Anti-meflin antibodies for treating cancer in a subject suffering from cancer and pharmaceutical compositions containing the same

Abstract

The present invention provides an anti-MEFLIN antibody and a pharmaceutical composition containing the same for treating cancer in a subject having cancer. According to the present invention, there is provided a pharmaceutical composition for treating cancer, for example, an antibody-drug conjugate (ADC) containing an antibody binding to MEFLIN and a cytotoxic agent.

Description

Technical Field

[0001] This invention relates to anti-MEFLIN antibodies for treating cancer in subjects with cancer and pharmaceutical compositions containing such antibodies. Background Technology

[0002] MEFLIN is a membrane protein containing leucine-rich repeat (LRR) sequences and immunoglobulin (Ig)-like domains. It is encoded by the ISLR (Immunoglobulin superfamily containing leucine-rich repeat) gene, derived from a human gene library, which is predominantly expressed in the retina compared to the brain (Non-Patent Literature 1, 2). It is also known by the same name as the gene, ISLR (Non-Patent Literature 1). MEFLIN is anchored to GPI (glycosylphosphatidylinositol) and exists on the cell membrane surface, but there are also reports of it being cleaved near the cell membrane and secreted extracellularly (Non-Patent Literature 2).

[0003] MEFLIN is a protein specifically expressed in mesenchymal stem cells (Non-Patent Literature 2). Various molecules are known as biomarkers for mesenchymal stem cells, including CD105, CD73, CD90, CD146, and CD271. MEFLIN has been reported to be the most specific biomarker for mesenchymal stem cells among these (Non-Patent Literature 2, Patent Literature 1).

[0004] Mesenchymal stem cells expressing MEFLIN are present in small quantities around blood vessels or in connective tissue of all organs, and have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, skeletal muscle cells, myofibroblasts, and nerve cells (Non-Patent Literature 2). It has also been reported that mesenchymal stem cells are cells substantially the same as pericytes (vascular peridermocytes) and perivascular fibroblasts (Non-Patent Literature 3).

[0005] It is known that fibroblasts derived from mesenchymal stem cells proliferate around cancer cells in cancer, and are referred to as cancer-associated fibroblasts (CAF) (Non-Patent Literature 4). CAFs are known to be observed in tissues of almost all cancer types, showing significant proliferation, particularly in refractory cancers such as pancreatic cancer, bile duct cancer, breast cancer, and poorly differentiated gastrointestinal cancers (Non-Patent Literature 4, 5). MEFLIN is positive in CAFs, which can be investigated by in situ hybridization detecting mRNA from the ISLR gene or by immunohistostaining using antibodies (Non-Patent Literature 5). In both in situ hybridization and immunohistostaining, biopsy or surgical materials from cancer patients are used. MEFLIN is known to be specifically expressed in cancer tissues in CAFs, but not in cancer cells, vascular endothelial cells, smooth muscle cells, blood cells, and nerve cells (Non-Patent Literature 5).

[0006] It has been reported that the abundance of MEFLIN-positive cancer-associated fibroblasts is correlated with the prognosis and treatment efficacy of cancer patients (Non-Patent Literature 5, Patent Literature 2). Specifically, pancreatic cancer patients with more than 20% MEFLIN-positive cancer-associated fibroblasts showed a better prognosis compared to patients with less than 20% (Non-Patent Literature 5). Furthermore, it has been reported that patients with less than 20% MEFLIN-positive cancer-associated fibroblasts showed lower efficacy (efficacy) of treatment with immune checkpoint inhibitors (Patent Literature 2).

[0007] MEFLIN-positive mesenchymal stem cells or fibroblasts are known to be important cells for tissue repair in various organs. For example, in a mouse model of myocardial infarction, a high aggregation of MEFLIN-positive cells can be observed in the acute phase following myocardial infarction (Non-Patent Literature 6). MEFLIN expression in these fibroblasts is essential for myocardial repair, and cardiac rupture has been observed in mice with ISLR gene deficiency (Non-Patent Literature 6). In addition, MEFLIN-positive fibroblasts have been reported to inhibit fibrosis and sclerosis after tissue repair (Non-Patent Literature 6).

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: WO2017 / 22472

[0011] Patent Document 2: WO2019 / 159825

[0012] Non-patent literature

[0013] Non-patent literature 1: Nagasawa, A. et al., Genomics, 44:273-279, 1977

[0014] Non-patent literature 2: Maeda K., et al., Sci. Rep., 6:22288, 2016

[0015] Non-patent literature 3: Crisan M. et al., Cell Stem Cell., 3:301-313, 2008

[0016] Non-patent literature 4: Kobayashi H. et al., Nat Rev Gastroenterol Hepatol., 16:282-295, 2019

[0017] Non-patent literature 5: Mizutani Y. et al., Cancer Res., 79:5367-5381, 2019

[0018] Non-patent literature 6: Hara A. et al., Circ. Res., 125:414-430, 2019

[0019] Non-patent literature 7: Schaum, N. et al., Nature, 562(7727), 367-372, 2018 Summary of the Invention

[0020] This invention provides an anti-MEFLIN antibody for treating cancer in subjects with cancer and a pharmaceutical composition containing the antibody. In this invention, the cancer can be sarcoma or MEFLIN-negative carcinoma.

[0021] The inventors have discovered that antibody-drug conjugates (ADCs) binding to the MEFLIN protein and cytotoxic agents exert antitumor effects against sarcomas. Furthermore, the inventors have discovered that MEFLIN-positive cells exist in the stroma of MEFLIN-negative carcinomas, and that antibody-drug conjugates binding to the MEFLIN protein and cytotoxic agents exert antitumor effects against these carcinomas. This invention is based on these insights.

[0022] According to the present invention, for example, the following invention is provided.

[0023] [1] A pharmaceutical composition for treating cancer, comprising an antibody-drug conjugate (ADC) of an antibody that binds to MEFLIN and a cytotoxic agent.

[0024] [2] According to the pharmaceutical composition described in [1] above, wherein the aforementioned antibody has internalization activity.

[0025] [3] According to the pharmaceutical composition described in [1] or [2] above, wherein the ADC is an ADC formed by linking an antibody and a drug through a connector having a cleavage site for intracellular lysis.

[0026] [4] An antibody that binds to MEFLIN, selected from the group consisting of the following antibodies:

[0027] (1A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains a heavy chain CDR1 having the amino acid sequence described in sequence number 1, a heavy chain CDR2 having the amino acid sequence described in sequence number 2, and a heavy chain CDR3 having the amino acid sequence described in sequence number 3, and the light chain variable region contains a light chain CDR1 having the amino acid sequence described in sequence number 4, a light chain CDR2 having the amino acid sequence described in sequence number 5, and a light chain CDR3 having the amino acid sequence described in sequence number 6.

[0028] (1B) An antibody containing a heavy chain variable region having the amino acid sequence described in sequence number 7 and a light chain variable region having the amino acid sequence described in sequence number 8.

[0029] (1C) An antibody that competes with the antibody in (1B) above for binding to the MEFLIN protein; and

[0030] (1D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described in (1B) above;

[0031] (2A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 9, heavy chain CDR2 as described in sequence number 10 and heavy chain CDR3 as described in sequence number 11, and the light chain variable region contains light chain CDR1 as described in sequence number 12, light chain CDR2 as described in sequence number 13 and light chain CDR3 as described in sequence number 14.

[0032] (2B) An antibody having the heavy chain variable region described in sequence number 15 and the light chain variable region described in sequence number 16;

[0033] (2C) Antibodies that compete with the antibodies in (2B) above for binding to the MEFLIN protein; and

[0034] (2D) Antibodies that bind to epitopes on the MEFLIN protein that overlap with the antibodies described in (2B) above;

[0035] (3A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 17, heavy chain CDR2 as described in sequence number 18 and heavy chain CDR3 as described in sequence number 19, and the light chain variable region contains light chain CDR1 as described in sequence number 20, light chain CDR2 as described in sequence number 21 and light chain CDR3 as described in sequence number 22.

[0036] (3B) An antibody having the heavy chain variable region described in sequence number 23 and the light chain variable region described in sequence number 24;

[0037] (3C) Antibodies that compete with the antibodies in (3B) above for binding to the MEFLIN protein; and

[0038] (3D) Antibodies that bind to epitopes on the MEFLIN protein that overlap with the antibodies described above (3B);

[0039] (4A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 25, heavy chain CDR2 as described in sequence number 26 and heavy chain CDR3 as described in sequence number 27, and the light chain variable region contains light chain CDR1 as described in sequence number 28, light chain CDR2 as described in sequence number 29 and light chain CDR3 as described in sequence number 30.

[0040] (4B) An antibody having the heavy chain variable region described in sequence number 31 and the light chain variable region described in sequence number 32;

[0041] (4C) Antibodies that compete with the antibodies in (4B) above for binding to the MEFLIN protein; and

[0042] (4D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (4B);

[0043] (5A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 33, heavy chain CDR2 as described in sequence number 34 and heavy chain CDR3 as described in sequence number 35, and the light chain variable region contains light chain CDR1 as described in sequence number 36, light chain CDR2 as described in sequence number 37 and light chain CDR3 as described in sequence number 38.

[0044] (5B) An antibody having the heavy chain variable region described in sequence number 39 and the light chain variable region described in sequence number 40;

[0045] (5C) Antibodies that compete with the antibodies in (5B) above for binding to the MEFLIN protein; and

[0046] (5D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (5B);

[0047] (6A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as recorded in sequence number 41, heavy chain CDR2 as recorded in sequence number 42 and heavy chain CDR3 as recorded in sequence number 43, and the light chain variable region contains light chain CDR1 as recorded in sequence number 44, light chain CDR2 as recorded in sequence number 45 and light chain CDR3 as recorded in sequence number 46.

[0048] (6B) An antibody having the heavy chain variable region described in sequence number 47 and the light chain variable region described in sequence number 48;

[0049] (6C) An antibody that competes with the antibody in (6B) above for binding to the MEFLIN protein; and

[0050] (6D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (6B);

[0051] (7A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 49, heavy chain CDR2 as described in sequence number 50 and heavy chain CDR3 as described in sequence number 51, and the light chain variable region contains light chain CDR1 as described in sequence number 52, light chain CDR2 as described in sequence number 53 and light chain CDR3 as described in sequence number 54.

[0052] (7B) An antibody having the heavy chain variable region described in sequence number 55 and the light chain variable region described in sequence number 56;

[0053] (7C) Antibodies that compete with the antibodies in (7B) above for binding to the MEFLIN protein; and

[0054] (7D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (7B);

[0055] (8A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 57, heavy chain CDR2 as described in sequence number 58 and heavy chain CDR3 as described in sequence number 59, and the light chain variable region contains light chain CDR1 as described in sequence number 60, light chain CDR2 as described in sequence number 61 and light chain CDR3 as described in sequence number 62.

[0056] (8B) An antibody having the heavy chain variable region described in sequence number 63 and the light chain variable region described in sequence number 64;

[0057] (8C) An antibody that competes with the antibody in (8B) above for binding to the MEFLIN protein; and

[0058] (8D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (8B);

[0059] (9A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 65, heavy chain CDR2 as described in sequence number 66 and heavy chain CDR3 as described in sequence number 67, and the light chain variable region contains light chain CDR1 as described in sequence number 68, light chain CDR2 as described in sequence number 69 and light chain CDR3 as described in sequence number 70.

[0060] (9B) An antibody having the heavy chain variable region described in sequence number 71 and the light chain variable region described in sequence number 72;

[0061] (9C) An antibody that competes with the antibody in (9B) above for binding to the MEFLIN protein; and

[0062] (9D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (9B);

[0063] (10A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 73, heavy chain CDR2 as described in sequence number 74 and heavy chain CDR3 as described in sequence number 75, and the light chain variable region contains light chain CDR1 as described in sequence number 76, light chain CDR2 as described in sequence number 77 and light chain CDR3 as described in sequence number 78.

[0064] (10B) An antibody having the heavy chain variable region described in sequence number 79 and the light chain variable region described in sequence number 80;

[0065] (10C) An antibody that competes with the antibody in (10B) above for binding to the MEFLIN protein; and

[0066] (10D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (10B).

[0067] (11A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as recorded in sequence number 81, heavy chain CDR2 as recorded in sequence number 82 and heavy chain CDR3 as recorded in sequence number 83, and the light chain variable region contains light chain CDR1 as recorded in sequence number 84, light chain CDR2 as recorded in sequence number 85 and light chain CDR3 as recorded in sequence number 86.

[0068] (11B) An antibody having the heavy chain variable region described in sequence number 87 and the light chain variable region described in sequence number 88;

[0069] (11C) An antibody that competes with the antibody in (11B) above for binding to the MEFLIN protein; and

[0070] (11D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (11B).

[0071] (12A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 89, heavy chain CDR2 as described in sequence number 90 and heavy chain CDR3 as described in sequence number 91, and the light chain variable region contains light chain CDR1 as described in sequence number 92, light chain CDR2 as described in sequence number 93 and light chain CDR3 as described in sequence number 94.

[0072] (12B) An antibody having the heavy chain variable region described in sequence number 95 and the light chain variable region described in sequence number 96;

[0073] (12C) An antibody that competes with the antibody in (12B) above for binding to the MEFLIN protein; and

[0074] (12D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (12B).

[0075] (13A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 97, heavy chain CDR2 as described in sequence number 98 and heavy chain CDR3 as described in sequence number 99, and the light chain variable region contains light chain CDR1 as described in sequence number 100, light chain CDR2 as described in sequence number 101 and light chain CDR3 as described in sequence number 102;

[0076] (13B) An antibody having the heavy chain variable region described in sequence number 103 and the light chain variable region described in sequence number 104;

[0077] (13C) An antibody that competes with the antibody in (13B) above for binding to the MEFLIN protein; and

[0078] (13D) an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (13B);

[0079] (14A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 105, heavy chain CDR2 as described in sequence number 106 and heavy chain CDR3 as described in sequence number 107, and the light chain variable region contains light chain CDR1 as described in sequence number 108, light chain CDR2 as described in sequence number 109 and light chain CDR3 as described in sequence number 110;

[0080] (14B) An antibody having the heavy chain variable region described in sequence number 111 and the light chain variable region described in sequence number 112;

[0081] (14C) An antibody that competes with the antibody described in (14B) for binding to the MEFLIN protein; and

[0082] (14D) an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (14B);

[0083] (15A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 113, heavy chain CDR2 as described in sequence number 114 and heavy chain CDR3 as described in sequence number 115, and the light chain variable region contains light chain CDR1 as described in sequence number 116, light chain CDR2 as described in sequence number 117 and light chain CDR3 as described in sequence number 118;

[0084] (15B) An antibody having the heavy chain variable region described in sequence number 119 and the light chain variable region described in sequence number 120;

[0085] (15C) An antibody that competes with the antibody described in (15B) for binding to the MEFLIN protein; and

[0086] (15D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (15B).

[0087] (16A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 121, heavy chain CDR2 as described in sequence number 122 and heavy chain CDR3 as described in sequence number 123, and the light chain variable region contains light chain CDR1 as described in sequence number 124, light chain CDR2 as described in sequence number 125 and light chain CDR3 as described in sequence number 126;

[0088] (16B) An antibody having the heavy chain variable region described in sequence number 127 and the light chain variable region described in sequence number 128;

[0089] (16C) is an antibody that competes with the antibody described above (16B) for binding to the MEFLIN protein; and

[0090] (16D) and an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (16B); and

[0091] (17A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 129, heavy chain CDR2 as described in sequence number 130 and heavy chain CDR3 as described in sequence number 131, and the light chain variable region contains light chain CDR1 as described in sequence number 132, light chain CDR2 as described in sequence number 133 and light chain CDR3 as described in sequence number 134;

[0092] (17B) An antibody having the heavy chain variable region described in sequence number 135 and the light chain variable region described in sequence number 136;

[0093] (17C) An antibody that competes with the antibody in (17B) above for binding to the MEFLIN protein; and

[0094] (17D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (17B).

[0095] [5] A pharmaceutical composition comprising an antibody-drug conjugate (ADC) of the antibody and cytotoxic agent described above [4].

[0096] [6] The pharmaceutical composition described in [5] above is used to treat cancer.

[0097] [7] The pharmaceutical composition according to any one of [1] to [3] and [6] above, wherein the carcinoma is a sarcoma.

[0098] [8] The pharmaceutical composition according to [7] above, wherein the carcinoma is a MEFLIN-positive sarcoma.

[0099] [9] The pharmaceutical composition according to [7] or [8] above, wherein the sarcoma is selected from the group consisting of myxofibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, malignant peripheral schwannoma, Ewing sarcoma, epithelioid sarcoma, clear cell sarcoma, synovial sarcoma and osteosarcoma.

[0100]

[10] The pharmaceutical composition according to any one of [1] to [3] and [6] above, wherein the cancer is a malignant epithelial tumor.

[0101]

[11] According to the pharmaceutical composition described in

[10] above, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, lung cancer, colorectal cancer, gastric cancer, bile duct cancer, ovarian cancer, bladder cancer and esophageal cancer.

[0102]

[12] The pharmaceutical composition according to any one of [1] to [3], [6],

[10] and

[11] above, wherein the cancer is MEFLIN negative and the stroma surrounding the cancer contains MEFLIN positive cells.

[0103]

[13] The pharmaceutical composition according to any one of

[10] to

[12] above, wherein the antibody does not have internalizing activity.

[0104]

[14] The pharmaceutical composition according to any one of [7] to [9] above, wherein the antibody has internalization activity.

[0105]

[15] According to the pharmaceutical composition described in

[13] above, wherein the ADC is an ADC formed by linking an antibody and a drug through a connector, and the connector is a cleavage connector.

[0106]

[16] The pharmaceutical composition according to

[15] above, wherein the linker is a cleavage linker cleaved by cathepsin K.

[0107]

[17] The pharmaceutical composition according to

[16] above, wherein the linker contains a valine-citrulline dipeptide that is cleaved in the presence of cathepsin K.

[0108]

[18] The pharmaceutical composition according to any one of the above claims, wherein the connector is a non-disintegrating connector. Attached Figure Description

[0109] Figure 1 The results of Western blotting were used to demonstrate the binding of human MEFLIN protein to monoclonal antibodies derived from various clones. Figure 1 The text shows information about the clone name and the MEFLIN fragment (the region of the fragment is indicated by amino acid numbers).

[0110] Figure 2 The results of Western blotting were used to demonstrate the binding of human MEFLIN protein to monoclonal antibodies derived from various clones. Figure 2 The text shows information about the clone name and the MEFLIN fragment (the region of the fragment is indicated by amino acid numbers).

[0111] Figure 3 Fluorescence microscopy images illustrating the intracellular uptake (internalization) activity of monoclonal antibodies derived from various clones. Cell nuclei were stained with DAPI, and antibodies were detected using Alexa488-labeled antibodies.

[0112] Figure 4 Fluorescence microscopy images illustrating the intracellular uptake (internalization) activity of monoclonal antibodies derived from various clones. Cell nuclei were stained with DAPI, and antibodies were detected using Alexa488-labeled antibodies.

[0113] Figure 5 Fluorescence microscopy images illustrating the intracellular uptake (internalization) activity of monoclonal antibodies derived from various clones. Cell nuclei were stained with DAPI, and antibodies were detected using Alexa488-labeled antibodies.

[0114] Figure 6 The antibody-drug conjugate (ADC) produced is shown to inhibit cell proliferation in vitro in human MEFLIN protein-overexpressing cells (HEK293 cells). Figure 6 Additionally, FACS analysis results for human MEFLIN protein overexpressing cells (HEK293 cells) are shown for the anti-MEFLIN monoclonal antibody treatment group and the untreated group (negative control). The horizontal axis represents the expression level of MEFLIN protein, and the vertical axis represents the cell frequency.

[0115] Figure 7 This study demonstrates the inhibitory activity of ADC on the cell proliferation of human rhabdomyosarcoma cell line (KYM-1 cells) in vitro. Figure 7 Additionally, FACS analysis results for the human rhabdomyosarcoma cell line (KYM-1 cells) used are shown for the anti-MEFLIN monoclonal antibody treated group and the untreated group (negative control). The horizontal axis represents the expression level of MEFLIN protein, and the vertical axis represents the cell frequency.

[0116] Figure 8 This figure illustrates the antitumor effect of ADC on tumor-bearing mice with subcutaneous transplantation of the rhabdomyosarcoma cell line (KYM-1). The arrows in the figure indicate the timing of drug administration. Figure 8 Additionally, the expression of human MEFLIN in the transplanted rhabdomyosarcoma tissue is shown.

[0117] Figure 9 The figure illustrates the antitumor effect of ADC on tumor-bearing mice with subcutaneous transplantation of osteosarcoma cell line (HsOs1). The arrows in the figure indicate the timing of drug administration. Figure 9 Additionally, the expression of human MEFLIN in the transplanted osteosarcoma tissue is shown.

[0118] Figure 10 This figure illustrates the antitumor effect of ADC on tumor-bearing mice with subcutaneous transplantation of pancreatic cancer cell line (BxPC-3). The arrows in the figure indicate the timing of drug administration. Figure 10 Additionally, the expression of mouse MEFLIN in the transplanted pancreatic cancer tissue and stroma is shown.

[0119] Figure 11 The figure illustrates the antitumor effect of ADC on tumor-bearing mice with subcutaneous transplantation of lung cancer cell line (A549). The arrows in the figure indicate the timing of drug administration. Figure 11The expression of mouse MEFLIN in the tissue and stroma of the transplanted lung cancer is also shown.

[0120] Figure 12 This figure illustrates the antitumor effect of ADC on tumor-bearing mice with subcutaneous transplantation of neuroblastoma cell line (NB-1). The arrows in the figure indicate the timing of drug administration. Figure 12 Additionally, the expression of human MEFLIN in the transplanted neuroblastoma tissue is shown.

[0121] Figure 13 This figure illustrates the antitumor effect of ADC on tumor-bearing mice with subcutaneous transplantation of the colorectal cancer cell line (DLD-1). The arrows in the figure indicate the timing of drug administration. Figure 13 Additionally, the expression of mouse MEFLIN in the transplanted colorectal cancer tissue and stroma is shown.

[0122] Figure 14 This figure illustrates the antitumor effect of ADC on tumor-bearing mice with subcutaneous transplantation of gastric cancer cell line (MKN45). The arrows in the figure indicate the timing of drug administration. Figure 14 Additionally, the expression of mouse MEFLIN in the transplanted gastric cancer tissue and stroma is shown.

[0123] Figure 15 The results show the analysis of single-cell RNA sequencing data (TabulaMuris) from mouse pancreas stored on the Internet. The MEFLIN-positive cell population in the mouse pancreas is consistent with the cathepsin K-positive cell population (arrow).

[0124] Figure 16 The results show the analysis of single-cell RNA sequencing data (Tabula Muris) from mouse lungs stored on the internet. The data shows that the MEFLIN-positive cell population in the mouse lungs is consistent with the cathepsin K-positive cell population (arrow).

[0125] Figure 17 The expression of cathepsin K in the CHO cell line expressing exogenous mouse MEFLIN is shown.

[0126] Figure 18 The results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody are shown in tumor tissues from tumor-bearing mice with subcutaneous transplantation of osteosarcoma cell line (HsOs1). Cathepsin K (red) is shown secreted around MEFLIN-positive cells (green) (arrow).

[0127] Figure 19The results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody are shown in tumor tissues from a tumor-bearing mouse model with subcutaneously transplanted pancreatic cancer cell line (BxPC-3). Cathepsin K (red) is shown secreted around MEFLIN-positive cells (green) (arrow).

[0128] Figure 20 This figure illustrates the antitumor effect of ADC on tumor tissues of a tumor-bearing mouse model with subcutaneous transplantation of the cholangiocarcinoma cell line (HuCCT1). The arrows in the figure indicate the timing of drug administration. Figure 20 Additionally, the expression of mouse MEFLIN in the transplanted cholangiocarcinoma tissue and stroma is shown.

[0129] Figure 21 This figure illustrates the antitumor effect of ADC on tumor tissues of a mouse model of bladder cancer cells (T24) transplanted subcutaneously. The arrows in the figure indicate the timing of administration. Figure 21 Additionally, the expression of mouse MEFLIN in the transplanted bladder cancer tissue and stroma is shown.

[0130] Figure 22 This figure illustrates the antitumor effect of ADC on tumor tissues of tumor-bearing mice with subcutaneous transplantation of ovarian cancer cell line (OV-90). The arrows in the figure indicate the timing of drug administration. Figure 22 Additionally, the expression of mouse MEFLIN in the transplanted ovarian cancer tissue and stroma is shown.

[0131] Figure 23 This figure illustrates the antitumor effect of ADC on tumor tissues of a tumor-bearing mouse model mouse with a subcutaneous transplantation of the esophageal cancer cell line (KYSE). The arrows in the figure indicate the timing of drug administration. Figure 23 Additionally, the expression of mouse MEFLIN in the transplanted esophageal cancer tissue and stroma is shown.

[0132] Figure 24 This figure illustrates the antitumor effect of ADC on tumor tissues of tumor-bearing mice subcutaneously transplanted with osteosarcoma cell line (HsOs1). The arrows in the figure indicate the timing of drug administration.

[0133] Figure 25 This figure illustrates the antitumor effect of ADC on tumor tissues of a mouse model of breast cancer cell line (MCF7) transplanted subcutaneously. The arrows in the figure indicate the timing of drug administration. Figure 25 Additionally, the expression of mouse MEFLIN in the transplanted breast cancer tissue and stroma is shown.

[0134] Figure 26The figures show the results of immunohistochemical (IHC) staining with anti-MEFLIN antibody and in situ hybridization (ISH) staining of tissue sections from human pancreatic cancer surgical samples. Arrows in the figures indicate the same location in both the upper and lower images. Detailed Implementation

[0135] In this invention, "object" refers to mammals, and in particular, can be humans.

[0136] In this instruction manual, the term "treatment" is used to include both therapeutic (therapeutic treatment) and preventative (preventative treatment). In this instruction manual, "treatment" refers to the cure, prevention, or mitigating improvement of a disease or disorder, or a reduction in the rate of progression of a disease or disorder. In this instruction manual, "prevention" refers to reducing the likelihood of a disease or condition occurring, or delaying its onset.

[0137] In this instruction manual, "disease" refers to a symptom that benefits from treatment. In this instruction manual, "cancer" refers to a malignant tumor.

[0138] In this specification, "antibody" refers to immunoglobulin, including polyclonal antibodies and monoclonal antibodies. Monoclonal antibodies are preferred. The source of the antibody is not particularly limited, and examples include antibodies from non-human animals, non-human mammals, and humans. Furthermore, the antibody can be a chimeric antibody, a humanized antibody, or a human antibody. Additionally, the antibody can be a bispecific antibody. The antibody used as a drug is preferably a chimeric antibody, more preferably a humanized antibody, and even more preferably a human antibody. The bispecific antibody is a monoclonal antibody, preferably a chimeric antibody, more preferably a humanized antibody, and even more preferably a human antibody.

[0139] In this instruction manual, "therapeuticly effective dose" refers to a dosage of medicine that is effective in treating (preventing or treating) a disease or condition. A therapeutically effective dose of medicine can reduce the rate of worsening of the symptoms of a disease or condition, prevent the worsening of the aforementioned symptoms, improve the aforementioned symptoms, cure the aforementioned symptoms, or inhibit the onset or development of the aforementioned symptoms.

[0140] In this specification, "competition" means that, in relation to binding to an antigen, an antibody competes with other binding antibodies for binding. Competition may occur when the binding sites of two antibodies for a given antigen overlap. Such antibodies can be obtained, as described above, through epitope immunization, and / or by using competition analysis to confirm whether the binding of one antibody to an antigen is reduced by another antibody.

[0141] In this specification, "antibody-drug conjugate" (hereinafter also referred to as "ADC") refers to a substance formed by linking an antibody to a cytotoxic agent. In an ADC, the antibody and the cytotoxic agent can be linked through a suitable adapter. Cytotoxic agents can include chemotherapy agents, radioactive isotopes, and toxins. ADCs also include conjugates of antibody antigen-binding fragments with drugs.

[0142] In this specification, "antigen-binding fragment" refers to a portion of an antibody that maintains its binding affinity to an antigen. The antigen-binding fragment may contain the heavy chain variable region or the light chain variable region of the antibody of the present invention, or both. The antigen-binding fragment may be chimeric or humanized. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), double-chain antibody, and sc(Fv)2 (single-chain (Fv)2). Such antibody fragments are not particularly limited; for example, they can be obtained by enzymatic treatment of the antibody. For example, digesting the antibody with papain yields Fab. Alternatively, digesting the antibody with pepsin yields F(ab')2, which is then reduced to obtain Fab'. Such antigen-binding fragments of antibodies can be used in the present invention.

[0143] In this specification, "MEFLIN" or "Meflin" refers to proteins also known as the immunoglobulin superfamily (ISLR) containing leucine-rich repeat sequences. Human MEFLIN may have the amino acid sequence registered under GenBank accession number BAA85970.1. MEFLIN can be a MEFLIN with an amino acid sequence corresponding to the amino acid sequence registered under GenBank accession number BAA85970.1 (e.g., human MEFLIN). When it is desired to specify the animal species of origin, it is referred to as human MEFLIN (or hMEFLIN) and mouse MEFLIN (mMEFLIN), etc.

[0144] In this invention, in antibody-drug conjugates (ADCs), the antibody and the cytotoxic agent are linked via a linker. Examples of cytotoxic agents include chemotherapeutic agents (e.g., commercially available anticancer agents such as auristatin (aurestatin E, auristatin F phenylenediamine (AFP), monomethylaurestatin E, monomethylaurestatin F, and their derivatives); maytansine DM1 and DM4, and their derivatives), camptothecin (SN-38, irinotecan, letopecan, DB67, BMP1350, ST1481, CKD602, topotecan and esaxatecan, and their derivatives), DNA minor groove binding agents (enediyne, lexitropsin, pyruvicin, and their derivatives), taxanes (paclitaxel and docetaxel, and their derivatives), polyketides (spongein and its derivatives), anthraquinone derivatives (mitoxantrone and its derivatives), and benzodiazepines. (pyrrolobenzodiazepine) Indolinebenzodiazepine and Azoline benzodiazepine And their derivatives), vinca alkaloids (vincristine, vinca alkaloid, vinorelbine and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholinodoxorubicin and cyanomorpholinodoxorubicin and their derivatives), cardiac glycosides (digitoxin and its derivatives), carlicycin, epothilone, candidone, cimadolicin, cimadolicin, rhizobium, fusiformin, cobustatin, soft coral alcohol, etoposide, T67 (tullaric) and nocodazole), radioactive isotopes (e.g. 32 P, 60 C 90 Y、 111 In、 131 I, 125 I, 153 Sm、 186 Re、 188 Re and 212 Bi) and toxins (e.g., diphtheria toxin A, pseudomonadine, ricin, saposhnikovia toxin, etc.) can be used as cytotoxic agents in the ADC of the present invention. Camptothecin, particularly SN-38 or ethatecan, is preferably used as the cytotoxic agent in the ADC of the present invention. Regarding the cytotoxic agent, any cytotoxic agent used for treating cancer can be used. Pharmaceutically permissible salts, solvates (e.g., hydrates), esters, or prodrugs of the aforementioned cytotoxic agents can be used as cytotoxic agents.

[0145] In this invention, the adapter for the ADC can be either a non-cleaving adapter or a cleaving adapter. Such adapters can be suitably selected and synthesized by those skilled in the art during the fabrication of the ADC. As cleaving adapters, adapters having decomposable bonds such as ester bonds can also be listed. As cleaving adapters, adapters having protease-cleaving sites such as cleavable regions composed of peptides consisting of valine-citrulline or valine-alanine can be listed. The peptide region composed of valine-citrulline can be cleaved by proteases such as cathepsin B. In one embodiment, a first spacer group can be introduced between the antibody and the cleavable region, for example, polyethylene glycol (PEG), such as PEG with approximately 5 to 40 repeating units per molecule, can be used as the first spacer group. A second spacer group can be introduced between the cleavable region and the cytotoxic agent, for example, p-aminobenzyloxycarbonyl (PABC) can be used as the second spacer group. The cleaving adapter is physiologically stable except at the cleavage site in cancer tissue (especially physiologically stable before reaching cancer tissue).

[0146] In one embodiment, the connector comprises a first spacer group and a cleavable portion. In another embodiment, the connector comprises a first spacer group, a cleavable portion, and a second spacer group. In a particular embodiment, the connector comprises PEG, a cleavable portion, and PABC.

[0147] In the binding of antibodies to linkers, for example, the maleimide group can be linked to the thiol group of the antibody.

[0148] In one embodiment, the antibody is linked to an anticancer agent via its thiol group using a linker having a maleimide-PEG-cleavable moiety. In another embodiment, the antibody is linked to an anticancer agent via its thiol group using a linker having a maleimide-PEG-cleavable moiety-PABC.

[0149] In one embodiment, the ADC may have the structure shown in Equation (II).

[0150] The ADC of the present invention is considered useful as a cancer treatment drug.

[0151] According to the present invention, antibodies having the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 of antibodies produced by clones selected from the following can be provided. This antibody is preferably a human chimeric antibody, and may preferably be a humanized antibody. In this embodiment, according to the present invention, antibodies having the amino acid sequences of the heavy chain variable region and light chain variable region of antibodies produced by clones selected from the following can be provided. This antibody may preferably be a human chimeric antibody.

[0152] [Table 1]

[0153] Table 1: Rats with hybridoma The amino acid sequence of CDR1–CDR3 of the heavy chain variable region (VH) of the antibody produced by cloning List

[0154]

[0155] [Table 2]

[0156] Table 2: Amino acid sequence of CDR1–CDR3 of the light chain variable region (VL) of antibodies produced by rat hybridoma clones. List

[0157]

[0158] [Table 3]

[0159] Table 3: Amino acid sequences of the heavy chain variable region (VH) of antibodies produced by rat hybridoma clones

[0160]

[0161] [Table 4]

[0162] Table 4: Amino acid sequences of the light chain variable region (VL) of antibodies produced by rat hybridoma clones

[0163]

[0164] [Table 5]

[0165] Table 5: Small The amino acid sequence of CDR1–CDR3 of the heavy chain variable region (VH) of antibodies produced by mouse hybridoma clones. List

[0166]

[0167] [Table 6]

[0168] Table 6: Amino acid sequence of CDR1–CDR3 of the light chain variable region (VL) of antibodies produced by mouse hybridoma clones. List

[0169]

[0170] [Table 7]

[0171] Table 7: Amino acid sequences of the heavy chain variable region (VH) of antibodies produced by mouse hybridoma clones

[0172]

[0173] [Table 8]

[0174] Table 8: Amino acid sequences of the light chain variable region (VL) of antibodies produced by mouse hybridoma clones

[0175]

[0176] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0177] (1A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 1, heavy chain CDR2 as described in sequence number 2 and heavy chain CDR3 as described in sequence number 3, and the light chain variable region contains light chain CDR1 as described in sequence number 4, light chain CDR2 as described in sequence number 5 and light chain CDR3 as described in sequence number 4.

[0178] (1B) An antibody having the heavy chain variable region described in sequence number 7 and the light chain variable region described in sequence number 8;

[0179] (1C) An antibody that competes with the antibody in (1B) above for binding to the MEFLIN protein; and

[0180] (1D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described in (1B) above.

[0181] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0182] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0183] (2A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 9, heavy chain CDR2 as described in sequence number 10 and heavy chain CDR3 as described in sequence number 11, and the light chain variable region contains light chain CDR1 as described in sequence number 12, light chain CDR2 as described in sequence number 13 and light chain CDR3 as described in sequence number 14.

[0184] (2B) An antibody having the heavy chain variable region described in sequence number 15 and the light chain variable region described in sequence number 16;

[0185] (2C) Antibodies that compete with the antibodies in (2B) above for binding to the MEFLIN protein; and

[0186] (2D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described in (2B) above.

[0187] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0188] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0189] (3A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 17, heavy chain CDR2 as described in sequence number 18 and heavy chain CDR3 as described in sequence number 19, and the light chain variable region contains light chain CDR1 as described in sequence number 20, light chain CDR2 as described in sequence number 21 and light chain CDR3 as described in sequence number 22.

[0190] (3B) An antibody having the heavy chain variable region described in sequence number 23 and the light chain variable region described in sequence number 24;

[0191] (3C) Antibodies that compete with the antibodies in (3B) above for binding to the MEFLIN protein; and

[0192] (3D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (3B).

[0193] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0194] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0195] (4A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 25, heavy chain CDR2 as described in sequence number 26 and heavy chain CDR3 as described in sequence number 27, and the light chain variable region contains light chain CDR1 as described in sequence number 28, light chain CDR2 as described in sequence number 29 and light chain CDR3 as described in sequence number 30.

[0196] (4B) An antibody having the heavy chain variable region described in sequence number 31 and the light chain variable region described in sequence number 32;

[0197] (4C) Antibodies that compete with the antibodies in (4B) above for binding to the MEFLIN protein; and

[0198] (4D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (4B).

[0199] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0200] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0201] (5A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 33, heavy chain CDR2 as described in sequence number 34 and heavy chain CDR3 as described in sequence number 35, and the light chain variable region contains light chain CDR1 as described in sequence number 36, light chain CDR2 as described in sequence number 37 and light chain CDR3 as described in sequence number 38.

[0202] (5B) An antibody having the heavy chain variable region described in sequence number 39 and the light chain variable region described in sequence number 40;

[0203] (5C) Antibodies that compete with the antibodies in (5B) above for binding to the MEFLIN protein; and

[0204] (5D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (5B).

[0205] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0206] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0207] (6A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as recorded in sequence number 41, heavy chain CDR2 as recorded in sequence number 42 and heavy chain CDR3 as recorded in sequence number 43, and the light chain variable region contains light chain CDR1 as recorded in sequence number 44, light chain CDR2 as recorded in sequence number 45 and light chain CDR3 as recorded in sequence number 46.

[0208] (6B) An antibody having the heavy chain variable region described in sequence number 47 and the light chain variable region described in sequence number 48;

[0209] (6C) An antibody that competes with the antibody in (6B) above for binding to the MEFLIN protein; and

[0210] (6D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (6B).

[0211] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0212] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0213] (7A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 49, heavy chain CDR2 as described in sequence number 50 and heavy chain CDR3 as described in sequence number 51, and the light chain variable region contains light chain CDR1 as described in sequence number 52, light chain CDR2 as described in sequence number 53 and light chain CDR3 as described in sequence number 54.

[0214] (7B) An antibody having the heavy chain variable region described in sequence number 55 and the light chain variable region described in sequence number 56;

[0215] (7C) Antibodies that compete with the antibodies in (7B) above for binding to the MEFLIN protein; and

[0216] (7D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (7B).

[0217] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0218] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0219] (8A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 57, heavy chain CDR2 as described in sequence number 58 and heavy chain CDR3 as described in sequence number 59, and the light chain variable region contains light chain CDR1 as described in sequence number 60, light chain CDR2 as described in sequence number 61 and light chain CDR3 as described in sequence number 62.

[0220] (8B) An antibody having the heavy chain variable region described in sequence number 63 and the light chain variable region described in sequence number 64;

[0221] (8C) An antibody that competes with the antibody in (8B) above for binding to the MEFLIN protein; and

[0222] (8D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (8B).

[0223] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0224] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0225] (9A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 65, heavy chain CDR2 as described in sequence number 66 and heavy chain CDR3 as described in sequence number 67, and the light chain variable region contains light chain CDR1 as described in sequence number 68, light chain CDR2 as described in sequence number 69 and light chain CDR3 as described in sequence number 70.

[0226] (9B) An antibody having the heavy chain variable region described in sequence number 71 and the light chain variable region described in sequence number 72;

[0227] (9C) An antibody that competes with the antibody in (9B) above for binding to the MEFLIN protein; and

[0228] (9D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (9B).

[0229] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0230] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0231] (10A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 73, heavy chain CDR2 as described in sequence number 74 and heavy chain CDR3 as described in sequence number 75, and the light chain variable region contains light chain CDR1 as described in sequence number 76, light chain CDR2 as described in sequence number 77 and light chain CDR3 as described in sequence number 78.

[0232] (10B) An antibody having the heavy chain variable region described in sequence number 79 and the light chain variable region described in sequence number 80;

[0233] (10C) An antibody that competes with the antibody in (10B) above for binding to the MEFLIN protein; and

[0234] (10D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (10B).

[0235] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0236] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0237] (11A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as recorded in sequence number 81, heavy chain CDR2 as recorded in sequence number 82 and heavy chain CDR3 as recorded in sequence number 83, and the light chain variable region contains light chain CDR1 as recorded in sequence number 84, light chain CDR2 as recorded in sequence number 85 and light chain CDR3 as recorded in sequence number 86.

[0238] (11B) An antibody having the heavy chain variable region described in sequence number 87 and the light chain variable region described in sequence number 88;

[0239] (11C) An antibody that competes with the antibody in (11B) above for binding to the MEFLIN protein; and

[0240] (11D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (11B).

[0241] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0242] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0243] (12A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 89, heavy chain CDR2 as described in sequence number 90 and heavy chain CDR3 as described in sequence number 91, and the light chain variable region contains light chain CDR1 as described in sequence number 92, light chain CDR2 as described in sequence number 93 and light chain CDR3 as described in sequence number 94.

[0244] (12B) An antibody having the heavy chain variable region described in sequence number 95 and the light chain variable region described in sequence number 96;

[0245] (12C) An antibody that competes with the antibody in (12B) above for binding to the MEFLIN protein; and

[0246] (12D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (12B).

[0247] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0248] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0249] (13A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 97, heavy chain CDR2 as described in sequence number 98 and heavy chain CDR3 as described in sequence number 99, and the light chain variable region contains light chain CDR1 as described in sequence number 100, light chain CDR2 as described in sequence number 101 and light chain CDR3 as described in sequence number 102;

[0250] (13B) An antibody having the heavy chain variable region described in sequence number 103 and the light chain variable region described in sequence number 104;

[0251] (13C) An antibody that competes with the antibody in (13B) above for binding to the MEFLIN protein; and

[0252] (13D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (13B).

[0253] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0254] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0255] (14A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 105, heavy chain CDR2 as described in sequence number 106 and heavy chain CDR3 as described in sequence number 107, and the light chain variable region contains light chain CDR1 as described in sequence number 108, light chain CDR2 as described in sequence number 109 and light chain CDR3 as described in sequence number 110;

[0256] (14B) An antibody having the heavy chain variable region described in sequence number 111 and the light chain variable region described in sequence number 112;

[0257] (14C) An antibody that competes with the antibody described in (14B) for binding to the MEFLIN protein; and

[0258] (14D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (14B).

[0259] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0260] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0261] (15A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 113, heavy chain CDR2 as described in sequence number 114 and heavy chain CDR as described in sequence number 115, and the light chain variable region contains light chain CDR1 as described in sequence number 116, light chain CDR2 as described in sequence number 117 and light chain CDR3 as described in sequence number 118;

[0262] (15B) An antibody having the heavy chain variable region described in sequence number 119 and the light chain variable region described in sequence number 120;

[0263] (15C) An antibody that competes with the antibody described in (15B) for binding to the MEFLIN protein; and

[0264] (15D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (15B).

[0265] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0266] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0267] (16A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 121, heavy chain CDR2 as described in sequence number 122 and heavy chain CDR3 as described in sequence number 123, and the light chain variable region contains light chain CDR1 as described in sequence number 124, light chain CDR2 as described in sequence number 125 and light chain CDR3 as described in sequence number 126;

[0268] (16B) An antibody having the heavy chain variable region described in sequence number 127 and the light chain variable region described in sequence number 128;

[0269] (16C) is an antibody that competes with the antibody described above (16B) for binding to the MEFLIN protein; and

[0270] (16D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (16B).

[0271] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0272] According to the present invention, an antibody that binds to the MEFLIN protein is provided:

[0273] (17A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain CDR1 as described in sequence number 129, heavy chain CDR2 as described in sequence number 130 and heavy chain CDR3 as described in sequence number 131, and the light chain variable region contains light chain CDR1 as described in sequence number 132, light chain CDR2 as described in sequence number 133 and light chain CDR3 as described in sequence number 134;

[0274] (17B) An antibody having the heavy chain variable region described in sequence number 135 and the light chain variable region described in sequence number 136;

[0275] (17C) An antibody that competes with the antibody in (17B) above for binding to the MEFLIN protein; and

[0276] (17D) is an antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody described above (17B).

[0277] The antibody is preferably a human chimeric antibody, and may be preferably a humanized antibody.

[0278] In one embodiment of the present invention, the antibody that binds to the MEFLIN protein binds to the human MEFLIN protein. In another embodiment of the present invention, the antibody that binds to the MEFLIN protein specifically binds to the human MEFLIN protein. "Specific binding" means binding to the MEFLIN protein with a strong affinity compared to binding to at least one other protein. "Specific binding" also includes binding to the human MEFLIN protein with a strong affinity compared to binding to the mouse MEFLIN protein.

[0279] Whether antibodies compete with each other can be confirmed by in vitro competition assays. In a competition assay, an antibody that can block the binding of, for example, at least 20%, preferably at least 20-50%, and more preferably at least 50%, of the desired antibody can be considered a competing antibody in binding to the same antigen. Competing antibodies can be confirmed by cross-blocking assays, preferably competitive ELISA assays. In a cross-blocking assay, the antigen is coated onto, for example, a microtiter plate, and a competing antibody, which is a candidate antibody, is added to it and incubated to form a binding between the antigen and the candidate antibody. Then, the desired antibody is labeled and added to the wells and incubated, washed, and the amount of the desired antibody bound is quantified, thereby determining whether the antibodies are competing. In the case of competition, the amount of label remaining in the wells should be reduced.

[0280] Whether antibodies bind to overlapping epitopes on proteins that serve as binding partners can be confirmed by checking whether their interaction surfaces overlap. For example, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a known method for epitope identification. In HDX-MS, the exchange of hydrogen to deuterium from amide protons in a protein complex is detected in the presence of heavy water. In the presence of heavy water, amide protons on the interaction surfaces of the protein complex are difficult to exchange for deuterium (or the exchange rate is slow), while amide protons exposed on the surface are easily exchanged for deuterium (or the exchange rate is fast). Therefore, using this phenomenon, regions where the rate of hydrogen-to-deuterium exchange decreases can be identified as antibody-binding regions, thereby determining the antibody's interaction surface (epitaphage) on its binding partner. If the epitope regions of two antibodies are determined using the above method, it can be determined whether the epitope regions overlap. The hydrogen-to-deuterium exchange can be detected by those skilled in the art using mass spectrometry.

[0281] In one embodiment of the present invention, the antibody binding to the MEFLIN protein is an antibody with internalization activity that allows it to enter cells. In another embodiment of the present invention, the antibody binding to the MEFLIN protein is an antibody without internalization activity that allows it to enter cells. In another embodiment of the present invention, the cancer cells are MEFLIN protein positive, and the antibody binding to the MEFLIN protein is an antibody with internalization activity that allows it to enter cells. In another embodiment of the present invention, the cancer stromal cells are MEFLIN protein positive, and the antibody binding to the MEFLIN protein is an antibody with internalization activity that allows it to enter cells. In another embodiment of the present invention, the cancer stromal cells are MEFLIN protein positive, and the antibody binding to the MEFLIN protein is an antibody without internalization activity that allows it to enter cells. In these embodiments, the ADC may have a cleavable linker. In these embodiments, the ADC may have a non-cleavable linker. When the ADC payload is pre-drugated in a manner that the cytotoxicity is exerted only after the linker is cleaved, the linker is preferably a cleavable linker. In one embodiment, the ADC of the present invention has internalizing activity and has a lytic linker that lyses within cells. In another embodiment, the ADC of the present invention does not have internalizing activity but has a lytic linker that lyses outside cells.

[0282] Whether an antibody possesses internalization activity can be confirmed through in vitro assays. For example, for cells with MEFLIN protein on their cell surface, this can be confirmed as follows: expose the cell to an antibody bound to MEFLIN protein, incubate for a sufficient time to allow antibody internalization (e.g., approximately 15 minutes), remove the antibody bound to the cell surface from the culture medium by washing (e.g., with an aqueous solution containing 0.5 M NaCl and 3% acetic acid), and then stain the internalized antibody. Staining of internalized antibodies can be performed in the same manner as staining intracellular proteins. For example, staining of internalized antibodies can be detected using labeled secondary antibodies based on the label.

[0283] Cancerous tissue includes cancer cells (i.e., malignant tumor cells) and stromal cells of cancer. MEFLIN is also expressed in both malignant (sarcoma) and benign tumors derived from mesenchymal stem cells or similar cells. Specifically, expression has been confirmed on cancer cells in almost all non-epithelial tumor cases, including osteosarcoma, chondrosarcoma, liposarcoma, rhabdomyosarcoma, undifferentiated pleomorphic sarcoma, desmoidoma, and meningioma. Therefore, the ADC of the present invention can be used to treat malignant tumors (sarcoma) derived from mesenchymal stem cells or similar cells. The ADC of the present invention can also be used to treat benign tumors. Furthermore, in cancers arising from epithelial cells (malignant epithelial tumors), even when the cancer cells themselves are MEFLIN-negative, MEFLIN protein is expressed in stromal cells (e.g., CAF) in almost all cancers. Therefore, the ADC of the present invention can be used to treat malignant epithelial tumors by targeting stromal cells of malignant epithelial tumors. MEFLIN expression can be investigated by methods such as in situ hybridization or immunohistochemical staining using antibodies, or by other methods such as RT-PCR, Western blotting, DNA microarray, and RNA sequencing.

[0284] The cancers that can be treated as the ADC or pharmaceutical composition of the present invention are not particularly limited, and can be listed as cancers, such as cancers arising from epithelial cells (malignant epithelial tumors), such as 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, bile duct cancer, biliary tract cancer, and metastatic cancers (or cancer cells) thereof. Other cancers that can be treated as the ADC or pharmaceutical composition of the present invention include retroperitoneal tumors, angiosarcomas / lymphangiosarcomas, and metastatic cancers (or cancer cells) thereof. Brain tumors and soft tissue tumors of bone can also be listed as cancers that can be treated as the ADC or pharmaceutical composition of the present invention. In these cancers, the cancer cells are MEFLIN protein positive, or the cancer cells are MEFLIN protein negative but the cancer stroma contains MEFLIN protein positive cells. According to the present invention, MEFLIN protein positive cells are gradually recruited into the stroma of the above-mentioned cancers. Therefore, according to the present invention, the cancer (or cancer cells) that can be treated as the ADC or pharmaceutical composition of the present invention can be MEFLIN negative. MEFLIN-positive cells are recruited into the stroma of MEFLIN-negative cancer cells. The ADC of the present invention targets these MEFLIN-positive cells recruited into the stroma, and exerts anti-tumor activity against the cancer (or cancer cells) regardless of whether the cancer itself is MEFLIN-positive or MEFLIN-negative. This can be illustrated by the bystander effect. The presence of MEFLIN-positive cells in the cancer stroma can be confirmed by examining the expression of MEFLIN protein in tissue samples. In one aspect of the present invention, the cancer targeted for treatment is MEFLIN-negative cancer with MEFLIN-positive cells present in the cancer stroma. According to the present invention, the cancer targeted for treatment by the ADC or pharmaceutical composition of the present invention can be MEFLIN-positive cancer (e.g., sarcoma, such as myxofibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor, Ewing sarcoma, epithelioid sarcoma, clear cell sarcoma, synovial sarcoma, and osteosarcoma). In one embodiment of the present invention, the cancer targeted for treatment is MEFLIN protein-positive and has MEFLIN protein-positive cells present in the cancer stroma. In another embodiment, the cancer targeted for treatment is MEFLIN protein-positive and no MEFLIN protein-positive cells are detected in the cancer stroma. In yet another embodiment, the cancer targeted for treatment may be a cancer evaluated or presumed to be MEFLIN-positive, or a cancer whose cancer cells themselves are MEFLIN-negative but are evaluated or presumed to have MEFLIN-positive cells present in the stroma.

[0285] Bispecific antibodies can be, for example, antibodies that have binding affinity for cancer cells and immune cells respectively. Bispecific antibodies can be, for example, antibodies that bind to T cell surface antigens such as MEFLIN protein and CD3 protein. Bispecific antibodies can be IgG type and double-chain antibody type. This approach can be effective against MEFLIN protein-positive cancer cells, for example.

[0286] In one embodiment of the present invention, the pharmaceutical composition comprises the ADC and excipients of the present invention. The pharmaceutical composition of the present invention can be administered via intravenous, subcutaneous, intratumoral, intraperitoneal, intraventricular, or intramuscular methods. The dosage can be appropriately determined by a physician, taking into account factors such as the patient's age, sex, weight, and the severity of the disease.

[0287] According to the present invention, the use of the ADC of the present invention in the manufacture of a medicament for treating cancer is provided.

[0288] According to the present invention, a method for treating cancer in a subject with this need is provided, comprising the step of administering a therapeutically effective amount of the ADC of the present invention to the subject. In this manner, the cancer being treated is a MEFLIN protein-negative cancer with MEFLIN protein-positive cells present in the cancer stroma (e.g., a malignant epithelial tumor). Alternatively, in another manner, the cancer may be a MEFLIN protein-positive cancer (e.g., a sarcoma). Such subjects can be identified or selected by detecting MEFLIN mRNA in tissue samples or tissue sections obtained from the subject using immunohistochemical staining or in situ hybridization with anti-MEFLIN antibodies.

[0289] According to the present invention, the application of the ADC of the present invention in a method for treating cancer is provided. According to the present invention, an ADC of the present invention can be provided for use in a method for treating cancer.

[0290] Example

[0291] Example 1: Preparation of antibodies and characterization of the obtained monoclonal antibodies

[0292] In this embodiment, a monoclonal antibody that recognizes the human MEFLIN protein was prepared, and the binding properties of the antibody were confirmed through various analyses.

[0293] [Cell Preparation]

[0294] Using Flp-In TMThe Thermo Fisher system established a stable human fetal kidney (HEK293) cell line expressing mouse Meflin (hereinafter referred to as the mMeflin HEK293 cell line) and a stable Chinese hamster ovary (CHO) cell line expressing mouse Meflin (hereinafter referred to as the "mMeflin CHO cell line"). Similarly, a stable human fetal kidney (HEK293) cell line expressing human MEFLIN (hereinafter referred to as the hMeflin HEK293 cell line) and a stable Chinese hamster ovary (CHO) cell line expressing human MEFLIN (hereinafter referred to as the "hMEFLIN CHO cell line") were established.

[0295] [Preparation of rat anti-human MEFLIN monoclonal antibody]

[0296] In animal immunizations used to produce human MEFLIN antibodies, recombinant human MEFLIN protein is used as the antigen. The recombinant human MEFLIN protein is obtained from a mammalian cell expression system. For example, the human MEFLIN gene is introduced into human fetal kidney cell lines (Expi293F cells, Thermo Fisher) acclimated in suspension culture via liposome transfection (lipofectamine2000, Invitrogen). To facilitate subsequent purification, the GPI anchor sequence at the C-terminus of the MEFLIN gene is removed, and a histidine tag is introduced therein. Cell culture medium, collected 5–8 days after gene introduction, is recovered, filtered (0.22 μm), and fed into a column filled with a vector (Ni Excel, GE Healthcare) specifically binding to histidine-tagged proteins, thereby binding the MEFLIN protein in the culture medium. After column washing, the bound MEFLIN protein is competitively eluted using a buffer containing imidazole (500 mM). Imidazole was removed from the eluent containing MEFLIN protein by ultrafiltration (dialysis using phosphate buffer), thus obtaining the final human MEFLIN recombinant protein. The purity of the purified human MEFLIN recombinant protein was determined by SDS-PAGE electrophoresis followed by Coomassie brilliant blue staining.

[0297] One immunization with 100 μg of recombinant human MEFLIN protein (95% purification) was performed on the hind limbs of WKY rats. Two weeks later, plasma cells and myeloma (SP2 / 0) cell lines present in the iliac lymph nodes were electrofused to create hybridomas. For the culture medium, HAT medium (MPB) and BM-condimed H1 (Roche) were added to high-glucose DMEM (Nakkari) for hybridoma screening.

[0298] [Preparation of mouse anti-human MEFLIN monoclonal antibody]

[0299] 50 μg of recombinant human MEFLIN protein (95% purification) was immunized once in the tail root muscle of C57BL / 6 mice. Otherwise, monoclonal antibodies were prepared using the same method as that used for the preparation of rat anti-human MEFLIN monoclonal antibodies.

[0300] [Screening of hybridomas]

[0301] Hybridomas that generate monoclonal antibodies that recognize antigens were screened using various methods, including ELISA with recombinant human MEFLIN protein, immunofluorescence staining (IF) with hMEFLIN CHO cell line, flow cytometry with hMEFLIN CHO cell line, immunoprecipitation with cell lysate prepared from hMeflin HEK293 cell line, and Western blotting.

[0302] [Western blot]

[0303] Human fetal kidney (HEK293) cell line was transiently expressed using a pRP-CMV vector containing DNA encoding human MEFLIN protein or a fragment thereof via liposome transfection. Cell lysate was prepared afterward. The fragment was... Figure 1 The fragment shown in Figure 2 has a G196 tag and a His tag at the C-terminus. SDS-PAGE was performed using the cell lysate to transfer the electrophoretically deposited protein onto a PVDF membrane. The PVDF membrane was reacted with 5% skim milk / PBS at room temperature for 1 hour. As the primary antibody, a solution prepared by diluting the culture supernatant of each hybridoma clone 50-fold with PBS was used. For positive subjects, anti-G196 antibody was used as the primary antibody. The membrane was reacted with the primary antibody at 4°C overnight at 4°C. The PVDF membrane was then washed with 0.05% Tween / PBS. As the secondary antibody, an anti-rat or anti-mouse antibody-horseradish peroxidase (HRP) conjugate was used. The secondary antibody was diluted 1000-fold with PBS and reacted with the PVDF membrane at room temperature for 45 minutes. After washing with 0.05% Tween / PBS, the HRP labeled with the antibody was luminescent using an ECL (GE Healthcare), and the images were imported into a computer using a CCD imager (Las4000, GE Healthcare).

[0304] [Analysis of the internalization activity of monoclonal antibodies]

[0305] hMeflin CHO cell lines or mMeflin CHO cell lines were cultured at 37°C and 5% CO2 for 3 hours in an analytical solution prepared by adding 20 mM HEPS and 0.1% BSA to F-12 Glutamax medium (Gibco). The supernatant from each hybridoma was diluted 10-fold with the analytical solution and added to the CHO cell lines for 15 minutes. The CHO cell lines were washed with PBS (4°C) and then with a washing solution (MilliQ-H2O containing 0.5 M NaCl and 3% acetic acid) to remove antibodies bound to the cell membrane. The CHO cell lines were fixed by reacting in 4% paraformaldehyde (PFA) for 10 minutes, followed by cell membrane permeation treatment in 0.1% Tiriton X-100 for 5 minutes. The secondary antibody, using an anti-rat or anti-mouse antibody-Alexa488 conjugate, was diluted 400-fold with PBS and reacted with the permeabilized cells at room temperature for 30 minutes. After washing with PBS, react with DAPI at room temperature for 5 minutes, wash with PBS, and obtain images using a confocal microscope (LSM700, ZEISS).

[0306] [In situ hybridization method]

[0307] From pathological tissue samples obtained from sarcoma patients or malignant epithelial tumor patients, the areas with the highest tumor components were selected under microscopic examination, and the expression of MEFLIN (also known as "ISLR") was investigated using in situ hybridization (hereinafter referred to as "ISH staining") (refer to WO2017 / 22472). Within the investigated tissues, the tissues were observed at medium magnification (20x objective). For sarcoma tissues, tissues with more than 20% of cells exhibiting tumor cell morphology being ISLR positive were classified as ISLR-positive tumor tissues, and those with less than 20% being ISLR-negative tumor tissues. Similarly, in malignant epithelial tumors, tissues with more than 20% of cells infiltrating the cancer stroma and exhibiting fibroblast-like morphology being ISLR-positive were classified as ISLR-positive tumor tissues, and those with less than 20% being ISLR-negative tumor tissues. ISLR positivity was defined as the presence of a signal in at least a portion of the cytoplasm.

[0308] [Double immunofluorescence staining method]

[0309] Tumor tissues from tumor-bearing mice subcutaneously transplanted with osteosarcoma cell line (HsOs1) and pancreatic cancer cell line (BxPC-3) were sampled, fixed in 10% formalin, and embedded in paraffin. The tissues were thinly sliced ​​to 2 μm thickness, placed on glass slides, and deparaffined using xylene and ethanol. Antigen activation was then performed using an antigen activation solution (Leica) at pH 6.0. After blocking, double immunofluorescence staining was performed using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody (ab37259, abcam) as primary antibodies, and anti-rat antibody-Alexa488 conjugate and anti-mouse antibody-Alexa594 conjugate as secondary antibodies (Invitrogen). Intracellular nuclei were stained with DAPI. MEFLIN (green), cathepsin K (red), and nuclei (blue). Images were obtained using a confocal microscope (LSM700, ZEISS).

[0310] [Immunohistochemical staining]

[0311] From pathological tissue samples obtained from patients with malignant epithelial tumors, the areas with the highest tumor components were selected under microscopic examination, and the expression of MEFLIN (also known as "ISLR") was investigated using immunohistochemical staining (hereinafter referred to as "IHC staining"). Within the investigated tissues, the tissues were observed at medium magnification (20x objective). Tissues with more than 20% of fibroblast-like cells infiltrating the cancer stroma and exhibiting ISLR morphology were classified as ISLR-positive tumor tissues, while tissues with less than 20% were classified as ISLR-negative tumor tissues. ISLR positivity was defined as when a signal was observed in at least a portion of the cytoplasm. Anti-rat or anti-mouse antibody-horseradish peroxidase (HRP) conjugates were used as secondary antibodies (ImmPRESS, VECTORSLABORATORIES).

[0312] [result]

[0313] The results obtained by confirming the binding of each monoclonal antibody to the respective human MEFLIN protein fragment using Western blotting are as follows: Figure 1 As shown. Figure 1 As shown, the rat or mouse monoclonal antibody derived from clone 27-7 (hereinafter referred to as "27-7 antibody") does not bind to amino acid positions 1-343 of human MEFLIN, but rather to amino acid positions 1-399. This suggests that the 27-7 antibody binds to amino acid positions 344-399 of human MEFLIN. Similarly, Figure 1This suggests that antibody 34-4 binds to amino acid positions 232-343; antibody 35-9 binds to amino acid positions 1-146; and antibodies 27-8, 41-10, and 46-3 bind to amino acid positions 344-399 of human MEFLIN. Additionally, as... Figure 2 As shown, this suggests that antibodies 3-2, 11-8, 12-2, 19-7, and 23-1 bind to amino acid regions 344–399 of human MEFLIN; antibody 13-1 binds to amino acid regions 147–231; and antibodies 16-5, 22-3, and 32-1 bind to amino acid regions 1–146.

[0314] The internalization activity of each antibody clone was confirmed. The results are as follows: Figures 3-5 As shown. Figures 3-5 As shown, strong signals from the antibodies were observed intracellularly in the presence of 21-3, 25-1, 8-2, 27-7, 27-8, 34-4, 35-9, 46-3, 3-2, 11-8, 12-2, 16-5, 19-7, 22-3, 23-1, and 32-1 antibodies.

[0315] The results are shown in Table 9.

[0316] [Table 9]

[0317] Table 9: Overview of the characteristics of various monoclonal antibodies

[0318]

[0319] In Table 9, the symbol "S" indicates strong internalization activity, "M" indicates moderate internalization activity, and "N" indicates no internalization activity detected. "?" indicates undetermined. Additionally, in Table 9, "host" indicates the species from which the antibody originates: rat or mouse. In Table 9, antibodies 21-3, 25-1, and 8-2 bind to mouse MEFLIN protein; all other antibodies bind to human MEFLIN protein.

[0320] The expression of MEFLIN protein in various cancer tissues is shown in Table 10. Table 10 shows the number of confirmed tissue samples, the number of positive samples, and the positive rate (%).

[0321] [Table 10]

[0322] Table 10: Expression of human MEFLIN (ISLR) in tumor cells

[0323]

[0324] The expression of human MEFLIN (ISLR) in the tumor stroma is shown in Table 11. Table 11 shows the number of confirmed tissue samples, the number of positive samples, and the positive rate (%). In addition, 2 out of 7 gastric cancer cases were positive.

[0325] [Table 11]

[0326] Table 11: Expression of human MEFLIN (ISLR) in tumor stroma

[0327]

[0328] It should be noted that human MEFLIN expression is negative in tumor cells of pancreatic cancer, lung cancer, breast cancer, colorectal cancer, gastric cancer, bile duct cancer, ovarian cancer, bladder cancer, and esophageal cancer.

[0329] Example 2: Preparation of antibody-drug conjugates (ADCs) and their in vivo efficacy testing

[0330] In this embodiment, a conjugate of the antibody and cytotoxic agent prepared in Example 1 was fabricated. Furthermore, the fabricated conjugate was administered to a tumor-bearing mouse model, and its antitumor effect was confirmed.

[0331] [ADC fabrication]

[0332] Antibodies were purified from antibody-containing hybridoma supernatants concentrated through high-density hybridoma culture using Mono Spin L ProG (GL Sciense). The antibodies were then reduced at 25°C for 30 minutes using 1.0 mM DTT, thereby cleaving the disulfide bonds. VcMMAE (mc-vc-PAB-MMAE, MedChemExpress) or mc-PEG was then added to the exposed thiol groups at room temperature. 12 -vc-PABC-MMAE was purified by ultrafiltration. VcMMAE is a compound registered under CAS number 646502-53-6. VcMMAE is monomethylaurestatin E (MMAE), which binds to the valine-citrulline linker via a p-aminobenzoyloxycarbonyl (PBAC) group. The mechanism involves the release of MMAE when the valine-citrulline linker is cleaved by proteases such as cathepsin B. The valine-citrulline linker is modified with a maleimide hexanoyl group, which can react with the thiol group of the antibody's cysteine ​​residues via the maleimide group. mc-PEG 12-vc-PABC-MMAE is identical to VcMMAE except for the inclusion of PEG, and the MMAE release mechanism is also the same as VcMMAE. The drug-antibody ratio (DAR, the number of cytotoxic agents conjugated to one antibody molecule) is determined by calculating the difference between the number of thiol groups on the reduced antibody and the number of thiol groups after adding VcMMAE to the antibody. Specifically, 5,5'-dithiobis(2-nitrobenzoic acid) (Dōjin Chemical Research Institute) is reacted with exposed thiol groups to generate stable 5-mercapto-2-nitrobenzoic acid. The absorbance (λ) of the generated thiol is measured. max =412nm, ε=1.55×10 4 To quantify thiol groups.

[0333] mc-vc-PAB-MMAE has the structure of the following formula (I).

[0334]

[0335] The resulting ADC can have the structure of the following formula (II).

[0336]

[0337] In the formula, n is any natural number from 1 to 8.

[0338] mc-PEG 12 -vc-PABC-MMAE has the structure of the following formula (III).

[0339]

[0340] The prepared ADC was used to test its cytotoxicity against various cell types.

[0341] ADC cytotoxicity against HEK293 cells expressing human MEFLIN

[0342] First, the cytotoxicity of the ADC on HEK293 cells expressing human MEFLIN was tested. Specifically, the hMeflin HEK293 cell line was injected with 1.0 × 10⁻⁶ cells. 4Cells were seeded in each well on agar plates and cultured at 37°C and 5% CO2 for one day. Then, ADCs of various concentrations were added and the cells were cultured for two days. Cell proliferation was evaluated using the MTT assay. Cell proliferation was assessed by measuring the absorbance of the samples at 590 nm using a spectrophotometer (POWERSCAN4, DS PHARMA BIOMEDICAL) in the MMT assay. Additionally, Meflin expression in the hMeflin HEK293 cell line was evaluated by FACS analysis. In the following text, when the clone number is simply listed in the figure, it indicates that the monoclonal antibody itself was used. When the clone number is listed immediately before or after the ADC, it indicates that the ADC containing that clone's antibody was used. CTL ADC or allotype control IgG ADC indicates that the ADC containing the allotype control IgG antibody was used. Rat IgG2a (BioLegend) was used as the allotype control antibody.

[0343] The results are as follows Figure 6 As shown. Figure 6 As shown in the figure, FACS analysis revealed that HEK293 cells expressing human MEFLIN strongly expressed the human MEFLIN protein. Furthermore, ADC27-7 (drug-antibody ratio (DAR) = 2.4) significantly reduced the proliferation of HEK293 cells expressing human MEFLIN. The EC50 of ADC27-7... 50 The concentration was 0.72 nM. ADC34-4 (DAR = 3.4), which exhibits moderate internalizing activity in hMEFLIN CHO cells, also has weak inhibitory activity against cell proliferation.

[0344] ADC cytotoxicity to human rhabdomyosarcoma cell lines

[0345] Similarly, the cytotoxicity of the ADC against the rhabdomyosarcoma (KYM-1) cell line was tested. The results are as follows: Figure 7 As shown. Figure 7 As shown in the figure, FACS analysis revealed that the rhabdomyosarcoma cell line strongly expressed human MEFLIN protein. Furthermore, ADC27-7 and ADC34-4 significantly reduced the proliferation of the KYM-1 cell line.

[0346] Cytotoxicity of ADCs in a subcutaneous transplantation model of rhabdomyosarcoma cell lines

[0347] Then, the cytotoxicity induced by the ADC was tested in vitro using a tumor-bearing mouse model. 1.0 × 10⁻⁶ ADCs were used. 7 One KYM-1 cell line was subcutaneously transplanted into the back of a female NOD SCID mouse (n=5 in each group). For mice with approximately 100 mm... 3Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting KYM cell lines were subjected to ISH staining.

[0348] The results are as follows Figure 8 As shown. Figure 8 As shown, 27-7 ADC and 34-4 ADC exhibit strong antitumor activity against rhabdomyosarcoma. Tissue images also show that the tumor strongly expresses human MEFLIN.

[0349] Cytotoxicity of ADCs in a subcutaneous transplantation model of osteosarcoma cell lines

[0350] Furthermore, 1.0 × 10 7 One endogenous Meflin-positive osteosarcoma (HsOs1) cell line was subcutaneously transplanted into the back of female NODSCID mice (n=5 in each group). For mice with approximately 75 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting HsOs1 cell lines were stained with ISH.

[0351] The results are as follows Figure 9 As shown. Figure 9 As shown, 27-7ADC and 34-4ADC exhibit strong antitumor activity against osteosarcoma. Tissue images indicate that the tumor expresses human MEFLIN to a moderate degree.

[0352] Cytotoxicity of ADCs in a subcutaneous transplantation model of pancreatic cancer cell lines

[0353] 1.0×10 7 One endogenous Meflin-negative human pancreatic cancer (BxPC-3) cell line was subcutaneously transplanted into the back of female nude mice (BALB / cSlc nu / nu) (n=5 per group). For mice with approximately 150 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting BxPC-3 cell lines were stained with ISH.

[0354] The results are as follows Figure 10 As shown. Figure 10 The tissue images show that pancreatic cancer cells are MEFLIN-negative, but mouse MEFLIN-positive cells were identified in the tumor stromal cells. Furthermore, 21-3ADC and 25-1ADC exhibited potent antitumor activity against pancreatic cancer. Since the original tissue was MEFLIN-negative, it is evident that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0355] Cytotoxicity of ADCs in a subcutaneous transplantation model of lung cancer cell lines

[0356] 1.0×10 7 One endogenous Meflin-negative human lung cancer (A549) cell line was subcutaneously transplanted into the back of female nude mice (BALB / cSlc nu / nu) (n=5 per group). For mice with approximately 100 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, ISH staining was performed on tumors formed by transplanting A549 cell lines.

[0357] The results are as follows Figure 11 As shown. Figure 11 As shown, 25-1 ADCs exhibit strong antitumor activity against lung cancer, while 21-3 ADCs exhibit weak antitumor activity. Furthermore, tissue images reveal that while lung cancer cells are initially MEFLIN-negative, mouse MEFLIN-positive cells were identified within the tumor stroma. Since the original tissue was MEFLIN-negative, this indicates that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0358] Cytotoxicity of ADCs in a subcutaneous transplantation model of neuroblastoma cell lines

[0359] Furthermore, 1.0 × 10 7 One endogenous Meflin-positive neuroblastoma (NB-1) cell line was subcutaneously transplanted into the back of female NOD SCID mice (n=5 per group). For mice with approximately 250 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2The volume was calculated using ×0.52. Additionally, tumors formed by transplanting NB-1 cell lines were stained with ISH.

[0360] The results are as follows Figure 12 As shown. Figure 12 As shown, 27-7ADC and 34-4ADC exhibit weak antitumor activity against neuroblastoma. Tissue images indicate that the tumor expresses human MEFLIN to a moderate degree.

[0361] Cytotoxicity of ADC in a subcutaneous transplantation model of colorectal cancer cell lines

[0362] 1.0×10 7 One endogenous Meflin-negative human colorectal cancer (DLD-1) cell line was subcutaneously transplanted into the back of female nude mice (BALB / cSlc nu / nu) (n=5 per group). For mice with approximately 200 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting the DLD-1 cell line were subjected to ISH staining.

[0363] The results are as follows Figure 13 As shown. Figure 13 As shown, 25-1 ADCs exhibited weak antitumor activity against colorectal cancer, while 21-3 ADCs showed no antitumor activity. Furthermore, tissue images revealed that while the colorectal cancer cells themselves were human MEFLIN-negative, mouse MEFLIN-positive cells were identified in the tumor stromal cells. Since the original tissue was MEFLIN-negative, it indicates that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0364] Cytotoxicity of ADCs in a subcutaneous transplantation model of gastric cancer cell lines

[0365] 1.0×10 7 One endogenous Meflin-negative human gastric cancer (MKN45) cell line was subcutaneously transplanted into the back of female nude mice (BALB / cSlc nu / nu) (n=5 per group). For mice with approximately 200 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting MKN45 cell lines were subjected to ISH staining.

[0366] The results are as follows Figure 14 As shown. Figure 14 As shown, 21-3ADC and 25-1ADC exhibit strong antitumor activity against gastric cancer. Furthermore, tissue images reveal that while the gastric cancer cells themselves are human MEFLIN-negative, mouse MEFLIN-positive cells were identified in the tumor stromal cells. Since the original tissue was MEFLIN-negative, it indicates that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0367] Single-cell analysis of mouse pancreas

[0368] Figure 15 In this study, single-cell RNA sequencing data from mouse pancreas cell populations publicly available on the internet (Tabula Muris) were analyzed (refer to Non-Patent Literature 7). The vertical and horizontal axes, respectively, represent tSNE (t-distributed Stochastic Neighbor Embedding), which compresses multidimensional data consisting of large amounts of RNA expression data into a one-dimensional representation through nonlinear transformation. That is, Figure 15 In this method, by unfolding the large amount of RNA expression data expressed in each cell into a two-dimensional plane formed by tSNE1 and tSNE2, cells can be clustered based on gene expression profiles. This method can be understood, for example, as identifying the type of cell clusters formed by the circular symbols. Therefore, this clustering method can confirm whether cell populations expressing specific factors are consistent with cell populations expressing other specific factors, and further, determine which cell species corresponds to that cell population. Figure 15 The image shows that mouse pancreatic stellate cells express both MEFLIN and cathepsin K, indicating that the MEFLIN-positive cell population is consistent with the cathepsin K-positive cell population in the mouse pancreas (see arrows in the figure).

[0369] Single-cell analysis of mouse lungs

[0370] Figure 16 In this study, single-cell RNA sequencing data from mouse lung cell populations publicly available on the internet (Tabula Muris) were analyzed (refer to Non-Patent Literature 7). The results showed that mouse lung stromal cells expressed both MEFLIN and cathepsin K, indicating that the MEFLIN-positive cell population was consistent with the cathepsin K-positive cell population in mouse lungs (see arrows in the figure).

[0371] We induced full-length mouse MEFLIN protein expression in CHO cells and attempted Western blotting of their cell lysate. The results were as follows: Figure 17 As shown. Figure 17 As shown, by inducing CHO cells to express the full-length mouse MEFLIN protein, the expression of endogenous cathepsin K was enhanced.

[0372] Immunofluorescence double staining of osteosarcoma tissue

[0373] Figure 18 The results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody are shown in tumor tissues from tumor-bearing mice subcutaneously transplanted with osteosarcoma cell line (HsOs1). Cathepsin K secretion (red) is visible around MEFLIN-positive cells (green) (arrows).

[0374] The results are as follows Figure 18 As shown. Figure 18 As shown, cathepsin K (red) can be confirmed to be present around MEFLIN-positive cells (green) (arrow in magnified view).

[0375] Immunofluorescence double staining of pancreatic cancer tissue

[0376] Figure 19 The results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody are shown in tumor tissues from a tumor-bearing mouse model with subcutaneously transplanted pancreatic cancer cell line (BxPC-3). Cathepsin K secretion (red) is visible around MEFLIN-positive cells (green) (arrows).

[0377] The results are as follows Figure 19 As shown. Figure 19 As shown, cathepsin K (red) is confirmed to be present around MEFLIN-positive cells (green) (arrow in magnified view).

[0378] This indicates that when human MEFLIN-negative cancer cells are transplanted into mice, MEFLIN-positive cells are recruited to the tissue containing these cancer cells. Furthermore, it suggests that treating cancer with ADCs targeting recruited MEFLIN-positive cells is beneficial. It is also known that internalization activity is not essential for ADCs targeting human MEFLIN protein. Additionally, it is believed that enzymes such as cathepsins that cleave the valine-citrulline linker are present in the stroma of MEFLIN-positive cells; therefore, ADCs not taken into the cells exhibit cytotoxicity.

[0379] Cytotoxicity of ADC in a subcutaneous transplantation model of cholangiocarcinoma cell lines

[0380] 1.0×107 One endogenous Meflin-negative human cholangiocarcinoma (HuCCT1) cell line was subcutaneously transplanted into the back of female NODSCID mice (n=5 per group). For mice with approximately 250 mm... 3 Mice with tumors of approximately [amount missing] were administered ADC (5 mg / kg body weight, administered three times at 4-day intervals) via tail vein. Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting the HuCCT1 cell line were subjected to ISH staining.

[0381] The results are as follows Figure 20 As shown. Figure 20 The tissue images show that the cholangiocarcinoma cells themselves are MEFLIN-negative, but mouse MEFLIN-positive cells were identified in the tumor stroma cells. Furthermore, 21-3ADC and 25-1ADC exhibited potent antitumor activity against cholangiocarcinoma. Since the original tissue was MEFLIN-negative, it is evident that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0382] Cytotoxicity of ADCs in a subcutaneous transplantation model of bladder cancer cell lines

[0383] 1.0×10 7 One endogenous Meflin-negative human bladder cancer (T24) cell line was subcutaneously transplanted into the back of female NOD SCID mice (n=4 per group). For mice with approximately 300 mm... 3 Mice with tumors of approximately [amount missing] were administered ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals) via tail vein. Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting T24 cell lines were subjected to ISH staining.

[0384] The results are as follows Figure 21 As shown. Figure 21 The tissue images show that the bladder cancer cells themselves are MEFLIN-negative, but mouse MEFLIN-positive cells were identified in the tumor stromal cells. Furthermore, 21-3ADC and 25-1ADC exhibited weak antitumor activity against bladder cancer. Since the original tissue was MEFLIN-negative, it is evident that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0385] Cytotoxicity of ADC in a subcutaneous transplantation model of ovarian cancer cell lines

[0386] 1.0×10 7 One endogenous Meflin-negative human ovarian cancer (OV-90) cell line was subcutaneously transplanted into the back of female NODSCID mice (n=5 in each group). For mice with approximately 100 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, ISH staining was performed on tumors formed by transplanting the OV-90 cell line.

[0387] The results are as follows Figure 22 As shown. Figure 22 The tissue images show that ovarian cancer cells are MEFLIN-negative, but mouse MEFLIN-positive cells were identified in the tumor stromal cells. Furthermore, 21-3ADC and 25-1ADC exhibited potent antitumor activity against ovarian cancer. Since the original tissue was MEFLIN-negative, it is evident that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0388] Cytotoxicity of ADC in a subcutaneous transplantation model of esophageal cancer cell lines

[0389] 1.0×10 7 One endogenous Meflin-negative human esophageal cancer (KYSE) cell line was subcutaneously transplanted into the back of female NODSCID mice (n=5 per group). For mice with approximately 200 mm... 3 Mice with tumors of approximately [amount missing] were administered an intraperitoneal dose of ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, ISH staining was performed on tumors formed by transplanting KYSE cell lines.

[0390] The results are as follows Figure 23 As shown. Figure 23 The tissue images show that the esophageal cancer cells themselves are MEFLIN-negative, but mouse MEFLIN-positive cells were identified in the tumor stromal cells. Furthermore, 21-3ADC and 25-1ADC exhibited potent antitumor activity against esophageal cancer. Since the original tissue was MEFLIN-negative, it is evident that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0391] Cytotoxicity of ADCs in a subcutaneous transplantation model of osteosarcoma cell lines

[0392] 1.0×10 7 One endogenous Meflin-positive osteosarcoma (HsOs1) cell line was subcutaneously transplanted into the back of female NOD SCID mice (n=4 in each group). For mice with approximately 250 mm... 3 Mice with tumors of approximately [amount missing] were administered ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals) via tail vein. Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, tumors formed by transplanting HsOs1 cell lines were stained with ISH.

[0393] The results are as follows Figure 24 As shown. Figure 24 As shown, 46-3ADC exhibits potent antitumor activity against osteosarcoma. Additionally, the tumor expresses human MEFLIN at moderate levels (reference: Figure 9 ).

[0394] Cytotoxicity of ADCs in a subcutaneous transplantation model of breast cancer cell lines

[0395] 1.0×10 7 One endogenous Meflin-negative human breast cancer (MCF-7) cell line was subcutaneously transplanted into the back of female nude mice (BALB / cSlc nu / nu) (n=5 per group). For mice with approximately 300 mm... 3 Mice with tumors of approximately [amount missing] were administered ADC (5 mg / kg body weight, administered a total of 5 times at 4-day intervals) via tail vein. Tumor length (L) and width (W) were measured using electronic calipers according to the formula V = L × W. 2 The volume was calculated using ×0.52. Additionally, ISH staining was performed on tumors formed by transplanting MCF-7 cell lines.

[0396] The results are as follows Figure 25 As shown. Figure 25 The tissue images show that the breast cancer cells themselves are MEFLIN-negative, but mouse MEFLIN-positive cells were identified in the tumor stroma. Furthermore, 21-3ADC and 25-1ADC exhibited weak antitumor activity against breast cancer. Since the original tissue was MEFLIN-negative, it is evident that MEFLIN-positive cells were recruited to the vicinity of the tumor. This further suggests that ADCs exert antitumor activity against the tumor.

[0397] Appropriateness evaluation of immunohistostaining of human pancreatic cancer tissue

[0398] Serial sections of human pancreatic cancer surgical samples were used as the target samples. Immunohistochemical staining with anti-MEFLIN antibody was performed on these serial sections to confirm MEFLIN expression in human tissue. Additionally, in situ hybridization was used to confirm the expression of human MEFLIN mRNA in these serial sections.

[0399] The results are as follows Figure 26 As shown. Cells that become MEFLIN-positive in immunohistoscopy of human pancreatic cancer tissue are the same as those that become MEFLIN-positive in in situ hybridization. MEFLIN expression can be confirmed by immunohistoscopy of human tissue using anti-MEFLIN antibody. The arrows indicate the MEFLIN levels confirmed by immunohistoscopy in human pancreatic cancer tissue. Figure 26 The image above), and MEFLIN confirmed by in situ hybridization. Figure 26 (See the image below). Figure 26 The arrows in the top and bottom images are in the same position, pointing to the same cell. sequence list <110> National University Corporation, Donghai National University Medical Institution Innovation Association <120> Anti-MEFLIN antibody and pharmaceutical composition containing the antibody for the treatment of cancer in cancerous subjects. <130> PT86-9009WO <150> JP 2020-016535 <151> 2020-02-03 <160> 136 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 27-7 HCDR1 <400> 1 Lys Tyr Trp Met Asp 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 27-7 HCDR2 <400> 2 Glu Ile Asn Thr Asp Gly Ser Lys Thr Asn Tyr Ala Pro Ser Ile Lys 1 5 10 15 Asp <210> 3 <211> 4 <212> PRT <213> Artificial sequence <220> <223> 27-7 HCDR3 <400> 3 Trp Glu Asp Tyr 1 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 27-7 LCDR1 <400> 4 Lys Ser Ser Gln Ser Leu Val Tyr Ser Asp Gly Lys Thr Tyr Leu His 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 27-7 LCDR2 <400> 5 Gln Val Ser Asn Leu Asp Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 27-7 LCDR3 <400> 6 Ala Gln Thr Thr His Phe Pro Tyr Thr 1 5 <210> 7 <211> 113 <212> PRT <213> Artificial sequence <220> <223> 27-7 Heavy Chain Variable Region <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Tyr Thr Phe Ser Lys Tyr 20 25 30 Trp Met Asp Trp Val Arg Gln Thr Pro Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Thr Asp Gly Ser Lys Thr Asn Tyr Ala Pro Ser Ile 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Asn Val Lys Ser Asp Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Thr Asn Trp Glu Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser 100 105 110 Ser <210> 8 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 27-7 Light Chain Variable Region <400> 8 Asp Val Val Met Thr Gln Thr Pro Pro Ser Leu Ser Val Ala Ile Gly 1 5 10 15 Gln Ser Val Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Lys Thr Tyr Leu His Trp Leu Leu Gln Ser Ser Gly Arg Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Gln Val Ser Asn Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Thr Gly Ser Gln Lys Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Lys Asp Leu Gly Val Tyr Tyr Cys Ala Gln Thr 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 9 <211> 8 <212> PRT <213> artificial sequence <220> <223> 34-4 HCDR1 <400> 9 Gly Phe Ser Leu Thr Ser Tyr Asp 1 5 <210> 10 <211> 7 <212> PRT <213> artificial sequence <220> <223> 34-4 HCDR2 <400> 10 Ile Trp Gly Asn Gly Asn Thr 1 5 <210> 11 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 34-4 HCDR3 <400> 11 Thr Gly Gly Tyr Phe Tyr 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 34-4 LCDR1 <400> 12 Gln Ser Leu Val Gly Thr Gly Gly Lys Thr Tyr 1 5 10 <210> 13 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 34-4 LCDR2 <400> 13 Leu Val Ser 1 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 34-4 LCDR3 <400> 14 Leu Gln Gly Thr His Phe Pro Phe Thr 1 5 <210> 15 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 34-4 Heavy Chain Variable Region <400> 15 Gln Val Gln Leu Met Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Trp Gly Asn Gly Asn Thr His Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Glu Asn Thr Ala Ile Tyr Phe Cys Thr 85 90 95 Gly Gly Tyr Phe Tyr Trp Gly Gln Gly Val Thr Val Thr Val Ser Ser 100 105 110 <210> 16 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 34-4 Light Chain Variable Region <400> 16 Asp Val Val Met Thr Gln Thr Pro Val Ser Leu Ser Val Ala Ile Gly 1 5 10 15 His Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val Gly Thr 20 25 30 Gly Gly Lys Thr Tyr Leu Ser Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Ile Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Asp Asp Leu Gly Val Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Gln Leu Glu Met Lys 100 105 110 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 27-8 HCDR1 <400> 17 Gly Tyr Thr Ile Thr Ser Gly Tyr 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 27-8 HCDR2 <400> 18 Ile Thr Tyr Ser Gly Ser Thr 1 5 <210> 19 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 27-8 HCDR3 <400> 19 Ser Arg Asp Ile Ala Ala Ile Ser Phe Ala Tyr 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 27-8 LCDR1 <400> 20 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> twenty one <211> 3 <212> PRT <213> Artificial sequence <220> <223> 27-8 LCDR2 <400> twenty one Lys Val Ser 1 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> 27-8 LCDR3 <400> twenty two Phe Gln Ala Thr His Val Pro Leu Thr 1 5 <210> twenty three <211> 117 <212> PRT <213> Artificial sequence <220> <223> 27-8 Heavy Chain Variable Region <400> twenty three Val Gln Leu Thr Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser 1 5 10 15 Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Thr Ile Thr Ser Gly Tyr 20 25 30 Asp Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met 35 40 45 Gly Tyr Ile Thr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ser 85 90 95 Arg Asp Ile Ala Ala Ile Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> twenty four <211> 112 <212> PRT <213> Artificial sequence <220> <223> 27-8 light chain variable region <400> 24 Asp Val Val Met Thr Gln Thr Pro Val Ala Gln Pro Val Thr Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ile Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 46-3 HCDR1 <400> 25 Gly Tyr Thr Ile Thr Ser Gly Tyr 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 46-3 HCDR2 <400> 26 Ile Thr Tyr Ser Gly Ser Thr 1 5 <210> 27 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 46-3 HCDR3 <400> 27 Ala Arg Asp Phe Ser Gly Ile Ser Phe Ala Tyr 1 5 10 <210> 28 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 46-3 LCDR1 <400> 28 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 29 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 46-3 LCDR2 <400> 29 Lys Val Ser 1 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 46-3 LCDR3 <400> 30 Phe Gln Ala Thr His Asp Pro Leu Thr 1 5 <210> 31 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 46-3 Heavy Chain Variable Region <400> 31 Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser 1 5 10 15 Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Thr Ile Thr Ser Gly Tyr 20 25 30 Asp Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr His His Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Arg Asn Gln Phe Phe Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Phe Ser Gly Ile Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Ser Val Ser Ser 115 <210> 32 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 46-3 light chain variable region <400> 32 Asp Val Val Leu Thr Gln Thr Pro Val Ala Gln Pro Val Thr Leu Gly 1 5 10 15 Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ile Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Ala 85 90 95 Thr His Asp Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 21-3 HCDR1 <400> 33 Gly Phe Thr Phe Ser Asn Tyr Gly 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 21-3 HCDR2 <400> 34 Ile Asn Tyr Asp Gly Ser Ser Thr 1 5 <210> 35 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 21-3 HCDR3 <400> 35 Ala Arg Val Glu Tyr Arg Phe Ser Pro Phe Asp Tyr 1 5 10 <210> 36 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 21-3 LCDR1 <400> 36 Gln Asn Ile Asn Lys Tyr 1 5 <210> 37 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 21-3 LCDR2 <400> 37 Asn Thr Asn 1 <210> 38 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 21-3 LCDR3 <400> 38 Leu Gln Arg Asn Ser Trp Leu Thr 1 5 <210> 39 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 21-3 Heavy chain variable region <400> 39 Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser 1 5 10 15 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Gly 20 25 30 Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala 35 40 45 Thr Ile Asn Tyr Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu 65 70 75 80 Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Val Glu Tyr Arg Phe Ser Pro Phe Asp Tyr Trp Gly Gln Gly Val 100 105 110 Thr Ala Thr Val Ser Ser 115 <210> 40 <211> 106 <212> PRT <213> Artificial sequence <220> <223> 21-3 Light Chain Variable Region <400> 40 Asp Val Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Asn Ile Asn Lys Tyr 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Thr Asn Asn Leu His Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Leu Gln Arg Asn Ser Trp Leu Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 41 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 25-1 HCDR1 <400> 41 Gly Phe Asp Phe Lys Thr Tyr Ala 1 5 <210> 42 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 25-1 HCDR2 <400> 42 Ile Ser Ile Lys Thr Gln Asn Tyr Pro Thr 1 5 10 <210> 43 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 25-1 HCDR3 <400> 43 Thr Val Gly Ala Arg Tyr 1 5 <210> 44 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 25-1 LCDR1 <400> 44 Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 45 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 25-1 LCDR2 <400> 45 Leu Val Ser 1 <210> 46 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 25-1 LCDR3 <400> 46 Val Gln Ser Thr His Ala Pro Leu Thr 1 5 <210> 47 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> 25-1 Heavy chain variable region <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Lys Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ala Ser Ile Ser Ile Lys Thr Gln Asn Tyr Pro Thr Leu Tyr Ala Asp 50 55 60 Ser Val Lys Glu Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Val Tyr Leu His Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Leu Tyr 85 90 95 Tyr Cys Thr Val Gly Ala Arg Tyr Trp Gly Gln Gly Val Met Thr Thr 100 105 110 Val Ser Ser 115 <210> 48 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 25-1 Light Chain Variable Region <400> 48 Asp Val Val Met Thr Gln Thr Pro Pro Thr Leu Ser Ala Thr Ile Gly 1 5 10 15 Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Pro 35 40 45 Pro Gln Val Leu Ile Tyr Leu Val Ser Arg Leu Glu Ser Gly Val Pro 50 55 60 Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Gly Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Ser 85 90 95 Thr His Ala Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 49 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 41-10 HCDR1 <400> 49 Gly Phe Ser Leu Thr Ser Tyr Asp 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 41-10 HCDR2 <400> 50 Ile Trp Gly Asn Gly Asn Thr 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 41-10 HCDR3 <400> 51 Thr Arg Ser Leu Gly Val Gly 1 5 <210> 52 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 41-10 LCDR1 <400> 52 Gln Asp Ile Gly Asp Tyr 1 5 <210> 53 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 41-10 LCDR2 <400> 53 Gly Ala Thr 1 <210> 54 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 41-10 LCDR3 <400> 54 Leu Gln Ser Lys Glu Ser Pro Cys Ser Arg 1 5 10 <210> 55 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 41-10 Heavy chain variable region <400> 55 Gln Val Gln Leu Thr Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Trp Gly Asn Gly Asn Thr His Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Arg Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Ile Tyr Phe Cys Thr 85 90 95 Arg Ser Leu Gly Val Gly Trp Gly Gln Gly Val Met Ile Ile Val Ser 100 105 110 Ser <210> 56 <211> 107 <212> PRT <213> Artificial sequence <220> <223> 41-10 Light chain variable region <400> 56 Val Gln Met Thr Gln Ala Pro Ser Ser Leu Pro Ala Ser Leu Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asp Tyr Leu 20 25 30 Arg Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Leu Met Ile Tyr 35 40 45 Gly Ala Thr Asn Leu Ala Ala Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser Glu 65 70 75 80 Asp Met Ala Asp Tyr Tyr Cys Leu Gln Ser Lys Glu Ser Pro Cys Ser 85 90 95 Arg Ser Val Leu Gly Pro Asn Trp Arg Ser Asn 100 105 <210> 57 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 35-9 HCDR1 <400> 57 Gly Phe Ser Leu Ser Ser Tyr Gly 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 35-9 HCDR2 <400> 58 Ile Trp Gly Asn Gly Asn Thr 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 35-9 HCDR3 <400> 59 Ala Arg Trp Glu Ser Gly Asp Tyr 1 5 <210> 60 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 35-9 LCDR1 <400> 60 Gln Ser Leu Val Gly Ser Gly Gly Lys Thr Tyr 1 5 10 <210> 61 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 35-9 LCDR2 <400> 61 Leu Val Ser 1 <210> 62 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 35-9 LCDR3 <400> 62 Leu Gln Gly Thr His Phe Pro Trp Thr 1 5 <210> 63 <211> 114 <212> PRT <213> Artificial sequence <220> <223> 35-9 Heavy chain variable region <400> 63 Gln Val Gln Leu Thr Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Val Ile Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Trp Gly Asn Gly Asn Thr Asn Tyr Asn Ser Thr Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ile Leu Gln Thr Glu Asp Thr Ala Met Tyr Phe Cys Ala 85 90 95 Arg Trp Glu Ser Gly Asp Tyr Trp Gly Gln Gly Val Thr Val Thr Val 100 105 110 Ser Ser <210> 64 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 35-9 Light chain variable region <400> 64 Asp Val Val Met Thr Gln Thr Pro Val Ser Leu Ser Val Ala Val Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Leu Val Gly Ser 20 25 30 Gly Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Ser Leu Val Ser Lys Leu Asp Ser Gly Ile Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Asp Asp Leu Gly Val Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Gln Glu Leu Lys 100 105 110 <210> 65 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 3-2 HCDR1 <400> 65 Gly Phe Thr Phe Ser Asp Tyr Gly 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 3-2 HCDR2 <400> 66 Ile Ser Ser Leu Ala Tyr Thr Val 1 5 <210> 67 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 3-2 HCDR3 <400> 67 Ala Arg Phe Pro Val Glu Ala Met Asp Tyr 1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 3-2 LCDR1 <400> 68 Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe 1 5 10 <210> 69 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 3-2 LCDR2 <400> 69 Ala Ala Ser 1 <210> 70 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 3-2 LCDR3 <400> 70 Gln Gln Ser Lys Glu Val Pro Pro Thr 1 5 <210> 71 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 3-2 Heavy chain variable region <400> 71 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Gly 20 25 30 Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ala 35 40 45 Phe Ile Ser Ser Leu Ala Tyr Thr Val Tyr Tyr Ala Asp Thr Val Thr 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Phe Leu 65 70 75 80 Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Phe Pro Val Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 72 <211> 97 <212> PRT <213> Artificial sequence <220> <223> 3-2 Light Chain Variable Region <400> 72 Ile Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp 1 5 10 15 Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln 20 25 30 Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val 35 40 45 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn 50 55 60 Ile His Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln 65 70 75 80 Ser Lys Glu Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 85 90 95 Lys <210> 73 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 11-8 HCDR1 <400> 73 Gly Tyr Ser Phe Thr Asp Tyr Ile 1 5 <210> 74 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 11-8 HCDR2 <400> 74 Ile Asn Pro Tyr Ser Gly Asn Thr 1 5 <210> 75 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 11-8 HCDR3 <400> 75 Ala Arg Asp Gly Asp Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 76 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 11-8 LCDR1 <400> 76 Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 77 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 11-8 LCDR2 <400> 77 Lys Val Ser 1 <210> 78 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 11-8 LCDR3 <400> 78 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 <210> 79 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 11-8 Heavy Chain Variable Region <400> 79 Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Ile 20 25 30 Ile Leu Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 35 40 45 Asn Ile Asn Pro Tyr Ser Gly Asn Thr Arg Tyr Asn Leu Lys Phe Lys 50 55 60 Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Asp Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 80 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 11-8 Light chain variable region <400> 80 Asp Ile Val Ile Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 81 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 16-5 HCDR1 <400> 81 Gly Phe Thr Phe Ser Asp Tyr Gly 1 5 <210> 82 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 16-5 HCDR2 <400> 82 Ile Ser Ser Leu Ala Tyr Thr Val 1 5 <210> 83 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 16-5 HCDR3 <400> 83 Ala Arg Phe Pro Val Glu Ala Met Asp Tyr 1 5 10 <210> 84 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 16-5 LCDR1 <400> 84 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 85 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 16-5 LCDR2 <400> 85 Lys Val Ser 1 <210> 86 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 16-5 LCDR3 <400> 86 Ser Gln Ser Thr His Phe Leu Thr 1 5 <210> 87 <211> 116 <212> PRT <213> Artificial sequence <220> <223> 16-5 Heavy Chain Variable Region <400> 87 Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Gly 20 25 30 Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ala 35 40 45 Phe Ile Ser Ser Leu Ala Tyr Thr Val Tyr Tyr Ala Asp Thr Val Thr 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Phe Leu 65 70 75 80 Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Phe Pro Val Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 88 <211> 111 <212> PRT <213> Artificial sequence <220> <223> 16-5 Light chain variable region <400> 88 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 89 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 19-7 HCDR1 <400> 89 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 90 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 19-7 HCDR2 <400> 90 Ile Tyr Pro Cys Asn Gly Arg Thr 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 19-7 HCDR3 <400> 91 Ala Arg Trp Asp Trp Val Phe Asp Tyr 1 5 <210> 92 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 19-7 LCDR1 <400> 92 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 93 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 19-7 LCDR2 <400> 93 Leu Val Ser 1 <210> 94 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 19-7 LCDR3 <400> 94 Gln His Ile Arg Glu Leu Thr Arg 1 5 <210> 95 <211> 116 <212> PRT <213> Artificial sequence <220> <223> 19-7 heavy chain variable region <400> 95 Gln Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Cys Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asp Trp Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 96 <211> 85 <212> PRT <213> Artificial sequence <220> <223> 19-7 Light Chain Variable Region <400> 96 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp 1 5 10 15 Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val 20 25 30 Ser Asn Pro Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 35 40 45 Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala 50 55 60 Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly 65 70 75 80 Gly Pro Ser Trp Lys 85 <210> 97 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 22-3 HCDR1 <400> 97 Gly Phe Thr Phe Ser Asp Tyr Gly 1 5 <210> 98 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 22-3 HCDR2 <400> 98 Ile Ser Ser Leu Ala Tyr Thr Val 1 5 <210> 99 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 22-3 HCDR3 <400> 99 Ala Arg Phe Pro Val Glu Ala Met Asp Tyr 1 5 10 <210> 100 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 22-3 LCDR1 <400> 100 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 101 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 22-3 LCDR2 <400> 101 Leu Val Ser 1 <210> 102 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 22-3 LCDR3 <400> 102 Gln His Ile Arg Glu Leu Thr Arg 1 5 <210> 103 <211> 116 <212> PRT <213> Artificial sequence <220> <223> 22-3 Heavy Chain Variable Region <400> 103 Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Gly 20 25 30 Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ala 35 40 45 Phe Ile Ser Ser Leu Ala Tyr Thr Val Tyr Tyr Ala Asp Thr Val Thr 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Phe Leu 65 70 75 80 Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Phe Pro Val Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 104 <211> 85 <212> PRT <213> Artificial sequence <220> <223> 22-3 Light Chain Variable Region <400> 104 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp 1 5 10 15 Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val 20 25 30 Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 35 40 45 Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala 50 55 60 Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly 65 70 75 80 Gly Pro Ser Trp Lys 85 <210> 105 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 23-1 HCDR1 <400> 105 Gly Tyr Ser Phe Thr Asp Tyr Ile 1 5 <210> 106 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 23-1 HCDR2 <400> 106 Ile Asn Pro Tyr Tyr Gly Ser Thr 1 5 <210> 107 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 23-1 HCDR3 <400> 107 Ala Arg Asp Gly Thr Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 108 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 23-1 LCDR1 <400> 108 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 109 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 23-1 LCDR2 <400> 109 Leu Val Ser 1 <210> 110 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 23-1 LCDR3 <400> 110 Gln His Ile Arg Glu Leu Thr Arg 1 5 <210> 111 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 23-1 Heavy Chain Variable Region <400> 111 Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Ile 20 25 30 Met Leu Trp Leu Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 35 40 45 Asn Ile Asn Pro Tyr Tyr Gly Ser Thr Arg Tyr Asn Leu Lys Phe Lys 50 55 60 Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Thr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 112 <211> 85 <212> PRT <213> Artificial Sequence <220> <223> 23-1 Light chain variable region <400> 112 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp 1 5 10 15 Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val 20 25 30 Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 35 40 45 Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala 50 55 60 Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly 65 70 75 80 Gly Pro Ser Trp Lys 85 <210> 113 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 8-2 HCDR1 <400> 113 Gly Tyr Thr Ile Thr Asp Tyr Tyr 1 5 <210> 114 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 8-2 HCDR2 <400> 114 Ile Asn Pro Tyr Asn Gly Gly Thr 1 5 <210> 115 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 8-2 HCDR3 <400> 115 Ala Arg Ser Arg Thr Gly Gly Asn Gly Met Asp Tyr 1 5 10 <210> 116 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 8-2 LCDR1 <400> 116 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 117 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 8-2 LCDR2 <400> 117 Leu Val Ser 1 <210> 118 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 8-2 LCDR3 <400> 118 Gln His Ile Arg Glu Leu Thr Arg 1 5 <210> 119 <211> 118 <212> PRT <213> Artificial sequence <220> <223> 8-2 Heavy Chain Variable Region <400> 119 Val Gln Leu Gln Glu Ser Gly Pro Val Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Tyr Tyr 20 25 30 Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 35 40 45 Val Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys 50 55 60 Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ser Arg Thr Gly Gly Asn Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 120 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 8-2 light chain variable region <400> 120 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys 100 105 <210> 121 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 13-1 HCDR1 <400> 121 Gly Tyr Ser Phe Thr Gly Tyr Phe 1 5 <210> 122 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 13-1 HCDR2 <400> 122 Ile Asn Pro Tyr Asn Gly Asp Thr 1 5 <210> 123 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 13-1 HCDR3 <400> 123 Ala Lys Trp Asp Leu Tyr Asp Gly Tyr Phe Arg Asp Tyr 1 5 10 <210> 124 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 13-1 LCDR1 <400> 124 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 125 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 13-1 LCDR2 <400> 125 Leu Val Ser 1 <210> 126 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 13-1 LCDR3 <400> 126 Gln His Ile Arg Glu Leu Thr Arg 1 5 <210> 127 <211> 119 <212> PRT <213> Artificial sequence <220> <223> 13-1 Heavy Chain Variable Region <400> 127 Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Phe 20 25 30 Met Asn Trp Val Met Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 35 40 45 Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe Lys 50 55 60 Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His Met 65 70 75 80 Glu Leu Arg Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Trp Asp Leu Tyr Asp Gly Tyr Phe Arg Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 128 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 13-1 light chain variable region <400> 128 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys 100 105 <210> 129 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 32-1 HCDR1 <400> 129 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 130 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 32-1 HCDR2 <400> 130 Ile Ser Ser Gly Gly Ser Tyr Thr 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 32-1 HCDR3 <400> 131 Val Ile Asp Tyr Asp Val Met Asp Tyr 1 5 <210> 132 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 32-1 LCDR1 <400> 132 Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr 1 5 10 <210> 133 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 32-1 LCDR2 <400> 133 Trp Ala Ser 1 <210> 134 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 32-1 LCDR3 <400> 134 Lys Gln Ser Tyr Asn Leu Arg Thr 1 5 <210> 135 <211> 115 <212> PRT <213> Artificial sequence <220> <223> 32-1 Heavy Chain Variable Region <400> 135 Val Gln Leu Gln Gln Ser Gly Gly Asp Leu Val Lys Pro Gly Gly Ser 1 5 10 15 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly 20 25 30 Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala 35 40 45 Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val 85 90 95 Ile Asp Tyr Asp Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser 115 <210> 136 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 32-1 Light chain variable region <400> 136 Asp Ile Val Met Thr Gln Thr Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110

Claims

1. An antibody that binds to MEFLIN, selected from the group consisting of: (4A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 consisting of the amino acid sequence described in sequence number 25, a heavy chain CDR2 consisting of the amino acid sequence described in sequence number 26, and a heavy chain CDR3 consisting of the amino acid sequence described in sequence number 27, and the light chain variable region comprises a light chain CDR1 consisting of the amino acid sequence described in sequence number 28, a light chain CDR2 consisting of the amino acid sequence described in sequence number 29, and a light chain CDR3 consisting of the amino acid sequence described in sequence number 30. (4B) An antibody having a heavy chain variable region consisting of the amino acid sequence described in sequence number 31 and a light chain variable region consisting of the amino acid sequence described in sequence number 32. (8A) An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 consisting of the amino acid sequence described in sequence number 57, a heavy chain CDR2 consisting of the amino acid sequence described in sequence number 58, and a heavy chain CDR3 consisting of the amino acid sequence described in sequence number 59, and the light chain variable region comprises a light chain CDR1 consisting of the amino acid sequence described in sequence number 60, a light chain CDR2 consisting of the amino acid sequence described in sequence number 61, and a light chain CDR3 consisting of the amino acid sequence described in sequence number 62; and (8B) An antibody having a heavy chain variable region consisting of the amino acid sequence described in sequence number 63 and a light chain variable region consisting of the amino acid sequence described in sequence number 64.

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