] above, further comprising producing, selecting or identifying, from the obtained group of antibodies or antigen-binding fragments of the antibodies, an antibody or an antigen-binding fragment of the antibody that does not significantly react with HER2 expressed on non-cancer cells but can specifically react with HER2 expressed on cancer cells.

[20] A method for producing, selecting, or identifying an antibody or an antigen-binding fragment of an antibody, comprising the steps of: A method comprising producing, selecting or identifying an antibody or an antigen-binding fragment of an antibody that does not significantly react with HER2 expressed on non-cancer cells but can specifically react with HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments of antibodies having any of (iii), (iv), and (xii) and at least one amino acid mutation (e.g., 1 to 10, for example, 1, 2, 3, 4, 5, 6, etc.) selected from the group consisting of substitution, addition, insertion, and deletion in at least one CDR.

[0010]

[20] The method according to any one of

[16] to

[19] above, comprising producing the selected or identified antibody or an antigen-binding fragment of the antibody in an antibody-producing cell (e.g., a CHO cell).

[21] An antibody obtained by the method of

[20] above.

[22] The method according to any one of

[16] to

[20] above, wherein the antibody is a monoclonal antibody (e.g., an isolated monoclonal antibody).

[23] A pharmaceutical composition comprising a HER2 targeting agent having the antibody or antigen-binding fragment thereof according to any one of [1] to [7] above.

[24] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in [9] above.

[25] The pharmaceutical composition according to

[23] or

[24] above for use in treating cancer.

[0011]

[0101] (xi) The antibody or antigen-binding fragment thereof described in [0] or [1] above, which binds to a peptide comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NO: 37.

[0102] (xii) The antibody or antigen-binding fragment thereof according to any one of the above [0], [1] to [3], and

[0101] , A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 81; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 82, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 83, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 84; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 85, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 86; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 87; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 88, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 89, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 90; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 91, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:92; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 93; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 94, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 95, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 96; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 97, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 98; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 99; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 100, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 101, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 102; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 103, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 104; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 105; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 106, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 107, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 108; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 109, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 110; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 111; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 112, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 113, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 114; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 115, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 116; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 117; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 118, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 119, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 120; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 121, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 122; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 123; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 124, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 125, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 126; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 127, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 128; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 129; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 130, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 131, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 132; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 133, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 134; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 135; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 136, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 137, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 138; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 139, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 140; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 141; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 142, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 143, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 144; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 145, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 146; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 147; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 148, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 149, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 150; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 151, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 152; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 153; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 154, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 155, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 156; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 157, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 158; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 159; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 160, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 161, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 162; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 163, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 164; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 165; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 166, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 167, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 168; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 169, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 170; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 171; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 172, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 173, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 174; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 175, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 176; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 177; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 178, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 179, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 180; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 181, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 182; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 183; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 184, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 185, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 186; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 187, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 188; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 189; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 190, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 191, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 192; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 193, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 194; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 195; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 197, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 198; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 199, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 200; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 201; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 202, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 203, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 204; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and An antibody or antigen-binding fragment thereof having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 206.

[0103] (xiii) (a) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 39 and a light chain variable region having the amino acid sequence of SEQ ID NO: 40; (b) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41 and a light chain variable region having the amino acid sequence of SEQ ID NO: 42; (c) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 and a light chain variable region having the amino acid sequence of SEQ ID NO: 44; (d) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 45 and a light chain variable region having the amino acid sequence of SEQ ID NO: 46; (e) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (f) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50; (g) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; (h) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO: 54; (i) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 55 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; (j) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57 and a light chain variable region having the amino acid sequence of SEQ ID NO: 58; (k) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 59 and a light chain variable region having the amino acid sequence of SEQ ID NO: 60; (l) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 61 and a light chain variable region having the amino acid sequence of SEQ ID NO: 62; (m) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and a light chain variable region having the amino acid sequence of SEQ ID NO: 64; (n) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and a light chain variable region having the amino acid sequence of SEQ ID NO: 66; (o) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 68; (p) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 70; (q) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (r) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 73 and a light chain variable region having the amino acid sequence of SEQ ID NO: 74; (s) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 75 and a light chain variable region having the amino acid sequence of SEQ ID NO: 76; (t) an antibody or an antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 77 and a light chain variable region having the amino acid sequence of SEQ ID NO: 78; (u) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 79 and a light chain variable region having the amino acid sequence of SEQ ID NO: 80; or (xiv) An antibody or antigen-binding fragment thereof described in any one of [0], [1] to [3],

[0101] and

[0102] above, which has at least one substitution, addition or deletion in at least one or more variable regions of the antibody or antigen-binding fragment thereof described in any one of (xiii)(a) to (u) above.

[0104] 1. A method for producing, selecting or identifying an antibody or a binding fragment thereof, comprising: A method comprising producing, selecting or identifying an antibody or antigen-binding fragment thereof that satisfies the following (xi) from a group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof.

[0105] 1. A method for producing, selecting or identifying an antibody or antigen-binding fragment thereof, comprising: A method comprising producing, selecting or identifying an antibody or antigen-binding fragment thereof that satisfies the following (xii) from a group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof.

[0106] 1. A method for producing, selecting or identifying an antibody or antigen-binding fragment thereof, comprising: A method comprising producing, selecting or identifying an antibody or antigen-binding fragment thereof that satisfies the following (xiii) from a group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof.

[0107] 1. A method for producing, selecting or identifying an antibody or antigen-binding fragment thereof, comprising: A method comprising producing, selecting or identifying an antibody or antigen-binding fragment thereof that satisfies the following (xiv) from a group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof.

[0012]

[0110] A method according to any one of the above-mentioned

[0101] to

[0103] , comprising producing the selected or identified antibody or antigen-binding fragment thereof in an antibody-producing cell (e.g., a CHO cell).

[0111] An antibody obtained by any of the methods

[0104] to

[0107] above.

[0112] The method according to any one of the above-mentioned

[0104] to

[0107] , wherein the antibody is a monoclonal antibody (e.g., an isolated monoclonal antibody).

[0113] A pharmaceutical composition comprising a HER2 targeting agent having an antibody or an antigen-binding fragment thereof described in any one of

[0101] to

[0103] above.

[0114] A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof described in any one of

[0101] to

[0103] above.

[0115] A pharmaceutical composition described in

[0113] or

[0114] above for use in treating cancer.

[0013]

[0120] The method described in any of the above-mentioned

[0104] to

[0107] , wherein the HER2 is HER2 expressed on LN229 cells.

[0121] The method according to any one of the above-mentioned

[0104] to

[0107] , wherein the HER2 fragment is a peptide consisting of any one of the amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 31 to 37 (for example, the amino acid sequences set forth in SEQ ID NOs: 31 to 36).

[0014] According to the present invention, the antibody or antigen-binding fragment thereof of the present invention can be used as a HER2 targeting agent. According to the present invention, the antibody or antigen-binding fragment thereof of the present invention can be used, for example, in detecting cancer cells and / or treating cancer. [Brief description of the drawings]

[0015] [Figure 1A] FIG. 1A is a vector map of pCAG / PA-HER2-RAP-MAP used in the Examples. [Figure 1B] FIG. 1B is a vector map of pCAG / PA-HER2ec-RAP-MAP used in the Examples. [Diagram 2] FIG. 2 shows that examples of the antibodies of the present invention (H2Mab-214 and H2Mab-250) bind to HER2 expressed on a breast cancer cell line (SK-BR-3 cells) while showing no reaction to HER2 expressed on a normal human epidermal keratinocyte cell line (HaCaT cells). [Diagram 3] FIG. 3 shows the results of immunohistochemical staining (IHC) demonstrating that examples of the antibodies of the present invention (H2Mab-214 and H2Mab-250) stain cancer cells diagnosed as HER2 negative by conventional diagnostic agents that examine the HER2 expression status of cancer cells. [Figure 4] FIG. 4 shows the binding of H2Mab-214 and H2Mab-250 to various partial peptides of HER2. [Diagram 5]Figure 5 shows the results of flow cytometry (FACS) analysis of CHO-K1 cells expressing various HER2 alanine substitution mutants (W614A, K615A, F616A) with an antibody against the PA tag (NZ-1), H2Mab-214 antibody, H2Mab-250 antibody, and a positive control antibody (trastuzumab). On the far left, CHO-K1 cells expressing wild-type HER2 were used as a positive control for the cells. [Figure 6A] FIG. 6A shows the results of epitope analysis of the antibody group obtained in Example 6. [Figure 6B] FIG. 6B shows the results of epitope analysis of the antibody group obtained in Example 6. [Figure 6C] FIG. 6C shows the results of epitope analysis of the antibody group obtained in Example 6. [Figure 6D] FIG. 6D shows the results of epitope analysis of the antibody group obtained in Example 6. [Figure 6E] FIG. 6E shows the results of epitope analysis of the antibody group obtained in Example 6. [Figure 7A] FIG. 7A shows the results of flow cytometry (FACS) analysis of the antibody group obtained in Example 6. [Figure 7B] FIG. 7B shows the results of flow cytometry (FACS) analysis of the antibody group obtained in Example 6. [Figure 7C] FIG. 7C shows the results of flow cytometry (FACS) analysis of the antibody group obtained in Example 6. [Figure 7D] FIG. 7D shows the results of flow cytometry (FACS) analysis of the antibody group obtained in Example 6. [Figure 7E] FIG. 7E shows the results of flow cytometry (FACS) analysis of the antibody group obtained in Example 6. [Figure 7F] FIG. 7F shows the results of flow cytometry (FACS) analysis of the antibody group obtained in Example 6. [Figure 7G] FIG. 7G shows the results of flow cytometry (FACS) analysis of the antibody group obtained in Example 6. [Figure 8A]FIG. 8A shows the results of immunohistochemical staining (IHC) of cancer cells and normal cells using the antibody group obtained in Example 6. [Figure 8B] FIG. 8B shows the results of immunohistochemical staining (IHC) of cancer cells and normal cells using the antibody group obtained in Example 6. [Figure 8C] FIG. 8C shows the results of immunohistochemical staining (IHC) of cancer cells and normal cells using the antibody group obtained in Example 6. [Figure 8D] FIG. 8D shows the results of immunohistochemical staining (IHC) of cancer cells and normal cells using the antibody group obtained in Example 6. [Figure 8E] FIG. 8E shows the results of immunohistochemical staining (IHC) of cancer cells and normal cells using the antibody group obtained in Example 6. [Figure 8F] FIG. 8F shows the results of immunohistochemical staining (IHC) of cancer cells and normal cells using the antibody group obtained in Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] <Definition> As used herein, the term "subject" refers to a "patient" that may be a mammal, and is preferably a human subject. More preferably, the subject may be a "cancer patient" that is suffering from or at risk of suffering from a tumor or cancer.

[0017] In this specification, the term "antibody" refers to an immunoglobulin, a protein having a structure in which two heavy chains (H chains) stabilized by disulfide bonds are associated with two light chains (L chains). The heavy chain is composed of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain is composed of a light chain variable region VL and a light chain constant region CL. Among these, the variable region fragment (Fv) composed of VH and VL is a region that is directly involved in antigen binding and provides diversity to the antibody. The antigen-binding region composed of VL, CL, VH, and CH1 is called the Fab region, and the region composed of the hinge region, CH2, and CH3 is called the Fc region. Among the variable regions, the region that directly contacts the antigen changes greatly and is called the complementarity-determining region (CDR). The portion other than the CDR that has relatively few mutations is called the framework region (FR). The light chain and heavy chain variable regions each have three CDRs, which are called heavy chain CDR1-3 and light chain CDR1-3, respectively, from the N-terminus. The antibody may be a recombinant protein (recombinant antibody), and may be produced in animal cells such as Chinese hamster ovary cells (CHO cells). The origin of the antibody is not particularly limited, and examples of the antibody include antibodies of non-human animals, antibodies of non-human mammals (e.g., mouse antibodies, rat antibodies, and camel antibodies), and human antibodies. The antibody may also be a chimeric antibody, a humanized antibody, or a fully human antibody. The antibody may be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody. A "chimeric antibody" is an antibody in which the heavy chain variable region and the light chain variable region are linked to heavy chain constant regions and light chain constant regions of different species, respectively. A humanized antibody means an antibody in which the corresponding positions of a human antibody are replaced with amino acid sequences characteristic of an antibody of non-human origin, and examples thereof include an antibody having heavy chain CDR1-3 and light chain CDR1-3 of an antibody produced by immunizing a mouse or rat, and all other regions, including the four framework regions (FR) of the heavy chain and the light chain, are derived from a human antibody. Such an antibody may also be called a CDR-grafted antibody. A "humanized antibody" may also include a human chimeric antibody. A "human chimeric antibody" is an antibody of non-human origin in which the constant region of the non-human antibody is replaced with the constant region of a human antibody. In the case of a human chimeric antibody, in terms of enhancing the ADCC activity, for example, the subtype of the human antibody used for the constant region may be IgG1. A "fully human antibody" means an antibody in which both the variable region consisting of FR and CDR and the constant region are derived from a human antibody. In addition, the antibody may be in the form of an antibody-drug conjugate in order to enhance the cytotoxicity of the antibody. The antibody may also be a multispecific antibody, for example, a bispecific antibody or a trispecific antibody. The antibody may be an isolated antibody or a purified antibody.

[0018] As used herein, the term "antigen-binding fragment" refers to a portion of an antibody that maintains its ability to bind to an antigen. The antigen-binding fragment may include the heavy chain variable region or the light chain variable region, or both, of the antibody of the present invention. The antigen-binding fragment may be chimerized or humanized. Examples of the antigen-binding fragment include Fab, Fab', F(ab')2, and Fv. The antibody-binding fragment may also include recombinantly produced conjugates or functional equivalents (e.g., other antibody portions having the form of scFv (single-chain Fv), diabody, scDb, tandem scFv, leucine zipper type, sc(Fv)2 (single-chain (Fv)2), etc.). Such an antigen-binding fragment of an antibody can be obtained, for example, by treating the antibody with an enzyme, without particular limitation. For example, Fab can be obtained by digesting the antibody with papain. Alternatively, F(ab')2 can be obtained by digesting the antibody with pepsin, and Fab' can be obtained by further reducing this. In the present specification, an antigen-binding fragment of such an antibody can be used. In scFv, VL and VH are linked by an artificial polypeptide linker, and the same antigen specificity as that of the original antibody can be maintained. VL and VH can be linked in the order of VH and VL or VL and VH from the N-terminus. The linker has a length of about 10 to 25 amino acids, is rich in glycine, and may contain amino acids such as serine and threonine to increase water solubility.

[0019] As used herein, the term "binding rate constant" (ka) and "dissociation rate constant" (kd) refer to the rate constant in the binding and dissociation reaction of two molecules. ka and kd are constants well known to those skilled in the art and can be determined appropriately using well-known techniques, such as surface plasmon resonance. The binding and dissociation constant (KD) of an antibody is calculated by kd / ka. The antibody may have a KD value for the target molecule of, for example, 10-7M or less, 10-8M or less, 10-9M or less, 10-10M or less, 10-11M or less, or 10-12M or less.

[0020] As used herein, strong (or high) binding affinity means that the antibody binds strongly to an antigen, for example, that the KD value is relatively small. Weak (or low) binding affinity means that the antibody binds weakly to an antigen, and high or low binding affinity can be, for example, when the binding partner is a cell, the amount of label of the labeled antibody bound to the cell by flow cytometry.

[0021] As used herein, an antibody that binds to HER2 (or a fragment thereof) can be an antibody that specifically binds to HER2 (or a fragment thereof). Here, specific means that the binding affinity is stronger when binding to a specified molecule (e.g., HER2) than when binding to other molecules (other types of molecules, mutants, molecules with other modifications, or molecules without modifications). An antibody that specifically binds can have a KD value of, for example, 10-7M or less, 10-8M or less, 10-9M or less, 10-10M or less, 10-11M or less, or 10-12M or less for HER2. In terms of the strength of binding affinity, the binding affinity for HER2 can be 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 100-fold or more, 1,000-fold or more, 10,000-fold or more, or 100,000-fold or more, in terms of KD value, than the binding affinity for any of the mutants specified below. The difference between stronger and weaker binding affinities can be statistically significant (eg, p<0.05, p<0.01, p<0.005, or p<0.0001).

[0022] As used herein, the term "antibody-drug conjugate" (ADC) refers to a substance in which an antibody and a drug are linked (conjugated). In an ADC, a monoclonal antibody or an antigen-binding fragment thereof can be advantageously used as the antibody. In an ADC, a monoclonal antibody and a drug can be linked via a suitable linker. An ADC binds to a membrane component (e.g., a transmembrane protein such as a receptor) on a cell membrane, is taken up into a cell by endocytosis or internalization, and is released into the cell after being detached from the antibody. By introducing a cleavable linker between the antibody and the drug in a cell, it is possible to cleave the linker in the cell, for example, in an endosome, to dissociate the drug from the antibody and release it into the cytoplasm. If a cytotoxic agent is used as the drug, it is possible to kill the cell to which the drug is delivered. As the cytotoxic agent, a chemotherapeutic agent, a radioisotope, and a toxin can be used.

[0023] As used herein, the term "HER2 targeting agent" refers to a drug that includes a binding molecule for HER2 or a fragment thereof (e.g., an antibody or an antigen-binding fragment thereof that binds to HER2 or a fragment thereof, or a fusion protein containing the antigen-binding fragment), and targets HER2 to produce its medicinal effect. Examples of HER2 targeting agents include an antibody or an antigen-binding fragment thereof that binds to HER2 or a fragment thereof, or a drug containing either of these. The drug may be an antibody itself having ADCC activity. The drug may be, for example, a cytotoxic agent. The drug may also be a cell having cytotoxic activity (e.g., an immune cell (e.g., T cell, NK cell, etc.)).

[0024] As used herein, "nucleic acid" refers to a polymer formed by a series of nucleotides, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Proteins are encoded by DNA and translated from mRNA. The DNA encoding a protein can be operably linked to a control sequence (e.g., a promoter). The DNA encoding a protein operably linked to a control sequence may be incorporated into a gene expression vector. The DNA encoding a protein may be cDNA produced by reverse transcription from mRNA. The mRNA encoding a protein may have an m7G cap at the 5' end and a poly(A) tail at the 3' end. Ribosomes bind to the mRNA encoding a protein, and the protein is translated from the mRNA.

[0025] As used herein, "cancer" refers to a disease characterized by uncontrolled proliferation of cells. Cancer cells may spread locally or throughout the body via the bloodstream or lymphatic system. Non-limiting examples of cancer include, for example, solid tumors. Non-limiting examples of cancer include, for example, hematopoietic tumors. Solid tumors include, for example, lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, uterine cancer, ovarian cancer, skin cancer, thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, biliary tract cancer, brain tumors, bone and soft tissue tumors, retroperitoneal tumors, angio-lymphangiosarcoma, and metastatic cancers thereof (e.g., metastatic solid tumors). Examples of hematopoietic tumors include leukemia, acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (asymptomatic and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. Examples of acute leukemia include acute lymphocytic leukemia, acute myelogenous leukemia, acute myelogenous leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia. Examples of chronic leukemia include chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia. The cancer may be, in particular, a HER2-positive cancer. Those skilled in the art can appropriately determine whether a cancer is HER2-positive using a well-known method. The antibody of the present invention or an antigen-binding fragment thereof may be used to determine whether a cancer is HER2-positive.

[0026] As used herein, "treatment" includes preventive treatment and therapeutic treatment, and means reducing or eliminating the cause of a disease in a patient having a disease, slowing or stopping its progression, alleviating, alleviating, improving or eliminating its symptoms, and / or inhibiting the worsening of its symptoms, and in the case where the disease is "cancer", it also includes inhibiting metastasis of the cancer.

[0027] In the present specification, "HER2" may be, for example, human HER2. Human HER2 may be encoded by, for example, the nucleotide sequence (SEQ ID NO: 1) registered under Genebank Accession No.: X03363. Human HER2 may also have the amino acid sequence (SEQ ID NO: 2) registered under UniprotKB ID: P04626. In the amino acid sequence of SEQ ID NO: 2, human HER2 has an amino acid region of amino acid numbers 1 to 22 that is a signal peptide, an amino acid region of amino acid numbers 23 to 652 that is an extracellular domain (which may have the amino acid sequence of SEQ ID NO: 3), an amino acid region of amino acid numbers 653 to 675 that is a transmembrane domain, and an amino acid region of amino acid numbers 676 to 1255 that is a cytoplasmic domain. HER2 may include naturally occurring HER2 variants. Since HER2 is a target, its function may be reduced or lost, but its function may be maintained or enhanced. HER2 that can be recognized as an epitope may, for example, have an amino acid region of amino acid numbers 611 to 618 in the amino acid sequence of SEQ ID NO: 2 (MPIWKFPD: SEQ ID NO: 34). In addition, HER2 that can be recognized as an epitope may, for example, have an amino acid region of amino acid numbers 612 to 618 in the amino acid sequence of SEQ ID NO: 2 (PIWKFPD: SEQ ID NO: 35). In this specification, a peptide consisting of an amino acid region of amino acid numbers 23 to 652 in the amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 3) is referred to as "HER2ec".

[0028] HER2 expression or gene amplification can be examined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). The IHC method can score expression levels based on the 2018 ASCO / CAP guidelines (see Wolff AC, et al: J Clin Oncol 36 (20), 2018: 2105-2122). The 2018 ASCO / CAP guidelines state that HER2 expression should be scored based on the following criteria: 3+: Strong, complete, circumferential membrane staining in more than 10% of tumor cells; 2+: Weak to moderate circumferential membrane staining in more than 10% of tumor cells; 1+: faint / barely discernible incomplete membrane staining in more than 10% of tumor cells; 0: no staining or incomplete, faint / barely perceptible membrane staining in 10% or less of the tumor cells; It can be scored on four points: In addition, FISH can evaluate the degree of gene amplification based on the 2007 ASCO / CAP guidelines. The HER2 gene is present on chromosome 17, and therefore the degree of gene amplification can be evaluated by measuring the ratio of the HER2 gene to the number of centromeres on chromosome 17. HER2 testing can be performed using a commercially available kit.

[0029] As used herein, "an amino acid sequence corresponding to" means an amino acid sequence that corresponds when aligned.

[0030] For example, the alignment of two amino acid sequences can be performed using the CLUSTAL W algorithm with default parameters to align two or more amino acid sequences. The alignment algorithm can be fast or slow, preferably slow. The default parameters can be, for example, one or more or all of the parameters selected from the group consisting of GAP OPEN: 10, GAP EXTENSION: 0.1, KTUP: 1, WINDOW LENGTH: 5, TOPDIAG: 5, and PAIRGAP: 3. This allows corresponding amino acid sequences to be identified. This also allows the identity of amino acid sequences to be determined.

[0031] As used herein, "antibodies compete for binding to HER2" means that both antibodies cannot bind to HER2 at the same time because their binding sites (epitopes) on HER2 overlap, competing for the binding site on HER2, etc. Whether or not antibodies compete for binding to HER2 can be determined by antibody competition tests.

[0032] As used herein, "epitope" refers to a site on the surface of a target molecule to which an antibody binds.

[0033] <Antibody or antigen-binding fragment thereof of the present invention> The antibody or antigen-binding fragment of the present invention is an antibody or antigen-binding fragment thereof that binds to HER2 or a fragment thereof. The antibody or antigen-binding fragment of the present invention may include an antibody or antigen-binding fragment thereof having a mutation selected from the group consisting of insertion, deletion, addition, and substitution of one to several amino acids. In one embodiment, an antibody or antigen-binding fragment thereof is provided that includes at least one CDR, at least two, at least three, or more CDRs that are substantially identical to at least one CDR, at least two, at least three, or more CDRs in the antibody or antigen-binding fragment of the present invention. In another embodiment, an antibody having at least two, three, four, five, or six CDRs that are substantially identical to at least two, three, four, five, or six CDRs in or derived from the antibody or antigen-binding fragment of the present invention is included. In some embodiments, at least one, two, three, four, five, or six CDRs are at least about 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two, three, four, five, or six CDRs in the antibody or antigen-binding fragment of the present invention. In some embodiments, at least one, two, three, four, five, or six CDRs include at least one insertion, deletion, addition, or substitution in at least one, two, three, four, five, or six CDRs in or from the antibody or antigen-binding fragment of the present invention. The antibody or antigen-binding fragment of the present invention may include an antibody or antigen-binding fragment having 80% or more, 85% or more, 90% or more, or 95% or more amino acid sequence identity and the antigen specificity of the antibody. The antibody or antigen-binding fragment thereof of the present invention may include an antibody or antigen-binding fragment thereof having a mutation selected from the group consisting of insertion, deletion, addition, and substitution of one to several amino acids in the framework (FR) region of the heavy chain variable region or light chain variable region. The antibody or antigen-binding fragment of the present invention may preferably be an antibody that specifically binds to HER2. The antibody or antigen-binding fragment of the present invention has binding affinity (or reactivity) to HER2 expressed on cancer cells. Therefore, it can be used for targeting to cancer cells. The antibody or antigen-binding fragment of the present invention may more preferably have stronger binding affinity (or reactivity) to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells. Therefore, it can be used for specific targeting to cancer cells. The antibody or antigen-binding fragment of the present invention may more preferably not significantly react (e.g., not react at all or substantially not react) to HER2 expressed on non-cancer cells, but may specifically react to HER2 expressed on cancer cells. Therefore, it can be used for targeting (particularly specific targeting) to cancer cells.

[0034] The antibody or antigen-binding fragment of the present invention may be preferably a monoclonal antibody from the viewpoint of use as a pharmaceutical ingredient. The antibody or antigen-binding fragment of the present invention is preferably an isolated monoclonal antibody or an antigen-binding fragment of an isolated monoclonal antibody from the viewpoint of use as a pharmaceutical ingredient. According to the present invention, a pharmaceutical composition comprising an isolated monoclonal antibody or an antigen-binding fragment of an isolated monoclonal antibody and a pharma- ceutical acceptable additive may be provided.

[0035] The antibody or antigen-binding fragment thereof of the present invention is (i) an antibody or an antigen-binding fragment thereof that binds to a peptide comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (for example, the amino acid sequences of SEQ ID NOs: 31 to 36); or (ii) A peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) has a stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A, for example, a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) has a point mutation consisting of W614A. an antibody or antigen-binding fragment thereof having stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having a point mutation consisting of F616A, a mutant of the extracellular domain of HER2 having a mutation consisting of two point mutations, W614A and F616A, or a mutant of the extracellular domain of HER2 having a mutation consisting of two point mutations, K615A and F616A {wherein the part of the extracellular domain of HER2 may be the region of amino acids 613 to 622 of the amino acid sequence set forth in SEQ ID NO: 2}. Here, W614A means that the amino acid residue corresponding to tryptophan (W) at amino acid number 614 in the amino acid sequence of HER2 described in SEQ ID NO: 2 is substituted with alanine (A), K615A means that the amino acid residue corresponding to lysine (K) at amino acid number 615 in the amino acid sequence of HER2 described in SEQ ID NO: 2 is substituted with alanine (A), and F616A means that the amino acid residue corresponding to phenylalanine (F) at amino acid number 616 in the amino acid sequence of HER2 described in SEQ ID NO: 2 is substituted with alanine (A). The peptide may be a synthetic polypeptide. In a preferred embodiment, the peptide is not glycosylated.

[0036] The binding reactivity to HER2 can also be evaluated by enzyme-linked immunosorbent assay (ELISA). In ELISA, for example, the presence or amount of an enzyme linked to an antibody can be evaluated by the amount of a reaction product of a substrate for the enzyme. For example, when horseradish peroxidase (HRP) is used as the enzyme and 3,3',5,5'-tetramethylbenzidine (TMB) is used as the substrate, TMB is oxidized to develop a blue color with maximum absorption at 370 nm and 652 nm. When the antibody is further contacted with a reaction stop solution containing sulfuric acid, a yellow color develops with maximum absorption at 450 nm. For example, the amount of the antibody that reacted can be estimated by measuring the absorbance at 652 nm, and the reactivity of the antibody to the antigen can be evaluated based on the amount of the antibody that reacted. The greater the amount of the antibody that reacted, the higher the reactivity of the antibody to the antigen can be evaluated. The binding reactivity (amount of antibody reacted) may be 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 100 times or more, 1,000 times or more, 10,000 times or more, or 100,000 times or more than the binding reactivity to the mutant. The strength of the binding reactivity may be a statistically significant difference (e.g., p<0.05, p<0.01, p<0.005, or p<0.0001).

[0037] The antibody or antigen-binding fragment thereof of the present invention is described in detail below. The antibodies or antigen-binding fragments of the present invention all bind to HER2 expressed on cancer cells (e.g., LN229 cells). The antibodies or antigen-binding fragments of the present invention preferably have stronger binding reactivity to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells. In one embodiment, the antibodies or antigen-binding fragments of the present invention more preferably bind to HER2 expressed on cancer cells (e.g., LN229 cells) and do not show significant binding to HER2 expressed on non-cancer cells (e.g., normal cells).

[0038] The antibody or antigen-binding fragment thereof of the present invention comprises: (i) It may be an antibody or an antigen-binding fragment thereof that binds to a peptide comprising any of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (for example, the amino acid sequences of SEQ ID NOs: 31 to 36). A peptide comprising any of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (for example, the amino acid sequences of SEQ ID NOs: 31 to 36) may be a partial peptide of the amino acid sequence of SEQ ID NO: 2. A peptide comprising any of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (for example, the amino acid sequences of SEQ ID NOs: 31 to 36) is a partial peptide of the amino acid sequence of SEQ ID NO: 2, and may be 10 to 20 amino acids long, 11 to 19 amino acids long, 12 to 18 amino acids long, 13 to 17 amino acids long, 14 to 16 amino acids long, or about 15 amino acids long. A peptide comprising any of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (for example, the amino acid sequences of SEQ ID NOs: 31 to 36) may be a peptide consisting of any of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (for example, the amino acid sequences of SEQ ID NOs: 31 to 36). This region is considered to have a conformation that is accessible to an antibody in HER2 expressed on the cancer cell membrane, and therefore an antibody targeting this region can bind to cancer cells with stronger binding affinity (or reactivity) than to non-cancer cells. In a preferred embodiment, an antibody targeting this region does not significantly react to HER2 expressed on non-cancer cells, but can specifically react to HER2 expressed on cancer cells. The peptide itself does not need to have glycosylation, and the peptide does not need to have glycosylation. Here, the peptide is not particularly limited, but may preferably be a synthetic peptide.

[0039] The antibody or antigen-binding fragment thereof of the present invention comprises: (ii) an antibody or an antigen-binding fragment thereof that has stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A in a peptide consisting of a part of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622); or The antibody or antigen-binding fragment thereof may have a stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having a point mutation of W614A in a peptide consisting of a part of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622), or to a mutant of the extracellular domain of HER2 having two point mutations, K615A and F616A; or The antibody or antigen-binding fragment thereof may have stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant peptide of the extracellular domain of HER2 having a point mutation of K615A in a peptide consisting of a part of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (region of amino acid numbers 603 to 622), or a mutant peptide of the extracellular domain of HER2 having a point mutation of F616A, or a mutant peptide of the extracellular domain of HER2 having two point mutations of W614A and F616A. An antibody that binds to this region or its vicinity can bind to cancer cells with stronger binding affinity (or reactivity) than to non-cancer cells. In the above, the part of the extracellular domain of HER2 may be the region of amino acid numbers 613 to 622 of the amino acid sequence set forth in SEQ ID NO: 2. Here, the extracellular domain may be a peptide expressed in a non-cancer cell or a cancer cell, or a synthetic peptide. The extracellular domain may preferably be a synthetic peptide. Furthermore, these antibodies may compete with each other for binding to a peptide consisting of a part of the extracellular domain of HER2 (the region of amino acid numbers 603 to 622). Furthermore, these antibodies may compete with each other for binding to HER2 expressed on a cancer cell (e.g., LN229 cell).

[0040] The antibody or antigen-binding fragment thereof of the present invention comprises: (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 18; A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20, and A light chain CDR1 having the amino acid sequence of SEQ ID NO: 21; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 22; and an antibody having a light chain variable region having a light chain CDR3 having the amino acid sequence of SEQ ID NO: 23, or an antigen-binding fragment thereof; (iv) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 24; A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 25, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 26, and A light chain CDR1 having the amino acid sequence of SEQ ID NO: 27; A light chain CDR2 having the amino acid sequence of SEQ ID NO: 28, and an antibody having a light chain variable region having a light chain CDR3 having the amino acid sequence of SEQ ID NO: 29, or an antigen-binding fragment thereof; (v) an antibody or an antigen-binding fragment thereof that competes with the antibody (iii) above for binding to HER2; or (vi) The antibody or antigen-binding fragment thereof may compete with the antibody of (iv) above for binding to HER2. The antibody or antigen-binding fragment thereof of the present invention may also be the antibody or antigen-binding fragment thereof of the present invention having at least one or more (e.g., several, e.g., 2, 3, 4, or 5) amino acid mutations selected from the group consisting of substitution, addition, insertion, and deletion in at least one CDR of (iii) or (iv) above. These antibodies or antigen-binding fragments thereof may bind to cancer cells with stronger binding affinity than to non-cancer cells. These antibodies or antigen-binding fragments thereof may preferably not significantly react with HER2 expressed on non-cancer cells, but may specifically react with HER2 expressed on cancer cells. The antibody or antigen-binding fragment thereof of the present invention may be a non-human animal antibody or antigen-binding fragment thereof. The non-human animal may be, for example, a rodent, for example, a mouse. The antibody or antigen-binding fragment thereof of the present invention may also preferably be a chimeric antibody or a human chimeric antibody. The antibody or antigen-binding fragment thereof of the present invention may more preferably be a humanized antibody or an antigen-binding fragment of a humanized antibody.

[0041] The antibody or antigen-binding fragment thereof of the present invention comprises: (vii) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 15; (viii) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 17; (ix) an antibody or an antigen-binding fragment thereof that competes with the antibody of (vii) above for binding to HER2 {e.g., a peptide consisting of a part of the extracellular domain of HER2 (the region of amino acid numbers 603 to 622), or HER2 expressed in LN229 cells}; or (x) The antibody may be an antibody or an antigen-binding fragment thereof that competes with the antibody (viii) above for binding to HER2 {e.g., a peptide consisting of a part of the extracellular domain of HER2 (the region of amino acid numbers 603 to 622), or HER2 expressed in LN229 cells}. The antibody or antigen-binding fragment thereof of the present invention may also be an antibody or antigen-binding fragment thereof having at least one or more (e.g., several) amino acid mutations selected from the group consisting of at least one substitution, addition, insertion, and deletion in each variable region of (vii) or (viii) above. In some embodiments, the at least one or more (e.g., several) amino acid mutations selected from the group consisting of substitution, addition, insertion, and deletion in each variable region may be mutations other than in the CDRs. The antibody or antigen-binding fragment thereof of the present invention comprises: (xi) It may be an antibody or an antigen-binding fragment thereof that binds to a peptide comprising the amino acid sequence of SEQ ID NO: 37. The antibody or antigen-binding fragment thereof of the present invention comprises: (xii) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 81; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 82, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 83, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 84; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 85, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 86; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 87; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 88, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 89, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 90; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 91, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:92; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 93; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 94, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 95, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 96; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 97, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 98; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 99; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 100, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 101, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 102; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 103, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 104; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 105; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 106, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 107, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 108; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 109, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 110; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 111; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 112, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 113, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 114; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 115, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 116; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 117; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 118, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 119, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 120; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 121, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 122; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 123; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 124, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 125, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 126; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 127, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 128; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 129; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 130, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 131, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 132; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 133, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 134; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 135; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 136, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 137, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 138; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 139, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 140; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 141; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 142, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 143, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 144; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 145, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 146; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 147; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 148, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 149, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 150; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 151, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 152; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 153; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 154, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 155, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 156; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 157, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 158; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 159; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 160, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 161, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 162; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 163, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 164; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 165; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 166, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 167, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 168; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 169, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 170; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 171; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 172, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 173, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 174; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 175, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 176; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 177; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 178, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 179, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 180; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 181, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 182; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 183; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 184, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 185, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 186; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 187, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 188; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 189; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 190, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 191, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 192; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 193, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 194; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 195; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 197, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 198; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 199, and having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 200; A heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 201; A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 202, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 203, and A light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 204; A light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and The antibody may be an antibody or an antigen-binding fragment thereof having a light chain variable region having a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:206. The antibody or antigen-binding fragment thereof of the present invention comprises: (xiii)(a) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 39 and a light chain variable region having the amino acid sequence of SEQ ID NO: 40; (b) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41 and a light chain variable region having the amino acid sequence of SEQ ID NO: 42; (c) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 and a light chain variable region having the amino acid sequence of SEQ ID NO: 44; (d) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 45 and a light chain variable region having the amino acid sequence of SEQ ID NO: 46; (e) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (f) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50; (g) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; (h) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO: 54; (i) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 55 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; (j) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57 and a light chain variable region having the amino acid sequence of SEQ ID NO: 58; (k) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 59 and a light chain variable region having the amino acid sequence of SEQ ID NO: 60; (l) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 61 and a light chain variable region having the amino acid sequence of SEQ ID NO: 62; (m) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and a light chain variable region having the amino acid sequence of SEQ ID NO: 64; (n) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and a light chain variable region having the amino acid sequence of SEQ ID NO: 66; (o) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 68; (p) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 70; (q) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71, and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (r) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 73 and a light chain variable region having the amino acid sequence of SEQ ID NO: 74; (s) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 75 and a light chain variable region having the amino acid sequence of SEQ ID NO: 76; (t) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 77 and a light chain variable region having the amino acid sequence of SEQ ID NO: 78; (u) an antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 79 and a light chain variable region having the amino acid sequence of SEQ ID NO: 80; or (xiv) The antibody or antigen-binding fragment thereof may compete with each of the antibodies (xii)(a) to (u) above for binding to HER2 {e.g., a peptide consisting of a part of the extracellular domain of HER2 (the region of amino acid numbers 603 to 622), or HER2 expressed in LN229 cells}. As will be apparent to those skilled in the art, the below-mentioned explanation of the antibody of the present invention or an antigen-binding fragment thereof can be applied to the above antibodies (xi) to (xiv) or antigen-binding fragments thereof.

[0042] The antibody or antigen-binding fragment thereof of the present invention is an isolated monoclonal antibody or an antigen-binding fragment thereof that has stronger binding affinity (or reactivity) to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells, and more preferably, an isolated monoclonal antibody or an antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells and can specifically react with HER2 expressed on cancer cells, The antibody or antigen-binding fragment thereof may satisfy any one or more of the above (i) to (xiv). In this embodiment, the antibody or antigen-binding fragment thereof of the present invention may have one or more of the profiles (i) and (ii) above, and more specifically, may have only the profile (i), only the profile (ii), or both the profiles (i) and (ii). In this embodiment, the antibody or antigen-binding fragment of the present invention may satisfy the above (i) and one or more selected from the group consisting of (v), (vi), (ix), (x), (xi), (xii), (xiii), and (xiv). In this embodiment, the antibody or antigen-binding fragment of the present invention may satisfy the above (ii) and one or more selected from the group consisting of (v), (vi), (ix), (x), (xi), (xii), (xiii), and (xiv). In this embodiment, the antibody or antigen-binding fragment of the present invention may satisfy the above (i) and (ii) and one or more selected from the group consisting of (v), (vi), (ix), (x), (xi), (xii), (xiii), and (xiv). These antibodies or antigen-binding fragments may bind to cancer cells with stronger binding affinity (or reactivity) than to non-cancer cells. These antibodies or antigen-binding fragments thereof preferably do not significantly react with HER2 expressed on non-cancer cells, and can specifically react with HER2 expressed on cancer cells.

[0043] The antibody or antigen-binding fragment thereof of the present invention may be an antibody or antigen-binding fragment thereof that satisfies any one or more of the above (i) to (xiv). In this embodiment, the antibody or antigen-binding fragment of the present invention may have any one or more of the profiles (i) and (ii) above. More specifically, the antibody or antigen-binding fragment may have only the profile (i), only the profile (ii), or both the profiles (i) and (ii). These antibodies or antigen-binding fragments may bind to cancer cells with stronger binding affinity (or reactivity) than to non-cancer cells. These antibodies or antigen-binding fragments may preferably not significantly react with HER2 expressed on non-cancer cells, but may specifically react with HER2 expressed on cancer cells. In this embodiment, the antibody or antigen-binding fragment of the present invention may satisfy the above (i) and one or more selected from the group consisting of (v), (vi), (ix), (x), (xi), (xii), (xiii), and (xiv). In this embodiment, the antibody or antigen-binding fragment of the present invention may satisfy the above (ii) and one or more selected from the group consisting of (v), (vi), (ix), (x), (xi), (xii), (xiii), and (xiv). In this embodiment, the antibody or antigen-binding fragment of the present invention may satisfy the above (i) and (ii) and one or more selected from the group consisting of (v), (vi), (ix), (x), (xi), (xii), (xiii), and (xiv).

[0044] The antibody of the present invention may have antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. ADCC activity refers to the activity of Fcγ receptor-bearing cells (effector cells) binding to the Fc portion of the antibody of the present invention via the Fcγ receptor, thereby damaging the target cell, when the antibody of the present invention binds to a cell surface antigen of the target cell. ADCC activity can be evaluated by mixing target cells expressing HER2 (cancer cells, for example, LN229 cells or SK-BR-3 cells), effector cells, and the antibody of the present invention, and measuring the degree of ADCC. For example, mouse splenocytes, or monocyte nuclei isolated from human peripheral blood or bone marrow can be used as effector cells. For example, HER2-positive breast cancer cells can be used as target cells. Target cells are labeled in advance with 51Cr or the like, and the antibody of the present invention is added thereto and incubated, and then effector cells (which may be activated) at an appropriate ratio to the target cells are added and incubated. After incubation, the supernatant is collected, and the above-mentioned labels in the supernatant are counted, whereby measurement is possible. CDC activity means cytotoxic activity by the complement system. CDC activity can be measured by using complement instead of effector cells in a test for ADCC activity.

[0045] The antibody of the present invention may be an antibody in which one or more N-linked glycans are bound to the Fc region, and fucose is not bound to the N-acetylglucosamine at the reducing end of the N-linked glycan. For example, the Fc region of an IgG antibody has two binding sites for N-linked glycans, and a complex glycan is bound to this site. An N-linked glycan refers to a glycan that binds to Asn in an Asn-X-Ser / Thr sequence, and has a common structure Man3GlcNAc2-Asn. Depending on the type of glycan that binds to two mannoses (Man) at the non-reducing end, it is classified into high mannose type, hybrid type, complex type, and the like. Fucose can be bound to the N-acetylglucosamine (GlcNAc) at the reducing end of the N-linked glycan, but it is known that when this fucose is not bound, the ADCC activity is significantly increased compared to when it is bound. This is described, for example, in International Publication No. WO 2002 / 031140, the disclosure of which is incorporated herein by reference in its entirety. By significantly improving ADCC activity, the dosage of the antibody can be reduced when used as a medicine, which makes it possible to reduce side effects and reduce treatment costs. In order to enhance ADCC activity, the human antibody subtype used in the antibody constant region can be IgG1.

[0046] <Antibody-drug conjugates> According to the present invention, there is provided a conjugate (antibody-drug conjugate; ADC) of the antibody or antigen-binding fragment thereof of the present invention and a drug. In the ADC of the present invention, the antibody or antigen-binding fragment thereof and a drug (e.g., a component having the property of damaging cells, e.g., a cytotoxic agent) may be linked via a linker. In the ADC of the present invention, the cytotoxic agent may be a chemotherapeutic agent (e.g., an anticancer agent such as a commercially available anticancer agent, for example, auristatin (auristatin E, auristatin F phenylenediamine (AFP), monomethylauristatin E, monomethylauristatin F and derivatives thereof), maytansinoids DM1 and DM4 and derivatives thereof), camptothecin (SN-38, topotecan and exotecan and derivatives thereof), DNA minor groove binders (enediyne, lexitropsin, duocarmycin and derivatives thereof), taxanes (paclitaxel and docetaxel and derivatives thereof), polyketides (discodermolide and derivatives thereof), anthraquinones (mitoxantrone and and their derivatives), benzodiazepines (pyrrolobenzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), cardiac glycosides (digitoxin and its derivatives), calechiamicin, epothilones, cryptophycins, cemadotin, rhizoxin, netropsin, combretastatin, erytherobin, etoposide, T67 (Tularik), and nocodazole), radioisotopes (e.g., Examples of cytotoxic agents include 32P, 60C, 90Y, 111In, 131I, 125I, 153Sm, 186Re, 188Re, and 212Bi, and toxins (e.g., diphtheria toxin A, Pseudomonas endotoxin, ricin, saporin, etc.) that can be used as cytotoxic agents in the ADCs of the invention. Any cytotoxic agent used in the treatment of cancer can be used.

[0047] It is preferable that the antibody be one that internalizes into cancer cells, but even antibodies that do not internalize can be used because the bystander effect can be used to kill cancer cells as long as the anticancer drug is delivered to the tissue surrounding the cancer.

[0048] In some embodiments of the present invention, the linker may be a non-cleavable linker or a cleavable linker. The antibody may be linked to the linker via, for example, a maleimide group to a sulfhydryl group of the antibody. The linker may optionally include a polyethylene glycol block.

[0049] For example, cleavable linkers include peptide linkers such as valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-lys) linkers, and hydrazone linkers that are cleaved in a pH-dependent manner. Cleavable linkers also include linkers that contain carbamate or ester bonds, which can be enzymatically degraded in cells. These linkers may be used in combination. In one embodiment of the present invention, the antibody and the cytotoxic agent can be linked via maleimide group-PEG-Val-Cit. In one embodiment of the present invention, the linker used in the examples of the present application (e.g., maleimide group-PEG-Val-Cit-PABA-cytotoxic agent) can be used. Furthermore, a spacer may be placed between the linker and the cytotoxic agent.

[0050] <Method of producing the antibody of the present invention> The antibody of the present invention can be obtained by (i) administering to an animal a peptide comprising any one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NO: 31 to 37 (for example, the amino acid sequences of SEQ ID NO: 31 to 36) as an immunogen. In one embodiment, the antibody of the present invention can be obtained by administering to an animal a peptide consisting of the amino acid sequence of SEQ ID NO: 31 as an immunogen. In one embodiment, the antibody of the present invention can be obtained by administering to an animal a peptide consisting of the amino acid sequence of SEQ ID NO: 32 as an immunogen. In one embodiment, the antibody of the present invention can be obtained by administering to an animal a peptide consisting of the amino acid sequence of SEQ ID NO: 33 as an immunogen. In one embodiment, the antibody of the present invention can be obtained by administering to an animal a peptide consisting of the amino acid sequence of SEQ ID NO: 34 as an immunogen. In one embodiment, the antibody of the present invention can be obtained by administering to an animal a peptide consisting of the amino acid sequence of SEQ ID NO: 35 as an immunogen. In one embodiment, the antibody of the present invention can be obtained by administering to an animal a peptide consisting of the amino acid sequence of SEQ ID NO: 36 as an immunogen. Furthermore, the antibody of the present invention can be obtained by administering to an animal as an immunogen a peptide consisting of the amino acid sequence of SEQ ID NO: 37. The peptide can be a synthetic peptide.

[0051] The antibody of the present invention can also be obtained by administering an extracellular domain portion of HER2, such as HER2ec, to an animal as an immunogen.The antibody of the present invention can also be obtained by methods well known to those skilled in the art, such as phage display.The extracellular domain portion of HER2 can be a synthetic peptide.The extracellular domain portion of HER2 can be expressed in a cell, for example, a non-cancer cell or a cancer cell, for example, a cancer cell line (for example, LN229 cell).

[0052] Immunization of an animal with an immunogen can be performed by a method well known to those skilled in the art. Whether an antibody against HER2 or a partial peptide thereof has been produced can be confirmed, for example, by using a body fluid (e.g., blood, plasma, or serum) or immune cells (e.g., spleen cells) obtained from an immunized animal. A monoclonal antibody can be produced by a method well known to those skilled in the art, such as a hybridoma method. A hybridoma can be produced by a method well known to those skilled in the art. After production, the hybridoma is made into a single clone by a limiting dilution method. The monoclonal antibody can be secreted into the hybridoma supernatant. Whether a monoclonal antibody has the desired binding properties can be confirmed by a binding assay (binding test) by those skilled in the art.

[0053] For example, whether an antibody binds to cancer cells with stronger binding affinity than to non-cancer cells, i.e., whether it does not significantly react to HER2 expressed on non-cancer cells, but can specifically react to HER2 expressed on cancer cells, can be easily tested by a person skilled in the art. The test can be preferably an in vitro test. For example, the test can use a cancer cell line, particularly a HER2-positive cancer cell line (e.g., SK-BR-3 cells), as the cancer cells. The test can also use, for example, a non-cancer cell line or a normal cell line, particularly a HER2-positive non-cancer cell line or a normal cell line (e.g., HaCaT cells), as the non-cancer cells. For example, the cells can be divided into single cells, and the interaction with the labeled antibody can be analyzed by flow cytometry to determine whether the antibody binds to cancer cells with stronger binding affinity than to non-cancer cells. The label can be a label well known to those skilled in the art, such as a fluorescent label. In addition, the binding reactivity (amount of antibody reacted) to cancer cells expressing HER2 can be 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 100 times or more, 1,000 times or more, 10,000 times or more, or 100,000 times or more than the binding reactivity to normal cells expressing HER2. The strength of the binding reactivity can be a statistically significant difference (e.g., p<0.05, p<0.01, p<0.005, or p<0.0001). The binding reactivity to cancer cells can be evaluated, for example, by using flow cytometry. When the binding affinity or reactivity is determined by flow cytometry, for example, peak values ​​can be compared. That an antibody does not exhibit significant binding to non-cancer cells (e.g., normal cells) can be determined by testing whether the antibody exhibits significant binding to a non-cancer cell line (e.g., a normal cell line, particularly a HER2-positive non-cancer cell line or a normal cell line (e.g., HaCaT cells)).

[0054] For example, whether the antibody or antigen-binding fragment thereof of the present invention satisfies the above (i) can be tested by assaying whether the hybridoma supernatant binds to each peptide containing any of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36). For example, the peptide is immobilized by a conventional method, and the binding of the antibody in the hybridoma supernatant to the peptide is assayed, and the antibody produced by the hybridoma can be detected using a labeled secondary antibody that specifically reacts with the antibody. Surface plasmon resonance (SPR) may also be used for detection. Similarly, whether or not an antibody or an antigen-binding fragment thereof of the present invention satisfies the above (ii) can be determined by assaying whether the antibody in the hybridoma supernatant binds to a peptide consisting of a part of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) and having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A (for example, a mutant extracellular domain of HER2 having a point mutation consisting of W614A; or a mutant extracellular domain of HER2 having a mutation consisting of two point mutations, K615A and F616A; or a mutant extracellular domain of HER2 having a point mutation consisting of K615A, or a mutant extracellular domain of HER2 having a point mutation consisting of F616A, or a mutant extracellular domain of HER2 having a mutation consisting of two point mutations, W614A and F616A), and by assaying whether the antibody in the hybridoma supernatant binds to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3. For example, the binding dissociation constant (KD) can be determined by using surface plasmon resonance (SPR), and the magnitude of the KD can be used as an index to easily determine whether or not the above (ii) is satisfied. Thus, according to the present invention, there can be provided a method for selecting or identifying an antibody or an antigen-binding fragment thereof, which comprises selecting or identifying an antibody or an antigen-binding fragment thereof that satisfies the above (i) from a group of antibodies or antigen-binding fragments thereof, for example, a group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof.The present invention also provides a method for selecting or identifying an antibody or an antigen-binding fragment thereof, comprising selecting or identifying an antibody or an antigen-binding fragment thereof that satisfies the above (ii) from a group of antibodies or antigen-binding fragments thereof, for example, an antibody or an antigen-binding fragment thereof that binds to HER2 or a fragment thereof. These methods may further comprise selecting or identifying an antibody or an antigen-binding fragment thereof that has stronger binding reactivity to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells from the group of antibodies or antigen-binding fragments thereof selected or identified by these methods, i.e., an antibody or an antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells and can specifically react with HER2 expressed on cancer cells. In the above, when a part of the extracellular domain of HER2 is the region of amino acid numbers 603 to 622 of the amino acid sequence set forth in SEQ ID NO: 2, the antibody or the antigen-binding fragment thereof can be tested by a similar assay using a mutant having the above-mentioned point mutation in the peptide.

[0055] Competitive assays can be used to verify that antibodies compete for binding to HER2 and that the antibodies have similar binding properties to each other. Antibodies that compete for binding to an antigen (e.g., HER2 or a fragment thereof) can be obtained by competitive assays well known to those skilled in the art. If an antibody can block the binding of a desired antibody by, for example, at least 20%, preferably at least 20-50%, more preferably at least 50%, more preferably 60%, more preferably 70%, more preferably 80%, and particularly preferably 90% or more in a competitive assay, it can be considered to be an antibody that competes for binding to the same antigen. Competitive antibodies can be confirmed by cross-blocking assays, flow cytometry, fluorescence energy transfer assay (FRET) or fluorescence microassay (FMAT (registered trademark)), and preferably competitive ELISA assays. In cross-blocking assays, an antigen is coated on, for example, a microtiter plate, and a candidate competing antibody is added thereto and incubated to form a bond between the antigen and the candidate antibody. The desired antibody can then be labeled and further added to the well, incubated, washed, and the amount of bound desired antibody quantified to determine whether the antibodies competed, which would result in less label remaining in the well.

[0056] Generally, in a competitive assay, just because antibody A dissociates the binding between antibody B and an antigen does not necessarily mean that antibody B dissociates the binding between antibody A and an antigen. This can be easily understood by considering a case in which antibody A exhibits much stronger binding to an antigen than antibody B. In order to obtain antibodies with similar binding characteristics, it is sufficient to confirm that antibody A dissociates the binding between antibody B and an antigen, and that antibody B dissociates the binding between antibody A and an antigen. In this specification, such a competitive state is referred to as "antibody A and antibody B compete with each other in binding to an antigen."

[0057] Therefore, whether the antibody or antigen-binding fragment of the present invention satisfies any of the above (v), (vi), (ix), and (x) can be determined by confirming whether the antibody or antigen-binding fragment competes with any of the above antibodies (iii), (iv), (vii), (viii), and (xiii) for binding to HER2 or a fragment thereof (epitope-containing peptide, etc.). Thus, according to the present invention, there is provided a method for selecting or identifying an antibody or antigen-binding fragment thereof, which comprises selecting or identifying an antibody or antigen-binding fragment thereof that competes with any of the above antibodies (iii), (iv), (vii), (viii), and (xiii) for binding to HER2 or a fragment thereof (epitope-containing peptide, etc.) from a group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof. This method may further comprise selecting or identifying an antibody or antigen-binding fragment thereof that has stronger binding reactivity to HER2 expressed on cancer cells than to HER2 expressed on non-cancer cells from the group of antibodies or antigen-binding fragments thereof selected or identified by these methods. As a fragment of HER2, a peptide containing any of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (for example, the amino acid sequences of SEQ ID NOs: 31 to 36) may be used. As a fragment of HER2, HER2ec may be used. As HER2, HER2 expressed on the membrane of LN229 cells may be used. According to the present invention, the antibody of the present invention or an antigen-binding fragment thereof can be obtained in this manner.

[0058] In this way, a monoclonal antibody having the binding characteristics of the antibody of the present invention can be selected and obtained. The amino acid sequence of the obtained monoclonal antibody can be determined after the antibody is obtained. For example, the nucleotide sequence of the gene encoding the obtained monoclonal antibody can be determined by extracting RNA from a hybridoma producing the monoclonal antibody, synthesizing cDNA, and determining the sequence of the cDNA encoding the immunoglobulin. The amino acid sequence of the monoclonal antibody can be determined from the nucleotide sequence. The amino acid sequence of the heavy chain CDR1 to 3 and the amino acid sequence of the light chain CDR1 to 3 can be deduced from the amino acid sequence of the monoclonal antibody. CDRs can be predicted, for example, by the Kabat numbering system (Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242), Chothia (Al-lazikani et al., (1997) J. Mol. Biol. 273: 927-948), Aho, IMGT, CCG, or a combination of these methods. It is also well known that the predicted CDR regions may vary depending on the numbering system. In this manner, an antibody having the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 can be designed or produced.

[0059] Antigen-binding fragments of antibodies can be produced by methods well known to those skilled in the art. For example, Fab fragments can be produced by digesting antibodies with papain. For example, F(ab')2 can be produced by digesting antibodies with pepsin. For example, Fab' can be produced by treating F(ab')2 with a reducing agent. For example, scFvs can be constructed in various ways. For example, the C-terminus of the heavy chain variable region can be linked to the N-terminus of the light chain variable region. Typically, a linker (e.g., (GGGGS)4, SEQ ID NO:38) is placed between the heavy chain variable region and the light chain variable region. However, the order in which the chains can be linked can be reversed, and the C-terminus of the light chain variable region can be linked to the N-terminus of the heavy chain variable region. A tag (e.g., Myc tag, His tag, or FLAG tag) that facilitates detection or purification of the scFv can be included.

[0060] According to the present invention, a method for producing an antibody or an antigen-binding fragment thereof is provided. The production method of the present invention may include administering to an animal a peptide of HER2 (e.g., a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 31 to 37, e.g., a peptide consisting of the amino acid sequence of SEQ ID NOs: 31 to 36, e.g., a peptide consisting of the amino acid sequence described in SEQ ID NO: 31, e.g., a peptide consisting of the amino acid sequence described in SEQ ID NO: 32, e.g., a peptide consisting of the amino acid sequence described in SEQ ID NO: 33, e.g., a peptide consisting of the amino acid sequence described in SEQ ID NO: 34, e.g., a peptide consisting of the amino acid sequence described in SEQ ID NO: 35, e.g., a peptide consisting of the amino acid sequence described in SEQ ID NO: 36, e.g., a peptide consisting of the amino acid sequence described in SEQ ID NO: 37) or HER2 expressed in a cancer cell (e.g., an extracellular domain of HER2) as an immunogen. The production method of the present invention may also include testing (or confirming) that the obtained antibody shows stronger reactivity to HER2 expressed in a cancer cell than to HER2 expressed in a non-cancer cell. The production method of the present invention may also include testing (or confirming) that the antibody obtained does not significantly react with HER2 expressed on non-cancer cells, but can specifically react with HER2 expressed on cancer cells. The production method of the present invention may also include testing (or confirming) that the antibody obtained is the antibody of the present invention (has the binding characteristics of the antibody of the present invention). Once it has been confirmed that the obtained antibody shows stronger reactivity with HER2 expressed on cancer cells than HER2 expressed on non-cancer cells, does not significantly react with HER2 expressed on non-cancer cells, but can specifically react with HER2 expressed on cancer cells, and / or is the antibody of the present invention (has the binding characteristics of the antibody of the present invention), the antibody can be selected. The antibody can be produced using recombinant antibody-producing cells (e.g., CHO cells, etc.) containing nucleic acid encoding the antibody. In the production method of the present invention, the produced antibody can be purified, if necessary.

[0061] According to the present invention, there is provided a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, comprising selecting or identifying an antibody or an antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity with HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments thereof that bind to HER2 (e.g., HER2 expressed in LN229 cells, a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 31 to 37, for example, a peptide consisting of the amino acid sequence of SEQ ID NOs: 31 to 36). In one embodiment, the group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof can bind to HER2ec protein produced by LN229 cells. In one embodiment, the group of antibodies or antigen-binding fragments thereof that bind to HER2 or a fragment thereof can bind to a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36).

[0062] According to the present invention, there is provided a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, the method comprising selecting or identifying, from any of the above-mentioned antibodies or antigen-binding fragments of the antibody, an antibody or antigen-binding fragment of the antibody that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on a cancer cell line (e.g., LN229 cells). For example, the present invention provides a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, the method comprising selecting or identifying an antibody or an antigen-binding fragment thereof that does not react significantly with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments thereof that can bind to HER2ec protein produced by LN229 cells. The present invention also provides a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, the method comprising selecting or identifying an antibody or an antigen-binding fragment thereof that does not react significantly with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells, from a group of antibodies or antigen-binding fragments thereof that can bind to a peptide comprising any amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36). The present invention further provides a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, the method comprising selecting or identifying an antibody or an antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells but has specific reactivity to HER2 expressed on cancer cells, from a group of antibodies or antigen-binding fragments thereof that have stronger binding affinity (or reactivity) to a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) than to a mutant of the extracellular domain of HER2 having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A in the peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622). The present invention further provides a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, the method comprising selecting or identifying an antibody or an antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells but has specific reactivity to HER2 expressed on cancer cells, from a group of antibodies or antigen-binding fragments thereof that have stronger reactivity to a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) than to a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) having a point mutation consisting of W614A, or a peptide consisting of a point mutation consisting of K615A or F616A. The present invention further provides a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, the method comprising selecting or identifying an antibody or an antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells but has specific reactivity to HER2 expressed on cancer cells, from a group of antibodies or antigen-binding fragments thereof that have stronger reactivity to a peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622) than to a mutant of the extracellular domain of HER2 having a point mutation of F616A in the peptide consisting of a portion of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622), or a peptide having two point mutations, W614A and F616A.

[0063] The antibody or antigen-binding fragment of the present invention can be obtained by selecting an antibody or antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments thereof having any of the above (iii), (iv), and (xii) and at least one or more (e.g., several) amino acid mutations selected from the group consisting of substitution, addition, insertion, and deletion in at least one CDR. Thus, according to the present invention, there is provided a method for selecting or identifying an antibody or antigen-binding fragment thereof, which does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments thereof having any of the above (iii), (iv), and (xii) and at least one or more (e.g., several) amino acid mutations selected from the group consisting of substitution, addition, insertion, and deletion in at least one CDR.

[0064] The antibody or antigen-binding fragment of the present invention can be obtained by selecting an antibody or antigen-binding fragment thereof that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments thereof having any of the above (vii), (viii), and (xiii) and at least one or more (e.g., several) amino acid mutations selected from the group consisting of substitution, addition, insertion, and deletion in at least one variable region. Thus, according to the present invention, there is provided a method for selecting or identifying an antibody or antigen-binding fragment thereof, which does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments thereof having any of the above (vii), (viii), and (xiii) and at least one or more (e.g., several) amino acid mutations selected from the group consisting of substitution, addition, insertion, and deletion in at least one variable region.

[0065] In this way, the antibody or antigen-binding fragment of the antibody of the present invention can be selected or identified. After the antibody or antigen-binding fragment of the antibody of the present invention is selected or identified, the genes encoding them can be introduced into antibody-producing cells (e.g., CHO cells, etc.), and the antibodies or antigen-binding fragments of the antibody can be recovered from the antibody-producing cells. The antibody or antigen-binding fragment of the antibody of the present invention can be humanized as necessary. The antibody or antigen-binding fragment of the antibody of the present invention can be isolated, for example, using a protein A or G column. Furthermore, the isolated antibody or antigen-binding fragment of the antibody of the present invention can be mixed with a pharma- ceutically acceptable excipient. Alternatively, the isolated antibody or antigen-binding fragment of the antibody of the present invention can be conjugated with a drug (e.g., a cytotoxic agent). This allows the antibody or antigen-binding fragment of the antibody of the present invention to target the drug to cancer cells expressing HER2.

[0066] According to the present invention there is provided a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, comprising the steps of: A method may be provided that includes selecting or identifying, from a group of antibodies or antigen-binding fragments of antibodies capable of binding to HER2 (e.g., HER2 expressed on LN229 cells) or a fragment thereof, an antibody or antigen-binding fragment of an antibody that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells.

[0067] The present invention also provides a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, comprising the steps of: A method is provided, comprising selecting or identifying an antibody or an antigen-binding fragment of an antibody that satisfies at least one of the above (i) or (ii) from a group of antibodies or antigen-binding fragments of antibodies that bind to HER2 or a fragment thereof: (i) binds to a peptide comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36); or (ii) has a stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A in a peptide consisting of a part of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622); or In a peptide consisting of a portion of the extracellular domain of HER2 described in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622), it has stronger reactivity to a peptide consisting of the extracellular domain of HER2 described in SEQ ID NO: 3 than to a peptide having a point mutation of W614A, or a peptide having a point mutation of K615A or F616A. The invention further provides a method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, comprising the steps of: A method is provided, comprising selecting or identifying an antibody or antigen-binding fragment thereof that binds to HER2 or a fragment thereof from a group of antibodies or antigen-binding fragments thereof that satisfy at least one selected from (i) or (ii) above: (i) binds to a peptide comprising any one of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 37 (e.g., the amino acid sequences of SEQ ID NOs: 31 to 36); or (ii) has a stronger binding affinity (or reactivity) to a peptide consisting of the extracellular domain of HER2 set forth in SEQ ID NO: 3 than to a mutant of the extracellular domain of HER2 having one or more amino acid mutations selected from the group consisting of W614A, K615A, and F616A in a peptide consisting of a part of the extracellular domain of HER2 set forth in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622); or In a peptide consisting of a portion of the extracellular domain of HER2 described in SEQ ID NO: 2 (the region of amino acid numbers 603 to 622), it has stronger reactivity to a peptide consisting of the extracellular domain of HER2 described in SEQ ID NO: 3 than to a peptide having a point mutation of W614A, or a peptide having a point mutation of K615A or F616A. In these embodiments, the present invention may further comprise selecting or identifying, from the obtained group of antibodies or antigen-binding fragments of the antibodies, an antibody or antigen-binding fragment of the antibody that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity with HER2 expressed on cancer cells.

[0068] The present invention also provides 1. A method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, comprising: The method includes selecting or identifying an antibody or an antigen-binding fragment of an antibody that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments of the antibody having either (iii) or (iv) above and at least one amino acid mutation selected from the group consisting of substitution, addition, insertion, and deletion in at least one CDR.

[0069] The present invention also provides 1. A method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, comprising: The method includes selecting or identifying an antibody or an antigen-binding fragment of an antibody that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments of the antibody having either (vii) or (viii) above and at least one amino acid mutation selected from the group consisting of substitution, addition, insertion, and deletion in at least one variable region.

[0070] The present invention also provides 1. A method for selecting or identifying an antibody or an antigen-binding fragment of an antibody, comprising: The method includes selecting or identifying an antibody or an antigen-binding fragment of an antibody that does not significantly react with HER2 expressed on non-cancer cells and has specific reactivity to HER2 expressed on cancer cells from a group of antibodies or antigen-binding fragments of the antibody having either (xii) or (xiii) above and at least one amino acid mutation selected from the group consisting of substitution, addition, insertion, and deletion in at least one CDR or variable region.

[0071] The method of selecting or identifying an antibody or antigen-binding fragment of an antibody of the invention may be a method of selecting or identifying a humanized antibody or antigen-binding fragment of an antibody, or a human antibody or antigen-binding fragment of a human antibody. The method of selecting or identifying an antibody or antigen-binding fragment of an antibody of the invention may be a method of selecting or identifying an isolated and / or purified antibody or antigen-binding fragment of an antibody.

[0072] According to the present invention, there is provided a method for producing an antibody or an antigen-binding fragment of an antibody, comprising selecting or identifying the above-mentioned antibody or antigen-binding fragment of the antibody of the present invention. In this production method, the antibody or the antigen-binding fragment of the antibody can be produced from an antibody-producing cell by introducing a gene encoding the antibody or the antigen-binding fragment into the antibody-producing cell. The antibody or the antigen-binding fragment of the antibody can also be humanized to produce a humanized antibody or an antigen-binding fragment of the humanized antibody. The antibody or the antigen-binding property of the antibody can be isolated and / or purified and used for applications. For example, the antibody or the antigen-binding fragment of the antibody can be mixed with a pharma-ceutically acceptable excipient, formulated, and made into a pharmaceutical composition.

[0073] <Pharmaceutical composition of the present invention> According to the present invention, there is provided a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention. The pharmaceutical composition of the present invention may comprise the antibody or antigen-binding fragment thereof of the present invention and a pharma- ceutical acceptable carrier or additive. Examples of the carrier and additive include, but are not limited to, water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharma- ceutical acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants, and the like.

[0074] According to the present invention, a pharmaceutical composition comprising a HER2 targeting agent (also called a HER2 targeting therapeutic agent) comprising the antibody of the present invention or its antigen-binding fragment can be provided. The pharmaceutical composition of the present invention can be used to treat cancer.

[0075] According to the present invention, a targeting agent comprising the antibody of the present invention or its antigen-binding fragment, and a pharmaceutical composition comprising the targeting agent are provided.For example, according to the present invention, a pharmaceutical composition comprising an antibody-drug conjugate (ADC) of the antibody of the present invention or its antigen-binding fragment and a drug (e.g., a cytotoxic agent) is provided.The pharmaceutical composition of the present invention can be used to treat cancer.

[0076] <Other> According to the present invention, there is provided a method for detecting HER2-positive cancer cells in a cancer sample obtained from a subject, comprising contacting the cancer sample with the antibody or antibody or antigen-binding fragment thereof of the present invention. In this method, when HER2 in a biological sample and an anti-HER2 antibody or antigen-binding fragment thereof form a complex, it can be determined that HER2-positive cancer cells have been detected. According to the present invention, there can also be provided a method for determining the effectiveness of HER2-targeting therapy using a HER2 targeting agent comprising the antibody or antibody or antigen-binding fragment thereof of the present invention, comprising contacting a biological sample obtained from a subject with an anti-HER2 antibody or antigen-binding fragment thereof (preferably, the antibody or antigen-binding fragment thereof of the present invention), and determining that HER2-targeting therapy is effective for the subject when HER2 in the biological sample and an anti-HER2 antibody or antigen-binding fragment thereof form a complex. The present invention also provides a method for determining the effectiveness of HER2 targeting therapy using a HER2 targeting agent comprising the antibody of the present invention or the antibody or its antigen-binding fragment, comprising contacting a biological sample obtained from a subject with an anti-HER2 antibody or its antigen-binding fragment (preferably the antibody or its antigen-binding fragment of the present invention), forming a complex between HER2 in the biological sample and the anti-HER2 antibody or its antigen-binding fragment, and detecting the complex, wherein the detection of the complex indicates that the HER2 targeting therapy is effective for the subject. Here, when the biological sample is a biopsy, the complex formation can be detected based on the binding of the biological sample to the biopsy sample, and when the biological sample is a liquid sample, it can be determined by a method well known to those skilled in the art, such as ELISA. In this embodiment, the method may be an in vitro method. Also, in this embodiment, the method is an industrially applicable method. In this embodiment, the method may not include a diagnostic step. According to the present invention, a diagnostic agent or a diagnostic kit comprising the HER2 antibody or its antigen-binding fragment of the present invention for use in this method may be provided.The HER2 antibody or antigen-binding fragment of the present invention may be labeled, thereby detecting the HER2 antibody or antigen-binding fragment of the present invention, or the HER2 antibody or antigen-binding fragment of the present invention may be detected by a labeled secondary antibody that recognizes the HER2 antibody or antigen-binding fragment of the present invention. Therefore, the diagnostic kit may further include a labeled secondary antibody that recognizes the HER2 antibody or antigen-binding fragment of the present invention. The label may be a label used in an enzyme labeling method such as alkaline phosphatase or horseradish peroxidase, and the presence of the label can be detected using a chromogenic substrate for these. Therefore, the diagnostic kit may further include a chromogenic substrate.

[0077] According to the present invention, a pharmaceutical composition can be provided that includes a HER2 targeting agent comprising the antibody or its antigen-binding fragment of the present invention for use in treating cancer in a subject for whom HER2 targeting therapy is determined to be effective by the above method. According to the present invention, a pharmaceutical composition can also be provided that includes a HER2 targeting agent comprising the antibody or its antigen-binding fragment of the present invention for use in treating cancer in a subject having a HER2-positive tumor reactive with the antibody or its antigen-binding fragment of the present invention. The HER2 targeting agent of the present invention can be used in combination with other anticancer agents, etc.

[0078] According to the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of a HER2 targeting agent comprising the antibody or antigen-binding fragment thereof of the present invention. According to the present invention, a HER2 targeting agent comprising the antibody or antigen-binding fragment thereof of the present invention for use in a method for treating cancer in a subject is also provided. According to the present invention, a HER2 targeting agent comprising the antibody or antigen-binding fragment thereof of the present invention for use in a method for treating cancer in a subject is further provided. According to these embodiments, the subject may be a subject for which HER2 targeting therapy is determined to be effective by the above method. The subject may also be a subject having a HER2-positive tumor that is reactive with the antibody or antibody or antigen-binding fragment thereof of the present invention. A therapeutically effective amount is an amount of a pharmaceutical component that provides a medically significant benefit.

[0079] According to the present invention, there is provided a method for binding an antibody or an antigen-binding fragment thereof to cancer cells in a subject having a HER2-positive cancer, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of the present invention. According to the present invention, there is also provided a method for binding a HER2 targeting agent comprising an antibody or antigen-binding fragment thereof to cancer cells in a subject having a HER2-positive cancer, the method comprising administering to the subject an effective amount of the HER2 targeting agent comprising the antibody or antigen-binding fragment thereof of the present invention. EXAMPLES

[0080] The following examples are described below. The reagents used in the following examples are specifically the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Fujifilm Wako Pure Chemical Industries, Ltd., Nacalai Tesque, Inc., R&D Systems, USCN Life Science INC., etc.) can also be used.

[0081] Example 1: Production of CasMab anti-HER2 antibodies (1) Cell Human glioblastoma cell line LN229, human breast cancer cell line SK-BR-3, Chinese hamster ovary (CHO)-K1, and mouse myeloma cell line P3U1 were purchased from the American Type Culture Collection (ATCC). Normal human epidermal keratinocyte cell line HaCaT was purchased from Cosmo Bio. Human HER2 having the amino acid sequence of SEQ ID NO: 2 was used as HER2. The extracellular domain of human HER2 (secreted HER2) having the amino acid sequence of SEQ ID NO: 3 was used as HER2ec. CHO-K1 cells were transfected with an expression plasmid (pCAG / PA-HER2-RAP-MAP; Fujii et al., 2016; see Fig. 1A) containing the HER2 protein with the PA tag (Protein Expr Purif., 95, 240-247, 2014), RAP tag (Fujii et al., Monoclon. Antib. Immunodiagn. Immunother. Apr;36(2):68-71, 2017), and MAP tag (Fujii et al., Monoclon. Antib. Immunodiagn. Immunother., 35(6), 293-299. 2016) at the N-terminus using Lipofectamine LTX (Thermo Fisher Scientific, Inc.). HER2-expressing cells were incubated with an anti-PA tag antibody (Protein Expr Purif., 95, 240-247, 2014), and then established a stable CHO / HER2 cell line under drug selection. In addition, LN229 was transfected with the expression plasmids (pCAG / PA-HER2-RAP-MAP or pCAG / PA-HER2ec-RAP-MAP) for HER2 protein tagged with PA at the N-terminus, RAP tag at the C-terminus, and MAP tag at the C-terminus (PA-HER2-RAP-MAP; see Fujii et al., 2016; Figure 1B) using the Neon transfection system (Thermo Fisher Scientific, Inc.).PA-HER2-RAP-MAP expressing cells were sorted with anti-PA tag antibody, and then the stable expressing cell line LN229 / HER2 was produced under drug selection. For the high expressing cell line of HER2ec, the culture supernatant was screened by sandwich ELISA with antibody against RAP tag (PMab-2) and antibody against PA tag (NZ-1), and LN229 / HER2ec (secretory type) was produced. RPMI 1640 medium (Nacalai Tesque, Inc.) was used as the basal medium for CHO-K1, CHO / HER2, and P3U1. Dulbecco's modified Eagle's medium (Nacalai Tesque, Inc.) was used as the basal medium for LN229, LN229 / HER2ec, LN229 / HER2, SK-BR-3, and HaCaT. The medium was supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), penicillin (100 units / mL), streptomycin (100 units / mL), and amphotericin B (0.25 mg / mL), and the cells were cultured at 37°C under 5% CO2.

[0082] (2) Preparation of monoclonal antibodies BALB / c mice (4 weeks old, female) used as immunized animals were purchased from CLEA Japan. The antibodies were produced using the CasMab method (Kato and Kaneko, Sci. Rep., 4: 5924, 2014). HER2ec protein produced by LN229 cells was affinity purified with an anti-MAP tag antibody (Fujii et al., 2016, supra). BALB / c mice were then given the first immunization with 100 μg of HER2ec protein mixed with Imject TM Alum (Thermo Fisher Scientific, Inc.). As booster immunizations (total of three times), 100 μg of HER2ec protein purified from LN229 / HER2ec was administered intraperitoneally every week. Then, 100 μg of HER2ec was administered as the final immunization two days before splenectomy. Splenocytes prepared from the resected spleen were fused with P3U1 cells using PEG1500 (Roche Diagnostics). After cell fusion, the cells were cultured in RPMI 1640 supplemented with hypoxanthine, aminopterin, and thymidine (Thermo Fisher Scientific, Inc.), and the culture supernatants were subjected to primary screening using HER2ec by the ELISA method described below. Hybridomas that were positive in the primary screening were cloned by limiting dilution. Monoclonal antibodies were prepared by using hybridoma supernatants cultured in serum-free medium (Hybridoma-SFM; Thermo Fisher Scientific, Inc.) and purified using Protein G Sepharose 4 Fast Flow (GE Healthcare UK Ltd). Flow cytometry, Western blotting, and immunohistochemistry were then performed as described below. As a result, from about 250 clones including the control antibody H2Mab-119 antibody, several clones of cancer-specific antibodies that are thought to recognize cancer-specific HER2 were obtained through narrowing down by flow cytometry, Western blot, and immunohistochemical staining. Hereinafter, two clones were named H2Mab-214 and H2Mab-250, respectively (hereinafter, these may be abbreviated as the antibody of the present invention).The antibodies obtained from these clones were designated H2Mab-214 antibody and H2Mab-250 antibody, respectively.

[0083] (3) ELISA HER2ec (diluted with phosphate-buffered saline (PBS)) was immobilized on a 96-well plate at a concentration of 1 μg / ml for 30 minutes at 37°C. Blocking was performed by reacting with 1% bovine serum albumin (BSA) / 0.05% Tween20 in PBS (PBST) for 30 minutes at 37°C. The culture supernatant was then reacted for 30 minutes at 37°C, and washed three times with 0.05% PBST. Furthermore, the secondary antibody (1 / 2000 dilution; Agilent Technologies, Inc.) was reacted for 30 minutes at 37°C, and washed three times with 0.05% PBST. Finally, the color was developed using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.), and the absorbance was measured at OD655nm on a microplate reader.

[0084] (4) Flow cytometry Various adherent cells were collected using 0.25% trypsin / 1mM EDTA (Nacalai Tesque, Inc.), washed with 0.1% BSA / PBS, and reacted with the antibody (1μg / mL) prepared in (2) above at 4℃. The cells were washed with 0.1% BSA / PBS, and reacted with Oregon Green-labeled anti-mouse IgG antibody (1000-fold dilution; Thermo Fisher Scientific, Inc.) for 30 minutes. Fluorescence intensity was measured using an EC800 cell Analyzer (Sony Corp.).

[0085] (5) Western blot The cell lysate prepared with SDS sample buffer (Nacalai Tesque, Inc.) containing 2-mercaptoethanol was electrophoresed on a 5-20% polyacrylamide gel (Fujifilm Wako Pure Chemical Industries, Ltd.) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck KGaA). After blocking the polyvinylidene difluoride (PVDF) membrane with 4% skim milk-added PBST, it was reacted with the antibody (5 μg / mL) prepared in (2) above or anti-b-actin antibody (clone AC-15; Sigma-Aldrich Corp.). It was then reacted with peroxidase-labeled anti-mouse IgG antibody (1000 dilution; Agilent Technologies, Inc.). A chemiluminescence reagent (ImmunoStar LD; Fujifilm Wako Pure Chemical Industries, Ltd.) was used for detection, and the signal was detected with a Sayaca-Imager (DRC Co., Ltd.).

[0086] (6) Immunohistochemical staining Breast cancer tissues purchased from BioChain (catalog number: B904111) and normal breast tissues purchased from BioChain Institute, Inc. (catalog number: B803077) were used. Tissue sections were deparaffinized with xylene, dehydrated, immersed in citrate buffer (pH 6.0; Agilent Technologies, Inc.), and autoclaved for 20 minutes. Next, the sections were reacted with the antibody (1 μg / mL) prepared in (2) above at room temperature for 1 hour, and then treated with Envision+ kit, mouse (Agilent Technologies, Inc.) for 30 minutes. The sections were treated with 3,3'-diaminobenzidine tetrahydrochloride for 2 minutes to develop color, and counterstained with hematoxylin. Staining intensity was evaluated as 0, 1+, 2+, and 3+ based on the 2018 ASCO / CAP guidelines mentioned above.

[0087] Example 2: Sequencing of CasMab anti-HER2 antibodies (1) Determination of the amino acid and nucleotide sequences of the H2Mab-214 and H2Mab-250 antibodies Total RNA was extracted from 1 × 10 6 H 2Mab-214 and H 2Mab-250 hybridoma cells using the RNeasy Plus mini kit (QIAGEN). cDNA synthesis was performed from 5 μg of total RNA using the SuperScript IV cDNA Syntheses System (Thermo Fisher Scientific, Inc.). The cDNA was used as a template in the following experiments. The following primers were used for amplifying the heavy chain (H chain). InF.HindIII-H2-214H (SEQ ID NO: 4) InF.HindIII-H2-250H (SEQ ID NO:5) InFr. IgG1terNotI (SEQ ID NO: 6)

[0088] HotStar HiFidelity DNA polymerase (QIAGEN) was used for the PCR reaction. The temperature conditions were 95°C for 5 minutes, followed by 35 cycles of 94°C for 15 seconds, 50°C for 1 minute, and 72°C for 1 minute, and finally 72°C for 10 minutes. The amplified PCR product was purified using FastGene Gel / PCR Extraction (Japan Genetics Co., Ltd.).

[0089] The PCR product of the H2Mab-214 antibody H chain was treated with the restriction enzymes HindIII and NotI at 37°C for 1 hour and then subcloned into the pCAG vector purified with the FastGene Gel / PCR Extraction kit (Nihon Genetics Co., Ltd.) using an InFusion-HD cloning kit (Takara Bio Inc.), and the base sequence was confirmed using the vector primers.

[0090] The PCR product of the H2Mab-250 antibody H chain was treated with the restriction enzymes HindIII and NotI at 37°C for 1 hour and then subcloned into the pCAG vector purified with the FastGene Gel / PCR Extraction kit (Nihon Genetics Co., Ltd.) using an InFusion-HD cloning kit (Takara Bio Inc.), and the base sequence was confirmed using the vector primers.

[0091] The following primers were used for amplifying the light chain (L chain): InF.HindIII-H2-214L (SEQ ID NO: 7) InF.HindIII-H2-250L (SEQ ID NO: 8) InF.mIgCKterNotI (SEQ ID NO: 9)

[0092] HotStar HiFidelity DNA polymerase (QIAGEN) was used for the PCR reaction. The temperature conditions were 95°C for 5 minutes, followed by 35 cycles of 94°C for 15 seconds, 50°C for 1 minute, and 72°C for 1 minute, and finally 72°C for 10 minutes. The amplified PCR product was purified using FastGene Gel / PCR Extraction (Japan Genetics Co., Ltd.).

[0093] The PCR product of the H2Mab-214 antibody L chain was treated with the restriction enzymes HindIII and NotI at 37°C for 1 hour and then subcloned into the pCAG vector purified with the FastGene Gel / PCR Extraction kit (Nihon Genetics Co., Ltd.) using an InFusion-HD cloning kit (Takara Bio Inc.), and the base sequence was confirmed using the vector primers.

[0094] The PCR product of the H2Mab-250 antibody L chain was treated with the restriction enzymes HindIII and NotI at 37°C for 1 hour and then subcloned into the pCAG vector purified with the FastGene Gel / PCR Extraction kit (Nihon Genetics Co., Ltd.) using an InFusion-HD cloning kit (Takara Bio Inc.), and the base sequence was confirmed using the vector primers. As a result, the base sequence of the DNA encoding the H chain of the H2Mab-214 antibody was shown in SEQ ID NO: 10, and the base sequence of the DNA encoding the L chain of the H2Mab-214 antibody was shown in SEQ ID NO: 11. Similarly, the base sequence of the DNA encoding the H chain of the H2Mab-250 antibody was shown in SEQ ID NO: 12, and the base sequence of the DNA encoding the L chain of the H2Mab-250 antibody was shown in SEQ ID NO: 13.

[0095] The amino acid sequence was predicted from each base sequence of the H2Mab-214 antibody. The H-chain amino acid sequence of the H2Mab-214 antibody is shown in SEQ ID NO: 14, and the L-chain amino acid sequence of the H2Mab-214 antibody is shown in SEQ ID NO: 15. Similarly, the amino acid sequence was predicted from each base sequence of the H2Mab-250 antibody. The H-chain amino acid sequence of the H2Mab-250 antibody is shown in SEQ ID NO: 16, and the L-chain amino acid sequence of the H2Mab-250 antibody is shown in SEQ ID NO: 17.

[0096] (2) Determination of CDRs (complementarity determining regions) of H2Mab-214 and H2Mab-250 antibodies From the base sequence determined in (1) above, the CDR sites were identified using immunoglobulin sequence prediction software (Kabat numbering) provided on the website at the following URL (abYsis; http: / / www.abysis.org / abysis / index.html).

[0097] As a result, the amino acid sequences of the heavy chain CDR1 to 3 and the light chain CDR1 to 3 of the H2Mab-214 antibody were identified as shown in SEQ ID NOs: 18 to 20 and 21 to 23, respectively. Similarly, the amino acid sequences of the heavy chain CDR1 to 3 and the light chain CDR1 to 3 of the H2Mab-250 antibody were identified as shown in SEQ ID NOs: 24 to 26 and 27 to 29, respectively.

[0098] Example 3: Evaluation of CasMab anti-HER2 antibodies (1) Flow cytometry Flow cytometry analysis was performed on the normal human epidermal keratinocyte cell line HaCaT (Cosmo Bio) and the breast cancer cell line SK-BR-3 (ATCC) using the following method. Various adherent cells were collected using 0.25% trypsin / 1 mM EDTA (Nacalai Tesque, Inc.), washed with 0.1% BSA / PBS, and then reacted with various anti-HER2 monoclonal antibodies (control antibody (H2Mab-119 antibody; see Monoclon. Antib. Immunodiagn. Immunother., vol.36(6), 287-290, 2017), H2Mab-214 antibody, and H2Mab-250 antibody, and each culture supernatant) at 4°C. The cells were washed with 0.1% BSA / PBS and then reacted with Oregon Green-labeled anti-mouse IgG antibody (1000-fold dilution; Thermo Fisher Scientific, Inc.) for 30 minutes. Fluorescence intensity was measured using an EC800 cell Analyzer (Sony Corp.). As a result, as shown in Figure 2, the control antibody (H2Mab-119 antibody) not only reacted with the cancer cell line SK-BR-3, but also showed moderate reactivity with the normal cell line HaCaT. On the other hand, the antibody of the present invention showed moderate reactivity with the cancer cell line SK-BR-3, but did not react at all with the normal cell line HaCaT. This demonstrated that the antibody of the present invention has cancer specificity.

[0099] (2) Immunohistochemical staining Immunohistochemical staining was performed on HER2-negative breast cancer tissue microarrays (catalog number: B904111) obtained from BioChain, Inc., using the following method. The tissue sections were deparaffinized and dehydrated in xylene, immersed in citrate buffer (pH 6.0; Agilent Technologies, Inc.), and autoclaved for 20 minutes. They were then reacted with the antibody of the present invention (1 μg / mL) at room temperature for 1 hour, and then treated with Envision+kit (Agilent Technologies, Inc.) for 30 minutes. They were treated with 3,3'-diaminobenzidine tetrahydrochloride for 2 minutes to develop color, and counterstained with hematoxylin. Staining intensity was evaluated as 0, 1+, 2+, or 3+ as described above. As a result, as shown in Figure 3 below, the control antibody (H2Mab-119 antibody) was negative in breast cancer tissues determined to be HER2 negative by a diagnostic agent that examines the HER2 expression status of cancer cells using immunohistochemical staining (IHC), whereas the antibody of the present invention had cores showing 1+ to 3+ positive images in the HER2-negative breast cancer tissues. This demonstrates that the antibody of the present invention exhibits high sensitivity in immunohistochemical staining.

[0100] Example 4: Epitope analysis of CasMab anti-HER2 antibody (1) The antibody of the present invention reacted with all of the deletion mutants prepared by a conventional method (WT-dN23 (a mutant in which the region from the N-terminus to amino acid number 22 of human HER2 (SEQ ID NO: 2) is deleted), dN200 (a mutant in which the region from the N-terminus to amino acid number 199 of human HER2 (SEQ ID NO: 2) is deleted), dN300 (a mutant in which the region from the N-terminus to amino acid number 299 of human HER2 (SEQ ID NO: 2) is deleted), dN400 (a mutant in which the region from the N-terminus to amino acid number 399 of human HER2 (SEQ ID NO: 2) is deleted), dN500 (a mutant in which the region from the N-terminus to amino acid number 499 of human HER2 (SEQ ID NO: 2) is deleted), and dN600 (a mutant in which the region from the N-terminus to amino acid number 599 of human HER2 (SEQ ID NO: 2) is deleted)). Thus, it was predicted that the antibody recognizes the amino acid sequence of amino acids 600-652 of human HER2 (SEQ ID NO: 2) (SEQ ID NO: 30). On the other hand, the control antibody (H2Mab-119 antibody) reacted with WT-dN23 but did not react with dN200, dN300, dN400, dN500, or dN600, and was therefore predicted to recognize the amino acid region of human HER2 from amino acid numbers 23 to 199. Note that the amino acid numbers in this and the following examples are indicated as the amino acid positions in the amino acid sequence of SEQ ID NO:2.

[0101] Next, HER2ec (23-652) and HER2ec 23-42, 33-52, 43-62, 53-72, 63-82, 73-92, 83-102, 93-112, 103-122, 113-132, 123-142, 133-152, 143-162, 153-172, 163-182, 173-192, 183- 202, 193-212, 203-222, 213-232, 223-242, 233-252, 243-262, 253-272, 263-282, 273-292, 283-302, 293-312, 303-322, 313-332, 323-342, 333-352, 343-362, 353-372, 363-382, 3 73-392, 383-402, 393-412, 403-422, 413-432, 423-442, 433-452, 443-462, 453-472, 463-482, 473-492, 483-502, 493-512, 503-522, 513-532, 523-542, 533-552, 543-562, 553-57 The reactivity of the antibody of the present invention to synthetic peptides consisting of amino acid regions of 2, 563-582, 573-592, 583-602, 593-612, 603-622, 613-632, 623-642, and 633-652 was confirmed by ELISA (here, the above amino acid numbers indicate the amino acid numbers in the amino acid sequence of SEQ ID NO: 2). That is, each peptide was immobilized on a 96-well plate at a concentration of 10 μg / ml at 37° C. for 30 minutes. Blocking was performed by reacting with 1% BSA / 0.05% PBST at 37° C. for 30 minutes. Thereafter, the antibody of the present invention at 10 μg / ml was reacted at 37° C. for 30 minutes, and then washed three times with 0.05% PBST. Furthermore, the secondary antibody (1 / 2000 dilution; Agilent Technologies, Inc.) was reacted at 37° C. for 30 minutes, and washed three times with 0.05% PBST. Finally, the color was developed using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.) for 15 minutes, and the absorbance was measured at OD655nm using a microplate reader (Bio-Rad).

[0102] As a result, the H2Mab-214 antibody and the H2Mab-250 antibody reacted strongly to the 603-622 peptide and the 613-632 peptide, suggesting that the epitope of the antibody of the present invention is the amino acid region from 603 to 622 of human HER2 (sequence number: 31), the amino acid region from 613 to 632 of human HER2 (sequence number: 32), and in particular the amino acid region from 613 to 622 (sequence number: 33).

[0103] Thus, using HER2ec as a positive control, the reactivity of the antibody of the present invention against 20 types of mutant peptides in which each of the 20 amino acids in the 603-622 peptide of human HER2 was replaced with alanine was confirmed by ELISA.

[0104] As a result, the reactivity of the H2Mab-214 antibody to the K615A peptide and the F616A peptide, in which K615 and F616 at amino acids 603 to 622 of human HER2 were replaced with alanine, was weaker than the reactivity to the wild-type HER2 peptide (amino acids 603 to 622 of human HER2), indicating that the two amino acids K615 and F616 and their surroundings are the epitope of the H2Mab-214 antibody. Furthermore, the reactivity of the H2Mab-250 antibody to the W614A peptide, in which W614 of human HER2 was replaced with alanine, was weaker than the reactivity to the wild-type HER2 peptide, indicating that the amino acid W614 and its surroundings are the epitope of the H2Mab-250 antibody.

[0105] The epitopes of the H2Mab-214 and H2Mab-250 antibodies were investigated in more detail. The reactivity of the antibody of the present invention against synthetic peptides (various deletion mutants) consisting of the amino acid region of human HER2, 0, 611-621, 611-622, 610-618, 610-619, 610-620, 610-621, 610-622, 609-618, 609-619, 609-620, 609-621, and 609-622, was confirmed by ELISA (here, the above amino acid numbers indicate the amino acid numbers in the amino acid sequence of SEQ ID NO: 2). ELISA was performed in the same manner as above. As the primary antibody, the antibody of the present invention was used at 50 μL / well, and as the secondary antibody, rabbit anti-mouse IgG / HRP (Agilent Technologies, Inc., 1% BSA / PBS-T, 1 / 2,000 dilution, 50 μL / well) was used. For detection, an ELISA POD substrate TMB kit (Nacalai Tesque, Inc.) was used. The absorbance at OD655 nm was measured using an iMark Microplate Reader. The results are shown in Figure 4. In Figure 4, the amino acid numbers are preceded by "p". In Figure 4, PC indicates the positive control and NC indicates the negative control.

[0106] As shown in Figure 4, the H2Mab-214 antibody showed strong reactivity to a peptide consisting of the amino acid region of 612 to 618 (SEQ ID NO: 35), and showed reactivity equivalent to a longer peptide containing the amino acid region of 612 to 618. Also, as shown in Figure 4, the H2Mab-250 antibody showed strong reactivity to a peptide consisting of the amino acid region of 611 to 618 (SEQ ID NO: 34), and showed reactivity equivalent to a longer peptide containing the amino acid region of 611 to 618.

[0107] Furthermore, as shown in Figure 4, when antibodies with an OD value of more than 0.1 were evaluated as being reactive, both the H2Mab-214 antibody and the H2Mab-250 antibody showed reactivity to a peptide consisting of the amino acid region of 613 to 619 (SEQ ID NO: 36). However, the reactivity was weaker than the reactivity to a peptide consisting of the amino acid region of 612 to 618 (SEQ ID NO: 35) and a peptide consisting of the amino acid region of 611 to 618 (SEQ ID NO: 34), demonstrating that the reactivity of the antibody of the present invention to the partial peptide of HER2 is strongly influenced by the amino acid at position 612 (and / or the amino acid at position 611).

[0108] Example 5: Epitope analysis of CasMab anti-HER2 antibody (2) (1) Flow cytometry Epitope analysis of the H2Mab-214 antibody or H2Mab-250 antibody was performed by flow cytometry (FACS) analysis using alanine substitution mutants. A HER2 gene (human HER2 having the amino acid sequence of SEQ ID NO: 2) in which an amino acid at a target position was substituted with alanine was prepared using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc.), and the introduction of the mutation was confirmed by determining the base sequence. The HER2 gene containing the target alanine substitution mutation was added with a PA tag at the N-terminus and a RAP tag and a MAP tag at the C-terminus, as with the wild-type HER2 gene, and incorporated into an expression vector (see Example 1). This plasmid was introduced into CHO-K1 cells using the Neon Transfection system (Thermo Fisher Scientific, Inc.), and a stable expression cell line was established by cell sorting using anti-PA antibody (cell sorter SA3800, Sony Corp.) and then drug selection using Zeocin (0.5 mg / mL, InVivogen).

[0109] CHO-K1 cells that forcibly and stably expressed the HER2 gene in which the amino acid at the target position was replaced with alanine were collected from the culture dish using 0.25% trypsin / 1 mM EDTA solution (Nacalai Tesque, Inc.) and washed with 0.1% BSA (Nacalai Tesque, Inc.) / PBS. The primary antibody (H 2Mab-214 antibody or H 2Mab-250 antibody) was adjusted to 10 μg / mL with 0.1% BSA / PBS, added to the collected cells, mixed, and reacted on ice for 30 minutes. Then, the cells were washed with 0.1% BSA / PBS. Subsequently, the fluorescently labeled secondary antibody (1 / 1000 dilution, anti-Mouse IgG Alexa Fluor 488, Thermo Fisher Scientific, Inc.) was adjusted with 0.1% BSA / PBS and reacted on ice for 30 minutes. The cells were washed again with 0.1% BSA / PBS. Antibody reactivity was detected using a cell analyzer EC800 (Sony Corp.).

[0110] FIG. 5 shows the results of flow cytometry (FACS analysis) of the antibody against the PA tag (NZ-1), the H2Mab-214 antibody, the H2Mab-250 antibody, and the positive control antibody (trastuzumab).

[0111] As a result, as shown in FIG. 5, H2Mab-214 did not show significant binding to HER2-K615A and HER2-F616A, and H2Mab-250 did not show significant binding to HER2-W614A. In contrast, the NZ-1 antibody detected all HER2 point mutants, so it is clear that the expression levels of each point mutant were sufficient. This suggests that the antibody of the present invention that recognizes the epitope (W614, K615, F616) does not react at all to HER2 expressed on non-cancer cells, but can specifically react to HER2 expressed on cancer cells. In addition, as shown in FIG. 5, trastuzumab showed significant binding to all point mutants. This suggests that, although trastuzumab is an antibody that recognizes domain IV of HER2 like the antibody of the present invention, it does not recognize the epitopes (W614, K615, F616) in domain IV that are recognized by the antibody of the present invention. Therefore, unlike trastuzumab, it is clear that the property of recognizing the epitopes (W614, K615, F616) is involved in the specific recognition of HER2 on cancer cells.

[0112] (2) Biacore measurement Using Biacore X100 (Cytiva), the interaction between the H2Mab-214 antibody or H2Mab-250 antibody and various peptides was analyzed. The peptides were the amino acid sequence from 603 to 622 of human HER2 (p603-622), p603-622 with one amino acid substituted with alanine (G603A, V604A, K605A, P606A, D607A, L608A, S609A, Y610A, M611A, P612A, I613A, W614A, K615A, F616A, P617A, D618A, E619A, E620A, G621A), p603-622 with one amino acid substituted with glycine (A622G), and a partial peptide of HER2 lacking part of the amino acids in p603-622 (p614-619A). , p614-620, p614-621, p614-622, p613-618, p613-619, p613-620, p613-621, p613-622, p612-618, p612-619, p612-620, p612-621, p612-622, p611-618, p6 The peptides were immobilized on a CM5 chip by the amine coupling method using the H2Mab-214 antibody or H2Mab-250 antibody, which were diluted with acetate buffer (pH 4.0). Unreacted NHS esters were blocked with ethanolamine. The interactions were measured by adding various peptides to the CM5 chip on which the H2Mab-214 antibody or H2Mab-250 antibody was immobilized. The running buffer was PBS containing 0.005% (v / v) Tween 20, or PBS containing 0.005% (v / v) Tween 20 and 1.19% DMSO, and the regeneration buffer was glycine-HCl (pH 1.5). All measurements were performed at 25°C. The measurement data was analyzed using the 1:1 binding model of BIAevaluation software (Cytiva), and the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD) were calculated.For those for which ka or kd could not be determined, KD was calculated using the equilibrium value analysis method of BIAevaluation software (Cytiva).

[0113] The results are shown in Tables 1 and 2 for the binding affinity of the H2Mab-214 antibody, and in Tables 3 and 4 for the H2Mab-250 antibody, as determined by Biocore. The amino acid numbers in Tables 2 and 4 indicate the amino acid regions used.

[0114] [Table 1]

[0115] [Table 2]

[0116] [Table 3]

[0117] [Table 4]

[0118] The results of Examples 4 and 5 showed that the binding affinity of the antibody of the present invention correlated in various alanine substitution mutants and various deletion mutants, indicating that the two amino acids K615 and F616 and their surroundings (I613, W614) are the epitope of the H2Mab-214 antibody.

[0119] In addition, it was found that the amino acid W614 and its surroundings (I613, K615, F616, P617) are the epitope of the H2Mab-250 antibody.

[0120] Example 6: Further antibody groups In this example, an attempt was made to obtain further anti-HER2 antibodies. mouse Six-week-old BALB / c mice were purchased from CLEA Japan.

[0121] immunogen As an immunogen, a 20-amino acid residue (HER2_604-622C; NH2-VKPDLSYMPIWKFPDEEGAC-COOH; SEQ ID NO: 37) was synthesized (Eurofin) by adding Cys to the C-terminus of HER2 604-622 amino acids (19 amino acids), and KLH was added by a standard method. The purity of the peptide was 90% or higher.

[0122] Immunization method HER2_604-622C was mixed at 100 μg / mouse (100 μl) with 100 μl of ImjectAlum (Thermo Fisher Scientific, Inc.) for the first immunization. Thereafter, 100 μg / mouse (100 μl) was used every week for the second, third, fourth, and fifth immunizations (without ImjectAlum). Two days after the fifth immunization, splenocytes were isolated from the spleen excised from the mouse by a standard method, and cell fusion was performed at a splenocyte:mouse myeloma cell (P3U1) ratio of 10:1. Cell fusion was performed by standard methods using PEG1500 (Roche Diagnostics).

[0123] Hybridoma culture The hybridomas were seeded onto a 96-well plate using RPMI (Nacalai Tesque, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.) containing HAT (Thermo Fisher Scientific, Inc.).

[0124] screening Six days later, screening was performed by ELISA. ELISA was performed as follows. HER2p604-622C was dissolved in DMSO at 10 mg / mL and then diluted with PBS (1 μg / mL). For peptide immobilization, HER2p604-622C was added to the plate at 50 ng / well (50 μL / well) and incubated at 37°C for 30 minutes. For blocking, 1% BSA / PBS-Tween (0.05%) was added at 100 μL / well and incubated at 37°C for 30 minutes. Hybridoma culture supernatant was added in 50 μL each and incubated at 37°C for 30 minutes. Secondary antibody was anti-mouse Immunoglobulins / HRP (Agilent, 1 / 2000 diluted with 1% BSA / PBS-0.05% Tween) at 50 μL each and incubated at 37°C for 30 minutes. Color development was performed by adding 50 μl of ELISA POD substrate TMB kit (Nacalai Tesque, Inc.) to each well and incubating for 10 minutes at room temperature. Measurement was performed for 15 minutes using an iMark Microplate Reader (OD 655 nm).

[0125] Single Cell Cloning Single cell cloning was performed on the positive wells. Clones were confirmed by visual inspection and positive clones were established by ELISA. The clones were named H2Mab-279~299.

[0126] Determining the Subclass Subclasses were determined by standard methods (similar to H2Mab-214,250).

[0127] [Table 5]

[0128] Purification of each antibody was performed as described in Example 1(1). The purification column used was Ab-Capcher TM (Protenova). The purified antibodies were subjected to epitope analysis, FACS, and immunohistochemical staining. FACS was performed as described in Example 1(4), and immunohistochemical staining was performed as described in Example 1(6). Epitope analysis was performed by ELISA as described in Example 4.

[0129] In epitope analysis, when an antibody tested against a peptide changed to alanine by alanine scanning reduced or eliminated binding affinity, the amino acid changed to alanine was determined to be the putative epitope of the antibody. The results of epitope analysis are shown in Figures 6A to 6E. In Figures 6A to 6E, the putative epitopes are underlined. As shown in Figures 6A to 6E, the putative epitopes of the above antibody clones were all found in the regions of W614, K615, and F616.

[0130] In the FACS analysis, the binding of each antibody clone to the live cells shown in the figure was tested. Met5A cells were used as a mesothelioma cell line, HaCaT cells were used as a human epidermal keratinocyte cell line, MCF10A-III was used as a non-tumor epithelial cell line, A431 was used as a human epidermoid carcinoma cell line, and SK-BR-3 was used as a breast cancer cell line. The results were as shown in Figures 7A to 7G. As shown in Figures 7A to 7G, most of the cell clones using the amino acid of SEQ ID NO: 37 as the immunogen did not bind to normal cells, but showed binding to positive cancer cells.

[0131] The results of immunohistochemical staining are shown in Figures 8A to 8F. As shown in Figures 8A to 8F, all of the antibody clones using the amino acid of SEQ ID NO: 37 as the immunogen reacted strongly with cancer tissues, but little reaction was observed with normal tissues.

[0132] Antibody gene cloning Antibody gene cloning, prediction of the heavy and light chain variable regions, and CDR prediction were performed as described in Example 2. The activity of the recombinant antibody was confirmed by FACS.

[0133] The amino acid numbers of the heavy chain variable region and the light chain variable region in the amino acid sequences of SEQ ID NOs: 14 to 17 and 39 to 80 are as shown in the table below. The predicted CDR sequences are shown in SEQ ID NOs: 81 to 206, respectively. [Table 6]

[0134] SEQ ID NO: 1: Human c-erb-B-2 (HER2) nucleotide sequence (Genebank accession number: X03363) SEQ ID NO: 2: Human c-erb-B-2 (HER2) amino acid sequence (UniprotKB ID: P04626) SEQ ID NO: 3: Human c-erb-B-2 (HER2) ec (secreted extracellular domain) amino acid sequence (23-652aa) SEQ ID NO: 4: Primer (InF.HindIII-H2-214H) SEQ ID NO: 5: Primer (InF.HindIII-H2-250H) SEQ ID NO: 6: Primer (InFr. IgG1terNotI) SEQ ID NO: 7: Primer (InF.HindIII-H2-214L) SEQ ID NO: 8: Primer (InF.HindIII-H2-250L) SEQ ID NO: 9: Primer (InF.mIgCKterNotI) SEQ ID NO: 10: Base sequence of DNA encoding the H chain of the H2Mab-214 antibody SEQ ID NO: 11: Base sequence of DNA encoding the L chain of the H2Mab-214 antibody SEQ ID NO: 12: Base sequence of DNA encoding the H chain of the H2Mab-250 antibody SEQ ID NO: 13: Base sequence of DNA encoding the L chain of the H2Mab-250 antibody SEQ ID NO: 14: H-chain amino acid sequence of H2Mab-214 antibody SEQ ID NO: 15: L-chain amino acid sequence of H2Mab-214 antibody SEQ ID NO: 16: H-chain amino acid sequence of H2Mab-250 antibody SEQ ID NO: 17: L-chain amino acid sequence of H2Mab-250 antibody SEQ ID NO: 18: Amino acid sequence of H chain CDR1 of H2Mab-214 antibody SEQ ID NO: 19: Amino acid sequence of H chain CDR2 of H2Mab-214 antibody SEQ ID NO: 20: Amino acid sequence of the H chain CDR3 of the H2Mab-214 antibody SEQ ID NO: 21: Amino acid sequence of L chain CDR1 of H2Mab-214 antibody SEQ ID NO: 22: Amino acid sequence of L chain CDR2 of H2Mab-214 antibody SEQ ID NO: 23: Amino acid sequence of the L chain CDR3 of the H2Mab-214 antibody SEQ ID NO: 24: Amino acid sequence of H chain CDR1 of H2Mab-250 antibody SEQ ID NO: 25: Amino acid sequence of H chain CDR2 of H2Mab-250 antibody SEQ ID NO: 26: Amino acid sequence of the H chain CDR3 of the H2Mab-250 antibody SEQ ID NO: 27: Amino acid sequence of L chain CDR1 of H2Mab-250 antibody SEQ ID NO: 28: Amino acid sequence of L chain CDR2 of H2Mab-250 antibody SEQ ID NO: 29: Amino acid sequence of the L chain CDR3 of the H2Mab-250 antibody SEQ ID NO: 30: Amino acid sequence of amino acid numbers 600 to 652 of human HER2 of SEQ ID NO: 2 SEQ ID NO: 31: Amino acid sequence of amino acid numbers 603 to 622 of human HER2 of SEQ ID NO: 2 SEQ ID NO: 32: Amino acid sequence of amino acid numbers 613 to 632 of human HER2 of SEQ ID NO: 2 SEQ ID NO: 33: Amino acid sequence of amino acid numbers 613 to 622 of human HER2 of SEQ ID NO: 2 SEQ ID NO: 34: Amino acid sequence of amino acid numbers 611 to 618 of human HER2 of SEQ ID NO: 2 SEQ ID NO: 35: Amino acid sequence of amino acid numbers 612 to 618 of human HER2 of SEQ ID NO: 2 SEQ ID NO: 36: Amino acid sequence of amino acid numbers 613 to 619 of human HER2 of SEQ ID NO: 2 SEQ ID NO: 37: Amino acid sequence of HER2p604-622C SEQ ID NO: 38: An example of a linker used in scFv SEQ ID NO: 39: H-chain amino acid sequence of H2Mab-279 antibody SEQ ID NO: 40: L-chain amino acid sequence of H2Mab-279 antibody SEQ ID NO: 41: H-chain amino acid sequence of H2Mab-280 antibody SEQ ID NO: 42: L-chain amino acid sequence of H2Mab-280 antibody SEQ ID NO: 43: H-chain amino acid sequence of H2Mab-281 antibody SEQ ID NO: 44: L-chain amino acid sequence of H2Mab-281 antibody SEQ ID NO: 45: H-chain amino acid sequence of H2Mab-282 antibody SEQ ID NO: 46: L-chain amino acid sequence of H2Mab-282 antibody SEQ ID NO: 47: H-chain amino acid sequence of H2Mab-283 antibody SEQ ID NO: 48: L-chain amino acid sequence of H2Mab-283 antibody SEQ ID NO: 49: H chain amino acid sequence of H2Mab-284 antibody SEQ ID NO: 50: L-chain amino acid sequence of H2Mab-284 antibody SEQ ID NO: 51: H2Mab-285 antibody H chain amino acid sequence SEQ ID NO: 52: L-chain amino acid sequence of H2Mab-285 antibody SEQ ID NO: 53: H-chain amino acid sequence of H2Mab-286 antibody SEQ ID NO: 54: L-chain amino acid sequence of H2Mab-286 antibody SEQ ID NO: 55: H2Mab-287 antibody H chain amino acid sequence SEQ ID NO: 56: L-chain amino acid sequence of H2Mab-287 antibody SEQ ID NO: 57: H-chain amino acid sequence of H2Mab-288 antibody SEQ ID NO: 58: L-chain amino acid sequence of H2Mab-288 antibody SEQ ID NO: 59: H-chain amino acid sequence of H2Mab-289 antibody SEQ ID NO: 60: L-chain amino acid sequence of H2Mab-289 antibody SEQ ID NO: 61: H-chain amino acid sequence of H2Mab-290 antibody SEQ ID NO: 62: L-chain amino acid sequence of H2Mab-290 antibody SEQ ID NO: 63: H-chain amino acid sequence of H2Mab-291 antibody SEQ ID NO: 64: L-chain amino acid sequence of H2Mab-291 antibody SEQ ID NO: 65: H2Mab-292 antibody H chain amino acid sequence SEQ ID NO: 66: L-chain amino acid sequence of H2Mab-292 antibody SEQ ID NO: 67: H-chain amino acid sequence of H2Mab-293 antibody SEQ ID NO: 68: L-chain amino acid sequence of H2Mab-293 antibody SEQ ID NO: 69: H-chain amino acid sequence of H2Mab-294 antibody SEQ ID NO: 70: L-chain amino acid sequence of H2Mab-294 antibody SEQ ID NO: 71: H2Mab-295 antibody H chain amino acid sequence SEQ ID NO: 72: L-chain amino acid sequence of H2Mab-295 antibody SEQ ID NO: 73: H-chain amino acid sequence of H2Mab-296 antibody SEQ ID NO: 74: L-chain amino acid sequence of H2Mab-296 antibody SEQ ID NO: 75: H-chain amino acid sequence of H2Mab-297 antibody SEQ ID NO: 76: L-chain amino acid sequence of H2Mab-297 antibody SEQ ID NO: 77: H-chain amino acid sequence of H2Mab-298 antibody SEQ ID NO: 78: L-chain amino acid sequence of H2Mab-298 antibody SEQ ID NO: 79: H-chain amino acid sequence of H2Mab-299 antibody SEQ ID NO: 80: L-chain amino acid sequence of H2Mab-299 antibody SEQ ID NO: 81: Amino acid sequence of H chain CDR1 of H2Mab-279 antibody SEQ ID NO: 82: Amino acid sequence of H chain CDR2 of H2Mab-279 antibody SEQ ID NO: 83: Amino acid sequence of the H chain CDR3 of the H2Mab-279 antibody SEQ ID NO: 84: Amino acid sequence of L chain CDR1 of H2Mab-279 antibody SEQ ID NO: 85: Amino acid sequence of the L chain CDR2 of the H2Mab-279 antibody SEQ ID NO: 86: Amino acid sequence of the L chain CDR3 of the H2Mab-279 antibody SEQ ID NO: 87: Amino acid sequence of H chain CDR1 of H2Mab-280 antibody SEQ ID NO: 88: Amino acid sequence of H chain CDR2 of H2Mab-280 antibody SEQ ID NO: 89: Amino acid sequence of the H chain CDR3 of the H2Mab-280 antibody SEQ ID NO: 90: Amino acid sequence of L chain CDR1 of H2Mab-280 antibody SEQ ID NO: 91: Amino acid sequence of the L chain CDR2 of the H2Mab-280 antibody SEQ ID NO: 92: Amino acid sequence of the L chain CDR3 of the H2Mab-280 antibody SEQ ID NO: 93: Amino acid sequence of H chain CDR1 of H2Mab-281 antibody SEQ ID NO: 94: Amino acid sequence of H chain CDR2 of H2Mab-281 antibody SEQ ID NO: 95: Amino acid sequence of the H chain CDR3 of the H2Mab-281 antibody SEQ ID NO: 96: Amino acid sequence of L chain CDR1 of H2Mab-281 antibody SEQ ID NO: 97: Amino acid sequence of L chain CDR2 of H2Mab-281 antibody SEQ ID NO: 98: Amino acid sequence of the L chain CDR3 of the H2Mab-281 antibody SEQ ID NO: 99: Amino acid sequence of H chain CDR1 of H2Mab-282 antibody SEQ ID NO: 100: Amino acid sequence of the H chain CDR2 of the H2Mab-282 antibody SEQ ID NO: 101: Amino acid sequence of the H chain CDR3 of the H2Mab-282 antibody SEQ ID NO: 102: Amino acid sequence of L chain CDR1 of H2Mab-282 antibody SEQ ID NO: 103: Amino acid sequence of the L chain CDR2 of the H2Mab-282 antibody SEQ ID NO: 104: Amino acid sequence of the L chain CDR3 of the H2Mab-282 antibody SEQ ID NO: 105: Amino acid sequence of H chain CDR1 of H2Mab-283 antibody SEQ ID NO: 106: Amino acid sequence of H chain CDR2 of H2Mab-283 antibody SEQ ID NO: 107: Amino acid sequence of the H chain CDR3 of the H2Mab-283 antibody SEQ ID NO: 108: Amino acid sequence of L chain CDR1 of H2Mab-283 antibody SEQ ID NO: 109: Amino acid sequence of the L chain CDR2 of the H2Mab-283 antibody SEQ ID NO: 110: Amino acid sequence of the L chain CDR3 of the H2Mab-283 antibody SEQ ID NO: 111: Amino acid sequence of H chain CDR1 of H2Mab-284 antibody SEQ ID NO: 112: Amino acid sequence of H chain CDR2 of H2Mab-284 antibody SEQ ID NO: 113: Amino acid sequence of the H chain CDR3 of the H2Mab-284 antibody SEQ ID NO: 114: Amino acid sequence of L chain CDR1 of H2Mab-284 antibody SEQ ID NO: 115: Amino acid sequence of the L chain CDR2 of the H2Mab-284 antibody SEQ ID NO: 116: Amino acid sequence of the L chain CDR3 of the H2Mab-284 antibody SEQ ID NO: 117: Amino acid sequence of H chain CDR1 of H2Mab-285 antibody SEQ ID NO: 118: Amino acid sequence of H chain CDR2 of H2Mab-285 antibody SEQ ID NO: 119: Amino acid sequence of the H chain CDR3 of the H2Mab-285 antibody SEQ ID NO: 120: Amino acid sequence of L chain CDR1 of H2Mab-285 antibody SEQ ID NO: 121: Amino acid sequence of the L chain CDR2 of the H2Mab-285 antibody SEQ ID NO: 122: Amino acid sequence of the L chain CDR3 of the H2Mab-285 antibody SEQ ID NO: 123: Amino acid sequence of H chain CDR1 of H2Mab-286 antibody SEQ ID NO: 124: Amino acid sequence of H chain CDR2 of H2Mab-286 antibody SEQ ID NO: 125: Amino acid sequence of the H chain CDR3 of the H2Mab-286 antibody SEQ ID NO: 126: Amino acid sequence of L chain CDR1 of H2Mab-286 antibody SEQ ID NO: 127: Amino acid sequence of the L chain CDR2 of the H2Mab-286 antibody SEQ ID NO: 128: Amino acid sequence of the L chain CDR3 of the H2Mab-286 antibody SEQ ID NO: 129: Amino acid sequence of H chain CDR1 of H2Mab-287 antibody SEQ ID NO: 130: Amino acid sequence of H chain CDR2 of H2Mab-287 antibody SEQ ID NO: 131: Amino acid sequence of the H chain CDR3 of the H2Mab-287 antibody SEQ ID NO: 132: Amino acid sequence of L chain CDR1 of H2Mab-287 antibody SEQ ID NO: 133: Amino acid sequence of the L chain CDR2 of the H2Mab-287 antibody SEQ ID NO: 134: Amino acid sequence of the L chain CDR3 of the H2Mab-287 antibody SEQ ID NO: 135: Amino acid sequence of H chain CDR1 of H2Mab-288 antibody SEQ ID NO: 136: Amino acid sequence of H chain CDR2 of H2Mab-288 antibody SEQ ID NO: 137: Amino acid sequence of the H chain CDR3 of the H2Mab-288 antibody SEQ ID NO: 138: Amino acid sequence of L chain CDR1 of H2Mab-288 antibody SEQ ID NO: 139: Amino acid sequence of L chain CDR2 of H2Mab-288 antibody SEQ ID NO: 140: Amino acid sequence of the L chain CDR3 of the H2Mab-288 antibody SEQ ID NO: 141: Amino acid sequence of H chain CDR1 of H2Mab-289 antibody SEQ ID NO: 142: Amino acid sequence of H chain CDR2 of H2Mab-289 antibody SEQ ID NO: 143: Amino acid sequence of the H chain CDR3 of the H2Mab-289 antibody SEQ ID NO: 144: Amino acid sequence of L chain CDR1 of H2Mab-289 antibody SEQ ID NO: 145: Amino acid sequence of the L chain CDR2 of the H2Mab-289 antibody SEQ ID NO: 146: Amino acid sequence of the L chain CDR3 of the H2Mab-289 antibody SEQ ID NO: 147: Amino acid sequence of H chain CDR1 of H2Mab-290 antibody SEQ ID NO: 148: Amino acid sequence of H chain CDR2 of H2Mab-290 antibody SEQ ID NO: 149: Amino acid sequence of the H chain CDR3 of the H2Mab-290 antibody SEQ ID NO: 150: Amino acid sequence of L chain CDR1 of H2Mab-290 antibody SEQ ID NO: 151: Amino acid sequence of the L chain CDR2 of the H2Mab-290 antibody SEQ ID NO: 152: Amino acid sequence of the L chain CDR3 of the H2Mab-290 antibody SEQ ID NO: 153: Amino acid sequence of H chain CDR1 of H2Mab-291 antibody SEQ ID NO: 154: Amino acid sequence of H chain CDR2 of H2Mab-291 antibody SEQ ID NO: 155: Amino acid sequence of the H chain CDR3 of the H2Mab-291 antibody SEQ ID NO: 156: Amino acid sequence of L chain CDR1 of H2Mab-291 antibody SEQ ID NO: 157: Amino acid sequence of the L chain CDR2 of the H2Mab-291 antibody SEQ ID NO: 158: Amino acid sequence of the L chain CDR3 of the H2Mab-291 antibody SEQ ID NO: 159: Amino acid sequence of H chain CDR1 of H2Mab-292 antibody SEQ ID NO: 160: Amino acid sequence of H chain CDR2 of H2Mab-292 antibody SEQ ID NO: 161: Amino acid sequence of the H chain CDR3 of the H2Mab-292 antibody SEQ ID NO: 162: Amino acid sequence of L chain CDR1 of H2Mab-292 antibody SEQ ID NO: 163: Amino acid sequence of the L chain CDR2 of the H2Mab-292 antibody SEQ ID NO: 164: Amino acid sequence of the L chain CDR3 of the H2Mab-292 antibody SEQ ID NO: 165: Amino acid sequence of H chain CDR1 of H2Mab-293 antibody SEQ ID NO: 166: Amino acid sequence of H chain CDR2 of H2Mab-293 antibody SEQ ID NO: 167: Amino acid sequence of the H chain CDR3 of the H2Mab-293 antibody SEQ ID NO: 168: Amino acid sequence of L chain CDR1 of H2Mab-293 antibody SEQ ID NO: 169: Amino acid sequence of the L chain CDR2 of the H2Mab-293 antibody SEQ ID NO: 170: Amino acid sequence of the L chain CDR3 of the H2Mab-293 antibody SEQ ID NO: 171: Amino acid sequence of H chain CDR1 of H2Mab-294 antibody SEQ ID NO: 172: Amino acid sequence of H chain CDR2 of H2Mab-294 antibody SEQ ID NO: 173: Amino acid sequence of the H chain CDR3 of the H2Mab-294 antibody SEQ ID NO: 174: Amino acid sequence of L chain CDR1 of H2Mab-294 antibody SEQ ID NO: 175: Amino acid sequence of the L chain CDR2 of the H2Mab-294 antibody SEQ ID NO: 176: Amino acid sequence of the L chain CDR3 of the H2Mab-294 antibody SEQ ID NO: 177: Amino acid sequence of H chain CDR1 of H2Mab-295 antibody SEQ ID NO: 178: Amino acid sequence of H chain CDR2 of H2Mab-295 antibody SEQ ID NO: 179: Amino acid sequence of the H chain CDR3 of the H2Mab-295 antibody SEQ ID NO: 180: Amino acid sequence of the L chain CDR1 of the H2Mab-295 antibody SEQ ID NO: 181: Amino acid sequence of the L chain CDR2 of the H2Mab-295 antibody SEQ ID NO: 182: Amino acid sequence of the L chain CDR3 of the H2Mab-295 antibody SEQ ID NO: 183: Amino acid sequence of H chain CDR1 of H2Mab-296 antibody SEQ ID NO: 184: Amino acid sequence of H chain CDR2 of H2Mab-296 antibody SEQ ID NO: 185: Amino acid sequence of the H chain CDR3 of the H2Mab-296 antibody SEQ ID NO: 186: Amino acid sequence of L chain CDR1 of H2Mab-296 antibody SEQ ID NO: 187: Amino acid sequence of the L chain CDR2 of the H2Mab-296 antibody SEQ ID NO: 188: Amino acid sequence of the L chain CDR3 of the H2Mab-296 antibody SEQ ID NO: 189: Amino acid sequence of H chain CDR1 of H2Mab-297 antibody SEQ ID NO: 190: Amino acid sequence of the H chain CDR2 of the H2Mab-297 antibody SEQ ID NO: 191: Amino acid sequence of the H chain CDR3 of the H2Mab-297 antibody SEQ ID NO: 192: Amino acid sequence of L chain CDR1 of H2Mab-297 antibody SEQ ID NO: 193: Amino acid sequence of the L chain CDR2 of the H2Mab-297 antibody SEQ ID NO: 194: Amino acid sequence of the L chain CDR3 of the H2Mab-297 antibody SEQ ID NO: 195: Amino acid sequence of H chain CDR1 of H2Mab-298 antibody SEQ ID NO: 196: Amino acid sequence of H chain CDR2 of H2Mab-298 antibody SEQ ID NO: 197: Amino acid sequence of the H chain CDR3 of the H2Mab-298 antibody SEQ ID NO: 198: Amino acid sequence of L chain CDR1 of H2Mab-298 antibody SEQ ID NO: 199: Amino acid sequence of the L chain CDR2 of the H2Mab-298 antibody SEQ ID NO: 200: Amino acid sequence of the L chain CDR3 of the H2Mab-298 antibody SEQ ID NO: 201: Amino acid sequence of H chain CDR1 of H2Mab-299 antibody SEQ ID NO: 202: Amino acid sequence of the H chain CDR2 of the H2Mab-299 antibody SEQ ID NO: 203: Amino acid sequence of the H chain CDR3 of the H2Mab-299 antibody SEQ ID NO: 204: Amino acid sequence of L chain CDR1 of H2Mab-299 antibody SEQ ID NO: 205: Amino acid sequence of the L chain CDR2 of the H2Mab-299 antibody SEQ ID NO: 206: Amino acid sequence of the L chain CDR3 of the H2Mab-299 antibody Industrial Applicability

[0135] The antibodies or antigen-binding fragments thereof of the present invention are useful for the detection and / or treatment of cancer.

Claims

1. A heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 24; A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 25, and A heavy chain variable region having a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 26, and A light chain CDR1 having the amino acid sequence of SEQ ID NO: 27; A light chain CDR2 having the amino acid sequence of SEQ ID NO: 28, and An isolated antibody that specifically binds to HER2 having a light chain variable region with a light chain CDR3 having the amino acid sequence of SEQ ID NO: 29, and having at least one substitution, addition or deletion in one or more of the CDRs, or a Fab, Fab', F(ab') ２ , Fv, scFv (single chain Fv), diabody, scDb, tandem scFv, leucine zipper type, and sc(Fv) ２ (Single chain (Fv) ２ ), wherein the cancer is a solid tumor or a HER2-positive cancer.

2. A heavy chain variable region having the amino acid sequence of SEQ ID NO: 16, and having a light chain variable region having the amino acid sequence of SEQ ID NO:17; or The pharmaceutical composition of claim 1, which is an isolated antibody that specifically binds to HER2, having at least one substitution, addition or deletion in each of the variable regions, or an antigen-binding fragment thereof selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), diabody, scDb, tandem scFv, leucine zipper type, and sc(Fv)2 (single-chain (Fv)2).

3. The pharmaceutical composition described in claim 1 or 2, which is an scFv.

4. The pharmaceutical composition described in claim 3, wherein the scFv is an scFv in which the light chain variable region and the heavy chain variable region are linked by an artificial polypeptide linker, and which can maintain the same antigen specificity as the original antibody.

5. The pharmaceutical composition described in claim 4, wherein the solid tumor is selected from the group consisting of lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, uterine cancer, ovarian cancer, skin cancer, thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, biliary tract cancer, brain tumors, bone and soft tissue tumors, retroperitoneal tumors, angio-lymphangiosarcoma, and metastatic cancers thereof.

6. The pharmaceutical composition described in claim 5, used in combination with other anticancer drugs.