Anti-MEFLIN antibody for use in treating cancer in subjects with cancer, and pharmaceutical composition containing said antibody

Abstract

The present invention provides an anti-MEFLIN antibody for use in treating cancer in a subject having cancer, and a pharmaceutical composition including said antibody. The present invention provides, for example, a pharmaceutical composition for use in treating cancer that includes an antibody-drug conjugate (ADC) of an antibody that binds to MEFLIN and a cytotoxic agent.

Description

[Technical Field]

[0001] The present invention relates to an anti-MEFLIN antibody for use in treating cancer in subjects with cancer, and to a pharmaceutical composition containing the antibody. [Background technology]

[0002] MEFLIN is a membrane protein containing a leucine-rich repeat (LRR) and an immunoglobulin (Ig)-like domain, encoded by the ISLR (Immunoglobulin superfamily containing leucine-rich repeat) gene, which was obtained from a library of human genes predominantly expressed in the retina compared to the brain (Non-Patent Literature 1, 2). It is also known by the name ISLR, which is the same as the gene name (Non-Patent Literature 1). MEFLIN exists bound to the cell membrane surface by a GPI (glycosylphosphatidylinositol) anchor, but it has also been reported that it can be 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). While numerous molecules such as CD105, CD73, CD90, CD146, and CD271 are known as markers for mesenchymal stem cells, MEFLIN has been reported to be the most specific marker molecule for mesenchymal stem cells among them (Non-Patent Literature 2, Patent Literature 1).

[0004] Mesenchymal stem cells expressing MEFLIN are present in small numbers around blood vessels or in connective tissue in all organs and have the ability to differentiate into osteoblasts, chondrocytes, lipoblasts, skeletal muscle cells, myofibroblasts, and nerve cells (Non-Patent Literature 2). Some reports suggest that mesenchymal stem cells are almost identical to pericytes (vascular pericytes) and perivascular fibroblasts (Non-Patent Literature 3).

[0005] In cancer, fibroblasts derived from mesenchymal stem cells are known to proliferate around cancer cells and are called cancer-associated fibroblasts (CAFs) (Non-Patent Literature 4). Cancer-associated fibroblasts are observed in tissues of almost all cancer types, but are known to proliferate particularly significantly in refractory cancers such as pancreatic cancer, cholangiocarcinoma, breast cancer, and poorly differentiated gastrointestinal cancers (Non-Patent Literature 4, 5). MEFLIN is positive for cancer-associated fibroblasts, and this can be investigated by in situ hybridization, which detects mRNA derived from the ISLR gene, or by immunohistochemistry using antibodies (Non-Patent Literature 5). Biopsy or surgical materials from cancer patients are used for in situ hybridization and immunohistochemistry. In cancer tissue, MEFLIN is known to be specifically expressed in cancer-associated fibroblasts and not in cancer cells, vascular endothelial cells, smooth muscle cells, hematopoietic cells, and nerve cells (Non-Patent Literature 5).

[0006] There are reports that the number of MEFLIN-positive cancer-associated fibroblasts correlates with the prognosis and response rate to treatment in cancer patients (Non-Patent Document 5, Patent Document 2). Specifically, pancreatic cancer patients in whom MEFLIN-positive cancer-associated fibroblasts account for 20% or more of all cancer-associated fibroblasts show a better prognosis compared to patients in whom this figure is less than 20% (Non-Patent Document 5). Furthermore, it has been reported that patients in whom MEFLIN-positive cancer-associated fibroblasts account for less than 20% of all cancer-associated fibroblasts show a lower therapeutic effect (response rate) with immune checkpoint inhibitors (Patent Document 2).

[0007] MEFLIN-positive mesenchymal stem cells or fibroblasts are known to be important cells for tissue repair in many organs. For example, in a mouse model of myocardial infarction, a high accumulation of MEFLIN-positive cells is observed in the acute phase after myocardial infarction (Non-Patent Literature 6). MEFLIN expression in these fibroblasts is essential for myocardial repair, and cardiac rupture is observed in mice lacking the ISLR gene (Non-Patent Literature 6). There are also reports that MEFLIN-positive fibroblasts function suppressively against fibrosis and histosclerosis after tissue repair (Non-Patent Literature 6). [Prior art documents]

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

[0010] The present invention provides an anti-MEFLIN antibody for use in treating cancer in subjects having cancer, and a pharmaceutical composition containing the antibody. In the present invention, cancer may be sarcoma and MEFLIN-negative cancer.

[0011] The inventors have found that an antibody-drug conjugate consisting of an antibody that binds to the MEFLIN protein and a cytotoxic agent exhibits an antitumor effect against sarcomas. They have also found that MEFLIN-positive cells are present in the stroma of MEFLIN-negative cancers, and that an antibody-drug conjugate consisting of an antibody that binds to the MEFLIN protein and a cytotoxic agent exhibits an antitumor effect against such cancers. This invention is based on these findings.

[0012] According to the present invention, for example, the following inventions are provided. [1] A pharmaceutical composition for use in treating cancer, comprising an antibody-drug conjugate (ADC) of an antibody that binds to MEFLIN and a cytotoxic agent. [2] The antibody is the pharmaceutical composition described in [1] above, having internalizing activity. [3] The pharmaceutical composition according to [1] or [2] above, wherein the ADC is an ADC in which an antibody and a drug are linked via a linker, and the linker has a cleavage site that is cleaved in a cell. [4] An antibody that binds to MEFLIN, selected from the group consisting of the following: (1A) An antibody having a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence described in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence described in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence described in SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence described in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence described in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence described in SEQ ID NO: 6; (1B) An antibody having a heavy chain variable region having the amino acid sequence described in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence described in SEQ ID NO: 8; (1C) Antibodies that compete with the antibody in (1B) above for binding to the MEFLIN protein; and (1D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody in (1B) above; (2A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 9, heavy chain CDR2 described in SEQ ID NO: 10, and heavy chain CDR3 described in SEQ ID NO: 11, A light chain variable region including light chain CDR1 described in SEQ ID NO: 12, light chain CDR2 described in SEQ ID NO: 13, and light chain CDR3 described in SEQ ID NO: 14. Antibodies that possess; (2B) An antibody having the heavy chain variable region described in SEQ ID NO: 15 and the light chain variable region described in SEQ ID NO: 16; (2C) Antibodies that compete with the antibody in (2B) above for binding to the MEFLIN protein; and, (2D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (2B) above; (3A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 17, heavy chain CDR2 described in SEQ ID NO: 18, and heavy chain CDR3 described in SEQ ID NO: 19, A light chain variable region including light chain CDR1 described in SEQ ID NO: 20, light chain CDR2 described in SEQ ID NO: 21, and light chain CDR3 described in SEQ ID NO: 22. Antibodies that possess; (3B) An antibody having the heavy chain variable region described in SEQ ID NO: 23 and the light chain variable region described in SEQ ID NO: 24; (3C) Antibodies that compete with the antibody in (3B) above for binding to the MEFLIN protein; and (3D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (3B) above; (4A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 25, heavy chain CDR2 described in SEQ ID NO: 26, and heavy chain CDR3 described in SEQ ID NO: 27, A light chain variable region including light chain CDR1 described in SEQ ID NO: 28, light chain CDR2 described in SEQ ID NO: 29, and light chain CDR3 described in SEQ ID NO: 30. Antibodies that possess; (4B) An antibody having the heavy chain variable region described in SEQ ID NO: 31 and the light chain variable region described in SEQ ID NO: 32; (4C) Antibodies that compete with the antibody in (4B) above for binding to the MEFLIN protein; and, (4D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (4B) above; (5A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 33, heavy chain CDR2 described in SEQ ID NO: 34, and heavy chain CDR3 described in SEQ ID NO: 35, A light chain variable region including light chain CDR1 described in SEQ ID NO: 36, light chain CDR2 described in SEQ ID NO: 37, and light chain CDR3 described in SEQ ID NO: 38. Antibodies that possess; (5B) An antibody having the heavy chain variable region described in SEQ ID NO: 39 and the light chain variable region described in SEQ ID NO: 40; (5C) Antibodies that compete with the antibody in (5B) above for binding to the MEFLIN protein; and, (5D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (5B) above; (6A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 41, heavy chain CDR2 described in SEQ ID NO: 42, and heavy chain CDR3 described in SEQ ID NO: 43, A light chain variable region including light chain CDR1 described in SEQ ID NO: 44, light chain CDR2 described in SEQ ID NO: 45, and light chain CDR3 described in SEQ ID NO: 46. Antibodies that possess; (6B) An antibody having the heavy chain variable region described in SEQ ID NO: 47 and the light chain variable region described in SEQ ID NO: 48; (6C) Antibodies that compete with the antibody in (6B) above for binding to the MEFLIN protein; and, (6D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (6B) above; (7A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 49, heavy chain CDR2 described in SEQ ID NO: 50, and heavy chain CDR3 described in SEQ ID NO: 51, A light chain variable region including light chain CDR1 described in SEQ ID NO: 52, light chain CDR2 described in SEQ ID NO: 53, and light chain CDR3 described in SEQ ID NO: 54. Antibodies that possess; (7B) An antibody having the heavy chain variable region described in SEQ ID NO: 55 and the light chain variable region described in SEQ ID NO: 56; (7C) Antibodies that compete with the antibody in (7B) above for binding to the MEFLIN protein; and, (7D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (7B) above; (8A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 57, heavy chain CDR2 described in SEQ ID NO: 58, and heavy chain CDR3 described in SEQ ID NO: 59, A light chain variable region including light chain CDR1 described in SEQ ID NO: 60, light chain CDR2 described in SEQ ID NO: 61, and light chain CDR3 described in SEQ ID NO: 62. Antibodies that possess; (8B) An antibody having the heavy chain variable region described in SEQ ID NO: 63 and the light chain variable region described in SEQ ID NO: 64; (8C) Antibodies that compete with the antibody in (8B) above for binding to the MEFLIN protein; and, (8D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (8B) above; (9A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 65, heavy chain CDR2 described in SEQ ID NO: 66, and heavy chain CDR3 described in SEQ ID NO: 67, A light chain variable region including light chain CDR1 described in SEQ ID NO: 68, light chain CDR2 described in SEQ ID NO: 69, and light chain CDR3 described in SEQ ID NO: 70. Antibodies that possess; (9B) An antibody having the heavy chain variable region described in SEQ ID NO: 71 and the light chain variable region described in SEQ ID NO: 72; (9C) Antibodies that compete with the antibody in (9B) above for binding to the MEFLIN protein; And, (9D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (9B) above; (10A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 73, heavy chain CDR2 described in SEQ ID NO: 74, and heavy chain CDR3 described in SEQ ID NO: 75, A light chain variable region including light chain CDR1 described in Sequence ID No. 76, light chain CDR2 described in Sequence ID No. 77, and light chain CDR3 described in Sequence ID No. 78. Antibodies that possess; (10B) An antibody having the heavy chain variable region described in SEQ ID NO: 79 and the light chain variable region described in SEQ ID NO: 80; (10C) Antibodies that compete with the antibody in (10B) above for binding to the MEFLIN protein; and, (10D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (10B) above; (11A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 81, heavy chain CDR2 described in SEQ ID NO: 82, and heavy chain CDR3 described in SEQ ID NO: 83, A light chain variable region including light chain CDR1 described in Sequence ID No. 84, light chain CDR2 described in Sequence ID No. 85, and light chain CDR3 described in Sequence ID No. 86. Antibodies that possess; (11B) An antibody having the heavy chain variable region described in SEQ ID NO: 87 and the light chain variable region described in SEQ ID NO: 88; (11C) Antibodies that compete with the antibody in (11B) above for binding to the MEFLIN protein; and, (11D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (11B) above; (12A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 89, heavy chain CDR2 described in SEQ ID NO: 90, and heavy chain CDR3 described in SEQ ID NO: 91, A light chain variable region including light chain CDR1 described in SEQ ID NO: 92, light chain CDR2 described in SEQ ID NO: 93, and light chain CDR3 described in SEQ ID NO: 94. Antibodies that possess; (12B) An antibody having the heavy chain variable region described in SEQ ID NO: 95 and the light chain variable region described in SEQ ID NO: 96; (12C) Antibodies that compete with the antibody in (12B) above for binding to the MEFLIN protein; and, (12D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (12B) above; (13A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 97, heavy chain CDR2 described in SEQ ID NO: 98, and heavy chain CDR3 described in SEQ ID NO: 99, A light chain variable region including light chain CDR1 described in SEQ ID NO: 100, light chain CDR2 described in SEQ ID NO: 101, and light chain CDR3 described in SEQ ID NO: 102. Antibodies that possess; (13B) An antibody having the heavy chain variable region described in SEQ ID NO: 103 and the light chain variable region described in SEQ ID NO: 104; (13C) Antibodies that compete with the antibody in (13B) above for binding to the MEFLIN protein; and, (13D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (13B) above; (14A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 105, heavy chain CDR2 described in SEQ ID NO: 106, and heavy chain CDR3 described in SEQ ID NO: 107, Light chain CDR1 described in Sequence ID No. 108, light chain CDR2 described in Sequence ID No. 109, and Light chain variable region including light chain CDR3 described in column number 110 and Antibodies that possess; (14B) An antibody having the heavy chain variable region described in SEQ ID NO: 111 and the light chain variable region described in SEQ ID NO: 112; (14C) Antibodies that compete with the antibody in (14B) above for binding to the MEFLIN protein; and, (14D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (14B) above; (15A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 113, heavy chain CDR2 described in SEQ ID NO: 114, and heavy chain CDR3 described in SEQ ID NO: 115, A light chain variable region including light chain CDR1 described in SEQ ID NO: 116, light chain CDR2 described in SEQ ID NO: 117, and light chain CDR3 described in SEQ ID NO: 118. Antibodies that possess; (15B) An antibody having the heavy chain variable region described in SEQ ID NO: 119 and the light chain variable region described in SEQ ID NO: 120; (15C) Antibodies that compete with the antibody in (15B) above for binding to the MEFLIN protein; and (15D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (15B) above; (16A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 121, heavy chain CDR2 described in SEQ ID NO: 122, and heavy chain CDR3 described in SEQ ID NO: 123, A light chain variable region including light chain CDR1 described in SEQ ID NO: 124, light chain CDR2 described in SEQ ID NO: 125, and light chain CDR3 described in SEQ ID NO: 126. Antibodies that possess; (16B) An antibody having the heavy chain variable region described in SEQ ID NO: 127 and the light chain variable region described in SEQ ID NO: 128; (16C) Antibodies that compete with the antibody in (16B) above for binding to the MEFLIN protein; and (16D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (16B) above; and (17A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 129, heavy chain CDR2 described in SEQ ID NO: 130, and heavy chain CDR3 described in SEQ ID NO: 131, A light chain variable region including light chain CDR1 described in SEQ ID NO: 132, light chain CDR2 described in SEQ ID NO: 133, and light chain CDR3 described in SEQ ID NO: 134. Antibodies that possess; (17B) An antibody having the heavy chain variable region described in SEQ ID NO: 135 and the light chain variable region described in SEQ ID NO: 136; (17C) Antibodies that compete with the antibody in (17B) above for binding to the MEFLIN protein; and (17D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (17B) above. [5] A pharmaceutical composition comprising an antibody-drug conjugate (ADC) of the antibody described in [4] above and a cytotoxic agent. [6] The pharmaceutical composition described in [5] above for use in treating cancer. [7] The pharmaceutical composition according to any one of [1] to [3] and [6] above, wherein the cancer is a sarcoma. [8] The pharmaceutical composition according to [7] above, wherein the cancer is a MEFLIN-positive sarcoma. [9] The pharmaceutical composition according to [7] or [8] above, wherein the sarcoma is a sarcoma selected from the group consisting of mucofibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, malignant peripheral nerve sheath tumor, Ewing's sarcoma, epithelioid sarcoma, clear cell sarcoma, synovial sarcoma, and osteosarcoma.

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

[11] The pharmaceutical composition according to

[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.

[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.

[13] A pharmaceutical composition according to any one of

[10] to

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

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

[15] The pharmaceutical composition according to

[13] above, wherein the ADC is an ADC in which an antibody and a drug are linked via a linker, and the linker is a cleavable linker.

[16] The pharmaceutical composition according to

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

[17] The pharmaceutical composition according to

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

[18] The pharmaceutical composition according to any of the above, wherein the linker is a non-cleavable linker. [Brief explanation of the drawing]

[0013] [Figure 1]Figure 1 shows the results of Western blotting demonstrating the binding affinity between human MEFLIN protein and monoclonal antibodies obtained from various clones. In Figure 1, the clone name and information about the MEFLIN fragment (the region of the fragment is indicated by the amino acid number) are shown. [Figure 2] Figure 2 shows the results of Western blotting demonstrating the binding affinity between human MEFLIN protein and monoclonal antibodies obtained from various clones. In Figure 2, the clone name and information about the MEFLIN fragment (the region of the fragment is indicated by the amino acid number) are shown. [Figure 3] Figure 3 shows fluorescence microscopy images illustrating the internalization activity of monoclonal antibodies obtained from various clones. Cell nuclei were stained with DAPI, and antibodies were detected using Alexa488-labeled antibodies. [Figure 4] Figure 4 shows fluorescence microscopy images illustrating the internalization activity of monoclonal antibodies obtained from various clones. Cell nuclei were stained with DAPI, and antibodies were detected using Alexa488-labeled antibodies. [Figure 5] Figure 5 shows fluorescence microscopy images illustrating the internalization activity of monoclonal antibodies obtained from various clones. Cell nuclei were stained with DAPI, and antibodies were detected using Alexa488-labeled antibodies. [Figure 6] Figure 6 shows the in vitro cell proliferation inhibitory activity of the prepared antibody-drug conjugate (ADC) against human MEFLIN protein overexpressing cells (HEK293 cells). Figure 6 also shows the FACS analysis results for human MEFLIN protein overexpressing cells (HEK293 cells) treated with anti-MEFLIN monoclonal antibody and untreated (negative control). The horizontal axis represents the expression level of MEFLIN protein, and the vertical axis represents the cell frequency. [Figure 7]Figure 7 shows the in vitro cell proliferation inhibitory activity of ADC against the human rhabdomyosarcoma cell line (KYM-1 cells). Figure 7 also shows the FACS analysis results for the human rhabdomyosarcoma cell line (KYM-1 cells) used, comparing the anti-MEFLIN monoclonal antibody-treated group with the untreated group (negative control). The horizontal axis represents the expression level of MEFLIN protein, and the vertical axis represents the cell frequency. [Figure 8] Figure 8 shows the in vivo antitumor effect of ADCs on tumor-bearing mouse models subcutaneously transplanted with the rhabdomyosarcoma cell line (KYM-1). Arrows in the graph indicate the timing of administration. Figure 8 also shows the expression of human MEFLIN in the transplanted rhabdomyosarcoma tissue. [Figure 9] Figure 9 shows the in vivo antitumor effect of ADCs in tumor-bearing mouse models subcutaneously transplanted with osteosarcoma cell line (HsOs1). Arrows in the graph indicate the timing of administration. Figure 9 also shows the expression of human MEFLIN in the transplanted osteosarcoma tissue. [Figure 10] Figure 10 shows the in vivo antitumor effect of ADC in tumor-bearing mouse models subcutaneously transplanted with a pancreatic cancer cell line (BxPC-3). Arrows in the graph indicate the timing of administration. Figure 10 also shows mouse MEFLIN expression in the tissue and stroma of the transplanted pancreatic cancer. [Figure 11] Figure 11 shows the in vivo antitumor effect of ADC in tumor-bearing mouse models subcutaneously transplanted with lung cancer cell line (A549). Arrows in the graph indicate the timing of administration. Figure 11 also shows mouse MEFLIN expression in the tissue and stroma of the transplanted lung cancer. [Figure 12] Figure 12 shows the in vivo antitumor effect of ADC in tumor-bearing mouse models subcutaneously transplanted with neuroblastoma cell line (NB-1). Arrows in the graph indicate the timing of administration. Figure 12 also shows the expression of human MEFLIN in transplanted neuroblastoma tissue. [Figure 13]Figure 13 shows the in vivo antitumor effect of ADCs in tumor-bearing mouse models subcutaneously transplanted with a colorectal cancer cell line (DLD-1). Arrows in the graph indicate the timing of administration. Figure 13 also shows mouse MEFLIN expression in the tissue and stroma of the transplanted colorectal cancer. [Figure 14] Figure 14 shows the in vivo antitumor effect of ADC in tumor-bearing mouse models subcutaneously transplanted with gastric cancer cell line (MKN45). Arrows in the graph indicate the timing of administration. Figure 14 also shows mouse MEFLIN expression in the tissue and stroma of the transplanted gastric cancer. [Figure 15] Figure 15 shows the results of analyzing single-cell RNA sequencing data (Tabula Muris) from mouse pancreas deposited on the internet. It shows that the MEFLIN-positive cell population and the cathepsin K-positive cell population in mouse pancreas are identical (arrow). [Figure 16] Figure 16 shows the results of analyzing single-cell RNA sequencing data (Tabula Muris) from mouse lungs deposited on the internet. It shows that the MEFLIN-positive cell population and the cathepsin K-positive cell population in mouse lungs are identical (arrow). [Figure 17] Figure 17 shows the expression of cathepsin K in an exogenous mouse MEFLIN-expressing CHO cell line. [Figure 18] Figure 18 shows the results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody on tumor tissue from tumor-bearing mouse models subcutaneously transplanted with osteosarcoma cell line (HsOs1). The arrows indicate the secretion of cathepsin K (red) around MEFLIN-positive cells (green). [Figure 19] Figure 19 shows the results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody on tumor tissue from tumor-bearing mouse models transplanted with pancreatic cancer cell line (BxPC-3). The arrows indicate that cathepsin K (red) is secreted around MEFLIN-positive cells (green). [Figure 20]Figure 20 shows the in vivo antitumor effect of ADC on tumor tissue in tumor-bearing mouse models subcutaneously transplanted with the cholangiocarcinoma cell line (HuCCT1). Arrows in the graph indicate the timing of administration. Figure 20 also shows mouse MEFLIN expression in the transplanted cholangiocarcinoma tissue and stroma. [Figure 21] Figure 21 shows the in vivo antitumor effect of ADC on tumor tissue in tumor-bearing mouse models subcutaneously transplanted with bladder cancer cell line (T24). Arrows in the graph indicate the timing of administration. Figure 21 also shows mouse MEFLIN expression in the transplanted bladder cancer tissue and stroma. [Figure 22] Figure 22 shows the in vivo antitumor effect of ADC on tumor tissue in tumor-bearing mouse models subcutaneously transplanted with ovarian cancer cell line (OV-90). Arrows in the graph indicate the timing of administration. Figure 22 also shows mouse MEFLIN expression in the transplanted ovarian cancer tissue and stroma. [Figure 23] Figure 23 shows the in vivo antitumor effect of ADC on tumor tissue in tumor-bearing mouse models subcutaneously transplanted with esophageal cancer cell line (KYSE). Arrows in the graph indicate the timing of administration. Figure 23 also shows mouse MEFLIN expression in the transplanted esophageal cancer tissue and stroma. [Figure 24] Figure 24 shows the in vivo antitumor effect of ADC on tumor tissue in tumor-bearing mouse models subcutaneously transplanted with the osteosarcoma cell line (HsOs1). The arrows in the graph indicate the timing of administration. [Figure 25] Figure 25 shows the in vivo antitumor effect of ADC on tumor tissue in tumor-bearing mouse models subcutaneously transplanted with the breast cancer cell line (MCF7). Arrows in the graph indicate the timing of administration. Figure 25 also shows mouse MEFLIN expression in the transplanted breast cancer tissue and stroma. [Figure 26] Figure 26 shows the results of immunohistochemistry (IHC) staining using anti-MEFLIN antibody and in situ hybridization (ISH) staining of tissue sections from human pancreatic cancer surgical specimens. The arrows in the figure indicate the same site in the upper and lower figures. Detailed description of the invention

[0014] In this invention, "subject" means mammal, and in particular may be human.

[0015] In this specification, “treatment” includes both therapeutic treatment and preventive treatment. In this specification, “treatment” means the treatment, cure, prevention, or improvement of remission of a disease or disorder, or the reduction of the rate of progression of a disease or disorder. In this specification, “prevention” means reducing the likelihood of developing a disease or condition, or delaying the onset of a disease or condition.

[0016] In this specification, “disease” means a condition for which treatment is beneficial. In this specification, “cancer” means a malignant tumor.

[0017] In this specification, "antibody" means immunoglobulin and includes polyclonal antibodies and monoclonal antibodies. A preferred antibody is a monoclonal antibody. The origin of the antibody is not particularly limited, but examples include antibodies from non-human animals, antibodies from non-human mammals, and human antibodies. The antibody may also be a chimeric antibody, a humanized antibody, or a human antibody. Furthermore, the antibody may be a bispecific antibody. Antibodies used as pharmaceuticals are preferably chimeric antibodies, more preferably humanized antibodies, and even more preferably human antibodies. Bispecific antibodies are monoclonal antibodies, preferably chimeric antibodies, more preferably humanized antibodies, and even more preferably human antibodies.

[0018] In this specification, “therapeutically effective dose” means the amount of drug that is effective in treating (preventing or curing) a disease or condition. A therapeutically effective dose of drug can slow the rate of progression of symptoms of a disease or condition, halt the progression of such symptoms, improve such symptoms, cure such symptoms, or suppress the onset or development of such symptoms.

[0019] In this specification, "competition" means that two antibodies compete with each other for binding to an antigen. Competition can occur when the binding sites of two antibodies overlap for a given antigen. Such antibodies can be obtained by immunization using an epitope as described above, and / or by a competition assay to determine whether the binding of one antibody to an antigen is reduced by the other antibody.

[0020] In this specification, “antibody-drug conjugate” (hereinafter also referred to as “ADC”) means a substance in which an antibody and a cytotoxic agent are linked. In an ADC, the antibody and the cytotoxic agent can be linked via an appropriate linker. As the cytotoxic agent, chemotherapeutic agents, radioisotopes, and toxins can be used. ADCs also include conjugates of an antigen-binding fragment of an antibody and a drug.

[0021] In this specification, “antigen-binding fragment” means a portion of an antibody whose ability to bind to an antigen is maintained. The antigen-binding fragment may include the heavy chain variable region, the light chain variable region, or both of the antibody of the present invention. 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), diabody, and sc(Fv)2 (single-chain (Fv)2). Such antibody fragments are not particularly limited, but can be obtained, for example, by treating the antibody with an enzyme. For example, digesting the antibody with papain can yield Fab. Alternatively, digesting the antibody with pepsin can yield F(ab')2, which can be further reduced to obtain Fab'. Such antibody antigen-binding fragments can be used in the present invention.

[0022] In this specification, "MEFLIN" or "Meflin" refers to a protein also known as the leucine-rich repeat-containing immunoglobulin superfamily (ISLR). Human MEFLIN may have an amino acid sequence registered under GenBank accession number BAA85970.1. MEFLIN may be MEFLIN (for example, human MEFLIN) having an amino acid sequence corresponding to the amino acid sequence registered under GenBank accession number BAA85970.1. If the animal species of origin is to be specified, it will be written as human MEFLIN (or hMEFLIN) and mouse MEFLIN (mMEFLIN), etc.

[0023] In the present invention, in an antibody-drug conjugate (ADC), an antibody and a cytotoxic agent are linked via a linker. Examples of the cytotoxic agent include chemotherapeutic agents (e.g., anticancer agents such as commercially available anticancer drugs, e.g., auristatins (auristatin E, auristatin F phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin F and their derivatives), maytansinoids DM1 and DM4 and their derivatives), camptothecins (SN-38, irinotecan, rutotecan, DB67, BMP1350, ST1481, CKD602, topotecan and exatecan, and their derivatives), DNA minor groove binders (engein, lexitropsin, duocarmycin and their derivatives), taxanes (paclitaxel and docetaxel and their derivatives), polyketides (discodermolide and its derivatives), anthraquinones (mitoxantrone and its derivatives), benzodiazepines (pyrrolobenzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), cardiac glycosides (digitoxin and its derivatives), calicheamicin, epothilone, cryptophycin, semadotin, semadotin, lysocine, netropsin, combretastatin, erythrobine, etoposide, T67 (tularik), and nocodazole), radioisotopes (e.g., 32 P, 60 C, 90 Y, 111 In, 131 I, 125 I, 153 Sm, 186 Re, 188 Re, and 212Examples of cytotoxic agents include Bi), and toxins (e.g., diphtheria toxin A, pseudomonas endotoxin, lysine, saporin, etc.), which can be used as cytotoxic agents in the ADC of the present invention. Preferably, the cytotoxic agent in the ADC of the present invention is, for example, camptothecin, particularly SN-38, or exatecan. Any cytotoxic agent used in the treatment of cancer can be used. As the cytotoxic agent, a pharmaceutically acceptable salt, solvate (e.g., hydrate), ester, or prodrug of the above cytotoxic agent may be used.

[0024] In the present invention, the linker of the ADC may be a non-cleavable linker or a cleavable linker. Such linkers can be appropriately selected and synthesized by those skilled in the art in the preparation of ADCs. Examples of cleavable linkers include linkers having degradable bonds such as ester bonds. Examples of cleavable linkers include linkers having protease cleavage sites such as cleavable regions of peptides consisting of valine-citrulline or valine-alanine. Peptide regions consisting of valine-citrulline can be cleaved by proteases such as cathepsin B. In some embodiments, the linker may have a first spacer introduced between the antibody and the cleavable region, for example, polyethylene glycol (PEG), for example, PEG with about 5 to 40 repeating units per molecule can be used as the first spacer. A second spacer may be introduced between the cleavable region and the cytotoxic agent, for example, p-aminobenzyloxycarbonyl (PABC) can be used as the second spacer. Fracturing linkers are physiologically stable in cancerous tissue except at the site of cleavage (in particular, they are physiologically stable until they reach the cancerous tissue). In some embodiments, the linker includes a first spacer and a cleavable portion. In some embodiments, the linker includes a first spacer, a cleavable portion, and a second spacer. In certain subjects, the linker includes a PEG, a cleavable portion, and a PABC. The binding of the antibody to the linker can be achieved, for example, by linking the sulfhydryl group of the antibody via a maleimide group. In one embodiment, the antibody is linked to the anticancer drug via its sulfhydryl group by a linker having a maleimide-PEG-cleavable moiety. In another embodiment, the antibody is linked to the anticancer drug via its sulfhydryl group by a linker having a maleimide-PEG-cleavable moiety-PABC. In one embodiment, the ADC may have the structure shown in the following equation (II). The ADC of the present invention is considered useful as a cancer treatment drug.

[0025] According to the present invention, an antibody is provided having the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of an antibody produced from a clone selected from the following. The antibody is preferably a human chimeric antibody, and more preferably a humanized antibody. In this embodiment, according to the present invention, an antibody is provided having the amino acid sequences of the heavy chain variable region and the light chain variable region of an antibody produced from a clone selected from the following. The antibody is preferably a human chimeric antibody.

[0026] [Table 1] [Table 2] [Table 3] [Table 4]

[0027] [Table 5]

[0028] [Table 6]

[0029] [Table 7] TIFF0007863808000008.tif110170

[0030] [Table 8] TIFF0007863808000010.tif195170

[0031] According to the present invention, an antibody that binds to the MEFLIN protein, (1A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 1, heavy chain CDR2 described in SEQ ID NO: 2, and heavy chain CDR3 described in SEQ ID NO: 3, A light chain variable region including light chain CDR1 described in Sequence ID No. 4, light chain CDR2 described in Sequence ID No. 5, and light chain CDR3 described in Sequence ID No. 4 Antibodies that possess; (1B) An antibody having the heavy chain variable region described in SEQ ID NO: 7 and the light chain variable region described in SEQ ID NO: 8; (1C) Antibodies that compete with the antibody in (1B) above for binding to the MEFLIN protein; and (1D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (1B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0032] According to the present invention, an antibody that binds to the MEFLIN protein, (2A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 9, heavy chain CDR2 described in SEQ ID NO: 10, and heavy chain CDR3 described in SEQ ID NO: 11, A light chain variable region including light chain CDR1 described in SEQ ID NO: 12, light chain CDR2 described in SEQ ID NO: 13, and light chain CDR3 described in SEQ ID NO: 14. Antibodies that possess; (2B) An antibody having the heavy chain variable region described in SEQ ID NO: 15 and the light chain variable region described in SEQ ID NO: 16; (2C) Antibodies that compete with the antibody in (2B) above for binding to the MEFLIN protein; and (2D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (2B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0033] According to the present invention, an antibody that binds to the MEFLIN protein, (3A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 17, heavy chain CDR2 described in SEQ ID NO: 18, and heavy chain CDR3 described in SEQ ID NO: 19, A light chain variable region including light chain CDR1 described in SEQ ID NO: 20, light chain CDR2 described in SEQ ID NO: 21, and light chain CDR3 described in SEQ ID NO: 22. Antibodies that possess; (3B) An antibody having the heavy chain variable region described in SEQ ID NO: 23 and the light chain variable region described in SEQ ID NO: 24; (3C) Antibodies that compete with the antibody in (3B) above for binding to the MEFLIN protein; and (3D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (3B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0034] According to the present invention, an antibody that binds to the MEFLIN protein, (4A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 25, heavy chain CDR2 described in SEQ ID NO: 26, and heavy chain CDR3 described in SEQ ID NO: 27, A light chain variable region including light chain CDR1 described in SEQ ID NO: 28, light chain CDR2 described in SEQ ID NO: 29, and light chain CDR3 described in SEQ ID NO: 30. Antibodies that possess; (4B) An antibody having the heavy chain variable region described in SEQ ID NO: 31 and the light chain variable region described in SEQ ID NO: 32; (4C) Antibodies that compete with the antibody in (4B) above for binding to the MEFLIN protein; and (4D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (4B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0035] According to the present invention, an antibody that binds to the MEFLIN protein, (5A) Heavy chain CDR1 described in Sequence ID No. 33 and heavy chain CDR2 described in Sequence ID No. 34, A heavy chain variable region including the heavy chain CDR3 described in Sequence ID No. 35, A light chain variable region including light chain CDR1 described in SEQ ID NO: 36, light chain CDR2 described in SEQ ID NO: 37, and light chain CDR3 described in SEQ ID NO: 38. Antibodies that possess; (5B) An antibody having the heavy chain variable region described in SEQ ID NO: 39 and the light chain variable region described in SEQ ID NO: 40; (5C) Antibodies that compete with the antibody in (5B) above for binding to the MEFLIN protein; and (5D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (5B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0036] According to the present invention, an antibody that binds to the MEFLIN protein, (6A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 41, heavy chain CDR2 described in SEQ ID NO: 42, and heavy chain CDR3 described in SEQ ID NO: 43, A light chain variable region including light chain CDR1 described in SEQ ID NO: 44, light chain CDR2 described in SEQ ID NO: 45, and light chain CDR3 described in SEQ ID NO: 46. Antibodies that possess; (6B) An antibody having the heavy chain variable region described in SEQ ID NO: 47 and the light chain variable region described in SEQ ID NO: 48; (6C) Antibodies that compete with the antibody in (6B) above for binding to the MEFLIN protein; and (6D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (6B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0037] According to the present invention, an antibody that binds to the MEFLIN protein, (7A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 49, heavy chain CDR2 described in SEQ ID NO: 50, and heavy chain CDR3 described in SEQ ID NO: 51, A light chain variable region including light chain CDR1 described in SEQ ID NO: 52, light chain CDR2 described in SEQ ID NO: 53, and light chain CDR3 described in SEQ ID NO: 54. Antibodies that possess; (7B) An antibody having the heavy chain variable region described in SEQ ID NO: 55 and the light chain variable region described in SEQ ID NO: 56; (7C) Antibodies that compete with the antibody in (7B) above for binding to the MEFLIN protein; and (7D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (7B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0038] According to the present invention, an antibody that binds to the MEFLIN protein, (8A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 57, heavy chain CDR2 described in SEQ ID NO: 58, and heavy chain CDR3 described in SEQ ID NO: 59, A light chain variable region including light chain CDR1 described in SEQ ID NO: 60, light chain CDR2 described in SEQ ID NO: 61, and light chain CDR3 described in SEQ ID NO: 62. Antibodies that possess; (8B) An antibody having the heavy chain variable region described in SEQ ID NO: 63 and the light chain variable region described in SEQ ID NO: 64; (8C) Antibodies that compete with the antibody in (8B) above for binding to the MEFLIN protein; and (8D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (8B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0039] According to the present invention, an antibody that binds to the MEFLIN protein, (9A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 65, heavy chain CDR2 described in SEQ ID NO: 66, and heavy chain CDR3 described in SEQ ID NO: 67, A light chain variable region including light chain CDR1 described in SEQ ID NO: 68, light chain CDR2 described in SEQ ID NO: 69, and light chain CDR3 described in SEQ ID NO: 70. Antibodies that possess; (9B) An antibody having the heavy chain variable region described in SEQ ID NO: 71 and the light chain variable region described in SEQ ID NO: 72; (9C) Antibodies that compete with the antibody in (9B) above for binding to the MEFLIN protein; and (9D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (9B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0040] According to the present invention, an antibody that binds to the MEFLIN protein, (10A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 73, heavy chain CDR2 described in SEQ ID NO: 74, and heavy chain CDR3 described in SEQ ID NO: 75, A light chain variable region including light chain CDR1 described in Sequence ID No. 76, light chain CDR2 described in Sequence ID No. 77, and light chain CDR3 described in Sequence ID No. 78. Antibodies that possess; (10B) An antibody having the heavy chain variable region described in SEQ ID NO: 79 and the light chain variable region described in SEQ ID NO: 80; (10C) Antibodies that compete with the antibody in (10B) above for binding to the MEFLIN protein; and (10D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (10B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0041] According to the present invention, an antibody that binds to the MEFLIN protein, (11A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 81, heavy chain CDR2 described in SEQ ID NO: 82, and heavy chain CDR3 described in SEQ ID NO: 83, A light chain variable region including light chain CDR1 described in Sequence ID No. 84, light chain CDR2 described in Sequence ID No. 85, and light chain CDR3 described in Sequence ID No. 86. Antibodies that possess; (11B) An antibody having the heavy chain variable region described in SEQ ID NO: 87 and the light chain variable region described in SEQ ID NO: 88; (11C) Antibodies that compete with the antibody in (11B) above for binding to the MEFLIN protein; and (11D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (11B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0042] According to the present invention, an antibody that binds to the MEFLIN protein, (12A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 89, heavy chain CDR2 described in SEQ ID NO: 90, and heavy chain CDR3 described in SEQ ID NO: 91, A light chain variable region including light chain CDR1 described in SEQ ID NO: 92, light chain CDR2 described in SEQ ID NO: 93, and light chain CDR3 described in SEQ ID NO: 94. Antibodies that possess; (12B) An antibody having the heavy chain variable region described in SEQ ID NO: 95 and the light chain variable region described in SEQ ID NO: 96; (12C) Antibodies that compete with the antibody in (12B) above for binding to the MEFLIN protein; and (12D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (12B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0043] According to the present invention, an antibody that binds to the MEFLIN protein, (13A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 97, heavy chain CDR2 described in SEQ ID NO: 98, and heavy chain CDR3 described in SEQ ID NO: 99, A light chain variable region including light chain CDR1 described in SEQ ID NO: 100, light chain CDR2 described in SEQ ID NO: 101, and light chain CDR3 described in SEQ ID NO: 102. Antibodies that possess; (13B) An antibody having the heavy chain variable region described in SEQ ID NO: 103 and the light chain variable region described in SEQ ID NO: 104; (13C) Antibodies that compete with the antibody in (13B) above for binding to the MEFLIN protein; and (13D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (13B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0044] According to the present invention, an antibody that binds to the MEFLIN protein, (14A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 105, heavy chain CDR2 described in SEQ ID NO: 106, and heavy chain CDR3 described in SEQ ID NO: 107, A light chain variable region including light chain CDR1 described in SEQ ID NO: 108, light chain CDR2 described in SEQ ID NO: 109, and light chain CDR3 described in SEQ ID NO: 110. Antibodies that possess; (14B) An antibody having the heavy chain variable region described in SEQ ID NO: 111 and the light chain variable region described in SEQ ID NO: 112; (14C) Antibodies that compete with the antibody in (14B) above for binding to the MEFLIN protein; and (14D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (14B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0045] According to the present invention, an antibody that binds to the MEFLIN protein, (15A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 113, heavy chain CDR2 described in SEQ ID NO: 114, and heavy chain CDR3 described in SEQ ID NO: 115, A light chain variable region including light chain CDR1 described in SEQ ID NO: 116, light chain CDR2 described in SEQ ID NO: 117, and light chain CDR3 described in SEQ ID NO: 118. Antibodies that possess; (15B) An antibody having the heavy chain variable region described in SEQ ID NO: 119 and the light chain variable region described in SEQ ID NO: 120; (15C) Antibodies that compete with the antibody in (15B) above for binding to the MEFLIN protein; and (15D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (15B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0046] According to the present invention, an antibody that binds to the MEFLIN protein, (16A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 121, heavy chain CDR2 described in SEQ ID NO: 122, and heavy chain CDR3 described in SEQ ID NO: 123, A light chain variable region including light chain CDR1 described in SEQ ID NO: 124, light chain CDR2 described in SEQ ID NO: 125, and light chain CDR3 described in SEQ ID NO: 126. Antibodies that possess; (16B) An antibody having the heavy chain variable region described in SEQ ID NO: 127 and the light chain variable region described in SEQ ID NO: 128; (16C) Antibodies that compete with the antibody in (16B) above for binding to the MEFLIN protein; and (16D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (16B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0047] According to the present invention, an antibody that binds to the MEFLIN protein, (17A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 129, heavy chain CDR2 described in SEQ ID NO: 130, and heavy chain CDR3 described in SEQ ID NO: 131, A light chain variable region including light chain CDR1 described in SEQ ID NO: 132, light chain CDR2 described in SEQ ID NO: 133, and light chain CDR3 described in SEQ ID NO: 134. Antibodies that possess; (17B) An antibody having the heavy chain variable region described in SEQ ID NO: 135 and the light chain variable region described in SEQ ID NO: 136; (17C) Antibodies that compete with the antibody in (17B) above for binding to the MEFLIN protein; and (17D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (17B) above. The antibody is provided. The antibody is preferably a human chimeric antibody, and preferably a humanized antibody.

[0048] In one aspect of the present invention, the antibody that binds to the MEFLIN protein binds to the human MEFLIN protein. In one aspect of the present invention, the antibody that binds to the MEFLIN protein specifically binds to the human MEFLIN protein. "Specifically binds" means that it binds to the MEFLIN protein with a stronger affinity than it binds to at least one other protein. "Specifically binds" includes binding to the human MEFLIN protein with a stronger affinity than it binds to the mouse MEFLIN protein.

[0049] Whether antibodies compete with each other can be confirmed by an in vitro competition assay. If the competition assay can block the binding of the desired antibody by, for example, at least 20%, preferably at least 20-50%, and more preferably at least 50%, then the antibody can be considered to compete for binding to the same antigen. Competing antibodies can be confirmed by a cross-blocking assay, preferably a competitive ELISA assay. In a cross-blocking assay, the antigen is coated, for example, on a microtiter plate, and the presence of a candidate competing antibody is added and incubated to form a bond between the antigen and the candidate antibody. Then, the desired antibody is labeled and added to the wells for further incubation, followed by washing. By quantifying the amount of the desired antibody bound, it can be determined whether the antibodies competed. If there is competition, the amount of labeled antibody remaining in the well should be small.

[0050] Whether antibodies bind to overlapping epitopes on their binding partner proteins can be determined by whether their interaction surfaces overlap. For example, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a known method for determining epitopes. HDX-MS can detect the exchange of hydrogen to deuterium in amide protons of protein complexes in the presence of heavy water. In the presence of heavy water, amide protons on the interaction surfaces of protein complexes are less likely to be exchanged for deuterium (or the exchange rate is slow), while amide protons in areas exposed to the surface are more likely to be exchanged for deuterium (or the exchange rate is fast). Therefore, by utilizing this phenomenon, regions where a decrease in the rate of hydrogen to deuterium exchange is observed can be considered as regions that bind to antibodies, thereby determining the interaction surface (epitope region) on the antibody's binding partner. By determining the epitope regions of two antibodies using the above method, it is possible to determine whether the epitope regions overlap. The exchange from hydrogen to deuterium can be detected by mass spectrometry by those skilled in the art.

[0051] In some embodiments of the present invention, the antibody that binds to the MEFLIN protein is an antibody that has intracellular internalization activity. In some embodiments of the present invention, the antibody that binds to the MEFLIN protein is an antibody that does not have intracellular internalization activity. In some embodiments of the present invention, cancer cells are MEFLIN protein positive, and the antibody that binds to the MEFLIN protein is an antibody that has intracellular internalization activity. In some embodiments of the present invention, cancer cells are MEFLIN protein positive, and the antibody that binds to the MEFLIN protein is an antibody that does not have intracellular internalization activity. In some embodiments of the present invention, cells in the cancer stroma are MEFLIN protein positive, and the antibody that binds to the MEFLIN protein is an antibody that has intracellular internalization activity. In some embodiments of the present invention, cells in the cancer stroma are MEFLIN protein positive, and the antibody that binds to the MEFLIN protein is an antibody that does not have intracellular internalization activity. In these embodiments, the ADC may have a cleavage linker. In these embodiments, the ADC may have a non-cleavage linker. If the ADC payload is prodrug-formulated so that it exhibits cytotoxicity only after linker cleavage, the linker is preferably a cleavable linker. In one embodiment, the ADC of the present invention has internalizing activity and a cleavable linker that cleaves inside the cell. In another embodiment, the ADC of the present invention does not have internalizing activity and has a cleavable linker that cleaves outside the cell.

[0052] Whether an antibody has internalizing activity can be confirmed by in vitro testing. For example, an antibody that binds to the MEFLIN protein is brought into contact with cells having the MEFLIN protein on their cell surface, incubated for a sufficient time for the antibody to internalize (e.g., about 15 minutes), the antibody bound to the culture medium and cell surface is removed by washing (e.g., with an aqueous solution containing 0.5 M NaCl and 3% by volume acetic acid), and then the internalized antibody is confirmed by staining. Staining of internalized antibodies can be performed in the same way as staining of intracellular proteins. Staining of internalized antibodies can be detected based on labeling, for example, using a labeled secondary antibody.

[0053] Cancer tissue includes cancer cells (i.e., malignant tumor cells) and cancer stromal cells. MEFLIN is expressed in malignant (sarcoma) and benign tumors derived from mesenchymal stem cells or similar cells. Specifically, it has been confirmed to be expressed on cancer cells in almost all cases of non-epithelial tumors, such as osteosarcoma, chondrosarcoma, liposarcoma, rhabdomyosarcoma, undifferentiated pleomorphic sarcoma, desmoid tumor, and meningioma. Therefore, the ADC of the present invention can be used to treat malignant (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 (carcinomas), even if the cancer cells themselves are MEFLIN protein-negative, the MEFLIN protein is expressed in stromal cells (e.g., CAF) in most cancers. Therefore, by targeting the stromal cells of carcinoma, the ADC of the present invention can be used to treat carcinomas. MEFLIN expression can be investigated by methods such as in situ hybridization or immunohistochemistry using antibodies, but it can also be investigated by other methods such as RT-PCR, Western blotting, DNA microarrays, and RNA sequencing.

[0054] Cancers that can be treated with the ADC or pharmaceutical composition of the present invention are not particularly limited, but include cancers that originate from epithelial cells (carcinomas), such as lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, stomach 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. Cancers that can be treated with the ADC or pharmaceutical composition of the present invention also include retroperitoneal tumors, angiovascular and lymphangiosarcomas, and metastatic cancers (or cancer cells) thereof. Cancers that can be treated with the ADC or pharmaceutical composition of the present invention also include brain tumors and bone and soft tissue tumors. These cancers either have MEFLIN protein-positive cancer cells, or even if the cancer cells are MEFLIN protein-negative, the tumor stroma contains MEFLIN protein-positive cells. According to the present invention, MEFLIN protein-positive cells are recruited into the tumor stroma. Accordingly, according to the present invention, the cancer (or cancer cells) to be treated by the ADC or pharmaceutical composition of the present invention may be MEFLIN-negative. MEFLIN-positive cells are recruited into the stroma of MEFLIN-negative cancer, and the ADC of the present invention can target these MEFLIN-positive cells recruited into the stroma, thereby exhibiting antitumor activity against the cancer (or cancer cells) regardless of whether the cancer itself is MEFLIN-positive or negative. This can be explained by the bystander effect. Whether or not MEFLIN-positive cells are present in the stroma of the cancer may be confirmed by examining the expression of MEFLIN protein in a tissue cadaver. In one embodiment of the present invention, the cancer to be treated is MEFLIN-negative, and MEFLIN-positive cells are present in the stroma of the cancer. According to the present invention, the cancers targeted for treatment by the ADC or pharmaceutical composition of the present invention may be MEFLIN protein-positive cancers (e.g., sarcomas, such as mucofibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor, Ewing's sarcoma, epithelioid sarcoma, clear cell sarcoma, synovial sarcoma, and osteosarcoma).In one aspect of the present invention, the cancer to be treated is MEFLIN protein-positive and MEFLIN protein-positive cells are present in the tumor stroma. In another aspect of the present invention, the cancer to be treated is MEFLIN protein-positive and MEFLIN protein-positive cells are not detected in the tumor stroma. In another aspect, the cancer to be treated may be a cancer that has been evaluated or suspected to be MEFLIN-positive, or a cancer in which the cancer cells themselves are MEFLIN-negative, but MEFLIN-positive cells are evaluated or suspected to be present in the stroma.

[0055] A bispecific antibody may be, for example, an antibody that has binding affinity to cancer cells and immune cells, respectively. A bispecific antibody may be, for example, an antibody that binds to T cell surface antigens such as MEFLIN protein and CD3 protein. A bispecific antibody may be IgG-type and diabody-type. This embodiment may be effective, for example, against MEFLIN protein-positive cancer cells.

[0056] In one embodiment of the present invention, the pharmaceutical composition comprises the ADC of the present invention and an excipient. The pharmaceutical composition of the present invention can be administered by methods such as intravenous administration, subcutaneous administration, intratumoral administration, intraperitoneal administration, intracerebroventricular administration, and intramuscular administration. The dosage can be appropriately determined by a physician taking into consideration the patient's age, sex, weight, and the severity of the disease.

[0057] The present invention provides for the use of the ADC in the manufacture of a pharmaceutical product for use in treating cancer.

[0058] The present invention provides a method for treating cancer in a subject requiring treatment, comprising administering a therapeutically effective amount of the ADC of the present invention to the subject. In this embodiment, the cancer to be treated may be a cancer (e.g., carcinoma) that is MEFLIN protein-negative and has MEFLIN protein-positive cells in the tumor stroma. In another embodiment, the cancer may be a MEFLIN protein-positive cancer (e.g., sarcoma). Such subjects can be identified or selected by detection of MEFLIN mRNA by immunohistochemical staining or in situ hybridization of tissue samples or tissue sections obtained from the subject using an anti-MEFLIN antibody.

[0059] According to the present invention, the use of the ADC of the present invention for use in a method of treating cancer is provided. According to the present invention, the ADC of the present invention for use in a method of treating cancer may be provided. [Examples]

[0060] Example 1: Antibody preparation and characterization of the obtained monoclonal antibody In this example, a monoclonal antibody that recognizes the human MEFLIN protein was prepared, and its binding characteristics were confirmed by various assays.

[0061] [Cell preparation] Flp-In TM Using the Thermo Fisher system, we established mouse Meflin-stable-expressing human fetal kidney (HEK293) cell lines (hereinafter referred to as the mMeflin HEK293 cell line) and mouse Meflin-stable-expressing Chinese hamster ovary (CHO) cell lines (hereinafter referred to as the mMeflin CHO cell line). Similarly, we established human MEFLIN-stable-expressing human fetal kidney (HEK293) cell lines (hereinafter referred to as the hMeflin HEK293 cell line) and human MEFLIN-stable-expressing Chinese hamster ovary (CHO) cell lines (hereinafter referred to as the hMEFLIN CHO cell line).

[0062] [Production of rat anti-human MEFLIN monoclonal antibodies] For animal immunization to produce human MEFLIN antibodies, recombinant human MEFLIN protein was used as the antigen. Recombinant human MEFLIN protein was obtained using a mammalian cell expression system. For example, the human MEFLIN gene was introduced into a human embryonic kidney-derived cell line (Expi293F cells, Thermo Fisher) adapted to suspension culture by lipofection (lipofectamine2000, invitrogen). To facilitate subsequent purification, the GPI anchor sequence at the carboxyl terminus of the MEFLIN gene was removed, and a histidine tag was introduced therein. After 5-8 days of gene introduction, the cell culture medium was collected, filtered (0.22 micrometers), and then subjected to a column packed with a carrier (Ni Excel, GE Healthcare) that specifically binds to histidine-tagged protein to conjugate the MEFLIN protein in the culture medium. After column washing, the conjugated MEFLIN protein was competitively eluted with a buffer solution containing imidazole (500 mM). The final human MEFLIN recombinant protein was obtained by removing imidazole from the eluate containing the MEFLIN protein by ultrafiltration (dialysis with phosphate buffer). The purity of the purified human MEFLIN recombinant protein was measured by subjecting the protein to SDS-PAGE electrophoresis followed by Coomassie Brilliant Blue staining. WKY rat hind limbs were immunized once with 100 μg of recombinant human MEFLIN protein (95% purity) on the soles of the feet. Two weeks later, plasma cells from the iliac lymph nodes were electrofused with myeloma (SP2 / 0) cell lines to create hybridomas. The culture medium was High glucose DMEM (Nacalai) supplemented with HAT medium (MPB) and BM condimed H1 (Roche), and hybridoma screening was performed.

[0063] [Production of mouse anti-human MEFLIN monoclonal antibodies] Monoclonal antibodies were produced using the same method as for rat anti-human MEFLIN monoclonal antibodies, except that C57BL / 6 mouse tail ridge muscles were immunized once with 50 μg of recombinant human MEFLIN protein (95% purity).

[0064] [Hybridoma Screening] Hybridomas producing monoclonal antibodies that recognize antigens were screened using each method: ELISA with recombinant human MEFLIN protein, immunofluorescence (IF) staining with the hMEFLIN CHO cell line, flow cytometry with the hMEFLIN CHO cell line, immunoprecipitation using cell lysates prepared from the hMeflin HEK293 cell line, and Western blotting.

[0065] [Western blotting] Human embryonic kidney (HEK293) cell lines were transiently expressed using a pRP-CMV vector containing DNA encoding human MEFLIN protein or a fragment thereof, via lipofection, to prepare cell lysates. The fragments are those shown in Figure 1 or 2, and have a G196 tag and a His tag at the C-terminus. SDS-PAGE was performed using the cell lysates, and the electrophoretic proteins were transferred to a PVDF membrane. The PVDF membrane was reacted with 5% skim milk / PBS at room temperature for 1 hour. As the primary antibody, the culture supernatant of each hybridoma clone was diluted 50-fold with PBS. For positive targets, an anti-G196 antibody was used as the primary antibody. The PVDF membrane was reacted overnight at 4°C in the presence of the primary antibody. Subsequently, the PVDF membrane was 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 used to label the antibody was made to emit light using ECL (GE Healthcare), and the image was captured by a computer using a CCD imager (Las4000, GE Healthcare).

[0066] [Analysis of internalization activity of monoclonal antibodies] hMeflin CHO cell lines or mMeflin CHO cell lines were cultured at 37°C and 5% CO2 for 3 hours in an assay solution prepared by adding 20 mM HEPS and 0.1% BSA to F-12 Glutamax culture medium (Gibco). The supernatant of each hybridoma was diluted 10-fold with the assay solution and added to the CHO cell lines, where they were cultured for 15 minutes. The CHO cell lines were washed with PBS (4°C) and then washed 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 with 4% paraformaldehyde (PFA) for 10 minutes, and then permeabilized by reacting with 0.1% Tiriton X-100 for 5 minutes. As a secondary antibody, a secondary antibody using an anti-rat or anti-mouse antibody-Alexa488 conjugate was diluted 400-fold in PBS and reacted with cells that had been permeabilized at room temperature for 30 minutes. After washing with PBS, the cells were reacted with DAPI at room temperature for 5 minutes, washed with PBS, and images were acquired using a confocal microscope (LSM700, ZEISS).

[0067] [In situ hybridization method] From pathological tissue specimens obtained from sarcoma or carcinoma patients, the region with the highest concentration of tumor components was selected under microscopic examination, and MEFLIN (also known as "ISLR") expression was examined using in situ hybridization (hereinafter referred to as "ISH staining") (see WO2017 / 22472). Of the examined tissues, the tissue was observed in a medium-magnification field (20x objective lens). For sarcoma tissue, tissues in which 20% or more of cells with tumor cell morphology were ISLR-positive were classified as ISLR-positive tumor tissue, and those with less than 20% were classified as ISLR-negative tumor tissue. Similarly, for carcinoma, tissues in which 20% or more of cells with fibroblast-like morphology infiltrating the cancer stroma were ISLR-positive were classified as ISLR-positive tumor tissue, and those with less than 20% were classified as ISLR-negative tumor tissue. ISLR positivity was defined as the presence of a signal in at least a portion of the cytoplasm.

[0068] [Histoimmunofluorescence Double Staining Method] Tumor tissue was sampled from tumor-bearing mouse models that had been subcutaneously transplanted with osteosarcoma cell lines (HsOs1) and pancreatic cancer cell lines (BxPC-3). The tissue was fixed with 10% formalin and then embedded in paraffin. The paraffin-embedded tissue was sectioned to a thickness of 2 μm, placed on a glass slide, deparaffinized with xylene and ethanol, and antigens were activated using a pH 6.0 antigen retrieval solution (Leica). Next, after blocking, immunofluorescence double 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). The nuclei within the cells were stained with DAPI. MEFLIN (green), cathepsin K (red), and nucleus (blue). Images were acquired using a confocal microscope (LSM700, ZEISS).

[0069] [Immunohistological staining method] From pathological tissue specimens obtained from cancer patients, the region with the highest concentration of tumor components was selected under microscopic examination, and MEFLIN (also known as "ISLR") expression was examined by immunohistochemistry (hereinafter referred to as "IHC staining"). Of the examined tissues, those in which 20% or more of the fibroblast-like cells infiltrating the cancer stroma were ISLR-positive were classified as ISLR-positive tumor tissue, and those with less than 20% were classified as ISLR-negative tumor tissue. ISLR positivity was defined as the presence of a signal in at least a portion of the cytoplasm. Anti-rat or anti-mouse antibody-horseradish peroxidase (HRP) conjugates were used as secondary antibodies (ImmPRESS, VECTORS LABORATORIES).

[0070] [result] The results of confirming the binding of each monoclonal antibody to each human MEFLIN protein fragment by Western blotting are shown in Figure 1. As shown in Figure 1, the rat or mouse-derived monoclonal antibody produced from clone 27-7 (hereinafter simply referred to as "27-7 antibody") did not bind to the amino acid region of human MEFLIN from amino acids 1 to 343, but bound to the amino acid region from amino acids 1 to 399. This suggests that the 27-7 antibody binds to the amino acid region of human MEFLIN from amino acids 344 to 399. Similarly, Figure 1 suggests that the 34-4 antibody binds to the amino acid region from amino acids 232 to 343; the 35-9 antibody binds to the amino acid region from amino acids 1 to 146; and the 27-8, 41-10, and 46-3 antibodies bind to the amino acid region of human MEFLIN from amino acids 344 to 399. Furthermore, as shown in Figure 2, the 3-2 antibody, 11-8 antibody, 12-2 antibody, 19-7 antibody, and 23-1 antibody were suggested to bind to the amino acid region of human MEFLIN from 344 to 399; the 13-1 antibody was suggested to bind to the amino acid region from 147 to 231; and the 16-5 antibody, 22-3 antibody, and 32-1 antibody were suggested to bind to the amino acid region from 1 to 146.

[0071] The internalization activity of each antibody clone was confirmed. The results are shown in Figures 3-5. As shown in Figures 3-5, strong antibody-derived signals were observed intracellularly for the 21-3 antibody, 25-1 antibody, 8-2 antibody, 27-7 antibody, 27-8 antibody, 34-4 antibody, 35-9 antibody, 46-3 antibody, 3-2 antibody, 11-8 antibody, 12-2 antibody, 16-5 antibody, 19-7 antibody, 22-3 antibody, 23-1 antibody, and 32-1 antibody.

[0072] The results are shown in Table 9. [Table 9]

[0073] In Table 9, the symbol "S" indicates strong internalizing activity, the symbol "M" indicates moderate internalizing activity, and the symbol "N" indicates no detected internalizing activity. "?" in the table indicates undetermined. Also in Table 9, "Host" indicates whether the antibody originates from rat or mouse. In Table 9, antibodies 21-3, 25-1, and 8-2 bound to mouse MEFLIN protein, while the other antibodies bound to human MEFLIN protein.

[0074] The expression levels of the MEFLIN protein in various cancer tissues are shown in Table 10. Table 10 shows the number of tissue samples examined, the number of positive samples among them, and the positive rate (%). [Table 10]

[0075] The expression of human MEFLIN (ISLR) in the tumor stroma is shown in Table 11. Table 11 shows the number of tissue samples examined, the number of positive samples among them, and the positive rate (%). In addition, two out of seven gastric cancer cases were positive. [Table 11]

[0076] Furthermore, tumor cells from pancreatic cancer, lung cancer, breast cancer, colorectal cancer, gastric cancer, bile duct cancer, ovarian cancer, bladder cancer, and esophageal cancer were negative for human MEFLIN expression.

[0077] Example 2: Preparation of antibody-drug conjugates (ADCs) and their in vivo efficacy testing. In this example, a conjugate was prepared using the antibody produced in Example 1 and a cytotoxic agent. The prepared conjugate was then administered to a tumor-bearing mouse model to confirm its antitumor effect.

[0078] [Preparing the ADC] Antibodies were purified from the antibody-containing hybridoma supernatant concentrated by high-density culture of hybridomas using Mono Spin™ L ProG (GL Science). Disulfide bonds of the antibodies were cleaved by reduction with 1.0 mM DTT at 25°C for 30 minutes, and the exposed thiol groups were treated with VcMMAE (mc-vc-PAB-MMAE, MedChemExpress) or mc-PEG. 12 -vc-PABC-MMAE was added at room temperature and purified by limit filtration. VcMMAE is a compound registered under CAS number 646502-53-6. In VcMMAE, monomethyl auristatin E (MMAE) is bound to a valine-citrulline linker via p-aminobenzoyloxycarbonyl (PBAC), and MMAE is released when valine-citrulline is cleaved by a protease such as cathepsin B. The valine-citrulline linker is modified with a maleimide-caproyl group, which can react with the thiol group of antibody cysteine ​​via the maleimide group. mc-PEG 12 -vc-PABC-MMAE is similar to VcMMAE except for the presence of PEG, and the MMAE release mechanism is also the same as VcMMAE. The drug-antibody ratio (DAR, the number of cytotoxic agents conjugated per antibody molecule) was determined by calculating the difference between the number of thiol groups of the antibody after reduction treatment and the number of thiol groups after VcMMAE was added to the antibody. Specifically, the exposed thiol groups were reacted with 5,5'-dithiobis(2-nitrobenzoic acid, Dojin Chemical Laboratories) to produce stable 5-mercapto-2-nitrobenzoic acid. The absorbance (λ) of this produced thiol was measured. max =412 nm, ε=1.55×10 4 The thiol group is quantified from the sample.

[0079] mc-vc-PAB-MMAE has the structure of formula (I) below. [ka]

[0080] The resulting ADC may have the structure of equation (II) below. [ka] {In the formula, n is a natural number between 1 and 8.}

[0081] mc-PEG 12 -vc-PABC-MMAE has the structure of equation (III) below. [ka]

[0082] The cytotoxicity of various cell types was tested using the prepared ADC.

[0083] Cytotoxicity of ADCs against human MEFLIN-expressing HEK293 cells First, the cytotoxicity of ADCs against human MEFLIN-expressing HEK293 cells was tested. Specifically, the hMeflin HEK293 cell line was subjected to a 1.0 × 10⁶ test. 4 Cells were seeded in plates at one well and cultured at 37°C and 5% CO2 for 1 day. Next, ADCs of each concentration were added and cultured for 2 days, and cell proliferation was evaluated by the MTT test. Cell proliferation was evaluated in the MMT test by measuring the absorbance of the sample at a wavelength of 590 nm using an absorbance meter (POWERSCAN4, DS PHARMA BIOMEDICAL). In addition, Meflin expression levels in the hMeflin HEK293 cell line were evaluated by FACS analysis. In the following figures, if only a clone number is listed, it means that the monoclonal antibody itself was used. If a clone number is listed immediately before or after an ADC, it means that an ADC whose antibody is that clone was used. CTL ADC or isotype control IgG ADC means that an ADC whose antibody is an isotype control IgG antibody was used. Rat IgG2a (BioLegend) was used as the isotype control antibody.

[0084] The results are shown in Figure 6. As shown in Figure 6, FACS analysis revealed that human MEFLIN-expressing HEK293 cells strongly expressed human MEFLIN protein. Furthermore, ADC27-7 (drug antibody ratio (DAR) = 2.4) significantly reduced the proliferation of human MEFLIN protein-expressing HEK293 cells. EC of ADC27-7 50 The concentration was 0.72 nM. ADC34-4 (DAR=3.4), which has moderate internalizing activity against hMEFLIN CHO cells, exhibited weak inhibitory activity on cell proliferation.

[0085] Cytotoxicity of ADCs against human rhabdomyosarcoma cell lines Similarly to the above, the cytotoxicity of ADCs against rhabdomyosarcoma (KYM-1) cell lines was tested. The results are shown in Figure 7. As shown in Figure 7, FACS analysis revealed that rhabdomyosarcoma cell lines strongly express human MEFLIN protein. In addition, ADC27-7 and ADC34-4 significantly reduced the proliferation of KYM-1 cell lines.

[0086] Cytotoxicity of ADCs in a subcutaneous transplantation model of rhabdomyosarcoma cell lines Next, the cytotoxicity of ADCs in vivo was tested using a tumor-bearing mouse model. KYM-1 cell line 1.0 × 10⁶ 7 Each individual was subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=5 in each group). Approximately 100 mm 3 ADC was administered intraperitoneally to mice with tumor volumes of varying sizes (5 mg / kg body weight, administered 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using a power caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of KYM cell lines.

[0087] The results are shown in Figure 8. As shown in Figure 8, 27-7ADC and 34-4ADC demonstrated potent antitumor activity against rhabdomyosarcoma. Histologically, the tumors strongly expressed human MEFLIN.

[0088] Cytotoxicity of ADCs in a subcutaneous transplantation model of osteosarcoma cell lines Furthermore, endogenous Meflin-positive osteosarcoma (HsOs1) cell line 1.0 × 10 7 The cells were subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=5 in each group). Approximately 75 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of the HsOs1 cell line.

[0089] The results are shown in Figure 9. As shown in Figure 9, 27-7ADC and 34-4ADC demonstrated potent antitumor activity against osteosarcoma. Histologically, the tumors expressed human MEFLIN to a moderate degree.

[0090] Cytotoxicity of ADCs in subcutaneous transplantation models of pancreatic cancer cell lines Endogenous Meflin-negative human pancreatic cancer (BxPC-3) cell line 1.0 × 10 7 Each individual was subcutaneously transplanted into the dorsal region of female nude mice (BALB / cSlc nu / nu) (n=5 in each group). Approximately 150 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of the BxPC-3 cell line.

[0091] The results are shown in Figure 10. As shown in the histological image in Figure 10, the pancreatic cancer cells themselves were MEFLIN-negative, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Furthermore, it was revealed that 21-3ADC and 25-1ADC exhibit potent antitumor activity against pancreatic cancer. Since the original tissue was MEFLIN-negative, it was clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exhibit antitumor activity against tumors.

[0092] Cytotoxicity of ADCs in subcutaneous transplantation models of lung cancer cell lines Endogenous Meflin-negative human lung cancer (A549) cell line 1.0 × 10 7 Each individual was subcutaneously transplanted into the dorsal region of female nude mice (BALB / cSlc nu / nu) (n=5 in each group). Approximately 100 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of the A549 cell line.

[0093] The results are shown in Figure 11. As shown in Figure 11, 25-1ADC exhibited strong antitumor activity against lung cancer, while 21-3ADC exhibited weak antitumor activity. Furthermore, as is evident from the histological findings, the lung cancer cells themselves were negative for human MEFLIN, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Since the original tissue was MEFLIN-negative, it became clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exhibit antitumor activity against tumors.

[0094] Cytotoxicity of ADCs in subcutaneous transplantation models of neuroblastoma cell lines Furthermore, endogenous Meflin-positive neuroblastoma (NB-1) cell line 1.0 × 10 7Each individual was subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=5 in each group). Approximately 250 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of NB-1 cell lines.

[0095] The results are shown in Figure 12. As shown in Figure 12, 27-7ADC and 34-4ADC were found to exhibit weak antitumor activity against neuroblastoma. As is evident from the histological findings, the tumors expressed human MEFLIN to a moderate degree.

[0096] Cytotoxicity of ADCs in a subcutaneous transplantation model of colorectal cancer cell lines Endogenous Meflin-negative human colorectal cancer (DLD-1) cell line 1.0 × 10 7 Each individual was subcutaneously transplanted into the dorsal region of female nude mice (BALB / cSlc nu / nu) (n=5 in each group). Approximately 200 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of DLD-1 cell lines.

[0097] The results are shown in Figure 13. As shown in Figure 13, 25-1ADC exhibited weak antitumor activity against colorectal cancer, while 21-3ADC showed no antitumor activity. Furthermore, as is evident from the histological findings, the colorectal cancer cells themselves were negative for human MEFLIN, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Since the original tissue was MEFLIN-negative, it became clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exhibit antitumor activity against tumors.

[0098] Cytotoxicity of ADCs in a subcutaneous transplantation model of gastric cancer cell lines Endogenous Meflin-negative human gastric cancer (MKN45) cell line 1.0 × 10 7 Each individual was subcutaneously transplanted into the dorsal region of female nude mice (BALB / cSlc nu / nu) (n=5 in each group). Approximately 200 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of the MKN45 cell line.

[0099] The results are shown in Figure 14. As shown in Figure 14, 21-3ADC and 25-1ADC demonstrated strong antitumor activity against gastric cancer. Furthermore, as is evident from the histological findings, the gastric cancer cells themselves were negative for human MEFLIN, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Since the original tissue was MEFLIN-negative, it became clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exert antitumor activity against tumors.

[0100] Single-cell analysis in mouse pancreas Figure 15 shows the results of an analysis of single-cell RNA sequencing data (Tabula Muris) (see Non-Patent Literature 7) from a cell population derived from mouse pancreas, which was publicly available on the internet. The tSNE (t-distributed Stochastic Neighbor Embedding) shown on the vertical and horizontal axes respectively represents numerical values ​​obtained by compressing multidimensional data composed of a large amount of RNA expression data into one dimension using a nonlinear transformation. In other words, Figure 15 shows that by unfolding the large amount of RNA expression data expressed in individual cells onto a two-dimensional plane using tSNE1 and tSNE2, cells can be clustered based on gene expression profiles. With this method, for example, it is possible to understand what kind of cell cluster each individual cell, indicated by a circle, forms. As a result, this clustering method makes it possible to check whether the cell group expressing a specific factor matches the cell group expressing other specific factors, and in addition, it is possible to determine which cell type corresponds to that cell group. Figure 15 shows that pancreatic stellate cells in the mouse pancreas express both MEFLIN and cathepsin K, indicating that the MEFLIN-positive cell population and the cathepsin K-positive cell population in the mouse pancreas coincide (see arrow in the figure).

[0101] Single-cell analysis in mouse lungs Figure 16 shows the results of an analysis of single-cell RNA sequencing data (Tabula Muris) from a cell population derived from mouse lung, which was publicly available on the internet (see Non-Patent Literature 7). It shows that both MEFLIN and cathepsin K are expressed in stromal cells of mouse lung, indicating that the MEFLIN-positive cell population and the cathepsin K-positive cell population in mouse lung coincide (see arrow in the figure). (See the arrow in the diagram).

[0102] CHO cells were expressed with the full-length mouse MEFLIN protein, and Western blotting was performed on the cell lysates. The results are shown in Figure 17. As shown in Figure 17, expressing the full-length mouse MEFLIN protein in CHO cells increased the expression of endogenous cathepsin K.

[0103] Immunofluorescence double staining in osteosarcoma tissue Figure 18 shows the results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody on tumor tissue from tumor-bearing mouse models subcutaneously transplanted with osteosarcoma cell line (HsOs1). The arrows indicate the secretion of cathepsin K (red) around MEFLIN-positive cells (green).

[0104] The results are shown in Figure 18. As shown in Figure 18, the presence of cathepsin K (red) was confirmed around MEFLIN-positive cells (green) (arrows in the enlarged view).

[0105] Immunofluorescence double staining method in pancreatic cancer tissue Figure 19 shows the results of double immunofluorescence staining using anti-MEFLIN monoclonal antibody and anti-cathepsin K antibody on tumor tissue from tumor-bearing mouse models subcutaneously transplanted with the pancreatic cancer cell line (BxPC-3). The arrows indicate that cathepsin K (red) is secreted around MEFLIN-positive cells (green).

[0106] The results are shown in Figure 19. As shown in Figure 19, the presence of cathepsin K (red) was confirmed around MEFLIN-positive cells (green) (arrows in the enlarged view).

[0107] Thus, it was revealed that when human MEFLIN-negative cancer cells are transplanted into mice, they recruit MEFLIN-positive cells into the tissue containing the cancer cells, and that these recruited MEFLIN-positive cells can be used to treat the cancer with ADCs (artificially administered diuretics) that target the cancer. It was also revealed that ADCs targeting human MEFLIN protein do not necessarily require internalization activity. Furthermore, it was thought that enzymes that cleave valine-citrulline linkers, such as cathepsin, are present in the stroma containing MEFLIN protein-positive cells, and that this is why ADCs that are not taken up into cells exhibit cytotoxicity.

[0108] Cytotoxicity of ADCs in subcutaneous transplantation models of cholangiocarcinoma cell lines Endogenous Meflin-negative human cholangiocarcinoma (HuCCT1) cell line 1.0 × 10⁻¹⁴ 7 Each individual was subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=5 in each group). Approximately 250 mm 3 Mice with tumor volumes before and after the initial administration were given ADC via tail vein (5 mg / kg body weight, administered 3 times at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of the HuCCT1 cell line.

[0109] The results are shown in Figure 20. As shown in the histological image of Figure 20, the cholangiocarcinoma cells themselves were MEFLIN-negative, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Furthermore, it was revealed that 21-3ADC and 25-1ADC exhibit potent antitumor activity against cholangiocarcinoma. Since the original tissue was MEFLIN-negative, it was clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exert antitumor activity against tumors.

[0110] Cytotoxicity of ADCs in subcutaneous transplantation models of bladder cancer cell lines Endogenous Meflin-negative human bladder cancer (T24) cell line 1.0 × 10 7The cells were subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=4 in each group). Approximately 300 mm 3 Mice with tumor volumes before and after the initial administration were given ADC via tail vein (5 mg / kg body weight, administered 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of T24 cell lines.

[0111] The results are shown in Figure 21. As shown in the histological image in Figure 21, the bladder cancer cells themselves were MEFLIN-negative, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Furthermore, it was revealed that 21-3ADC and 25-1ADC exhibited weak antitumor activity against bladder cancer. Since the original tissue was MEFLIN-negative, it was clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exert antitumor activity against tumors.

[0112] Cytotoxicity of ADCs in subcutaneous transplantation models of ovarian cancer cell lines Endogenous Meflin-negative human ovarian cancer (OV-90) cell line 1.0 × 10⁻¹⁴ 7 Each individual was subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=5 in each group). Approximately 100 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of OV-90 cell lines.

[0113] The results are shown in Figure 22. As shown in the histological image in Figure 22, the ovarian cancer cells themselves were MEFLIN-negative, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Furthermore, it was revealed that 21-3ADC and 25-1ADC exhibit potent antitumor activity against ovarian cancer. Since the original tissue was MEFLIN-negative, it was clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exert antitumor activity against tumors.

[0114] Cytotoxicity of ADCs in subcutaneous transplantation models of esophageal cancer cell lines Endogenous Meflin-negative human esophageal cancer (KYSE) cell line 1.0 × 10 7 Each individual was subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=5 in each group). Approximately 200 mm 3 Mice with tumor volumes before and after the specified size were administered ADC intraperitoneally (5 mg / kg body weight, 5 doses at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of KYSE cell lines.

[0115] The results are shown in Figure 23. As shown in the histological image in Figure 23, the esophageal cancer cells themselves were MEFLIN-negative, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Furthermore, it was revealed that 21-3ADC and 25-1ADC exhibit potent antitumor activity against esophageal cancer. Since the original tissue was MEFLIN-negative, it was clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exhibit antitumor activity against tumors.

[0116] Cytotoxicity of ADCs in a subcutaneous transplantation model of osteosarcoma cell lines Endogenous Meflin-positive osteosarcoma (HsOs1) cell line 1.0 × 10⁻¹⁴ 7 Each individual was subcutaneously transplanted into the dorsal region of female NOD SCID mice (n=4 in each group). Approximately 250 mm 3Mice with tumor volumes before and after the initial administration were given ADC via tail vein (5 mg / kg body weight, administered 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of the HsOs1 cell line.

[0117] The results are shown in Figure 24. As shown in Figure 24, 46-3ADC demonstrated potent antitumor activity against osteosarcoma. Furthermore, the tumors expressed human MEFLIN to a moderate degree (see Figure 9).

[0118] Cytotoxicity of ADCs in subcutaneous transplantation models of breast cancer cell lines Endogenous Meflin-negative human breast cancer (MCF-7) cell line 1.0 × 10⁻¹⁴ 7 Each individual was subcutaneously transplanted into the dorsal region of female nude mice (BALB / cSlc nu / nu) (n=5 in each group). Approximately 300 mm 3 Mice with tumor volumes before and after the initial administration were given ADC via tail vein (5 mg / kg body weight, administered 5 times at 4-day intervals). Tumor length (L) and width (W) were measured using a motorized caliper, and the volume was calculated as V = L × W. 2 The calculation was performed according to a multiplier of 0.52. In addition, ISH staining was performed on tumors formed by transplantation of the MCF-7 cell line.

[0119] The results are shown in Figure 25. As shown in the histological image of Figure 25, the breast cancer cells themselves were MEFLIN-negative, but mouse MEFLIN-positive cells were observed in the tumor stromal cells. Furthermore, it was revealed that 21-3ADC and 25-1ADC exhibited weak antitumor activity against breast cancer. Since the original tissue was MEFLIN-negative, it was clear that MEFLIN-positive cells were recruited to the vicinity of the tumor. In addition, it was suggested that ADCs exert antitumor activity against tumors.

[0120] Validation of immunohistochemistry in human pancreatic cancer tissue The study used serial sections of human pancreatic cancer surgical specimens. Immunohistochemical staining was performed on the serial sections of human pancreatic cancer surgical specimens using an anti-MEFLIN antibody, and MEFLIN expression was confirmed by using the anti-MEFLIN antibody on human tissue. In addition, human MEFLIN mRNA expression was confirmed on the same serial sections by in situ hybridization.

[0121] The results are shown in Figure 26. Cells that were MEFLIN-positive by immunohistochemistry in human pancreatic cancer tissue and cells that were MEFLIN-positive by in situ hybridization were the same, and MEFLIN expression could be confirmed by performing immunohistochemistry using an anti-MEFLIN antibody on human tissue. The arrows indicate MEFLIN confirmed by immunohistochemistry (Figure 26, top) and MEFLIN confirmed by in situ hybridization (Figure 26, bottom), respectively, in human pancreatic cancer tissue. The positions of the arrows in Figure 26, top and bottom, coincide and point to the same cells.

Claims

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

2. The pharmaceutical composition according to claim 1, wherein the antibody has internalizing activity.

3. The pharmaceutical composition according to claim 1 or 2, wherein the ADC is an ADC in which an antibody and a drug are linked via a linker, and the linker has a cleavage site that is cleaved in a cell.

4. An antibody that binds to MEFLIN, selected from the group consisting of the following: (1A) An antibody having a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence described in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence described in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence described in SEQ ID NO: 3, and a light chain variable region including a light chain CDR1 having the amino acid sequence described in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence described in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence described in SEQ ID NO: 6; (1B) An antibody having a heavy chain variable region having the amino acid sequence described in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence described in SEQ ID NO: 8; (1C) Antibodies that compete with the antibody in (1B) above for binding to the MEFLIN protein; and, (1D) An antibody that binds to an epitope on the MEFLIN protein that overlaps with the antibody in (1B) above; (2A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 9, heavy chain CDR2 described in SEQ ID NO: 10, and heavy chain CDR3 described in SEQ ID NO: 11, A light chain variable region including light chain CDR1 described in Sequence ID No. 12, light chain CDR2 described in Sequence ID No. 13, and light chain CDR3 described in Sequence ID No.

14. Antibodies that possess; (2B) An antibody having the heavy chain variable region described in SEQ ID NO: 15 and the light chain variable region described in SEQ ID NO: 16; (2C) Antibodies that compete with the antibody in (2B) above for binding to the MEFLIN protein; and, (2D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (2B) above; (3A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 17, heavy chain CDR2 described in SEQ ID NO: 18, and heavy chain CDR3 described in SEQ ID NO: 19, A light chain variable region including light chain CDR1 described in Sequence ID No. 20, light chain CDR2 described in Sequence ID No. 21, and light chain CDR3 described in Sequence ID No.

22. Antibodies that possess; (3B) An antibody having the heavy chain variable region described in SEQ ID NO: 23 and the light chain variable region described in SEQ ID NO: 24; (3C) Antibodies that compete with the antibody in (3B) above for binding to the MEFLIN protein; and (3D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (3B) above; (4A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 25, heavy chain CDR2 described in Sequence ID No. 26, and heavy chain CDR3 described in Sequence ID No. 27, A light chain variable region including light chain CDR1 described in Sequence ID No. 28, light chain CDR2 described in Sequence ID No. 29, and light chain CDR3 described in Sequence ID No.

30. Antibodies that possess; (4B) An antibody having the heavy chain variable region described in SEQ ID NO: 31 and the light chain variable region described in SEQ ID NO: 32; (4C) Antibodies that compete with the antibody in (4B) above for binding to the MEFLIN protein; and, (4D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (4B) above; (5A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 33, heavy chain CDR2 described in Sequence ID No. 34, and heavy chain CDR3 described in Sequence ID No. 35, A light chain variable region including light chain CDR1 described in Sequence ID No. 36, light chain CDR2 described in Sequence ID No. 37, and light chain CDR3 described in Sequence ID No.

38. Antibodies that possess; (5B) An antibody having the heavy chain variable region described in SEQ ID NO: 39 and the light chain variable region described in SEQ ID NO: 40; (5C) Antibodies that compete with the antibody in (5B) above for binding to the MEFLIN protein; and, (5D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (5B) above; (6A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 41, heavy chain CDR2 described in Sequence ID No. 42, and heavy chain CDR3 described in Sequence ID No. 43, A light chain variable region including light chain CDR1 described in Sequence ID No. 44, light chain CDR2 described in Sequence ID No. 45, and light chain CDR3 described in Sequence ID No.

46. Antibodies that possess; (6B) An antibody having the heavy chain variable region described in SEQ ID NO: 47 and the light chain variable region described in SEQ ID NO: 48; (6C) Antibodies that compete with the antibody in (6B) above for binding to the MEFLIN protein; and, (6D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (6B) above; (7A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 49, heavy chain CDR2 described in Sequence ID No. 50, and heavy chain CDR3 described in Sequence ID No. 51, A light chain variable region including light chain CDR1 described in Sequence ID No. 52, light chain CDR2 described in Sequence ID No. 53, and light chain CDR3 described in Sequence ID No. 54 Antibodies that possess; (7B) An antibody having the heavy chain variable region described in SEQ ID NO: 55 and the light chain variable region described in SEQ ID NO: 56; (7C) Antibodies that compete with the antibody in (7B) above for binding to the MEFLIN protein; and, (7D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (7B) above; (8A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 57, heavy chain CDR2 described in Sequence ID No. 58, and heavy chain CDR3 described in Sequence ID No. 59, A light chain variable region including light chain CDR1 described in Sequence ID No. 60, light chain CDR2 described in Sequence ID No. 61, and light chain CDR3 described in Sequence ID No.

62. Antibodies that possess; (8B) An antibody having the heavy chain variable region described in SEQ ID NO: 63 and the light chain variable region described in SEQ ID NO: 64; (8C) Antibodies that compete with the antibody in (8B) above for binding to the MEFLIN protein; and, (8D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (8B) above; (9A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 65, heavy chain CDR2 described in Sequence ID No. 66, and heavy chain CDR3 described in Sequence ID No. 67, A light chain variable region including light chain CDR1 described in Sequence ID No. 68, light chain CDR2 described in Sequence ID No. 69, and light chain CDR3 described in Sequence ID No. 70 Antibodies that possess; (9B) An antibody having the heavy chain variable region described in SEQ ID NO: 71 and the light chain variable region described in SEQ ID NO: 72; (9C) Antibodies that compete with the antibody in (9B) above for binding to the MEFLIN protein; and, (9D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (9B) above; (10A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 73, heavy chain CDR2 described in Sequence ID No. 74, and heavy chain CDR3 described in Sequence ID No. 75, A light chain variable region including light chain CDR1 described in Sequence ID No. 76, light chain CDR2 described in Sequence ID No. 77, and light chain CDR3 described in Sequence ID No.

78. Antibodies that possess; (10B) An antibody having the heavy chain variable region described in SEQ ID NO: 79 and the light chain variable region described in SEQ ID NO: 80; (10C) Antibodies that compete with the antibody in (10B) above for binding to the MEFLIN protein; and, (10D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (10B) above; (11A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 81, heavy chain CDR2 described in Sequence ID No. 82, and heavy chain CDR3 described in Sequence ID No. 83, A light chain variable region including light chain CDR1 described in Sequence ID No. 84, light chain CDR2 described in Sequence ID No. 85, and light chain CDR3 described in Sequence ID No.

86. Antibodies that possess; (11B) An antibody having the heavy chain variable region described in SEQ ID NO: 87 and the light chain variable region described in SEQ ID NO: 88; (11C) Antibodies that compete with the antibody in (11B) above for binding to the MEFLIN protein; and, (11D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (11B) above; (12A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 89, heavy chain CDR2 described in Sequence ID No. 90, and heavy chain CDR3 described in Sequence ID No. 91, A light chain variable region including light chain CDR1 described in Sequence ID No. 92, light chain CDR2 described in Sequence ID No. 93, and light chain CDR3 described in Sequence ID No. 94 Antibodies that possess; (12B) An antibody having the heavy chain variable region described in SEQ ID NO: 95 and the light chain variable region described in SEQ ID NO: 96; (12C) Antibodies that compete with the antibody in (12B) above for binding to the MEFLIN protein; and, (12D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (12B) above; (13A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 97, heavy chain CDR2 described in Sequence ID No. 98, and heavy chain CDR3 described in Sequence ID No. 99, A light chain variable region including light chain CDR1 described in SEQ ID NO: 100, light chain CDR2 described in SEQ ID NO: 101, and light chain CDR3 described in SEQ ID NO:

102. Antibodies that possess; (13B) An antibody having the heavy chain variable region described in SEQ ID NO: 103 and the light chain variable region described in SEQ ID NO: 104; (13C) Antibodies that compete with the antibody in (13B) above for binding to the MEFLIN protein; and, (13D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (13B) above; (14A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 105, heavy chain CDR2 described in SEQ ID NO: 106, and heavy chain CDR3 described in SEQ ID NO: 107, A light chain variable region including light chain CDR1 described in Sequence ID No. 108, light chain CDR2 described in Sequence ID No. 109, and light chain CDR3 described in Sequence ID No.

110. Antibodies that possess; (14B) An antibody having the heavy chain variable region described in SEQ ID NO: 111 and the light chain variable region described in SEQ ID NO: 112; (14C) Antibodies that compete with the antibody in (14B) above for binding to the MEFLIN protein; and, (14D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (14B) above; (15A) A heavy chain variable region including heavy chain CDR1 described in Sequence ID No. 113, heavy chain CDR2 described in Sequence ID No. 114, and heavy chain CDR3 described in Sequence ID No. 115, A light chain variable region including light chain CDR1 described in SEQ ID NO: 116, light chain CDR2 described in SEQ ID NO: 117, and light chain CDR3 described in SEQ ID NO: 118 Antibodies that possess; (15B) An antibody having the heavy chain variable region described in SEQ ID NO: 119 and the light chain variable region described in SEQ ID NO: 120; (15C) Antibodies that compete with the antibody in (15B) above for binding to the MEFLIN protein; and (15D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (15B) above; (16A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 121, heavy chain CDR2 described in SEQ ID NO: 122, and heavy chain CDR3 described in SEQ ID NO: 123, A light chain variable region including light chain CDR1 described in Sequence ID No. 124, light chain CDR2 described in Sequence ID No. 125, and light chain CDR3 described in Sequence ID No.

126. Antibodies that possess; (16B) An antibody having the heavy chain variable region described in SEQ ID NO: 127 and the light chain variable region described in SEQ ID NO: 128; (16C) Antibodies that compete with the antibody in (16B) above for binding to the MEFLIN protein; and (16D) An antibody that binds to the same epitope on the MEFLIN protein as the antibody in (16B) above; and (17A) A heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 129, heavy chain CDR2 described in SEQ ID NO: 130, and heavy chain CDR3 described in SEQ ID NO: 131, A light chain variable region including light chain CDR1 described in SEQ ID NO: 132, light chain CDR2 described in SEQ ID NO: 133, and light chain CDR3 described in SEQ ID NO: 134 Antibodies that possess; (17B) An antibody having the heavy chain variable region described in SEQ ID NO: 135 and the light chain variable region described in SEQ ID NO: 136; (17C) Antibodies that compete with the antibody in (17B) above for binding to the MEFLIN protein; and (17D) A pharmaceutical composition for use in treating cancer, comprising an antibody-drug conjugate (ADC) of the antibody described in (17B) above and an antibody that binds to an overlapping epitope on the MEFLIN protein, and a cytotoxic agent.

5. A pharmaceutical composition according to claim 4, for use in treating cancer.

6. The pharmaceutical composition according to any one of claims 1 to 3 and 5, wherein the cancer is a sarcoma.

7. The pharmaceutical composition according to claim 6, wherein the cancer is a sarcoma that is MEFLIN-positive.

8. The pharmaceutical composition according to claim 6 or 7, wherein the sarcoma is a sarcoma selected from the group consisting of mucofibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, malignant peripheral nerve sheath tumor, Ewing's sarcoma, epithelioid sarcoma, clear cell sarcoma, synovial sarcoma, and osteosarcoma.

9. The pharmaceutical composition according to any one of claims 1 to 3 and 5, wherein the cancer is a carcinoma.

10. The pharmaceutical composition according to any one of claims 1 to 3 and 5, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, lung cancer, colorectal cancer, stomach cancer, bile duct cancer, ovarian cancer, bladder cancer, and esophageal cancer.

11. The pharmaceutical composition according to any one of claims 1 to 3, 5, 9, and 10, wherein the cancer is MEFLIN-negative and the stroma surrounding the cancer contains MEFLIN-positive cells.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein the antibody does not have internalizing activity.

13. The pharmaceutical composition according to any one of claims 6 to 8, wherein the antibody has internalizing activity.

14. The pharmaceutical composition according to claim 12, wherein the ADC is an ADC in which an antibody and a drug are linked via a linker, and the linker is a cleavable linker.

15. The pharmaceutical composition according to claim 14, wherein the linker is a cleavable linker that is cleaved by cathepsin K.

16. The pharmaceutical composition according to claim 15, wherein the linker comprises a valine-citrulline dipeptide and is cleaved in the presence of cathepsin K.

17. The pharmaceutical composition according to claim 3, wherein the linker is a non-cleavable linker.

Images