Pharmaceutical composition for treatment and / or prevention of cancer

Abstract

An antibody or fragment thereof that is immunoreactive with a CAPRIN-1 protein having an amino acid sequence shown in any one of the items with an even-numbered sequence number among the sequence numbers 2 to 30, or an amino acid sequence having 80% or more sequence identity to the amino acid sequence, conjugated with a topoisomerase I inhibitor can be utilized as an antibody-drug conjugate (ADC) for the treatment and / or prevention of cancer.

Description

Technical Field

[0001] This invention relates to conjugates of antibodies or fragments thereof against CAPRIN-1 protein with topoisomerase I inhibitors, and the pharmaceutical use of such conjugates as treatments and / or preventative agents for cancer. Background Technology

[0002] Antibody drugs targeting specific antigenic proteins on cancer cells are used as cancer treatments with fewer side effects due to their cancer specificity. For example, cytoplasmic activation and proliferation-associateed protein 1 (CAPRIN-1) is expressed on the cell membrane surface of many solid cancers, and antibodies against CAPRIN-1 are known to have potential use as drugs for the treatment and / or prevention of cancer (Patent Document 1).

[0003] In recent years, research has been conducted on enhancing the efficacy of antibody drugs against cancer, especially as one of the means to enhance the efficacy of antibodies against CAPRIN-1 protein against cancer. It is known that the combination therapy of antibodies against CAPRIN-1 protein with topotecan and irinotecan, which are topoisomerase I inhibitors, is a combination therapy (Patent Document 2).

[0004] In addition, the development of antibody-drug conjugates (ADCs), which are formed by combining drugs with strong direct killing capabilities against cancer cells with antibodies, is prevalent. Successful examples of ADCs using topoisomerase I inhibitors are relatively few, but Trodelvy, an ADC that links an antibody against TROP-2 to SN38, has been successful. TM(sacituzumab govitecan-hziy) has been approved for use in patients with metastatic triple-negative breast cancer, achieving an effect of reducing the risk of death to about half (49%) (Non-patent literature 1). In addition, as an ADC that binds an antibody against HER2 to deruxtecan, one of the topoisomerase I inhibitors, trastuzumab deruxtecan (T-DXd) has been recognized in patients with HER2-positive unresectable or recurrent breast cancer with a history of chemotherapy, HER2-low expression unresectable or recurrent breast cancer with a history of chemotherapy, HER2-mutant unresectable advanced / recurrent non-small cell lung cancer that has worsened after chemotherapy, and HER2-positive unresectable advanced / recurrent gastric cancer that has worsened after chemotherapy. In patients with HER2-positive unresectable or recurrent breast cancer who received T-DXd and had a history of treatment with trastuzumab and taxane anti-tumor agents, progression-free survival was significantly prolonged compared to the ADC trastuzumab emtansine (T-DM1) which binds trastuzumab to emtansine (DM1) (Non-Patent Literature 2).

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: WO2010 / 016526

[0008] Patent Document 2: WO2022 / 270524

[0009] Non-patent literature

[0010] Non-patent document 1: GILEAD Press Releases, April 07, 2021

[0011] Non-patent literature 2: THE LANCET Vol. 401, May 27, 2023 Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] As mentioned earlier, successful commercially available examples of ADCs utilizing topoisomerase I inhibitors to enhance the efficacy of anti-cancer antibody drugs are limited to targeting a subset of cancers, and their anti-tumor effects are also limited. Furthermore, as illustrated in the embodiments of this application, the inventors have discovered that the efficacy of conjugates of trastuzumab and topoisomerase I inhibitors, which are already marketed as cancer antibody drugs, is only partially enhanced compared to trastuzumab alone in terms of anti-tumor effect. Therefore, ADC technology utilizing topoisomerase I inhibitors is not a technique that significantly enhances the anti-tumor effects of all cancer antibody drugs.

[0014] Therefore, the purpose of this invention is to discover cancer antibody drugs that can significantly enhance their efficacy by applying ADC technology utilizing topoisomerase I inhibitors, and to create new ADCs with excellent anti-tumor effects utilizing topoisomerase I inhibitors.

[0015] Methods for solving problems

[0016] The inventors' in-depth research revealed that conjugates of antibodies or fragments targeting CAPRIN-1 protein with topoisomerase I inhibitors exhibit significantly stronger antitumor effects compared to antibodies or fragments targeting CAPRIN-1 protein alone. Furthermore, the enhanced antitumor effect of conjugating topoisomerase I inhibitors to antibodies or fragments targeting CAPRIN-1 protein is significantly superior to the enhanced antitumor effect of conjugating topoisomerase I inhibitors to existing cancer antibody drugs, thus completing this invention.

[0017] Specifically, the present invention has the following features (1) to (12).

[0018] (1) An antibody or fragment thereof bound to a topoisomerase I inhibitor, wherein the antibody or fragment thereof is immunoreactive with CAPRIN-1 protein, wherein the CAPRIN-1 protein has an amino acid sequence represented by any one of the even-numbered sequence numbers 2 to 30, or an amino acid sequence having more than 80% sequence identity with such amino acid sequence.

[0019] (2) According to the conjugate of (1), the antibody or fragment thereof is immunoreactive with a portion of the CAPRIN-1 protein polypeptide, wherein the portion of the CAPRIN-1 protein polypeptide has an amino acid sequence represented by any one of sequence numbers 31-35, 296-299, 308, or 309, or an amino acid sequence having more than 80% sequence identity with such amino acid sequence.

[0020] (3) According to the conjugate described in (1) or (2), the antibody is a monoclonal antibody or a polyclonal antibody.

[0021] (4) The conjugate according to any one of (1) to (3), wherein the antibody or fragment thereof is any one of (A) to (M), (A) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 36, 37, and 38, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 40, 41, and 42, designated as CDR1, CDR2, and CDR3, respectively. (B) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 44, 45, and 46, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 48, 49, and 50, designated as CDR1, CDR2, and CDR3, respectively. (C) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 52, 53, and 54, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 56, 57, and 58, designated as CDR1, CDR2, and CDR3, respectively. (D) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 60, 61, and 62, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 64, 65, and 66, designated as CDR1, CDR2, and CDR3, respectively. (E) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 170, 171, and 172, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 173, 174, and 175, designated as CDR1, CDR2, and CDR3, respectively. (F) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 176, 177, and 178, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 179, 180, and 181, designated as CDR1, CDR2, and CDR3, respectively. (G) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 182, 183, and 184, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 185, 186, and 187, designated as CDR1, CDR2, and CDR3, respectively. (H) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 188, 189, and 190, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 191, 192, and 193, designated as CDR1, CDR2, and CDR3, respectively. (I) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 146, 147, and 148, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 149, 150, and 151, designated as CDR1, CDR2, and CDR3, respectively. (J) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 272, 273, and 274, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 275, 276, and 277, designated as CDR1, CDR2, and CDR3, respectively. (K) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 290, 291, and 292, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 293, 294, and 295, designated as CDR1, CDR2, and CDR3, respectively. (L) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 301, 302, and 303, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 305, 306, and 307, designated as CDR1, CDR2, and CDR3, respectively. (M) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 134, 135 and 136 as CDR1, CDR2 and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 137, 138 and 139 as CDR1, CDR2 and CDR3, respectively.

[0022] (5) The conjugate according to any one of (1) to (4), wherein the antibody or fragment thereof is any one of (a) to (al). (a) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 39 and whose light chain variable region contains the amino acid sequence of sequence number 43. (b) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 47 and whose light chain variable region contains the amino acid sequence of sequence number 51. (c) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 55 and whose light chain variable region contains the amino acid sequence of sequence number 59. (d) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 63 and whose light chain variable region contains the amino acid sequence of sequence number 67. (e) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 68 and whose light chain variable region contains the amino acid sequence of sequence number 69. (f) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 70 and whose light chain variable region contains the amino acid sequence of sequence number 71. (g) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 72 and whose light chain variable region contains the amino acid sequence of sequence number 73. (h) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 74 and whose light chain variable region contains the amino acid sequence of sequence number 75. (i) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 76 and whose light chain variable region contains the amino acid sequence of sequence number 77. (j) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 78 and whose light chain variable region contains the amino acid sequence of sequence number 79. (k) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 80 and whose light chain variable region contains the amino acid sequence of sequence number 81. (l) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 82 and whose light chain variable region contains the amino acid sequence of sequence number 83. (m) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 84 and whose light chain variable region contains the amino acid sequence of sequence number 85. (n) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 86 and whose light chain variable region contains the amino acid sequence of sequence number 87. (o) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 88 and whose light chain variable region contains the amino acid sequence of sequence number 89. (p) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 90 and whose light chain variable region contains the amino acid sequence of sequence number 91. (q) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 92 and whose light chain variable region contains the amino acid sequence of sequence number 93. (r) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 94 and whose light chain variable region contains the amino acid sequence of sequence number 95. (s) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 96 and whose light chain variable region contains the amino acid sequence of sequence number 97. (t) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 98 and whose light chain variable region contains the amino acid sequence of sequence number 99. (u) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 100 and whose light chain variable region contains the amino acid sequence of sequence number 101. (v) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 102 and whose light chain variable region contains the amino acid sequence of sequence number 103. (w) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 104 and whose light chain variable region contains the amino acid sequence of sequence number 105. (x) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 106 and whose light chain variable region contains the amino acid sequence of sequence number 107. (y) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 108 and whose light chain variable region contains the amino acid sequence of sequence number 109. (z) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 110 and whose light chain variable region contains the amino acid sequence of sequence number 111. (aa) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 112 and whose light chain variable region contains the amino acid sequence of sequence number 113. (ab) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 114 and whose light chain variable region contains the amino acid sequence of sequence number 115. (ac) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 116 and whose light chain variable region contains the amino acid sequence of sequence number 117. (ad) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 118 and whose light chain variable region contains the amino acid sequence of sequence number 119. (ae) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 120 and whose light chain variable region contains the amino acid sequence of sequence number 121. (af) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 122 and whose light chain variable region contains the amino acid sequence of sequence number 123. (ag) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 124 and whose light chain variable region contains the amino acid sequence of sequence number 125. (ah) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 126 and whose light chain variable region contains the amino acid sequence of sequence number 127. (ai) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 128 and whose light chain variable region contains the amino acid sequence of sequence number 129. (aj) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 130 and whose light chain variable region contains the amino acid sequence of sequence number 131. (ak) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 132 and whose light chain variable region contains the amino acid sequence of sequence number 133. (al) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 300 and whose light chain variable region contains the amino acid sequence of sequence number 304.

[0023] (6) The conjugate according to any one of (1) to (5), wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody or a single-chain antibody.

[0024] (7) The conjugate according to any one of (1) to (6), wherein the antibody or a fragment thereof binds to a topoisomerase I inhibitor via a linker.

[0025] (8) According to the conjugates described in (1) to (7), the topoisomerase I inhibitor is exatecan, SN38 or a derivative thereof.

[0026] (9) A pharmaceutical composition for the treatment and / or prevention of cancer, comprising any one of the conjugates described in (1) to (8) as an active ingredient.

[0027] (10) According to the pharmaceutical composition of (9), the cancer is a cancer that expresses CAPRIN-1 protein on the cell membrane surface.

[0028] (11) The pharmaceutical composition according to (9) or (10), wherein the cancer is breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor, stomach cancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, liver cancer, gallbladder cancer, bile duct cancer, sarcoma, mast cell tumor, melanoma, adrenocortical carcinoma, Ewing's tumor, Hodgkin's lymphoma, mesothelioma, multiple myeloma, testicular cancer, thyroid cancer, head and neck cancer, or urothelial carcinoma.

[0029] (12) A method of treating and / or preventing cancer, comprising administering to a subject the conjugate of any one of (1) to (8), or the pharmaceutical composition of any one of (9) to (11).

[0030] The effects of the invention

[0031] The conjugates of this invention not only exhibit significantly stronger antitumor effects compared to antibodies or fragments targeting the CAPRIN-1 protein alone, but also demonstrate significantly superior antitumor effects compared to conjugates of known cancer antibody drugs and topoisomerase I inhibitors to date. Furthermore, the enhanced antitumor effect resulting from conjugating antibodies or fragments targeting the CAPRIN-1 protein with topoisomerase I inhibitors is also significantly greater than the enhanced antitumor effect achieved by conjugating existing cancer antibody drugs with topoisomerase I inhibitors. Therefore, the conjugates of this invention are effective for the treatment and prevention of cancer. Detailed Implementation

[0032] The antitumor activity of the antibody or fragment thereof against CAPRIN-1 protein (hereinafter referred to as "anti-CAPRIN-1 antibody") used in this invention and the conjugate of topoisomerase I inhibitor is evaluated by investigating the inhibition of tumor proliferation in vitro or in vivo (specifically in cancer-bearing animals).

[0033] In this invention, the "conjugate" is formed by covalently linking an antibody and a drug. The binding of the antibody and the drug can be mediated by a linker.

[0034] The anti-CAPRIN-1 antibody used in this invention can be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. Furthermore, the conjugate of this invention can be any type of antibody as long as it can exert antitumor activity, including recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and non-human animal antibodies.

[0035] The topoisomerase I inhibitor used in this invention is known to be a substance that recognizes a specific DNA sequence and binds to the DNA minor groove, thereby inhibiting cell proliferation by hindering DNA synthesis and thus being toxic to cancer cells.

[0036] Subjects who are the recipients of cancer treatment and / or prevention in this invention are mammals such as humans, pets, livestock, and competitive animals, with humans being the preferred subject.

[0037] The following describes the anti-CAPRIN-1 antibody, topoisomerase I inhibitor, conjugate of anti-CAPRIN-1 antibody and topoisomerase I inhibitor, pharmaceutical composition using the conjugate, and methods for the treatment and / or prevention of cancer involved in this invention.

[0038] <Anti-CAPRIN-1 antibody>

[0039] The anti-CAPRIN-1 antibody used in this invention refers to an antibody or fragment thereof that is immunoreactive to the full length or a fragment of the CAPRIN-1 protein. Here, "immunoreactive" refers to the property of the antibody specifically binding to the CAPRIN-1 protein or a portion of its polypeptide in vivo.

[0040] The CAPRIN-1 protein that is immunoreactive with the anti-CAPRIN-1 antibody used in this invention, and has an amino acid sequence shown in any of the even-numbered sequence numbers 2 to 30, wherein the amino acid sequences shown in sequence numbers 6, 8, 10, 12, and 14 are the amino acid sequences of canine CAPRIN-1 protein, the amino acid sequences shown in sequence numbers 2 and 4 are the amino acid sequences of human CAPRIN-1 protein, the amino acid sequence shown in sequence number 16 is the amino acid sequence of bovine CAPRIN-1 protein, the amino acid sequence shown in sequence number 18 is the amino acid sequence of equine CAPRIN-1 protein, the amino acid sequences shown in sequence numbers 20 to 28 are the amino acid sequences of mouse CAPRIN-1 protein, and the amino acid sequence shown in sequence number 30 is the amino acid sequence of chicken CAPRIN-1 protein.

[0041] Furthermore, the anti-CAPRIN-1 antibody used in this invention can be an antibody that is immunoreactive to a variant of the CAPRIN-1 protein having sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more, with respect to any of the even-numbered sequence numbers in the sequence numbers 2 to 30. Here, "% sequence identity" refers to the percentage (%) of identical amino acids (or bases) relative to the total number of amino acids (or bases) (including the number of gaps) when two sequences are aligned (arranged) in a manner that maximizes similarity (or consistency) with or without a gap.

[0042] The anti-CAPRIN-1 antibody used in this invention can be a monoclonal antibody or a polyclonal antibody.

[0043] Polyclonal antibodies that are immunoreactive to the full length or fragments of CAPRIN-1 protein (anti-CAPRIN-1 polyclonal antibodies) can be obtained by, for example, immunizing mice, mice, rats, rabbits, chickens, etc. with natural CAPRIN-1 protein, fusion proteins with GST, etc., or partial peptides thereof, and then purifying the resulting serum through, for example, ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange columns, affinity columns coupled with CAPRIN-1 protein or partial peptides.

[0044] Regarding the full-length or fragmented CAPRIN-1 protein used in the aforementioned immunization, the base and amino acid sequences of CAPRIN-1 and its homologs can be obtained, for example, by logging into GenBank (NCBI, USA) and using algorithms such as BLAST and FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993; Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997). Furthermore, the method for preparing the CAPRIN-1 protein can be obtained by referring to WO2014 / 012479, or by using cells expressing the CAPRIN-1 protein.

[0045] Monoclonal antibodies that are immunoreactive to the full-length or fragments of the CAPRIN-1 protein (anti-CAPRIN-1 monoclonal antibodies) can be obtained, for example, by immunizing mice with breast cancer cells SK-BR-3 expressing the CAPRIN-1 protein, the full-length or fragments of the CAPRIN-1 protein, etc., and then selecting clones that produce anti-CAPRIN-1 monoclonal antibodies from the resulting fusion cells (hybridomas) isolated from these mice using spleen cells and myeloma cells. The antibodies produced by the selected hybridomas can be obtained using the same purification methods as described above for polyclonal antibodies.

[0046] The antibodies used in this invention include human antibodies, humanized antibodies, chimeric antibodies, and non-human animal antibodies.

[0047] Human antibodies can sensitize human lymphocytes infected with EB virus with proteins, protein-expressing cells or their lysates, and then fuse these sensitized lymphocytes with myeloma cells such as U266 cells derived from humans. Antibodies that are immunoreactive with the full length or fragments of the CAPRIN-1 protein can be obtained from the fused cells.

[0048] Humanized antibodies are modified antibodies, also known as reshaped human antibodies. Humanized antibodies are constructed by transplanting the complementarity-determining region (CDR) of an antibody derived from an immunized animal into the CDR of a human antibody. Genetic recombination, as a general method, is also a well-known technique. Specifically, using PCR, a DNA sequence designed to link the CDR of, for example, a mouse or rabbit antibody to the framework region of a human antibody is synthesized from several oligonucleotides produced with overlapping ends. The resulting DNA is linked to DNA encoding the constant region of the human antibody, then integrated into an expression vector, and introduced into the host to induce antibody production, thus obtaining a humanized antibody (see EP239400, WO96 / 02576). The framework region of the human antibody linked by the CDR can be selected to allow the CDR to form a good antigen-binding site. If necessary, amino acids in the framework region of the antibody's variable region can also be replaced to allow the CDR of the reshaped human antibody to form an appropriate antigen-binding site (Sato K. et al., Cancer Research 1993, 53: 851-856). Alternatively, it can be replaced with a framework region derived from various human antibodies (see WO99 / 51743).

[0049] Antibodies are typically heteropolymeric glycoproteins containing at least two heavy chains and two light chains. An antibody consists of two identical light chains and two identical heavy chains. The heavy chain has a variable region at one end, linked to several constant regions. The light chain has a variable region at one end, also linked to several constant regions. The variable regions exhibit specific variable areas called complementarity-determining regions (CDRs), which impart binding specificity to the antibody. Relatively conserved portions of the variable regions are called framework regions (FRs). The variable regions of both the complete heavy and light chains contain four FRs, each linked by three CDRs (CDR1–CDR3).

[0050] In addition, the sequences of constant and variable regions of human heavy and light chains can be obtained from NCBI (US: GenBank, UniGene, etc.). For example, the heavy chain constant region of human IgG1 can refer to the sequence of accession number J00228, the heavy chain constant region of human IgG2 can refer to the sequence of accession number J00230, the human light chain κ constant region can refer to the sequences of accession numbers V00557, X64135, X64133, etc., and the human light chain λ constant region can refer to the sequences of accession numbers X64132, X64134, etc.

[0051] Chimeric antibodies are antibodies created by combining sequences from different animals. Examples include antibodies containing the heavy and light chain variable regions of mouse antibodies and the constant regions of the heavy and light chain variable regions of human antibodies. Chimeric antibodies can be produced using known methods, such as by linking DNA encoding the V region of an antibody with DNA encoding the C region of a human antibody, integrating this into an expression vector, and introducing it into a host to induce antibody production.

[0052] Non-human animal antibodies can be obtained by sensitizing animals with sensitizing antigens using known methods. A common method is to inject the sensitizing antigen into the peritoneal cavity, intradermal cavity, or subcutaneous cavity of animals such as mice. When injecting the sensitizing antigen, it can be mixed with various adjuvants, such as CFA (Freud's complete adjuvant), and administered to the animal multiple times. After immunizing the animal and confirming the presence of anti-CAPRIN-1 antibodies in the serum, serum is obtained. As mentioned above, antibodies can be obtained through ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange columns, affinity columns coupled with CAPRIN-1 protein or partial peptides, etc. Alternatively, monoclonal antibodies can be obtained from non-human animals by collecting immune cells from immunized animals and fusing them with myeloma cells. The cell fusion of immune cells and myeloma cells can be performed according to known methods (see Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).

[0053] The antibodies used in this invention can also be cloned from hybridoma, integrated into a suitable vector, introduced into a host, and recombinant antibodies produced by the antibody using gene recombination technology (see Carl, AK, Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0054] The anti-CAPRIN-1 antibody used to obtain the conjugate of the present invention can also be an antibody obtained by replacing amino acids in the variable region (e.g., FR) or constant region with other amino acids. The amino acid replacement can be one or more, for example, less than 15, less than 10, 8 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, preferably 1 to 9 amino acids. The replaced antibody, compared to the unreplaced antibody, should have the same or better antigen-specific binding properties and antigen-binding affinity, and should be an antibody that does not cause rejection reactions when used in humans.

[0055] The higher the binding affinity of the anti-CAPRIN-1 antibody used in this invention to the CAPRIN-1 protein on the surface of cancer cells, the stronger the expected anti-tumor effect. The desired binding constant (affinity constant) Ka (kon / koff) is preferably at least 10. 7 M -1 At least 10 8 M -1 At least 5×10 8 M -1 At least 10 9 M -1 At least 5×10 9 M -1 At least 10 10 M -1 At least 5×10 10 M -1 At least 10 11 M -1 At least 5×10 11 M -1 At least 10 12 M -1 or at least 10 13 M -1 .

[0056] The anti-CAPRIN-1 antibody used in this invention enhances its binding affinity to effector cells by replacing one, two, or more amino acids in the constant region of the antibody's heavy chain, or by removing fucose that binds to N-acetylglucosamine in the N-glycosidic bond glycans bound to the constant region of the heavy chain. These replacements can be individual amino acid substitutions or combinations thereof with antibodies bound to fucose.

[0057] Antibodies that replace one, two, or more amino acids in the constant region of the heavy chain can be prepared according to, for example, WO2004 / 063351, WO2011 / 120135, US Patent 8388955, WO2011 / 005481, US Patent 6737056, and WO2005 / 063351.

[0058] Antibodies containing fucose attached to N-acetylglucosamine in the N-glycosidic bond glycan chain of the heavy chain constant region, or cells that produce such antibodies, can be prepared with reference to U.S. Patent No. 6,602,684, European Patent No. 1,914,244, and U.S. Patent No. 7,579,170. Compositions of antibodies containing fucose bound to N-acetylglucosamine in the N-glycosidic bond glycan chain of the heavy chain constant region and fucose-bound antibodies, or cells that produce such compositions, can be prepared with reference to, for example, U.S. Patent No. 8,642,292.

[0059] The anti-CAPRIN-1 polyclonal antibody, anti-CAPRIN-1 monoclonal antibody, antibody preparation method, purification method, and preparation method of CAPRIN-1 protein or its partial polypeptide for immunization used in this invention can be found in WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, and WO201... Obtained under the following numbers: 3 / 018886, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176, and WO2015 / 020212.

[0060] Specific examples of anti-CAPRIN-1 antibodies in this invention include the aforementioned WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, WO2013 / 018892, and WO201... Anti-CAPRIN-1 antibodies described in WO2013 / 018891, WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176 and WO2015 / 020212, but the following antibodies can be listed as preferred anti-CAPRIN-1 antibodies.

[0061] An antibody or fragment thereof that is immunoreactive with a portion of a polypeptide of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in Serial No. 2 or Serial No. 4 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and even more preferably 99% or more) sequence identity with respect to that amino acid sequence.

[0062] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 31 or an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%) sequence identity with respect to that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 36, 37, and 38, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 40, 41, and 42, and is immunoreactive with the CAPRIN-1 protein; or a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 140, 141, and 142, and a light chain variable region comprising sequences 143, 144, and 145, and is immunoreactive with the CAPRIN-1 protein. The antibody or fragment thereof contains a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequence numbers 45, and is immunoreactive to CAPRIN-1 protein; or a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequence numbers 164, 165, and 166, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequence numbers 167, 168, and 169, and is immunoreactive to CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises an amino acid sequence of sequence number 39 in the heavy chain variable region and an amino acid sequence of sequence number 43 in the light chain variable region; or an antibody or fragment thereof comprises an amino acid sequence of sequence number 70 in the heavy chain variable region and an amino acid sequence of sequence number 71 in the light chain variable region; or an antibody or fragment thereof comprises an amino acid sequence of sequence number 78 in the heavy chain variable region and an amino acid sequence of sequence number 79 in the light chain variable region.

[0063] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in Serial No. 33 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of Serial Nos. 60, 61, and 62, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of Serial Nos. 64, 65, and 66, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof whose heavy chain variable region comprises the amino acid sequence of Serial No. 63 and whose light chain variable region comprises the amino acid sequence of Serial No. 67.

[0064] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 32 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 52, 53, and 54, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 56, 57, and 58, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof having a heavy chain variable region comprising the amino acid sequence of sequence number 55 and a light chain variable region comprising the amino acid sequence of sequence number 59.

[0065] An antibody or fragment thereof that is immunoreactive with a portion of a polypeptide of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in Serial No. 34 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more) sequence identity with respect to that amino acid sequence. Preferably, the antibody or fragment thereof contains a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 170, 171, and 172, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 173, 174, and 175, and is immunoreactive to CAPRIN-1 protein; or an antibody or fragment thereof contains a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 176, 177, and 178, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 179, 180, and 181, and is immunoreactive to CAPRIN-1 protein. More preferably, an antibody or fragment thereof having an amino acid sequence of sequence number 80 in the heavy chain variable region and an amino acid sequence of sequence number 81 in the light chain variable region; or an antibody or fragment thereof having an amino acid sequence of sequence number 82 in the heavy chain variable region and an amino acid sequence of sequence number 83 in the light chain variable region.

[0066] An antibody or fragment thereof that is immunoreactive with a portion of a polypeptide of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in Serial No. 35 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more) sequence identity with respect to that amino acid sequence. Preferably, the antibody or fragment thereof contains a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 182, 183, and 184, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 185, 186, and 187, and is immunoreactive to CAPRIN-1 protein; or an antibody or fragment thereof contains a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 188, 189, and 190, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 191, 192, and 193, and is immunoreactive to CAPRIN-1 protein. More preferably, an antibody or fragment thereof having the amino acid sequence of sequence number 84 in the heavy chain variable region and the amino acid sequence of sequence number 85 in the light chain variable region; or an antibody or fragment thereof having the amino acid sequence of sequence number 86 in the heavy chain variable region and the amino acid sequence of sequence number 87 in the light chain variable region.

[0067] An antibody or fragment thereof contains a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 44, 45, and 46, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3) of sequence numbers 48, 49, and 50, and is immunoreactive to CAPRIN-1 protein. Preferably, the antibody or fragment thereof comprises an amino acid sequence of sequence number 47 in the heavy chain variable region and an amino acid sequence of sequence number 51 in the light chain variable region.

[0068] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 296 or an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 146, 147, and 148, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 149, 150, and 151, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof having a heavy chain variable region comprising the amino acid sequence of sequence number 72 and a light chain variable region comprising the amino acid sequence of sequence number 73.

[0069] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 297 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 272, 273, and 274, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 275, 276, and 277, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof containing an amino acid sequence of sequence number 114 in the heavy chain variable region and an amino acid sequence of sequence number 115 in the light chain variable region.

[0070] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 298 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 290, 291, and 292, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 293, 294, and 295, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof containing the amino acid sequence of sequence number 120 in the heavy chain variable region and the amino acid sequence of sequence number 121 in the light chain variable region.

[0071] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 299 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequence numbers 301, 302, and 303, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequence numbers 305, 306, and 307, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof having a heavy chain variable region comprising the amino acid sequence of sequence number 300 and a light chain variable region comprising the amino acid sequence of sequence number 304.

[0072] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 308 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 134, 135, and 136, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 137, 138, and 139, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof having a heavy chain variable region comprising the amino acid sequence of sequence number 68 and a light chain variable region comprising the amino acid sequence of sequence number 69.

[0073] An antibody or fragment thereof that is immunoreactive with a portion of the CAPRIN-1 protein having an amino acid sequence having the amino acid sequence shown in sequence number 309 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more) sequence identity with that amino acid sequence. Preferably, it is an antibody or fragment thereof containing a heavy chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 134, 135, and 136, and a light chain variable region comprising complementarity-determining regions (CDR1, CDR2, and CDR3, respectively) of sequences 137, 138, and 139, and is immunoreactive with the CAPRIN-1 protein. More preferably, it is an antibody or fragment thereof having a heavy chain variable region comprising the amino acid sequence of sequence number 68 and a light chain variable region comprising the amino acid sequence of sequence number 69.

[0074] In addition, the following anti-CAPRIN-1 antibody is preferred.

[0075] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 68 and the light chain variable region containing the amino acid sequence of sequence number 69.

[0076] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 70 and the light chain variable region containing the amino acid sequence of sequence number 71.

[0077] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 72 and the light chain variable region containing the amino acid sequence of sequence number 73.

[0078] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 74 and the light chain variable region containing the amino acid sequence of sequence number 75.

[0079] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 76 and the light chain variable region containing the amino acid sequence of sequence number 77.

[0080] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 78 and the light chain variable region containing the amino acid sequence of sequence number 79.

[0081] An antibody or fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 80 and the light chain variable region containing the amino acid sequence of sequence number 81.

[0082] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 82 and the light chain variable region containing the amino acid sequence of sequence number 83.

[0083] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 84 and the light chain variable region containing the amino acid sequence of sequence number 85.

[0084] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 86 and the light chain variable region containing the amino acid sequence of sequence number 87.

[0085] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 88 and the light chain variable region containing the amino acid sequence of sequence number 89.

[0086] An antibody or fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 90 and the light chain variable region containing the amino acid sequence of sequence number 91.

[0087] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 92 and the light chain variable region containing the amino acid sequence of sequence number 93.

[0088] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 94 and the light chain variable region containing the amino acid sequence of sequence number 95.

[0089] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 96 and the light chain variable region containing the amino acid sequence of sequence number 97.

[0090] An antibody or fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 98 and the light chain variable region containing the amino acid sequence of sequence number 99.

[0091] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 100 and the light chain variable region containing the amino acid sequence of sequence number 101.

[0092] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 102 and the light chain variable region containing the amino acid sequence of sequence number 103.

[0093] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 104 and the light chain variable region containing the amino acid sequence of sequence number 105.

[0094] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 106 and the light chain variable region containing the amino acid sequence of sequence number 107.

[0095] An antibody or fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 108 and the light chain variable region containing the amino acid sequence of sequence number 109.

[0096] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 110 and the light chain variable region containing the amino acid sequence of sequence number 111.

[0097] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 112 and the light chain variable region containing the amino acid sequence of sequence number 113.

[0098] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 114 and the light chain variable region containing the amino acid sequence of sequence number 115.

[0099] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 116 and the light chain variable region containing the amino acid sequence of sequence number 117.

[0100] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 118 and the light chain variable region containing the amino acid sequence of sequence number 119.

[0101] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 120 and the light chain variable region containing the amino acid sequence of sequence number 121.

[0102] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 122 and the light chain variable region containing the amino acid sequence of sequence number 123.

[0103] An antibody or fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 124 and the light chain variable region containing the amino acid sequence of sequence number 125.

[0104] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 126 and the light chain variable region containing the amino acid sequence of sequence number 127.

[0105] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 128 and the light chain variable region containing the amino acid sequence of sequence number 129.

[0106] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 130 and the light chain variable region containing the amino acid sequence of sequence number 131.

[0107] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 132 and the light chain variable region containing the amino acid sequence of sequence number 133.

[0108] An antibody or a fragment thereof having the heavy chain variable region containing the amino acid sequence of sequence number 300 and the light chain variable region containing the amino acid sequence of sequence number 304.

[0109] In the embodiments described later, conjugates with topoisomerase I inhibitors were prepared using the aforementioned polyclonal and monoclonal antibodies targeting the full-length CAPRIN-1 protein and a portion of the polypeptide expressed on the cell membrane surface of cancer cells, and their strong antitumor effects were confirmed.

[0110] <Topoisomerase I inhibitors>

[0111] Topoisomerase I inhibitors are substances that inhibit the action of topoisomerase I. Topoisomerase I reversibly splits the single strand of the DNA double helix and participates in DNA replication, DNA folding, unwinding-rewinding, and transcription. Through the action of topoisomerase I inhibitors, cell death is induced in cancer cells.

[0112] Specific examples of topoisomerase I inhibitors used in this invention include quinoline alkaloids, with camptothecin and its derivatives being representative compounds. Specific examples of camptothecin derivatives include compounds that retain the D-cyclic pyrrolidone, which is the active center of camptothecin.

[0113] In addition, specific examples of topoisomerase I inhibitors include eczemab (DX-8951), eczemab derivatives (eczemab mesylate 2-hydrate (DX-8951f), MAAA-1181a (DXd payload), delutecan, Dxd-d5), SN38 (7-ethyl-10-hydroxycamptothecin), SN38 derivatives (SN38 glucuronide, SN38-COOH, SN38 glucuronide-13C6, SN38-d3, SN38-CM2, SN38-CO-DMEDA TFA, phenol-linked MAC glucuronide, α-hydroxylactone-linked MAC glucuronide, PH-HG-005-5, and Antitumor). Agent (antitumor agent) - F10, TP3011 (CH0793011)), camptothecin, 10-hydroxycamptothecin, 7-n-butyl-10-aminocamptothecin, 7-n-butyl-9-amino-10,11-methylenedioxycamptothecin, T-2513, T-0128, irinotecan (CPT-11), topotecan (also known as noginotecan), letonotecan, ST1481, CKD602, DB67, BNP135, Antitumor agent-102, PNU-159682, 9-aminocamptothecin, acridine, belotecone, BN-80915, diflutamide, edocaline, epirubicin, gematocarbazin, aclarubicin A hydrochloride, becatecarine, β-lapaquinone, camptothecin-20(S)-O-propionic acid, CH-0793076(TP3076) 6-cyclic camptothecin analogue, choline chloride, DRF-1042, gematocarbazin, intecan, LMP-400, intolixin (RP 60475), carlemycin, LMP744 and its hydrochloride, Luotonin A, Luotonin F, Mauritianin, Namitecan (ST-1968), Podocarpusflavone, Proscillaridin, Pyrazoloacridine (NSC 366140), Butylrimycin, Rubitecan, SW044248, TAS-103 (BMS-247615), TAS-103 dihydrochloride (BMS-247615 dihydrochloride), Topoisomerase I inhibitor (CAS No.).210346-40-0), camptothecin derivatives (NSC100880, NSC603071, NSC107124, NSC643833, NSC629971, NSC295500, NSC249910, NSC606985, NSC74028, NSC176323, NSC295501, NSC606172, NSC606173, NSC610458, NSC618939, NSC610457, NSC610459, NSC606499, NSC610456, NSC3648) 30 and NSC606497), Morpholino-isoxorubicin (モルフォリンイソキソルビシン, (7S)-14-(3-aminobicyclo[1.1.1]pentan-1-yl)-7-ethyl-7-hydroxy-2H,10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H,13H)-dione, CT-2106, DX-310, PNU-159682, AZ14170132 or their derivatives.

[0114] Furthermore, the topoisomerase I inhibitor used in this invention can also be a complex compound with a linker. Specific examples of topoisomerase I inhibitors and complex compounds include camptothecin-like substances, such as WO1993 / 018045, WO2000 / 046228, WO2002 / 083682, WO2005 / 040170, WO2005 / 110423, WO2005 / 085251, WO2006 / 135687, WO2010 / 043880, WO2013 / 055990, WO2013 / 055993, WO2014 / 057072, WO2014 / 159981, WO2014 / 057120, and WO2015 / 0525. No. 32, WO2015 / 166289, WO2015 / 181559, WO2017 / 020972, WO2017 / 059289, WO2017 / 137555, WO2017 / 137556, WO2017 / 186894, WO2018 / 031662, WO2018 / 069490, WO2018 / 091646, WO2018 / 146188, WO2018 / 146189, WO2018 / 18234, WO2018 / 192944, WO2019 / 034764, WO2019 / 065964 WO2019 / 096788, WO2019 / 104289, WO2019 / 126691, WO2019 / 224340, WO2020 / 006722, WO2020 / 006722, WO2020 / 079229, WO2020 / 079239, WO2020 / 100954, WO2020 / 141923, WO2020 / 152462, WO2020 / 1964474, WO2020 / 196712, WO2021 / 137646, WO2022 / 063853, WO2021 / 188896 WO2021 / 067403, WO2018 / 217227, WO2018 / 187074, WO2018 / 183041, WO2018 / 156634, WO2018 / 102212, WO2018 / 031408, WO2017 / 189279, WO2021 / 173773, WO2017 / 139623, WO2017 / 034906, WO2017 / 004144, WO2016 / 210108, WO2016 / 201300, WO2016 / 172427, WO2016 / 111751WO2016 / 077061, WO2016 / 057398, WO2016 / 003869, WO2015 / 130416, WO2015 / 069430, WO2015 / 047510, WO2015 / 012904, WO2014 / 165506, WO2014 / 127200, WO2014 / 124227, WO2014 / 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WO2024 / 130161, WO2024 / 133845, WO2024 / 138000, WO2024 / 140443, WO2024 / 140447, WO2024 / 140449, WO2024 / No. 140450, WO2024 / 140933, WO2024 / 148241, WO2024 / 148431, WO2024 / 149345, WO2024 / 151890, WO2024 / 155728, WO2024 / 159067, WO2024 / 159079, WO2024 / 159081, WO2024 / 159088, WO2024 / 159103, WO2024 / 163630, WO2024 / 165045, WO2024 / 173761, WO2024 / 175069, WO2024 / 182311WO2024 / 187006, WO2024 / 189269, WO2024 / 189620, WO2024 / 191826, WO2024 / 193635, WO2024 / 194851, WO2024 / 199337, WO2024 / 205474, WO2024 / 206480, WO2024 / 206669, WO2024 / 208314, WO2024 / 208354, WO2024 / 208359, WO2024 / 210687, WO2024 / 212922, WO2024 / 213119, WO2024 / Nos. 215624, WO2024 / 218697, WO2024 / 225848, WO2024 / 226410, WO2024 / 227119, WO2024 / 227432, WO2024 / 229100, WO2024 / 230646, WO2024 / 230678, WO2024 / 230752, WO2024 / 231929, WO2024 / 231930, WO2024 / 235105, WO2024 / 238454, WO2024 / 239281, WO2024 / 240227, WO2024 / 243545 WO2024 / 244006, WO2024 / 251149, WO2024 / 251743, WO2024 / 255740, WO2024 / 259300, WO2024 / 261742, WO2025 / 012080, WO2025 / 014541, WO2025 / 014830, WO2025 / 016467, WO2025 / 019776, WO2025 / 019780, WO2025 / 019787, WO2025 / 021035, WO2025 / 021152, WO2025 / 027194, WO2025 / No. 029578, WO2025 / 036876, WO2025 / 038395, WO2025 / 038638, WO2005 / 081711, WO2024 / 116094, WO2024 / 023750, WO2023 / 228095, WO2023 / 218378, WO2023 / 209591, WO2023 / 176881, WO2023 / 100829, WO2022 / 102695, WO2022 / 102634, WO2022 / 014698, WO2020 / 240467, WO2020 / 130125WO2020 / 122034, WO2020 / 111234, WO2020 / 059772, WO2020 / 040245, WO2020 / 031936, WO2020 / 027100, WO2020 / 022475, WO2020 / 022363, WO2019 / 230645, WO2019 / 219891, WO2019 / 044947, WO2019 / 039483, WO2018 / 169092, WO2018 / 159582, WO2018 / 110515, WO2018 / 066626, WO2018 / No. 060714, WO2017 / 137503, WO2017 / 056012, WO2016 / 133194, WO2016 / 072440, WO2016 / 023894, WO2015 / 155998, WO2015 / 155976, WO2015 / 115091, WO2015 / 098099, WO2014 / 061277, WO2014 / 057687, WO2013 / 163229, WO2011 / 104981, WO2011 / 038159, WO2009 / 084614, WO2025 / 005240 WO2024 / 248123, WO2024 / 219442, WO2024 / 214685, WO2024 / 210162, WO2024 / 204679, WO2024 / 204629, WO2024 / 190815, WO2024 / 158047, WO2024 / 048490, WO2023 / 219072, WO2023 / 176863, WO2023 / 176862, WO2023 / 167238, WO2023 / 136342, WO2023 / 100829, WO2023 / 074843, WO2023 / No. 068226, WO2022 / 244825, WO2022 / 244815, WO2022 / 244801, WO2022 / 210485, WO2022 / 196719, WO2022 / 191313, WO2022 / 191312, WO2022 / 163846, WO2022 / 102695, WO2022 / 102694, WO2022 / 102652, WO2022 / 065344, WO2021 / 251453, WO2021 / 241730, WO2021 / 200857, WO2021 / 177438WO2021 / 112251, WO2021 / 095838, WO2021 / 079958, WO2021 / 025148, WO2021 / 020282, WO2021 / 006268, WO2020 / 240467, WO2020 / 196712, WO2020 / 196475, WO2020 / 196474, WO2020 / 179859, WO2020 / 153390, WO2020 / 130125, WO2020 / 122034, WO2020 / 116662, WO2020 / 111234, WO2020 / No. 100954, WO2020 / 095922, WO2020 / 095921, WO2020 / 050406, WO2020 / 032153, WO2020 / 013170, WO2020 / 010252, WO2020 / 009156, WO2019 / 245012, WO2019 / 244107, WO2019 / 240164, WO2019 / 235553, WO2019 / 230869, WO2019 / 230645, WO2019 / 219891, WO2019 / 172165, WO2019 / 156115 WO2019 / 070013, WO2019 / 065928, WO2019 / 054427, WO2019 / 049933, WO2019 / 044947, WO2019 / 039483, WO2019 / 023198, WO2019 / 022187, WO2019 / 017338, WO2019 / 009407, WO2019 / 009265, WO2019 / 004487, WO2018 / 235966, WO2018 / 212136, WO2018 / 181656, WO2018 / 159582, WO2018 / No. 147245, WO2018 / 139464, WO2018 / 135556, WO2018 / 135501, WO2018 / 123925, WO2018 / 117244, WO2018 / 117237, WO2018 / 110515, WO2018 / 097166, WO2018 / 097162, WO2018 / 070390, WO2018 / 066492, WO2018 / 047894, WO2018 / 003983, WO2017 / 172764, WO2017 / 137503, WO2017 / 069289WO2017 / 051888, WO2017 / 018499, WO2017 / 010559, WO2017 / 002776, WO2016 / 184909, WO2016 / 171107, WO2016 / 167276, WO2016 / 167236, WO2016 / 148115, WO2016 / 121908, WO2016 / 056673, WO2016 / 052697, WO2016 / 023894, WO2016 / 021674, WO2016 / 017693, WO2016 / 006706, WO2015 / No. 155998, WO2015 / 155976, WO2015 / 146132, WO2015 / 141616, WO2015 / 129812, WO2015 / 125956, WO2015 / 115091, WO2015 / 108175, WO2015 / 098099, WO2015 / 053407, WO2015 / 048802, WO2015 / 048793, WO2015 / 046505, WO2015 / 041318, WO2015 / 019976, WO2015 / 005473, WO2015 / 005253 WO2014 / 140210, WO2014 / 133071, WO2014 / 115797, WO2014 / 069672, WO2014 / 038606, WO2014 / 024914, WO2013 / 172379, WO2013 / 163229, WO2013 / 161895, WO2013 / 160879, WO2013 / 154206, WO2013 / 147213, WO2013 / 147212, WO2013 / 115280, WO2013 / 080934, WO2013 / 027680, WO2013 / No. 002248, WO2012 / 160464, WO2012 / 147713, WO2012 / 121361, WO2012 / 074038, WO2012 / 057288, WO2012 / 045481, WO2012 / 026434, WO2012 / 005378, WO2012 / 002274, WO2011 / 115264, WO2011 / 111824, WO2011 / 102404, WO2011 / 087102, WO2011 / 052715, WO2011 / 013728, WO2011 / 013656WO2010 / 117011, WO2010 / 093055, WO2010 / 082612, WO2010 / 044401, WO2010 / 016490, WO2010 / 001909, WO2009 / 151069, WO2009 / 119537, WO2009 / 075376, WO2009 / 057813, WO2009 / 048072, WO2009 / 034976, WO2009 / 028639, WO2009 / 020093, WO2008 / 072655, WO2008 / 053962, WO2008 / No. 016133, WO2007 / 132949, WO2007 / 129745, WO2024 / 243450, WO2024 / 042497, WO2022 / 056281, WO2021 / 127499, WO2020 / 223538, WO2019 / 246513, WO2019 / 191630, WO2019 / 090090, WO2019 / 090088, WO2019 / 090085, WO2019 / 090082, WO2019 / 090081, WO2019 / 090078, WO2019 / 090076 WO2019 / 090074, WO2019 / 090069, WO2018 / 188047, WO2018 / 107116, WO2018 / 107109, WO2018 / 068283, WO2018 / 068282, WO2018 / 053106, WO2017 / 214462, WO2017 / 214458, WO2017 / 214456, WO2017 / 214339, WO2017 / 214322, WO2017 / 214301, WO2017 / 214282, WO2017 / 214233, WO2017 / No. 205745, WO2017 / 205742, WO2017 / 205738, WO2017 / 201442, WO2017 / 193096, WO2017 / 193063, WO2017 / 193041, WO2017 / 193034, WO2017 / 193030, WO2017 / 184942, WO2017 / 177955, WO2017 / 112829, WO2017 / 112811, WO2017 / 112803, WO2017 / 096163, WO2017 / 095808, WO2017 / 095805WO2017 / 087800, WO2017 / 031458, WO2017 / 011342, WO2016 / 196314, WO2016 / 044770, WO2016 / 004389, WO2015 / 200635, WO2015 / 191934, WO2015 / 143382, WO2015 / 119712, WO2015 / 089075, WO2015 / 085925, WO2015 / 073884, WO2015 / 058173, WO2015 / 051379, WO2015 / 035044, WO2014 / No. 210425, WO2014 / 210209, WO2014 / 206150, WO2014 / 176415, WO2014 / 165143, WO2014 / 165127, WO2014 / 164780, WO2014 / 164771, WO2014 / 160028, WO2014 / 160017, WO2014 / 159999, WO2014 / 158528, WO2014 / 152199, WO2014 / 139328, WO2014 / 145000, WO2014 / 144960, WO2014 / 144935 WO2014 / 144299, WO2014 / 144280, WO2014 / 143765, WO2014 / 116846, WO2014 / 106015, WO2014 / 106001, WO2014 / 105810, WO2014 / 089209, WO2014 / 085654, WO2014 / 071074, WO2014 / 047222, WO2014 / 028381, WO2013 / 188381, WO2013 / 170118, WO2013 / 170115, WO2013 / 170113, WO2013 / No. 170112, WO2013 / 158952, WO2013 / 126656, WO2013 / 102042, WO2013 / 101993, WO2013 / 101972, WO2013 / 097601, WO2013 / 090666, WO2013 / 063095, WO2013 / 059485, WO2013 / 055897, WO2013 / 055895, WO2013 / 013031, WO2012 / 121758, WO2024 / 263630, WO2024 / 184349, WO2023 / 079057WO2023 / 023489, WO2023 / 015278, WO2017 / 015124, WO2017 / 015008, WO2003 / 076567, WO2002 / 090993, WO2002 / 020751, WO2023 / 201185, WO2022 / 232333, WO2022 / 011075, WO2020 / 210384, WO2020 / 181283, WO2020 / 135483, WO2019 / 101183, WO2019 / 101182, WO2019 / 001419, WO2018 / No. 009525, WO2017 / 053864, WO2016 / 127025, WO2016 / 081679, WO2016 / 057500, WO2015 / 061751, WO2015 / 061247, WO2014 / 093230, WO2014 / 085528, WO2014 / 031936, WO2014 / 008312, WO2013 / 096194, WO2013 / 078145, WO2013 / 042006, WO2013 / 040044, WO2013 / 039988, WO2012 / 177892 WO2012 / 170976, WO2012 / 143726, WO2012 / 135113, WO2012 / 061409, WO2012 / 005805, WO2011 / 140325, WO2011 / 140324, WO2011 / 138751, WO2011 / 135376, WO2011 / 098451, WO2011 / 086053, WO2011 / 062965, WO2011 / 060281, WO2010 / 151495, WO2010 / 132725, WO2010 / 077678, WO2010 / No. 054266, WO2010 / 048278, WO2010 / 045580, WO2010 / 038165, WO2010 / 021770, WO2009 / 151495, WO2009 / 140429, WO2009 / 140423, WO2009 / 140421, WO2009 / 137391, WO2009 / 098448, WO2009 / 091550, WO2009 / 055418, WO2009 / 047563, WO2009 / 036082, WO2009 / 016460, WO2008 / 157358WO2008 / 154333, WO2008 / 115478, WO2008 / 094896, WO2008 / 073922, WO2008 / 073921, WO2008 / 060866, WO2008 / 036776, WO2008 / 032162, WO2007 / 132308, WO2007 / 109211, WO2007 / 075567, WO2007 / 048088, WO2007 / 024754, WO2007 / 023382, WO2007 / 000676, WO2006 / 138155, WO2006 / No. 135721, WO2006 / 135629, WO2006 / 117669, WO2006 / 101937, WO2006 / 081327, WO2006 / 076442, WO2006 / 055676, WO2006 / 040646, WO2006 / 031330, WO2005 / 121124, WO2005 / 115145, WO2005 / 113494, WO2005 / 072731, WO2005 / 071096, WO2005 / 009435, WO2004 / 094463, WO2004 / 063334 WO2004 / 048598, WO2003 / 008410, WO2002 / 095000, WO2002 / 060919, WO2002 / 057414, WO2002 / 003997, WO2001 / 064251, WO1999 / 059565, WO2025 / 042953, WO2025 / 031307, WO2025 / 015281, WO2024 / 245382, WO2024 / 231224, WO2024 / 230742, WO2024 / 199462, WO2024 / 199432, WO2024 / No. 197082, WO2024 / 182454, WO2024 / 178155, WO2024 / 165057, WO2024 / 163920, WO2024 / 163630, WO2024 / 160176, WO2024 / 147097, WO2024 / 133763, WO2024 / 102523, WO2024 / 067811, WO2024 / 052333, WO2024 / 047654, WO2024 / 022165, WO2024 / 002330, WO2023 / 280092, WO2023 / 247731WO2023 / 247729, WO2023 / 232144, WO2023 / 225359, WO2023 / 201234, WO2023 / 180485, WO2023 / 180484, WO2023 / 174213, WO2023 / 173074, WO2023 / 143347, WO2023 / 138635, WO2023 / 121447, WO2023 / 118961, WO2023 / 109953, WO2023 / 109836, WO2023 / 107538, WO2023 / 077126, WO2023 / No. 017191, WO2022 / 205111, WO2022 / 187272, WO2022 / 187270, WO2022 / 161385, WO2022 / 105873, WO2022 / 093793, WO2022 / 078566, WO2022 / 068898, WO2019 / 218944, WO2019 / 055525, WO2018 / 095422, WO2017 / 222310, WO2017 / 214151, WO2011 / 073091, WO2008 / 130439, WO2008 / 079972 WO2008 / 079968, WO2008 / 070150, WO2008 / 048375, WO2008 / 045604, WO2007 / 064872, WO2007 / 059202, WO2006 / 064517, WO2005 / 110994, WO2004 / 078747, WO2009 / 079790, WO2009 / 076170, WO2009 / 074677, WO2009 / 070609, WO2009 / 061770, WO2009 / 059201, WO2009 / 055343, WO2009 / No. 054332, WO2009 / 049305, WO2009 / 038779, WO2009 / 037001, WO2009 / 030239, WO2009 / 026446, WO2009 / 026274, WO2009 / 020601, WO2009 / 018092, WO2009 / 012280, WO2009 / 012096, WO2009 / 008992, WO2009 / 005673, WO2009 / 002808, WO2009 / 002807, WO2009 / 002806, WO2009 / 002719WO2008 / 154927, WO2008 / 153207, WO2008 / 141111, WO2008 / 141110, WO2008 / 141107, WO2008 / 134761, WO2008 / 134679, WO2008 / 134088, WO2008 / 133866, WO2008 / 127656, WO2008 / 124735, WO2008 / 121949, WO2008 / 120098, WO2008 / 115556, WO2008 / 115499, WO2008 / 109432, WO2008 / No. 109163, WO2008 / 109154, WO2008 / 106491, WO2008 / 103470, WO2008 / 100922, WO2008 / 098789, WO2008 / 098788, WO2008 / 098138, WO2008 / 094834, WO2008 / 094319, WO2008 / 087643, WO2008 / 087488, WO2008 / 083027, WO2008 / 082887, WO2008 / 082602, WO2008 / 081914, WO2008 / 079735 WO2008 / 079346, WO2008 / 079240, WO2008 / 076278, WO2008 / 076257, WO2008 / 073817, WO2008 / 067027, WO2008 / 064133, WO2008 / 063663, WO2008 / 062466, WO2008 / 051421, WO2008 / 049633, WO2008 / 049105, WO2008 / 045184, WO2008 / 043576, WO2008 / 039254, WO2008 / 034776, WO2008 / No. 020827, WO2008 / 020045, WO2008 / 020024, WO2008 / 014574, WO2008 / 013987, WO2008 / 013840, WO2008 / 012511, WO2008 / 007122, WO2008 / 006883, WO2008 / 006535, WO2008 / 003317, WO2008 / 001115, WO2008 / 001101, WO2007 / 144384, WO2007 / 139569, WO2007 / 134037, WO2007 / 129062WO2007 / 126122, WO2007 / 124383, WO2007 / 123995, WO2007 / 123892, WO2007 / 114549, WO2007 / 113687, WO2007 / 112193, WO2007 / 109184, WO2007 / 107305, WO2007 / 106356, WO2007 / 106192, WO2007 / 105027, WO2007 / 098091, WO2007 / 098090, WO2007 / 098089, WO2007 / 097934, WO2007 / No. 096395, WO2007 / 096393, WO2007 / 095389, WO2007 / 092496, WO2007 / 089864, WO2007 / 080114, WO2007 / 076087, WO2007 / 075572, WO2007 / 067417, WO2007 / 064945, WO2007 / 064759, WO2007 / 062138, WO2007 / 056215, WO2007 / 056214, WO2007 / 056208, WO2007 / 054551, WO2007 / 046893 WO2007 / 041546, WO2007 / 041453, WO2007 / 041397, WO2007 / 039404, WO2007 / 039403, WO2007 / 038658, WO2007 / 027344, WO2007 / 024940, WO2007 / 019678, WO2006 / 136837, WO2006 / 135479, WO2006 / 129163, WO2006 / 125815, WO2006 / 125813, WO2006 / 124021, WO2006 / 121522, WO2006 / No. 121521, WO2006 / 121518, WO2006 / 113679, WO2006 / 112930, WO2006 / 109091, WO2006 / 107903, WO2006 / 107786, WO2006 / 107617, WO2006 / 105979, WO2006 / 105237, WO2006 / 099015, WO2006 / 097474, WO2006 / 088906, WO2006 / 086484, WO2006 / 084904, WO2006 / 084869, WO2006 / 083260WO2006 / 081337, WO2006 / 081331, WO2006 / 079645, WO2006 / 079644, WO2006 / 079478, WO2006 / 078711, WO2006 / 077425, WO2006 / 077424, WO2006 / 075012, WO2006 / 073430, WO2006 / 070024, WO2006 / 070023, WO2006 / 069063, WO2006 / 065780, WO2006 / 065234, WO2006 / 057429, WO2006 / No. 055676, WO2006 / 052767, WO2006 / 048892, WO2006 / 042146, WO2006 / 037230, WO2006 / 034128, WO2006 / 027346, WO2006 / 018182, WO2006 / 015318, WO2006 / 014361, WO2006 / 009805, WO2005 / 123676, WO2005 / 117980, WO2005 / 115391, WO2005 / 112919, WO2005 / 107712, WO2005 / 094830 WO2005 / 094818, WO2005 / 094802, WO2005 / 092097, WO2005 / 087221, WO2005 / 082889, WO2005 / 082422, WO2005 / 082353, WO2005 / 082023, WO2005 / 060663, WO2005 / 052143, WO2005 / 051444, WO2005 / 051452, WO2005 / 051432, WO2005 / 051430, WO2005 / 051229, WO2005 / 049105, WO2005 / No. 049091, WO2005 / 048822, WO2005 / 046747, WO2005 / 046746, WO2005 / 046637, WO2005 / 046599, WO2005 / 045055, WO2005 / 044365, WO2005 / 044302, WO2005 / 019435, WO2005 / 032492, WO2005 / 031002, WO2005 / 028539, WO2005 / 027907, WO2005 / 026328, WO2005 / 025508, WO2005 / 023294WO2005 / 020958, WO2005 / 019247, WO2005 / 018683, WO2005 / 007136, WO2005 / 003172, WO2005 / 003162, WO2005 / 000220, WO2004 / 111603, WO2004 / 110455, WO2004 / 103358, WO2004 / 098569, WO2004 / 096224, WO2004 / 093807, WO2004 / 089423, WO2004 / 089294, WO2004 / 087075, WO2004 / No. 085418, WO2004 / 085386, WO2004 / 080478, WO2004 / 076675, WO2004 / 073656, WO2004 / 072641, WO2004 / 069274, WO2004 / 069201, WO2004 / 060405, WO2004 / 060346, WO2004 / 056862, WO2004 / 054624, WO2004 / 050867, WO2004 / 050041, WO2004 / 043493, WO2004 / 038020, WO2004 / 037751 WO2004 / 032828, WO2004 / 020457, WO2004 / 018000, WO2004 / 014882, WO2004 / 014314, WO2004 / 010900, WO2004 / 009023, WO2004 / 008101, WO2004 / 006847, WO2004 / 004727, WO2003 / 103822, WO2003 / 103596, WO2003 / 098220, WO2003 / 093297, WO2003 / 090785, WO2003 / 090680, WO2003 / No. 089006, WO2003 / 086470, WO2003 / 082301, WO2003 / 080039, WO2003 / 074586, WO2003 / 074551, WO2003 / 070166, WO2003 / 066830, WO2003 / 059988, WO2003 / 057158, WO2003 / 055890, WO2003 / 055866, WO2003 / 055489, WO2003 / 053446, WO2003 / 050295, WO2003 / 044482, WO2003 / 043631WO2003 / 043583, WO2003 / 041643, WO2003 / 037860, WO2003 / 031579, WO2003 / 026577, WO2003 / 024967, WO2003 / 020262, WO2003 / 016500, WO2003 / 015698, WO2003 / 009881, WO2003 / 009777, WO2003 / 000928, WO2002 / 100353, WO2002 / 096367, WO2002 / 092826, WO2002 / 089772, WO2002 / No. 088714, WO2002 / 088327, WO2002 / 085459, WO2002 / 083180, WO2002 / 083067, WO2002 / 080849, WO2002 / 077036, WO2002 / 076486, WO2002 / 076448, WO2002 / 072151, WO2002 / 065136, WO2002 / 058694, WO2002 / 056790, WO2002 / 053138, WO2002 / 051862, WO2002 / 051444, WO2002 / 047611 WO2002 / 045737, WO2002 / 022159, WO2002 / 020500, WO2002 / 013843, WO2002 / 009720, WO2002 / 004409, WO2002 / 000168, WO2001 / 097855, WO2001 / 095945, WO2001 / 093836, WO2001 / 092584, WO2001 / 091807, WO2001 / 087307, WO2001 / 080843, WO2001 / 079187, WO2001 / 074402, WO2001 / No. 074401, WO2001 / 072721, WO2001 / 066708, WO2001 / 064687, WO2001 / 064252, WO2001 / 049284, WO2001 / 049268, WO2001 / 039815, WO2001 / 036007, WO2001 / 028542, WO2001 / 025407, WO2001 / 024763, WO2001 / 018013, WO2001 / 017563, WO2001 / 005437, WO2000 / 073340, WO2000 / 069390WO2000 / 062075, WO2000 / 061597, WO2000 / 050016, WO2000 / 005415, WO1999 / 065515, WO1999 / 049901, WO1999 / 046371, WO1999 / 030684, WO1999 / 029329, WO1999 / 003976, WO1998 / 054966, WO1998 / 0197 Compounds described in WO1997 / 044492, WO1997 / 044336, WO1997 / 044026, WO1997 / 041898, WO1997 / 009998, WO1996 / 010585, WO1996 / 007322, WO1996 / 003984, WO1996 / 001127, WO1992 / 020813, and WO1991 / 016904.

[0115] Specific examples of composite compounds of topoisomerase I inhibitor and linker preferred for use in this invention include CL2A-SN38, CL2-SN38, PAB-Lys(MMT)-oxydiacetamide-PEG8-N3-SN38, MC-SN38, PH-HG-005-5, Mc-VC-PAB-SN38, CL2E-SN38, and CL2E-SN38. TFA, CL2-MMT-SN38, delutec (DXd: a compound with Gly-Gly-Phe-Gly attached as a linker on MAAA-1181a), propargyl-PEG4-GGFG-DXd, DBCO-PEG4-GGFG-Dxd, Br-Val-Ala-NH2-bicyclo[1.1.1]pentane-7-MAD-MDCPT, MC-Gly-Gly-Phe-Gly-(R)-cyclopropane-eclonatecan, M C-Gly-Gly-Phe-Gly-(S)-cyclopropane-ecinotecan, Val-Cit-PAB-ecinotecan, MC-Val-Cit-PAB-ecinotecan, Val-Ala-PABC-ecinotecan, Val-Ala-PABC-ecinotecan trifluoroacetate, MC-VA-PAB-ecinotecan, MC-GGFG-ecinotecan, MC-Gly-Gly-Phe-Gly-cyclobutanecarboxylic acid-ecinotecan, PNU-EDA-Gly5 or their derivatives.

[0116] <Conjugate of anti-CAPRIN-1 antibody and topoisomerase I inhibitor>

[0117] In this invention, the binding mode of the anti-CAPRIN-1 antibody and the topoisomerase I inhibitor in the conjugate of the anti-CAPRIN-1 antibody and the topoisomerase I inhibitor is not particularly limited as long as it can maintain the anti-tumor activity against cancer, but it is preferred to be a binding mode in which a linker structure is formed between the anti-CAPRIN-1 antibody and the topoisomerase I inhibitor.

[0118] Here, the term "connector" refers to a compound capable of binding an anti-CAPRIN-1 antibody to a topoisomerase I inhibitor. Various known connectors can be used, or the topoisomerase I inhibitor side can be chemically modified in a manner similar to that of a connector to bind to the antibody.

[0119] The types of connectors and the details of the connection methods can be based on well-known methods (e.g., see Greg T. Hermanson Bioconjugate Techniques, Third Edition, WO2004 / 010957, WO2014 / 012479).

[0120] In embodiments of the present invention, the anti-CAPRIN-1 antibody, the topoisomerase I inhibitor, and the reactive group attached to the linker are listed below.

[0121] As reactive groups attached to the amino acid sequence of an antibody or to a glycoprotein with modified amino acids, these can include primary amines (ε-amino), carboxyl groups, thiohydroxy groups (thiol groups), carbonyl groups (ketones or aldehydes), and hydroxyl groups, provided they are not specifically chemically modified. Primary amines are located at the N-terminus of polypeptides, on the side chains of lysine residues, and are positively charged under physiological conditions. They are usually located on the outer side of proteins and can therefore be used for binding without altering the protein's structure. Carboxyl groups are located at the C-terminus of polypeptides, on the side chains of aspartic acid and glutamic acid. Thiol groups are located on the side chains of cysteine ​​residues, forming disulfide bonds that maintain the higher-order structure of proteins. Ketones or aldehydes are produced in glycoproteins by oxidizing glycosyl groups with sodium periodate.

[0122] The conjugates of the present invention are prepared by binding an agent to a linker that is bound to the reactive group located on an antibody, binding an antibody to a linker that is bound to an agent, or directly binding an agent to an antibody.

[0123] The following groups can be listed as reactive groups attached as linkers and topoisomerase inhibitors.

[0124] N-hydroxysuccinimide (NHS) ester, imide ester, pentafluorophenyl ester, hydroxymethylphosphine, isothiocyanate, isocyanate, acyl azide, N-hydroxy ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl, carbodiimide, and carboxylic anhydride are reactive groups capable of reacting with amines.

[0125] Carbodiimide, diazonium, diazonium acetyl compounds, and carbonyl diimidazole are reactive groups that can react with carboxyl and amine groups.

[0126] Maleimide, haloacetamide, pyridine disulfide, thiosulfone, vinyl sulfone, haloacetyl, aziridine, acryloyl, and aryl are reactive groups capable of reacting with thiohydroxyl groups.

[0127] Acylhydrazides and alkoxyamines are reactive groups capable of reacting with aldehydes. Epoxy groups, ethylene oxide, carbonyl diimidazole, N,N'-disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, and isocyanates are reactive groups capable of reacting with hydroxyl groups.

[0128] Isocyanates are reactive groups that can react with hydroxyl groups.

[0129] Diazirine, aryl azides, aryl groups, benzophenol, and diazo compounds are photoreactive reactive groups.

[0130] The following connectors are examples of connectors that have the above-mentioned reactive groups.

[0131] As a linker with the same reactive end group, linkers with N-hydroxysuccinimide esters as reactive groups can be used (e.g., disuccinimide glutarate (DSG), disuccinimide octanoate (DSS), bis(sulfosuccinimide) octanoate (BS3), trisuccinimide aminotriacetate (TSAT), PEGylated bis(sulfosuccinimide) octanoate (BS(PEG)5, BS(PEG)9), dithiobis(succinimide propionate) (DSP), 3,3'-dithiobis(sulfosuccinimide propionate) (DTSSP), ethylene glycol bis(succinimide succinate) (EGS), sulfonyl-ethylene glycol bis(succinimide succinate (sulfon-EGS), dimethyl hexamethyleneimine dihydrochloride (DMA), dimethyl heptamethine dihydrochloride (DMP)). Dimethyl octyl diimide dihydrochloride (DMS), 3,3'-dithiodipropylimide dimethyl dihydrochloride (DTBP), 1,5-difluoro-1,5-dinitrobenzene (DFDNB), disuccinimide tartrate (DST), bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone (BSOCOES) and maleimide-based linkers (e.g., bismaleimide ethane (BMOE), 1,4-bismaleimide butane (BMB), bismaleimide hexane (BMH), tris(2-maleimide ethyl)amine (TMEA), 1,8-bismaleimide-(PEG)2 (BM(PEG)2), 1,8-bismaleimide-(PEG)3 (BM(PEG)3), dithiobismaleimide ethane (DTME)).

[0132] As primary linkers with different reactive end groups, linkers with NHS esters and maleimides as reactive groups can be used (e.g., AMAS, BMPS, GMBS, sulfonated-MBS, MBS, sulfonated-MBS, SMCC, sulfonated-SMCC, EMCS, sulfonated-EMCS, SMPB, sulfonated-SMPB, SMPH, LC-SMCC, sulfonated-KMUS, SM(PEG)2, SM(PEG)4, SM(PEG)6, SM(PEG)8, SM(PEG)). 12 SM(PEG) 24Linkers using NHS esters and pyridyl dithiohydroxyl groups as reactants (e.g., SPDP, LC-SPDP, sulfonyl-LC-SPDP, SMPT, (PEG)4SPDP, PEG12-SPDP), linkers using NHS esters and haloacetyl groups as reactants (e.g., SIA, SBAP, SIAB, sulfonyl-SIAB), linkers using NHS esters and aryl azides as reactants (e.g., ANB-NOS, sulfonyl-SANPAH, ATFB), and linkers using NHS esters and diazirine as reactants (e.g., ...). Examples include SDA, sulfonyl-SDA, LC-SDA, SDAD, sulfonyl-SDAD), linkers with carbodiimide as the reactive group (e.g., DCC, EDC, EDAC, NHS, sulfonyl-NHS), linkers with maleimide and hydrazide as the reactive group (e.g., BMPH, EMCH, MPBH, KMUH), linkers with pyridyl dithiohydroxy and hydrazide as the reactive group (e.g., PDPH), linkers with isocyanate and maleimide as the reactive group (e.g., PMPI), and linkers with NHS ester and psoralen as the reactive group (e.g., SPB).

[0133] Other linkers can be peptide-containing linkers, such as Fmoc(fluorenylmethoxycarbonyl)-Ala-Ala-Asn-PAB, Fmoc-Ala-Ala-Asn(Trt)-PAB, Fmoc-PEG3-Ala-Ala-Asn(Trt)-PAB, Fmoc-PEG4-Ala-Ala-Asn(Trt)-PAB, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Ala-Ala-Asn(Trt)-PAB-PNP, and Fmoc-PE G3-Ala-Ala-Asn(Trt)-PAB-PNP, Azide-PEG4-Ala-Ala-Asn(Trt)-PAB-PNP, Mal-PEG4-Ala-Ala-Asn(Trt)-PAB-PNP, Fmoc-Val-Cit-PAB-OH, Val-Cit-PAB-OH, Fmoc-Val-Cit-PAB-PNP, MC-Val-Cit-PAB, MC-Val-Cit-PAB-PNP, etc.

[0134] Alternatively, Bis-PEG-acid (biscarboxylated polyethylene glycol) and PEG Acid (carboxylated polyethylene glycol) can be used (e.g., Acid-PEG-TEMPO, Amino-PEG-acid, Amino-PEG-CH2COOH, Aminoxy-PEG-acid, Azido-PEG-acid, Carboxy-PEG-sulfonic acid, Fmoc-N-amido-PEG-acid, Fmoc-N-amido-PEG-CH2COOH, Fmoc-aminooxy-PEG-acid, Hydroxy-PEG-acid, Hydroxy-PEG-CH2CO2H, m-PEG-acid, m-PEG-(CH2)3-acid, Methoxytrityl-N-PEG- acid, N-Methyl(methyl)-N-(t-Boc(tert-butyloxycarbonyl))-PEG-acid, Propargyl(propynyl)-PEG-acid, Propargyl-PEG-CH2COOH, Propargyl-PEG-(CH2)3-acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH2COOH, t-Boc-Aminooxy-PEG-acid, Acid-PEG-PFP esters, miscellaneous PEG acids, PEG PFP esters (e.g., Acid-PEG-PFP ester, Bis-PEG-PFP ester), Bis-PEG-NHS, PEG Aldehyde (e.g., m-PEG-aldehyde, m-PEG-benzaldehyde, Ald-PEG-acid, Ald-PEG-amine, Ald-PEG-azide, Ald-PEG-NH-Boc, Ald-PEG-NHS ester, Ald-PEG-TFP ester, Ald-PEG-t-butyl ester), PEG Tosylate (e.g.,Azido-PEG-Tos (toluenesulfonate), Hydroxy-PEG-Tos, m-PEG-Tos, t-Boc-Aminooxy-PEG-Tos, Trifluoroethyl-PEG-Tos, Tos-PEG-acid, Tos-PEG-CH2COOH, Tos-PEG-alkyne, Tos-PEG-t-butyl ester, Tos-PEG-CH2CO2tBu (tert-butyl), Tos-PEG-Tos, S-acetyl-PEG6-Tos, N-Tos-N-(t-butoxycarbonyl)-animooxy-PEG4-Tos, Ms-PEG-Ms, Ms-PEG-t-butyl (tert-butyl) ester, PEG-Ms, Propargyl-PEG-Ms), Boc-PEG (e.g., Amino-PEG-t-Boc-Hydrazide, Azido-PEG-t-Boc-Hydrazide, Boc-NH-PEG-NH-Boc, Bromoacetamido-PEG-Boc-amine, m-PEG-ONHBoc, Mal-Alkyl-t-Boc- amine, N-Boc-PEG-alcohol (alcoholic hydroxyl group), N-Boc-PEG-bromide (bromide), N-methyl-N-(t-Boc)-PEG-acid, t-Boc-N-amido-PEG-ac id, t-Boc-N-amido-PEG-CH2COOH, t-Boc-N-Amido-PEG-amine, t-Boc-N-amido-PEG-azide, t-Boc-N-amido-PEG-NHS ester, t-Boc-N-amido-PEG-sulfonic acid), PEGNHS ester (e.g., Acid-PEG-NHS ester, Azido-PEG-NHS ester, Bis-PEG-NHS, Fmoc-PEG-NHS ester, m-PEG-NHS ester, m-PEG-NHS Carbonate, Mal-PEG-NHS ester, Propargyl-PEG-NHS ester, t-Boc-N-amido-PEG-NHS ester, t-Butoxycarbonyl-PEG-NHS ester), Fmoc-PEG (e.g.,Fmoc-N-amido-PEG-acid, Fmoc-NH-PEG-CH2COOH, Fmoc-PEG-NHS ester), Biotin PEG (e.g., Biotin PEG-acid, Biotin PEG-alcohol, Biotin PEG-alkyne, Biotin PEG-amine, Biotin PEG-azide, Biotin PEG-DBCO, Biotin PEG-hydrazide, Biotin-PEG-Mal, Biotin-PEG-NHS, Biotin-EDA-PEG-NHS, Biotin-PEG-oxyamine, Biotin-PEG-PFP, Biotin-EDA-PEG-PFP, Biotin-PEG-Tetrazine, Biotin-PEG-TFP, Azide-SS-biotin, Biotin-PEG3-SS-azide, DBCO-S-S-PEG3-Biotin, Dde Biotin-PEG4-Alkyne, Dde Biotin-PEG4-Azide, Dde Biotin-PEG4-DBCO, Diazo Biotin-PEG3-Alkyne, Diazo Biotin-PEG3-Azide, Diazo Biotin-PEG3-DBCO, Diol Biotin-PEG3-Alkyne, Diol Biotin-PEG3-Azide, PC Biotin-PEG3-Alkyne, PC-Biotin-PEG4-PEG4-Alkyne, PC-Biotin-PEG4-PEG4-Alkyne, PC Biotin-PEG3-Azide, PC-Biotin-PEG4-PEG3-Azide, PC-Biotin-PEG4-NHS carbonate, PC DBCO-PEG3-Biotin, WSPCBiotin-PEG3-DBCO, Fmoc-Lys (biotin-PEG)-OH, Fmoc-N-amido-(PEG-biotin)-acid, TAMRA-Azide-PEG-Biotin), PEG Phosphonate, Aminooxy PEG (e.g.,Aminooxy-PEG-acid, Aminooxy-PEG-alcohol, Aminooxy-PEG-azide, Aminooxy-PEG-bromide, Aminooxy-PEG-methane, Aminooxy-PEG-Propargyl, Aminooxy-PEG-t-butyl ester, Aminooxy-PEG-Thiol, Bis-(Aminooxy)-PEG, t-Boc-Aminooxy-PEG-acid, t-Boc-Aminooxy-PEG-alcohol, t-Boc-Aminooxy-PEG-amine, t-Boc-Aminooxy-PEG-Azide, t-Boc-Aminooxy-PEG-Bromide, t-Boc-aminooxy-PEG-Methane, t-Boc-aminooxy-PEG-Propargyl, t-Boc-aminooxy-PEG-S-Ac, t-Boc-Aminooxy-PEG-Thiol, t-Boc-Aminooxy-PEG-Tos, Fmoc-aminooxy-PEG-acid, Trifluoroethyl-PEG-Aminooxy, Alkyne PEG (for example,endo-BCN-PEG, exo-BCN-PEG, Propargyl-PEG-acid, Propargyl-PEG-CH2COOH, Propargyl-PEG-(CH2)3-acid, Propargyl-PEG-(CH2)3-methyl ester, Propargyl-PEG-Acrylate, Propargyl-PEG-alcohol, Propargyl-PEG-amine, Propargyl-PEG-methylamine (methylamine), Aminooxy-PEG-Propargyl, Propargyl-PEG-azide, Propargyl-PEG-bromide, Propargyl-PEG-Maleimide (maleimide), Propargyl-PEG-Ms, Propargyl-PEG-NHS ester, Propargyl-PEG-sulfonic acid, Propargyl-PEG-t-butyl ester, Propargyl-PEG-CH2CO2tBu, Propargyl-PEG-thiol, Propargyl-PEG-5-nitrophenyl carbonate (nitrophenyl carbonate), t-Boc-aminooxy-PEG-Propargyl, Bis-Propargyl-PEG, m-PEG-Propargyl), AzidoPEG (for example,Azido-PEG-acid, Azido-PEG-CH2COOH, Azido-PEG-(CH2)3-methyl ester, Azido-PEG-Acrylate, Azido-PEG-alcohol, Azido-PEG-(CH2)3OH, Azido-PEG-amine, Azido-PEG-azide, Azido-PEG-Maleimide, Azido-PEG-methylamine, Azido-PEG-methylester, Azido-PEG-NHS ester, Azido-PEG-CH2CO2-NHS, Azido-PEG-oxazolidin-2-one, Azido-PEG-PFP ester, Azido-PEG-phosphonic acid, Azido-PEG-phosphonicacid ethyl ester, Azido-PEG-sulfonic acid, Azido-PEG-t-Boc-Hydrazide, Azido-PEG-t-butyl ester, Azido-PEG-CH2CO2-t-butyl ester, Azido-PEG-TFP ester, Azido-PEG-Tos, Aminooxy-PEG-azide, Bromo-PEG-azide, Bromoacetamido-PEG-azide, Carboxyrhodamine 110-PEG-Azide, Isothiocyanato-PEG-Azide, Isothiocyanato-PEG-Azide, m-PEG-azide, Propargyl-PEG-azide, TAMRA-PEG-Azide, t-Boc-N-Amido-PEG-Azide, t-Boc-Aminooxy-PEG-Azide, Thiol-PEG-Azide, Trifluoroethyl-PEG-Azide, Azido-PEG-amino acid, Azido-PEG4-4-nitrophenyl carbonate, S-Acetyl-PEG3-Azido, Azide, Trityl-PEG, 10-Azide), Alkyne PEG, DBCO-PEG, BCN-PEG, Propargyl-PEG, Bis-PEG-acid, Bis-PEG-NHS, Bis-PEG-PFP, Bis-Propargyl-PEG, Amine-PEG-Amine, Azido-PEG-azide, Bromo-PEG, Mal PEG.

[0135] In addition, as another implementation, by using polyethylene glycol (PEG) as described in WO2015 / 057699 and WO2017 / 165851, it is possible to obtain conjugates that are expected to have better pharmacokinetic properties in vivo.

[0136] The linker between the anti-CAPRIN-1 antibody and the topoisomerase I inhibitor can be composed of a single type or multiple types.

[0137] Methods for preparing conjugates of anti-CAPRIN-1 antibodies and topoisomerase I inhibitors include: methods using the ε-amino group of the lysine side chain of the antibody to bind the topoisomerase I inhibitor or a derivative thereof; and methods using a thiohydroxyl group formed by reducing a cysteine ​​residue that forms a disulfide bond in the antibody. Reduction methods and the methods for binding the reduced drug are described in WO2019 / 191630, WO2015 / 031698, WO2017 / 201204, WO2015 / 031541, and WO2015 / 031693.

[0138] When using the ε-amino group of lysine residues in an antibody, an amide bond can be formed, for example, by reacting an active ester (e.g., N-hydroxysuccinimide ester). In this case, the binding reaction proceeds nonspecifically due to the presence of a large number of lysine residues in the antibody.

[0139] When using thiohydroxyl groups that form disulfide bonds on the side chain of cysteine ​​in antibodies, a method can be used to generate thiohydroxyl groups from the disulfide bonds on the antibody using a reducing agent such as mercaptoethanol, allowing them to react with maleimide or α-haloamide. Alternatively, to stabilize the binding via thiohydroxyl groups, methods using, for example, sulfonephenyloxadiazole, 4-cyanoethynyloxy derivatives, etc., are employed. These bindings are more stable over longer periods than those utilizing the conjugated addition reaction of cysteine ​​to maleimide. Furthermore, the imide ring formed by the addition of thiohydroxyl groups to maleimide is opened by hydrolysis, and once an amide bond is formed, stability is increased; therefore, a linker with an amino group near the imide group can also be used. Another method involves forming disulfide bonds from the thiohydroxyl groups of cysteine ​​on the antibody, with a topoisomerase I inhibitor bound between the two thiohydroxyl groups. As an example, a cross-linked binding can be formed using a linker with two disulfide bond sites formed from an amide group having two sulfones at the β-position, or with dibromomaleimide.

[0140] The conjugates of the present invention can be, for example, using the THIOMAB (trademark) technique (see Nature Biotechnology 26, 925-932 (2008)) as a method for introducing a defined number of thiohydroxyl groups at a specific site on an antibody, or using the ThioBridge (trademark) technique (see Methods Mol Biol. 2078: 113-129 (2020)), or using the RESPECT (trademark) technique (see MAbs.9(6): 907-915 (2017)).

[0141] The conjugates of the present invention, for example, can be obtained by reducing an antibody with the reducing agent dithiothreitol (DTT) in phosphate buffer to obtain an antibody with a thiohydroxyl reactive group, thereby forming a conjugate with a topoisomerase I inhibitor or a derivative thereof. In addition to the method using a reducing agent, conjugates can also be obtained by adding a thiohydroxyl group to the primary amine of the lysine residue of the antibody by introducing Traut's Reagent (2-iminothione, N-succinimide S-acetylthioacetate (SATA)).

[0142] The amount of thiohydroxyl groups added to the antibody can be quantified, for example, by mixing a sample solution containing 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and SH groups with phosphate buffer (pH 8.0) and distilled water, adding DTNB solution dissolved in phosphate buffer, Good's buffer, or Tris buffer, incubating for a certain period of time, and then measuring the absorbance at 412 nm (see GLEllman, Arch. Biochem. Biophys., 82, 70 (1959)).

[0143] The added thiohydroxyl groups, which are added after the disulfide bonds of the antibody are cleaved by reduction treatment, are preferably subjected to a treatment to prevent the reformation of disulfide bonds (capping). Capping can be performed using, for example, N-ethylmaleimide (NEM) or 2-iodoacetamide (IAA).

[0144] The formation of conjugates by binding topoisomerase I inhibitors with the thiohydroxyl group added to the antibody can be carried out by known methods. Specifically, for example, binding can be performed by using a linker reagent having a maleimide group or a linker reagent having a bromoacetamide group as a linker reagent that specifically binds to the thiohydroxyl group of the reduced antibody. For example, N-succinimidyl-4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (SMCC) can be used as a linker having a maleimide group. In this case, if an amino group is present on the drug side, an amide bond can be formed with the N-succinimidyl group of the SMCC, thereby obtaining the conjugate.

[0145] As another implementation method, an amide bond can be formed on the amino group present on the drug side using SMCC, and then the thiohydroxy group added to the antibody side can be reacted with the maleimide group of the SMCC on the drug side to obtain a conjugate.

[0146] Alternatively, as another implementation, the conjugate can be formed using two linkers. For example, a substance is prepared by forming an amide bond between the primary amino group present on the lysine residue of the antibody side and the N-succinimide group of SATA (N-succinimide-S-acetylthioacetate). A thiohydroxyl group is added to the antibody, and the SMCC reacts with an agent containing an amino group or having an amino group added according to conventional methods. A substance that forms an amide bond with the N-succinimide group on the SMCC is synthesized. The maleimide group on the SMCC of the drug side reacts with the thiohydroxyl group on the SATA bound to the antibody, thereby obtaining the conjugate.

[0147] As another embodiment, a method using, for example, maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PAB) as a linker in the preparation of conjugates of antibodies with topoisomerase I inhibitors or their derivatives can be cited. mc-val-Cit-PAB (mc-vc-PAB) is a linker capable of being cleaved by intracellular proteases (e.g., cathepsin B). An antibody dissolved in phosphate buffer is pre-treated with a thiohydroxyl group such as DTT. Alternatively, a topoisomerase I inhibitor or its derivative having an amino group is reacted with the benzyloxycarbonyl (PAB) group on mc-val-Cit-PAB to prepare a topoisomerase I inhibitor or its derivative bound to mc-val-Cit-PAB. This conjugate is then reacted with the aforementioned antibody with the added thiohydroxyl group to obtain the conjugate.

[0148] As a further embodiment, the conjugate is obtained by binding a primary amino group on the lysine residue of the antibody to a SATA and adding a thiohydroxyl group, and by reacting succinimide 3-(2-pyridyl dithio)propionate (SPDP) with the amino group on the pharmaceutical side to form an amide bond with the N-succinimide group on the SPDP.

[0149] As the linker used in this invention, a linker that can be cleaved under intracellular conditions can be used. If such a linker is used, an intracellular topoisomerase I inhibitor or a substance containing an antitumor active agent comprising a portion of the linker is released. Examples include linkers cleaved by intracellular peptidases or proteases. Preferably, linkers cleaved by lysosomal, endosomal, cathepsin B, cathepsin D, or plasminogen cleavage are used. Examples include linkers containing polypeptides (Val-Cit, Phe-Leu, or Gly-Phe-Leu-Gly) that can be cleaved by cathepsin B. More specifically, the linker described in U.S. Patent No. 6,214,345 can be used.

[0150] Furthermore, as another embodiment, a linker having glucuronic acid (preferably β-D-glucuronic acid) as described in, for example, WO2007 / 011968 can be used as a means to improve the blood stability, solubility, metabolism, and binding affinity of the conjugate of the present invention to topoisomerase I inhibitors or their derivatives against CAPRIN-1 antibodies. Alternatively, the means described in WO2013 / 173337, WO2015 / 095755, WO2015 / 123679, and WO2018 / 031690 can be used.

[0151] Furthermore, as another implementation, in order to make the topoisomerase I inhibitor or its derivative specifically bind to the anti-CAPRIN-1 antibody, for example, the means described in WO2006 / 65533 and WO2018 / 160683 can be used.

[0152] In addition, as another embodiment, a conjugate of two or more drugs, including a topoisomerase I inhibitor, with an anti-CAPRIN-1 antibody can be obtained using the method described in WO2018 / 112253.

[0153] To obtain a composition containing the conjugate of the present invention, for example, it can be obtained by separating and extracting a more molecularly oriented peak compared to the antibody before binding the linker, by performing gel filtration chromatography or the like. To determine the quality of the conjugate while maintaining the integrity of the bivalent antibody, for example, the method described in WO2013 / 049410 can be used.

[0154] The number of topoisomerase I inhibitors bound to each antibody molecule in the conjugate of the present invention can be quantified using known methods such as mass spectrometry, ELISA, electrophoresis, and HPLC.

[0155] <Anti-tumor effects of conjugates>

[0156] The conjugates of the present invention exhibit antitumor activity in vitro or in vivo. Antitumor activity refers to the reduction, elimination, inhibition of proliferation, apoptosis, necrosis, or killing of target cancer cells. Therefore, the antitumor effect of the conjugates of the present invention can be determined by investigating their antitumor activity against cancer.

[0157] To determine the antitumor effect in vivo, it can be evaluated by administering the conjugate to an organism with cancer, measuring the size of the tumor after administration, and monitoring the tumor size over time. The antitumor effect of the present invention can also be evaluated by investigating survival rates. Additionally, it can be evaluated by investigating the production capacity of cytokines or chemokines. The antitumor effect of the conjugate of the present invention can be further determined by investigating its ability to prevent cancer, metastasis, or recurrence.

[0158] The higher the binding affinity of the conjugate of the present invention to the CAPRIN-1 protein on the surface of cancer cells, the stronger the anti-tumor effect can be expected. The binding constant (affinity constant) Ka (kon / koff) is preferably at least 10. 7 M -1 At least 10 8 M -1 At least 5×10 8 M -1 At least 10 9 M -1 At least 5×10 9 M -1 At least 10 10 M -1 At least 5×10 10 M -1 At least 10 11 M -1 At least 5×10 11 M -1 At least 10 12 M -1 or at least 10 13 M -1 .

[0159] The ability of the conjugates of the present invention to bind to CAPRIN-1 can be specifically determined using binding detection methods such as surface plasmon resonance (SPR), ELISA, Western blotting, immunofluorescence, and flow cytometry.

[0160] As described above, the conjugate of the present invention exhibits enhanced antitumor efficacy compared to the anti-CAPRIN-1 antibody alone, with an enhancement rate preferably of 30% or more, more preferably 40% or more, further preferably 50% or more, further more preferably 55% or more, further more preferably 60% or more, further more preferably 65% ​​or more, and most preferably 70% or more. The enhancement rate of the antitumor efficacy of the conjugate of the present invention relative to the anti-CAPRIN-1 antibody alone can be calculated by administering an effective amount to cancer-bearing mice under the same conditions and comparing the tumor volume after day 10 following the start of administration.

[0161] <Pharmaceutical compositions, methods of cancer treatment and / or prevention>

[0162] The target of the pharmaceutical composition of the present invention for the treatment and / or prevention of cancer is not particularly limited as long as it is a cancer (cell) expressing CAPRIN-1 protein.

[0163] The terms “tumor” and “cancer” used in this specification refer to malignant neoplasms and are used interchangeably.

[0164] The cancer targeted in this invention can be any cancer that expresses the CAPRIN-1 protein on its cell membrane surface. Preferred cancers include breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor, stomach cancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, liver cancer, gallbladder cancer, sarcoma, mast cell tumor, melanoma, adrenocortical carcinoma, Ewing's tumor, Hodgkin's lymphoma, mesothelioma, multiple myeloma, testicular cancer, thyroid cancer, head and neck cancer, and urothelial carcinoma.

[0165] More specifically, the cancers mentioned include, for example, breast cancer, complex breast cancer, mixed malignant tumors of the breast, intraductal papillary carcinoma, recurrent metastatic breast cancer, lung adenocarcinoma, non-small cell lung cancer (NSCLC), squamous cell non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, gliomas (as neuroepithelial tumors), glioblastomas, neuroblastomas, ependymomas, neuronal tumors, fetal neuroectodermal tumors, schwannomas, neurofibromas, meningiomas, chronic lymphocytic leukemia, lymphomas, and gastrointestinal lymphomas. Gastrointestinal lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor, pancreatic duct cancer, invasive pancreatic duct cancer, adenocarcinoma of pancreas, metastatic adenocarcinoma, acinar cell carcinoma, adenosquamous cell carcinoma, giant cell tumor, intraductal papillary mucinous tumor, mucinous cystadenocarcinoma, pancreatoblastoma, pancreatic head cell tumor, Frants' tumor, serous cystadenocarcinoma, solid papillary carcinoma, gastrinoma, glucagonoma, insulinoma, multiple endocrine adenoma syndrome 1 Wermer syndrome, nonfunctional islet cell tumors, growth hormone-inhibiting tumors, VIP-producing tumors, cervical cancer, endometrial cancer, fibrosarcoma, osteoarthritis, Ewing sarcoma, Wilms' tumor, hepatoblastoma, soft tissue sarcoma, acute leukemia, chronic leukemia, spinal cord tumors, soft tissue malignant tumors, teratoma, head and neck cancer including hypopharyngeal cancer, middle pharyngeal cancer, tongue cancer, upper pharyngeal cancer, oral cancer, lip cancer, sinus cancer, laryngeal cancer, recurrent brain tumors, etc., but not limited to these.

[0166] In addition, the preferred subjects (patients) to be subjects are mammals, such as mammals including primates, pets, livestock, and racing animals, with humans, dogs and cats being particularly preferred.

[0167] When the conjugates used in this invention are used as pharmaceutical compositions, they can be formulated using methods known to those skilled in the art. For example, they can be used non-orally as an injectable form of a sterile solution or suspension of water or a pharmaceutically acceptable liquid other than water. For example, formulation can be carried out by mixing with a suitable pharmacologically acceptable carrier or medium, specifically sterile water and / or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, binder, etc., in a unit dosage form as generally considered necessary for pharmaceutical implementation. The amount of active ingredient in these formulations is such that an appropriate dosage within the indicated range is obtained.

[0168] When using the conjugates of the present invention as pharmaceutical compositions, any salt, surfactant, buffer, sugar, or antifreeze (containing a portion of sugar) may be included, and the composition may be formulated in a freeze-dried state. For example, a pharmaceutical composition containing sucrose, polysorbate 20, polysorbate 80, cyclodextrin, glucose, glycerol, polyethylene glycol, mannitol, sodium chloride, and amino acids, as described in WO2007 / 019232, and including excipients, may be formulated into a pharmaceutical composition solution with a pH of 4 to 8, preferably 4.5 to 7.6.

[0169] Sterile compositions for injection can be formulated using a medium such as distilled water for injection, following standard pharmaceutical preparation practices. As an aqueous solution for injection, it can be mixed, for example, with isotonic solutions containing physiological saline, D-sorbitol, D-mannose, D-mannitol, other excipients, alcohols (specifically, ethanol), polyols (specifically, propylene glycol, polyethylene glycol), nonionic surfactants (specifically, polysorbate 80(TM), HCO-60), benzyl benzoate, benzyl alcohol, and other solubilizers, oily solutions (specifically, sesame oil, soybean oil), buffers (specifically, phosphate buffer, sodium acetate buffer), analgesics (specifically, procaine hydrochloride), stabilizers (specifically, benzyl alcohol, phenol), and antioxidants. The prepared injection solution is typically filled into appropriate ampoules.

[0170] The administration can be oral or non-oral, with non-oral administration being preferred. Specific examples include injectable dosage forms, nasal dosage forms, pulmonary dosage forms, and percutaneous dosage forms. Examples of injectable dosage forms include systemic or local administration via intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intratumoral injection.

[0171] In addition, an appropriate administration method can be selected based on the patient's age, weight, sex, symptoms, etc. The dosage of the drug composition containing an antibody or a polynucleotide encoding an antibody can be selected, for example, from 0.0001 mg to 1000 mg per kg of body weight, such as 0.5 mg, 1 mg, 2 mg, 3 mg, 5 mg, 10 mg, 20 mg, 50 mg, 75 mg, 100 mg, 200 mg, 500 mg, or 1000 mg per kg of body weight. Alternatively, the dosage can be selected from 0.001 to 100,000 mg per patient, but is not necessarily limited to these values.

[0172] The dosage and method of administration vary depending on the patient's weight, age, gender, symptoms, etc., and anyone skilled in the art can make appropriate choices.

[0173] By administering a pharmaceutical composition for the treatment and / or prevention of cancer, comprising the conjugate of the present invention as an active ingredient, to a subject, it is possible to treat and / or prevent the aforementioned cancers that express CAPRIN-1 on the cell membrane surface, preferably breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor, gastric cancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, liver cancer, gallbladder cancer, sarcoma, mast cell tumor, melanoma, adrenocortical carcinoma, Ewing's tumor, Hodgkin's lymphoma, mesothelioma, multiple myeloma, testicular cancer, thyroid cancer, head and neck cancer, and urothelial carcinoma.

[0174] Example

[0175] The present invention will now be described in detail with reference to specific embodiments, but the scope of the present invention is not limited to these specific embodiments.

[0176] (Example 1) Anti-CAPRIN-1 polyclonal antibody

[0177] The anti-CAPRIN-1 polyclonal antibody used in the conjugate of the present invention, which is immunoreactive to the CAPRIN-1 protein, was prepared by mixing 1 mg of recombinant human CAPRIN-1 protein (serial numbers 2 and 4) prepared according to Example 3 of WO2010 / 016526 with an equal volume of Fluorin's incomplete adjuvant (IFA) solution. This mixture was administered subcutaneously to rabbits every two weeks for a total of four administrations. Blood was then collected to obtain antiserum containing the polyclonal antibody. The obtained antiserum was purified using a protein G vector to obtain a polyclonal antibody against the CAPRIN-1 protein (anti-CAPRIN-1 polyclonal antibody #1). Additionally, serum from rabbits not treated with the antigen was purified using the same protein G vector as described above, serving as a rabbit control antibody.

[0178] In addition, using the same method as the above-mentioned method for producing polyclonal antibodies against CAPRIN-1 protein, the following polyclonal antibodies #2 to #6 targeting partial peptides of CAPRIN-1 protein were obtained.

[0179] Anti-CAPRIN-1 polyclonal antibody #2 against a subset of the polypeptides described in WO2011 / 096528 (serial number 31 of this specification).

[0180] Anti-CAPRIN-1 polyclonal antibody #3 against a subset of the polypeptides described in WO2013 / 018894 (serial number 32 in this specification).

[0181] Anti-CAPRIN-1 polyclonal antibody #4 against a subset of the polypeptides described in WO2013 / 125654 (serial number 33 of this specification).

[0182] Anti-CAPRIN-1 polyclonal antibody #5 against a subset of the polypeptides described in WO2011 / 096533 (serial number 34 in this specification).

[0183] Anti-CAPRIN-1 polyclonal antibody #6 against a subset of the polypeptide represented by Serial No. 37 (Serial No. 35 of this specification) as described in WO2011 / 096534.

[0184] (Example 2) Anti-CAPRIN-1 monoclonal antibody

[0185] The anti-CAPRIN-1 monoclonal antibody used in the conjugates of this invention uses the following monoclonal antibody.

[0186] As described in WO2011 / 096528, the anti-CAPRIN-1 monoclonal antibody consists of CDR1-3 of the heavy chain variable region composed of amino acid sequences of sequence numbers 36, 37 and 38, respectively, and CDR1-3 of the light chain variable region composed of sequence numbers 40, 41 and 42, respectively, and contains the amino acid sequence of the heavy chain variable region shown in sequence number 39 and the amino acid sequence of the light chain variable region shown in sequence number 43.

[0187] As described in WO2015 / 020212, the anti-CAPRIN-1 monoclonal antibody consists of CDR1-3 of the heavy chain variable region composed of amino acid sequences of sequence numbers 44, 45 and 46, respectively, and CDR1-3 of the light chain variable region composed of sequence numbers 48, 49 and 50, respectively, and contains the amino acid sequence of the heavy chain variable region shown in sequence number 47 and the amino acid sequence of the light chain variable region shown in sequence number 51.

[0188] As described in WO2011 / 096519, the anti-CAPRIN-1 monoclonal antibody consists of CDR1-3 of the heavy chain variable region composed of amino acid sequences of sequence numbers 52, 53 and 54, respectively, and CDR1-3 of the light chain variable region composed of sequence numbers 56, 57 and 58, respectively, and contains the amino acid sequence of the heavy chain variable region shown in sequence number 55 and the amino acid sequence of the light chain variable region shown in sequence number 59.

[0189] As described in WO2013 / 125654, the anti-CAPRIN-1 monoclonal antibody consists of CDR1-3 of the heavy chain variable region composed of amino acid sequences of sequence numbers 60, 61 and 62, respectively, and CDR1-3 of the light chain variable region composed of sequence numbers 64, 65 and 66, respectively, and contains the amino acid sequence of the heavy chain variable region shown in sequence number 63 and the amino acid sequence of the light chain variable region shown in sequence number 67.

[0190] As described in WO2011 / 096517, the anti-CAPRIN-1 monoclonal antibody comprises the amino acid sequence of the heavy chain variable region shown in sequence number 68 and the amino acid sequence of the light chain variable region shown in sequence number 69.

[0191] The anti-CAPRIN-1 monoclonal antibody described in WO2011 / 096528 comprises the following: an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 70 and the amino acid sequence of the light chain variable region shown in sequence number 71; an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 72 and the amino acid sequence of the light chain variable region shown in sequence number 73; an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 74 and the amino acid sequence of the light chain variable region shown in sequence number 75; an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 76 and the amino acid sequence of the light chain variable region shown in sequence number 77; and an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 78 and the amino acid sequence of the light chain variable region shown in sequence number 79.

[0192] The anti-CAPRIN-1 monoclonal antibody described in WO2011 / 096533 includes an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 80 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 81, and an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 82 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 83.

[0193] The anti-CAPRIN-1 monoclonal antibody described in WO2011 / 096534 includes an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 84 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 85, and an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 86 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 87.

[0194] As described in WO2010 / 016526, this antibody comprises the amino acid sequences of the heavy chain variable region shown in sequence number 88 and the light chain variable region shown in sequence number 89; the amino acid sequences of the heavy chain variable region shown in sequence number 90 and the light chain variable region shown in sequence number 91; the amino acid sequences of the heavy chain variable region shown in sequence number 92 and the light chain variable region shown in sequence number 93; and the amino acid sequence of sequence number 94... Antibodies containing the amino acid sequences of the heavy chain variable region shown in the sequence number 95 and the light chain variable region shown in the sequence number 96 and the light chain variable region shown in the sequence number 97; antibodies containing the amino acid sequences of the heavy chain variable region shown in the sequence number 98 and the light chain variable region shown in the sequence number 99; and antibodies containing the amino acid sequences of the heavy chain variable region shown in the sequence number 100 and the light chain variable region shown in the sequence number 101.

[0195] The anti-CAPRIN-1 monoclonal antibody described in WO2013 / 018894 includes an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 102 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 103, and an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 104 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 105.

[0196] As described in WO2013 / 018892, the anti-CAPRIN-1 monoclonal antibody comprises the amino acid sequence of the heavy chain variable region shown in sequence number 106 and the amino acid sequence of the light chain variable region shown in sequence number 107.

[0197] As described in WO2013 / 018891, the anti-CAPRIN-1 monoclonal antibody comprises the amino acid sequence of the heavy chain variable region shown in sequence number 108 and the amino acid sequence of the light chain variable region shown in sequence number 109.

[0198] As described in WO2013 / 018889, the anti-CAPRIN-1 monoclonal antibody comprises the amino acid sequence of the heavy chain variable region shown in sequence number 110 and the amino acid sequence of the light chain variable region shown in sequence number 111.

[0199] As described in WO2013 / 018883, the anti-CAPRIN-1 monoclonal antibody comprises the amino acid sequence of the heavy chain variable region shown in sequence number 112 and the amino acid sequence of the light chain variable region shown in sequence number 113.

[0200] As described in WO2013 / 125636, the anti-CAPRIN-1 monoclonal antibody comprises the amino acid sequence of the heavy chain variable region shown in sequence number 114 and the amino acid sequence of the light chain variable region shown in sequence number 115.

[0201] The anti-CAPRIN-1 monoclonal antibody described in WO2013 / 125654 includes an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 116 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 117, and an antibody containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 118 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 119.

[0202] As described in WO2013 / 125630, the anti-CAPRIN-1 monoclonal antibody comprises the amino acid sequence of the heavy chain variable region shown in sequence number 120 and the amino acid sequence of the light chain variable region shown in sequence number 121.

[0203] The anti-CAPRIN-1 monoclonal antibody described in WO2015 / 020212 comprises the following: an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 122 and the amino acid sequence of the light chain variable region shown in sequence number 123; an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 124 and the amino acid sequence of the light chain variable region shown in sequence number 125; an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 126 and the amino acid sequence of the light chain variable region shown in sequence number 127; an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 128 and the amino acid sequence of the light chain variable region shown in sequence number 129; an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 130 and the amino acid sequence of the light chain variable region shown in sequence number 131; and an antibody containing the amino acid sequence of the heavy chain variable region shown in sequence number 132 and the amino acid sequence of the light chain variable region shown in sequence number 133.

[0204] Base sequences were designed to express the heavy chain variable region CDR1-3 of the aforementioned anti-CAPRIN-1 monoclonal antibody, which consist of amino acid sequences of sequence numbers 36, 37, and 38, respectively, and whose frame region contains the sequence of the human antibody. These sequences were then inserted into a mammalian expression vector that already contains the heavy chain constant region of human IgG1. Similarly, base sequences were designed to express the light chain variable region CDR1-3, which consist of sequence numbers 40, 41, and 42, respectively, and whose frame region contains the sequence of the human antibody. These sequences were then inserted into a mammalian expression vector that already contains the light chain constant region of human IgG1. The two recombinant expression vectors were introduced into mammalian cells using conventional methods to obtain the culture supernatant of humanized anti-CAPRIN-1 monoclonal antibody #1 (humanized antibody #1), in which the CDR1-3 containing the heavy chain variable region is composed of amino acid sequences of sequence numbers 36, 37, and 38, respectively, and the CDR1-3 containing the light chain variable region is composed of sequence numbers 40, 41, and 42, respectively.

[0205] Similarly, base sequences were designed such that the heavy chain variable regions CDR1-3, which can express the heavy chain variable regions, consist of amino acid sequences of sequence numbers 44, 45, and 46, respectively, and the frame region contains the sequence of human antibody, as shown in sequence number 47. These sequences were then inserted into mammalian expression vectors that already have human IgG1 heavy chain constant regions inserted into them. Similarly, base sequences were designed to express the heavy chain variable region, with CDR1-3 consisting of amino acid sequences of sequence numbers 48, 49, and 50, respectively, and the frame region containing the heavy chain variable region shown in sequence number 51. These sequences were then inserted into mammalian expression vectors that already contained the heavy chain constant region of human IgG1. The two recombinant expression vectors were introduced into mammalian cells using conventional methods to obtain culture supernatants of humanized anti-CAPRIN-1 monoclonal antibody #2 (humanized antibody #2), which contains the heavy chain variable region consisting of CDR1-3 consisting of amino acid sequences of sequence numbers 44, 45, and 46, and the light chain variable region consisting of CDR1-3 consisting of amino acid sequences of sequence numbers 48, 49, and 50, respectively.

[0206] Similarly, the culture supernatant of humanized anti-CAPRIN-1 monoclonal antibody #3 (humanized antibody #3) was obtained, in which the CDR1-3 containing the heavy chain variable region is composed of amino acid sequences of sequence number 52, sequence number 53 and sequence number 54, respectively, and the CDR1-3 containing the light chain variable region is composed of sequence number 56, sequence number 57 and sequence number 58, respectively.

[0207] Similarly, the culture supernatant of humanized anti-CAPRIN-1 monoclonal antibody #4 (humanized antibody #4) was obtained, in which the CDR1-3 containing the heavy chain variable region is composed of amino acid sequences of sequence number 60, sequence number 61 and sequence number 62, respectively, and the CDR1-3 containing the light chain variable region is composed of sequence number 64, sequence number 65 and sequence number 66, respectively.

[0208] Similarly, culture supernatants containing the following humanized anti-CAPRIN-1 monoclonal antibodies #9 to #41 (humanized antibodies #9 to #41) were obtained.

[0209] Humanized anti-CAPRIN-1 monoclonal antibody #9 (humanized antibody #9) contains the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 68 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 69.

[0210] Humanized anti-CAPRIN-1 monoclonal antibody #10 (humanized antibody #10) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 70 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 71.

[0211] Humanized anti-CAPRIN-1 monoclonal antibody #11 (humanized antibody #11) containing the amino acid sequence of the heavy chain variable region shown in sequence number 72 and the amino acid sequence of the light chain variable region shown in sequence number 73.

[0212] Humanized anti-CAPRIN-1 monoclonal antibody #12 (humanized antibody #12) containing the amino acid sequence of the heavy chain variable region shown in sequence number 74 and the amino acid sequence of the light chain variable region shown in sequence number 75.

[0213] Humanized anti-CAPRIN-1 monoclonal antibody #13 (humanized antibody #13) containing the amino acid sequence of the heavy chain variable region shown in sequence number 76 and the amino acid sequence of the light chain variable region shown in sequence number 77.

[0214] Humanized anti-CAPRIN-1 monoclonal antibody #14 (humanized antibody #14) contains the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 78 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 79.

[0215] Humanized anti-CAPRIN-1 monoclonal antibody #15 (humanized antibody #15) containing the amino acid sequence of the heavy chain variable region shown in sequence number 80 and the amino acid sequence of the light chain variable region shown in sequence number 81.

[0216] Humanized anti-CAPRIN-1 monoclonal antibody #16 (humanized antibody #16) containing the amino acid sequence of the heavy chain variable region shown in sequence number 82 and the amino acid sequence of the light chain variable region shown in sequence number 83.

[0217] Humanized monoclonal antibody #17 (humanized antibody #17) contains the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 84 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 85.

[0218] Humanized anti-CAPRIN-1 monoclonal antibody #18 (humanized antibody #18) containing the amino acid sequence of the heavy chain variable region shown in sequence number 86 and the amino acid sequence of the light chain variable region shown in sequence number 87.

[0219] Humanized anti-CAPRIN-1 monoclonal antibody #19 (humanized antibody #19) containing the amino acid sequence of the heavy chain variable region shown in sequence number 88 and the amino acid sequence of the light chain variable region shown in sequence number 89.

[0220] Humanized anti-CAPRIN-1 monoclonal antibody #20 (humanized antibody #20) containing the amino acid sequence of the heavy chain variable region shown in sequence number 90 and the amino acid sequence of the light chain variable region shown in sequence number 91.

[0221] Humanized anti-CAPRIN-1 monoclonal antibody #21 (humanized antibody #21) containing the amino acid sequence of the heavy chain variable region shown in sequence number 92 and the amino acid sequence of the light chain variable region shown in sequence number 93.

[0222] Humanized anti-CAPRIN-1 monoclonal antibody #22 (humanized antibody #22) containing the amino acid sequence of the heavy chain variable region shown in sequence number 94 and the amino acid sequence of the light chain variable region shown in sequence number 95.

[0223] Humanized anti-CAPRIN-1 monoclonal antibody #23 (humanized antibody #23) containing the amino acid sequence of the heavy chain variable region shown in sequence number 96 and the amino acid sequence of the light chain variable region shown in sequence number 97.

[0224] Humanized anti-CAPRIN-1 monoclonal antibody #24 (humanized antibody #24) contains the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 98 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 99.

[0225] Humanized anti-CAPRIN-1 monoclonal antibody #25 (humanized antibody #25) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 100 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 101.

[0226] Humanized anti-CAPRIN-1 monoclonal antibody #26 (humanized antibody #26) containing the amino acid sequence of the heavy chain variable region shown in sequence number 102 and the amino acid sequence of the light chain variable region shown in sequence number 103.

[0227] Humanized anti-CAPRIN-1 monoclonal antibody #27 (humanized antibody #27) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 104 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 105.

[0228] Humanized anti-CAPRIN-1 monoclonal antibody #28 (humanized antibody #28) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 106 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 107.

[0229] Humanized anti-CAPRIN-1 monoclonal antibody #29 (humanized antibody #29) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 108 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 109.

[0230] Humanized anti-CAPRIN-1 monoclonal antibody #30 (humanized antibody #30) containing the amino acid sequence of the heavy chain variable region shown in sequence number 110 and the amino acid sequence of the light chain variable region shown in sequence number 111.

[0231] Humanized anti-CAPRIN-1 monoclonal antibody #31 (humanized antibody #31) containing the amino acid sequence of the heavy chain variable region shown in sequence number 112 and the amino acid sequence of the light chain variable region shown in sequence number 113.

[0232] Humanized anti-CAPRIN-1 monoclonal antibody #32 (humanized antibody #32) containing the amino acid sequence of the heavy chain variable region shown in sequence number 114 and the amino acid sequence of the light chain variable region shown in sequence number 115.

[0233] Humanized anti-CAPRIN-1 monoclonal antibody #33 (humanized antibody #33) containing the amino acid sequence of the heavy chain variable region shown in sequence number 116 and the amino acid sequence of the light chain variable region shown in sequence number 117.

[0234] Humanized anti-CAPRIN-1 monoclonal antibody #34 (humanized antibody #34) containing the amino acid sequence of the heavy chain variable region shown in sequence number 118 and the amino acid sequence of the light chain variable region shown in sequence number 119.

[0235] Humanized anti-CAPRIN-1 monoclonal antibody #35 (humanized antibody #35) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 120 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 121.

[0236] Humanized anti-CAPRIN-1 monoclonal antibody #36 (humanized antibody #36) containing the amino acid sequence of the heavy chain variable region shown in sequence number 122 and the amino acid sequence of the light chain variable region shown in sequence number 123.

[0237] Humanized anti-CAPRIN-1 monoclonal antibody #37 (humanized antibody #37) containing the amino acid sequence of the heavy chain variable region shown in sequence number 124 and the amino acid sequence of the light chain variable region shown in sequence number 125.

[0238] Humanized anti-CAPRIN-1 monoclonal antibody #38 (humanized antibody #38) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 126 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 127.

[0239] Humanized anti-CAPRIN-1 monoclonal antibody #39 (humanized antibody #39) containing the amino acid sequence of the heavy chain variable region shown in sequence number 128 and the amino acid sequence of the light chain variable region shown in sequence number 129.

[0240] Humanized monoclonal antibody #40 (humanized antibody #40) contains the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 130 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 131.

[0241] Humanized anti-CAPRIN-1 monoclonal antibody #41 (humanized antibody #41) containing the amino acid sequence of the heavy chain variable region shown in the amino acid sequence of sequence number 132 and the amino acid sequence of the light chain variable region shown in the amino acid sequence of sequence number 133.

[0242] Furthermore, for the humanized antibody #1 in the aforementioned anti-CAPRIN-1 monoclonal antibody, a base sequence was designed to express the heavy chain variable region, where CDR1-3 consist of amino acid sequences of sequence numbers 36, 37, and 38, respectively, and the frame region contains the sequence of the human antibody. This sequence was then inserted into a mammalian expression vector containing the heavy chain constant region of human IgG1, where the amino acid serine (Ser) at EU label 239 was replaced with aspartic acid (Asp), and the amino acid isoleucine (Ile) at EU label 332 was replaced with glutamic acid (Glu). Additionally, a base sequence was designed to express the light chain variable region, where CDR1-3 consist of amino acid sequences of sequence numbers 40, 41, and 42, respectively, and the frame region contains the sequence of the human antibody. This sequence was then inserted into a mammalian expression vector containing the light chain constant region of human IgG1. The two recombinant expression vectors were introduced into mammalian cells using conventional methods to obtain culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #5 (humanized antibody #5). Humanized antibody #5 contains a full-length heavy chain amino acid sequence consisting of the heavy chain variable region prepared above and the heavy chain constant region of human IgG1, in which serine (Ser) at EU label 239 is replaced with aspartic acid (Asp) and isoleucine (Ile) at EU label 332 is replaced with glutamate (Glu), and a full-length light chain amino acid sequence consisting of the light chain variable region prepared above and the human light chain constant region.

[0243] Similarly, a culture supernatant containing the amino acid sequence of the heavy chain variable region of the humanized antibody #2 prepared above and the amino acid sequence of the light chain variable region of the humanized antibody #2 was obtained.

[0244] Similarly, a culture supernatant containing the amino acid sequence of the heavy chain variable region of the humanized antibody #3 prepared above and the amino acid sequence of the light chain variable region was obtained.

[0245] Similarly, a culture supernatant containing the amino acid sequence of the heavy chain variable region of the humanized antibody #4 prepared above and the amino acid sequence of the light chain variable region was obtained.

[0246] Similarly, culture supernatants containing the amino acid sequences of the heavy chain variable regions and the light chain variable regions of the humanized antibodies #9 to #41 prepared above were obtained.

[0247] The culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibodies #1 to #74 was purified using Hitrap Protein A SepharoseFF (manufactured by GE Health) according to standard methods. The supernatant was then replaced with PBS (-) and filtered through a 0.22 μm filter (manufactured by Mirapore).

[0248] (Example 3) Preparation of a conjugate of anti-CAPRIN-1 antibody and topoisomerase I inhibitor (SN38)

[0249] As a conjugate of anti-CAPRIN-1 antibody and topoisomerase I inhibitor, an anti-CAPRIN-1 and SN38 conjugate (SN38 conjugate) was prepared according to conventional methods.

[0250] The conjugates of anti-CAPRIN-1 polyclonal antibodies #1 to #6 described in Example 1 with SN38 were prepared using conventional methods. A solution was prepared by dissolving the purified anti-CAPRIN-1 polyclonal antibody #1 in PBS(-) at a concentration of 10 mg / ml. The solution containing polyclonal antibody #1 was then conjugated to SN38, one of the topoisomerase I inhibitors, by reducing the anti-CAPRIN-1 polyclonal antibody #1 with TCEP (tris(2-carboxyethyl)phosphine) using conventional methods. Specifically, using the compound CL2A-SN38 (CAS No. 1279680-68-0) with a linker attached to SN38, it was conjugated to anti-CAPRIN-1 polyclonal antibody #1 using conventional methods to obtain a solution containing the anti-CAPRIN-1 polyclonal antibody #1-CL2A-SN38 (SN38 conjugate 1) of the present invention.

[0251] Similarly, solutions containing conjugates using CL2A-SN38 were also obtained for anti-CAPRIN-1 polyclonal antibodies #2 to #6 (the above-mentioned conjugate using anti-CAPRIN-1 polyclonal antibody #2: SN38 conjugate 2; the above-mentioned conjugate using anti-CAPRIN-1 polyclonal antibody #3: SN38 conjugate 3; the above-mentioned conjugate using anti-CAPRIN-1 polyclonal antibody #4: SN38 conjugate 4; the above-mentioned conjugate using anti-CAPRIN-1 polyclonal antibody #5: SN38 conjugate 5; the above-mentioned conjugate using anti-CAPRIN-1 polyclonal antibody #6: SN38 conjugate 6). Furthermore, by the same procedure described above, solutions containing conjugates with CL2A-SN38 (SN38 control conjugate 1) were also obtained for the rabbit control antibody described in Example 1, which does not react with CAPRIN-1 protein.

[0252] In addition, using the same method as described above, a solution containing a conjugate of CL2A-SN38 (SN38 conjugate 10) was obtained using humanized antibody #4, which is described in Example 2 as an anti-CAPRIN-1 monoclonal antibody.

[0253] In addition, a conjugate of humanized antibody #1, which is the anti-CAPRIN-1 monoclonal antibody described in Example 2, and CL2A-SN38 was prepared according to conventional methods. A solution was prepared by dissolving the purified humanized antibody #1 in PBS(-) at a concentration of 10 mg / ml. After replacing the PBS(-) buffer, the solution was filtered through a 0.22 μm sterile filter membrane to obtain a solution containing the humanized antibody #1-CL2A-SN38 (SN38 conjugate 7) of the present invention. Similarly, solutions containing conjugates using SN38 (SN38 conjugate 8, SN38 conjugate 9, SN38 conjugate 11 to SN38 conjugate 80) were also obtained for humanized antibodies #2, #3, #5 to #74.

[0254] In addition, by the same procedure described above, a solution containing a conjugate with an SN38 linker (SN38 control conjugate 2) was also obtained for a human IgG control antibody that does not react with CAPRIN-1 protein using the same method.

[0255] The above conjugates were analyzed using conventional methods to measure the number of SN38 molecules bound to each antibody molecule, with an average of approximately 4 to 5.

[0256] (Example 4) Specific responsiveness of the conjugate to CAPRIN-1 protein and CAPRIN-1-expressing cancer cells

[0257] The specific reactivity of the conjugate (anti-CAPRIN-1 antibody-CL2A-SN38 conjugate) prepared in Example 3 to CAPRIN-1 protein, and whether it showed reactivity on the cell membrane surface of human cancer cells and mouse cancer cells expressing CAPRIN-1 protein, were evaluated.

[0258] The specific reactivity to CAPRIN-1 protein was confirmed using an ELISA method. 100 μL of 1 μg / mL CAPRIN-1 protein solution was added to each well of a 96-well plate and incubated at 4°C for 18 hours. After washing each well three times with PBS-T, 400 μL of 0.5% bovine serum albumin (BSA) solution was added to each well and incubated at room temperature for 3 hours. After removing the solution, each well was washed three times with 400 μL of PBS-T, and 100 μL of a solution containing conjugates 1–6 and control conjugate 1 was added to each well and incubated at room temperature for 2 hours. After washing each well three times with PBS-T, 100 μL of HRP-labeled anti-rabbit IgG antibody diluted 5000 times with PBS was added to each well and incubated at room temperature for 1 hour. After washing the wells three times with PBS-T, 100 μL of TMB substrate solution was added to each well, and the mixture was allowed to stand for 15–30 minutes for colorimetric development. After development, 100 μL of 1N sulfuric acid was added to each well to stop the reaction, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorbance meter. The results confirmed that SN38 conjugates 1–6 had higher absorbance values ​​than the negative control SN38 conjugate 1, and specifically showed a reaction to CAPRIN-1 protein.

[0259] Similarly, the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (SN38 conjugates 7 to SN38 conjugates 80) prepared in Example 3 using humanized antibodies #1 to #74 were tested for specificity against CAPRIN-1 protein using an ELISA assay with HRP-labeled anti-human antibodies. The results confirmed that the absorbance values ​​were higher than those of human IgG (SIGMA) used as a negative control and SN38 control conjugate 2, indicating a specific reaction with CAPRIN-1 protein.

[0260] Next, the reactivity of the cell membrane surface of cancer cells expressing CAPRIN-1 protein was confirmed by flow cytometry. 2 × 10⁶ cells were used. 5Personal breast cancer cells BT-474 (obtained from ATCC) and mouse breast cancer cells 4T1 (obtained from ATCC) were separated by centrifugation in 1.5 ml microcentrifuge tubes. 100 μL of solutions containing conjugates 1–6 and control conjugate 1 were added to separate tubes, and the mixtures were incubated at 4°C for 1 hour. After washing with PBS, Alexa488-labeled anti-rabbit IgG (H+L) diluted 100-fold with PBS(-) containing 0.5% FBS (0.5% FBS-PBS(-)) was added, and the mixtures were incubated at 4°C for 1 hour. After washing with 0.5% FBS-PBS(-), the dead cells were stained with BD Horizon Fixable ViabilityStain (FVS) Reagents (FVS450, ベクトン / ディッキンソンアンドカンパニー) and then the fluorescence intensity was measured using FACSFortessa (trademark). The results confirmed that the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (conjugates 1-6) had higher fluorescence intensity than the control conjugate 1 used as a negative control, indicating a stronger reaction with the cell surface of human breast cancer cells BT474 and mouse breast cancer 4T1 expressing CAPRIN-1 protein.

[0261] Similarly, the specific reactivity of anti-CAPRIN-1 antibody-CL2A-SN38 conjugates using humanized antibodies #1 to #74 against human cancer cells BT474 and mouse cancer cells 4T1, which express CAPRIN-1, was detected using Alexa488-labeled anti-human IgG (H+L) antibody. The results confirmed that the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (SN38 conjugates 7 to SN38 conjugate 80) using humanized antibodies #1 to #80 exhibited higher fluorescence intensity than control conjugate 2 (used as a negative control), indicating a stronger reaction with the cell surface of human breast cancer cells BT474 and mouse breast cancer cells 4T1 expressing CAPRIN-1 protein.

[0262] Similarly, the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (SN38 conjugates 7 to SN38 conjugates 80) prepared in Example 3 using anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74 were confirmed to be responsive to various human and mouse cancer cells. This study targeted human cancer cells for which CAPRIN-1 gene expression and protein expression on the cell membrane surface of cancer cells were confirmed, including breast cancer cells (BT-474), colorectal cancer cells (HT-29, HCT116, and Lovo), lung cancer cells (A549), gastric cancer cells (NCI-N87, NU-GC-3), uterine cancer cells (HEC-1-A), prostate cancer cells (22Rv1), pancreatic cancer cells (Panc10.5), liver cancer cells (HepG2), ovarian cancer cells (MCAS, SKOV3), kidney cancer cells (Caki-2), brain tumor cells (U-87MG), bladder cancer cells (T24, UMUC3), bile duct cancer cells (KKU213), and fibrosarcoma. Tumor cells (HT-1080), esophageal cancer cells (OE33), leukemia cells (OCI-AML5), lymphoma cells (Ramos), gallbladder cancer cells (TGBC14TKB), melanoma cells (Malme-3M), head and neck cancer cells (FaDu), mouse renal cancer cells (Renca) and mouse breast cancer cells (4T1), which confirmed the expression of CAPRIN-1 protein on the cell membrane surface of cancer cells, all showed stronger fluorescence intensity and reacted more strongly with the cell membrane surface of cancer cells expressing CAPRIN-1 protein compared to SN38 control conjugate 1 and SN38 control conjugate 2, which served as negative controls.

[0263] (Example 5) Internalization of SN38 conjugate

[0264] The internalization of the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (SN38 conjugates 1 to SN38 conjugate 80) prepared in Example 3, which used anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74, into cancer cells was confirmed using laser confocal microscopy. The anti-CAPRIN-1 antibody-CL2A-SN38 conjugates were reacted at 100 μg / mL with HT29 cancer cells cultured for 24 hours on a pre-culture plate under ice-cooled conditions for 1 hour, followed by reaction at 37°C and 5% CO2 for 1 to 6 hours. After the reaction, the cells were washed with PBS and fixed at room temperature using PBS containing 4% paraformaldehyde. The fixed cells were washed with PBS and then permeabilized with PBS containing 0.3% Triton-X. After permeabilization, the cells were washed with PBS and blocked with PBS containing 5% BSA. After blocking, the samples were reacted with Alexa488-labeled anti-rabbit IgG secondary antibody or anti-human IgG secondary antibody (A11013, Invitrogen) at a concentration of 10 μg / ml at room temperature for 1 hour. The internalization of the conjugates of this invention into cancer cells was observed using a NiconA1R inverted microscopy after the secondary antibody reaction and washing with PBS.

[0265] The observations showed that the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (SN38 conjugates 1 to SN38 conjugate 80) prepared in Example 3, which used anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74, were clearly present in the cells, indicating that the conjugates of the present invention had undergone internalization. On the other hand, no internalization into cancer cells was observed for the control conjugates 1 and 2 prepared in Example 3.

[0266] (Example 6) Antitumor activity of SN38 conjugate

[0267] The antitumor activity of the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (SN38 conjugate 1 to SN38 conjugate 80) prepared in Example 3 using anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 against cancer cells was evaluated in vitro. The antitumor activity against human cancer cells, including breast cancer cells (BT-474), colorectal cancer cells (HT-29, HCT116), lung cancer cells (NCI-H322, NCI-H2228), gastric cancer cells (NCI-N87, NU-GC-3), uterine cancer cells (Ishikawa, HEC-1-A), pancreatic cancer cells (Panc10.5), ovarian cancer cells (MCAS), head and neck cancer cells (FaDu), bladder cancer cells (UMUC3), prostate cancer cells (22Rv1), liver cancer cells (HepG2), kidney cancer cells (Caki-2), brain tumor cells (U-87MG), cholangiocarcinoma cells (KKU213), fibrosarcoma cells (HT-1080), gallbladder cancer cells (TGBC14TKB), and melanoma cells (Malme-3M), which were confirmed to express CAPRIN-1 protein on the cell membrane surface of cancer cells in Example 4, was evaluated.

[0268] The cancer cells obtained by standard methods were seeded at a rate of 90 μL / well (1–4 × 10⁶ cells per well) in black 96-well plates (96well optical Btm Plt PolymerBase Black w / Lid Cell Culture Sterile PS 165305, Thermo Scientific) capable of cell culture, at a rate of 1–4 × 10⁶ cells per well. 4 Individual samples were incubated for 18–24 hours in a 5% CO2 incubator at 37°C. Then, they were cultured to become 10... -2 μg / mL~10 2 Using a range of μg / mL, 10 μL of the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (SN38 conjugate 1 to SN38 conjugate 80) prepared in Example 3, containing anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74, were added to each well and incubated in a 5% CO2 incubator at 37°C. Cell viability was measured from 3 to 4 days after the start of incubation using the Cell Titer Glow Luminescent Cell Viability Assay (#7573, Promega). 100 μL of substrate was added to each well, and the cells were shaken for 2 minutes using a microplate shaker. After incubation for 10 minutes, the luminescence signal intensity was measured using a SpectraMax (registered trademark) iD3 (MOLECULAR DEVICE) to calculate the ATP content of surviving cells.

[0269] As a result, when the number of cancer cells surviving in the wells without the conjugate was taken as 100%, the number of cancer cells surviving at concentrations above 1 μg / mL using the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates (conjugates 1 to 80) described in this invention, which use anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74, was less than 70%. These results indicate that the anti-CAPRIN-1 antibody-CL2A-SN38 conjugates exhibit antitumor activity against cancer cells.

[0270] (Example 7) Antitumor effect of the conjugate in cancer-bearing mice

[0271] The antitumor effects of SN38 conjugates 1 and SN38 conjugate 7 (both conjugates in which approximately four SN38 molecules are bound per antibody molecule), which were prepared as anti-CAPRIN-1 antibody-CL2A-SN38 conjugates in Example 3, were evaluated in cancer-bearing mice. The antitumor effects of the conjugates of the present invention were studied using SCID mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. For each mouse, 10... 7 FaDu, a personal head and neck cancer cell line, was mixed with Matrigel (SIGMA) and transplanted subcutaneously to create a tumor that had grown to 50mm. 3 The above-mentioned cancer-bearing mice. FaDu expresses CAPRIN-1 protein on the cell membrane surface, as shown in Example 4. SN38 conjugates 7 and 10 bind to FaDu. SN38 conjugates 7 and 10 were administered to FaDu-bearing mice via tail vein at a dose of 20 mg / kg, once a week, for a total of 8 administrations.

[0272] Furthermore, following the method described in Example 3, a conjugate of trastuzumab (Chugai Pharmaceutical Co., Ltd.) as an anti-HER2 antibody and CL2A-SN38 was prepared, with the same number of drug-binding molecules per antibody molecule as SN38 conjugate 1 and SN38 conjugate 7. This conjugate was administered to the aforementioned cancer-bearing mice in the same amount as a control. FaDu expresses the HER2 protein, which is the target antigen of trastuzumab, on the cell membrane surface and specifically binds to the aforementioned conjugate of trastuzumab and topoisomerase I inhibitor.

[0273] The size of the tumor in the cancer-bearing mice was measured over time using vernier calipers. The tumor volume was calculated using the formula: (length of the major axis of the tumor) × (length of the minor axis of the tumor) according to standard methods. 2The result was calculated as ×0.5. The evaluation results showed that, on day 7 of administration, with the tumor volume of cancer-bearing mice administered the unconjugated SN38 anti-CAPRIN-1 antibody (comparative control) as 100%, the tumor volume of cancer-bearing mice administered the above conjugate was less than 60%.

[0274] On the other hand, the tumor volume of mice given the trastuzumab-CL2A-SN38 conjugate was approximately 75% of that of cancer-bearing mice given the unconjugated trastuzumab, taking the tumor volume as 100%.

[0275] The results of this evaluation indicate that the anti-CAPRIN-1 antibody-CL2A-SN38 conjugate has a significantly enhanced anti-tumor effect compared to the unconjugated SN38 anti-CAPRIN-1 antibody (comparison control), but the enhancement of the anti-tumor effect of the trastuzumab-CL2A-SN38 conjugate compared to the unconjugated SN38 trastuzumab is minimal.

[0276] (Example 8) Synthesis of PEG9-EVA-PAB-Ecinotecan

[0277] As an inhibitor of topoisomerase I conjugated to antibodies, a compound with the following chemical structure, using eczema as the antitumor active agent, was synthesized: (S)-1-((2,5-dioxopyrrolidone-1-yl)oxy)-33-(((S)-1-(((S)-1-((4-((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de] ]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamoyl)-1,31-dioxo-4,7,10,13,16,19,22,25,28-nonoxa-32-azahexahexadecane-36-acid (hereinafter referred to as "PEG9-EVA-PAB-Ecinotecan").

[0278]

[0279] Specifically, 4-((S)-2-((S)-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (CAS: 2943010-48-6, 9.2) Dissolve N-(tert-butoxycarbonyl)-L-glutamic acid 5-tert-butyl ester (4.3 mg, 0.0141 mmol) in dimethylformamide (270 μL), then add diethylamine (30 μL) and stir at room temperature for 2 hours. Remove the reactants by vacuum distillation, then add 1-[di(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (5.2 mg, 0.0094 mmol) and diethylamine (30 μL). A solution of diisopropylethylamine (4.7 μL, 0.0273 mmol) dissolved in dimethylformamide (200 μL) was stirred at room temperature for 1 hour. The reaction mixture was then removed by vacuum distillation and purified by silica gel column chromatography. The resulting compound was dissolved in dichloromethane (150 μL), and trifluoroacetic acid (150 μL) was added under ice-cooling. The mixture was then stirred under ice-cooling for 4.5 hours, and the reaction mixture was removed by vacuum distillation. Half of the resulting residue (0.0047 mmol) was added to di(N-succinimide) 4,7,10,13,16,19,22,25,28-nonoxatranecarbamate di(N-succinimide) ester (13.3 mg, 0.0136 mmol). The mixture was prepared by adding dimethylformamide (200 μL) and triethylamine (3.9 μL, 0.0282 mmol) under ice-cooling. The mixture was then stirred at room temperature for 1 hour, and the reaction solution was removed by vacuum distillation. For purification, high-performance liquid chromatography (HPLC) (Nexera Prep, Shimadzu Corporation) was used to obtain PEG9-EVA-PAB-ecetane (1.5 mg).

[0280] (Example 9) Preparation of a conjugate of anti-CAPRIN-1 antibody and topoisomerase I inhibitor (ezetenecan)

[0281] As a conjugate of anti-CAPRIN-1 antibody and topoisomerase I inhibitor, anti-CAPRIN-1 polyclonal antibodies #1 to #6 described in Example 1 and a conjugate of PEG9-EVA-PAB-ecinotecan with ecinotecan as the antitumor active agent described in Example 8 (ecinotecan conjugate) were prepared according to conventional methods.

[0282] A solution was prepared by dissolving the purified anti-CAPRIN-1 polyclonal antibody #1 in PBS(-) at a concentration of 2.5 mg / mL. 78 μL of DMSO solution prepared in the manner described in Example 8 (PEG9-EVA-PAB-ezetene at 10 mM) was added to the solution containing polyclonal antibody #1, and the solution was allowed to stand at room temperature for 1 hour. The reaction solution was then replaced with PBS, desalted twice using a Zeba spincolumn (40K MWCO), and filtered through a 0.22 μm filter to obtain a solution containing the anti-CAPRIN-1 polyclonal antibody #1-PEG9-EVA-PAB-ezetene (ezetene conjugate 1) of the present invention.

[0283] Similarly, solutions containing eccentrica conjugates using PEG9-EVA-PAB-eccentrica were also obtained for anti-CAPRIN-1 polyclonal antibodies #2 to #6 (eccentrica conjugate 2 using anti-CAPRIN-1 polyclonal antibody #2; eccentrica conjugate 3 using anti-CAPRIN-1 polyclonal antibody #3; eccentrica conjugate 4 using anti-CAPRIN-1 polyclonal antibody #4; eccentrica conjugate 5 using anti-CAPRIN-1 polyclonal antibody #5; and eccentrica conjugate 6 using anti-CAPRIN-1 polyclonal antibody #6). Furthermore, by the same procedure described above, solutions containing conjugates with PEG9-EVA-PAB-eccentrica (eccentrica control conjugate 1) were also obtained for the rabbit control antibody described in Example 1, which does not react with CAPRIN-1 protein.

[0284] In addition, using the same method as described above, a solution containing a conjugate of PEG9-EVA-PAB-ecitecan (ecitecan conjugate 10) was obtained using humanized antibody #4, which is described in Example 2 as an anti-CAPRIN-1 monoclonal antibody.

[0285] In addition, a conjugate of humanized antibody #1, which is the anti-CAPRIN-1 monoclonal antibody described in Example 2, and PEG9-EVA-PAB-eciletcan was prepared according to conventional methods. A solution was prepared by dissolving the purified humanized antibody #1 in PBS(-) at a concentration of 10 mg / ml. After replacing the PBS(-) buffer, the solution was filtered through a 0.22 μm sterile filter membrane to obtain a solution containing the humanized antibody #1-PEG9-EVA-PAB-eciletcan (eciletcan conjugate 7). Similarly, solutions containing conjugates using eciletcan (eciletcan conjugate 8, eciletcan conjugate 9, eciletcan conjugate 11 to eciletcan conjugate 80) were also obtained for humanized antibodies #2, #3, and #5 to #74.

[0286] In addition, by the same procedure described above, a solution containing a conjugate with an ixotecan linker (ixotecan control conjugate 2) was also obtained for a human IgG control antibody that does not react with the CAPRIN-1 protein using the same method.

[0287] The number of eczema binding to each antibody molecule was measured using a UV spectrophotometer (NanoDrop One, Thermo Scientifics) according to standard methods, and the average result was about 4 to 5.

[0288] (Example 10) Specific reactivity of eczemaconjugates to CAPRIN-1 protein and CAPRIN-1-expressing cancer cells

[0289] The specific reactivity of the eczetidine conjugate (anti-CAPRIN-1 antibody-PEG9-EVA-PAB-eczetidine conjugate) prepared in Example 8 to CAPRIN-1 protein, and whether it showed reactivity on the cell membrane surface of human cancer cells and mouse cancer cells expressing CAPRIN-1 protein, were evaluated.

[0290] The specific reactivity to CAPRIN-1 protein was confirmed using an ELISA method. 100 μL of 1 μg / mL CAPRIN-1 protein solution was added to each well of a 96-well plate and incubated at 4°C for 18 hours. After washing each well three times with PBS-T, 400 μL of 0.5% bovine serum albumin (BSA) solution was added to each well and incubated at room temperature for 3 hours. After removing the solution, each well was washed three times with 400 μL of PBS-T, and 100 μL of a solution containing eczemaconjugates 1–6 and eczemaconjugate control conjugate 1 was added to each well and incubated at room temperature for 2 hours. After washing each well three times with PBS-T, 100 μL of HRP-labeled anti-rabbit IgG antibody diluted 5000 times with PBS was added to each well and incubated at room temperature for 1 hour. After washing the wells three times with PBS-T, 100 μL of TMB substrate solution was added to each well, and the mixture was allowed to stand for 15–30 minutes for colorimetric development. After development, 100 μL of 1N sulfuric acid was added to each well to stop the reaction, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorbance meter. The results confirmed that eczemaconjugates 1–6 had higher absorbance values ​​than eczemaconjugate control 1 (used as a negative control), and specifically showed a reaction to CAPRIN-1 protein.

[0291] Similarly, the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-eciletane conjugates (eciletane conjugates 7-80) prepared in Example 8 using humanized antibodies #1 to #74 were tested for specificity against CAPRIN-1 protein using an ELISA method with HRP-labeled anti-human antibodies. The results confirmed that the absorbance values ​​were higher than those of human IgG (SIGMA) used as a negative control and eciletane control conjugate 2, indicating a specific reaction with CAPRIN-1 protein.

[0292] Next, the reactivity of the cell membrane surface of cancer cells expressing CAPRIN-1 protein was confirmed by flow cytometry. 2 × 10⁶ cells were used. 5Personal breast cancer cells BT-474 (obtained from ATCC) and mouse breast cancer cells 4T1 (obtained from ATCC) were separated by centrifugation in 1.5 ml microcentrifuge tubes. 100 μL of solutions containing eczemaconjugates 1–6 and eczemaconjugate control conjugate 1 were added to separate tubes, and the mixtures were incubated at 4°C for 1 hour. After washing with PBS, Alexa488-labeled anti-rabbit IgG (H+L) diluted 100-fold with PBS(-) containing 0.5% FBS (0.5% FBS-PBS(-)) was added, and the mixtures were incubated at 4°C for 1 hour. After washing with 0.5% FBS-PBS(-), the dead cells were stained using BD Horizon Fixable Viability Stain (FVS) Reagents (FVS450, BECKON / DIKINGSONANDICANPANI). The fluorescence intensity was then measured using FACSFortessa (trademark). The results confirmed that the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ecilecan conjugates (ecilecan conjugates 1-6) exhibited higher fluorescence intensity compared to the negative control ecilecan control conjugate 1, indicating a stronger reaction with the cell surface of human breast cancer cells BT474 and mouse breast cancer 4T1 expressing CAPRIN-1 protein.

[0293] Similarly, the specific reactivity of anti-CAPRIN-1 antibody-PEG9-EVA-PAB-eciletane conjugates using humanized antibodies #1 to #74 against human cancer cells BT474 and mouse cancer cells 4T1, which express CAPRIN-1, was detected using Alexa488-labeled anti-human IgG (H+L) antibody. The results confirmed that the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-eciletane conjugates (eciletane conjugates 7 to 80) using humanized antibodies #1 to #80 exhibited higher fluorescence intensity than the negative control eciletane control conjugate 2, indicating a stronger reaction with the cell surface of human breast cancer cells BT474 and mouse breast cancer cells 4T1 expressing CAPRIN-1 protein.

[0294] Similarly, the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ecithecan conjugates (ecithecan conjugates 7 to ecithecan conjugates 80) prepared in Example 8 using anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74 were confirmed to be responsive to various human and mouse cancer cells. This study investigated human cancer cells for which CAPRIN-1 gene expression and protein expression on the cell membrane surface of cancer cells were confirmed, including breast cancer cells (BT-474), colorectal cancer cells (HT-29, HCT116, and Lovo), lung cancer cells (A549), gastric cancer cells (NCI-N87, NU-GC-3), uterine cancer cells (HEC-1-A), prostate cancer cells (22Rv1), pancreatic cancer cells (Panc10.5), liver cancer cells (HepG2), ovarian cancer cells (MCAS, SKOV3), kidney cancer cells (Caki-2), brain tumor cells (U-87MG), bladder cancer cells (T24, UMUC3), bile duct cancer cells (KKU213), and fibrosarcoma cells. The anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ecirotecan conjugate prepared in Example 8 showed stronger fluorescence intensity and a stronger reaction with the cell membrane surface of cancer cells expressing CAPRIN-1 protein compared to ecirotecan control conjugate 1 and ecirotecan control conjugate 2, which were used as negative controls. The conjugates included HT-1080, esophageal cancer cells (OE33), leukemia cells (OCI-AML5), lymphoma cells (Ramos), gallbladder cancer cells (TGBC14TKB), melanoma cells (Malme-3M), head and neck cancer cells (FaDu), mouse renal cancer cells (Renca), and mouse breast cancer cells (4T1), all of which were confirmed to express CAPRIN-1 protein on the cell membrane surface of cancer cells.

[0295] (Example 11) Internalization of eczemaconjugate

[0296] The internalization of the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-eciletane conjugates (eciletane conjugates 1 to eciletane conjugate 80) prepared in Example 8, using anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74, into cancer cells was confirmed using laser confocal microscopy. The anti-CAPRIN-1 antibody-CL2A-SN38 conjugate was reacted at 100 μg / mL with HT29 cancer cells cultured for 24 hours on a pre-culture plate under ice-cooled conditions for 1 hour, followed by reaction at 37°C and 5% CO2 for 1 to 6 hours. After the reaction, the cells were washed with PBS and fixed at room temperature using PBS containing 4% paraformaldehyde. The fixed cells were washed with PBS and then permeabilized with PBS containing 0.3% Triton-X. After permeabilization, the cells were washed with PBS and blocked with PBS containing 5% BSA. After blocking, the samples were reacted with Alexa488-labeled anti-rabbit IgG secondary antibody or anti-human IgG secondary antibody (A11013, Invitrogen) at a concentration of 10 μg / ml at room temperature for 1 hour. The internalization of the conjugates of this invention into cancer cells was observed using a NiconA1R inverted microscopy after the secondary antibody reaction and washing with PBS.

[0297] The observations showed that the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-eciletane conjugates (eciletane conjugates 1-80) prepared in Example 8, which used anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74, were clearly present in the cells, indicating that the eciletane conjugates of the present invention had been internalized. On the other hand, no internalization into cancer cells was observed for the eciletane control conjugates 1 and 2 prepared in Example 8.

[0298] (Example 12) Antitumor activity of eczemaconjugate

[0299] The antitumor activity of the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ecithecan conjugates (ecithecan conjugate 1 to ecithecan conjugate 80) prepared in Example 8 using anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 against cancer cells was evaluated in vitro. The antitumor activity against human cancer cells, including breast cancer cells (BT-474), colorectal cancer cells (HT-29, HCT116), lung cancer cells (NCI-H322, NCI-H2228), gastric cancer cells (NCI-N87), uterine cancer cells (Ishikawa, HEC-1-A), pancreatic cancer cells (Panc10.5), ovarian cancer cells (MCAS), head and neck cancer cells (FaDu), gastric cancer cells (NU-GC-3), bladder cancer cells (UMUC3), prostate cancer cells (22Rv1), liver cancer cells (HepG2), kidney cancer cells (Caki-2), brain tumor cells (U-87MG), cholangiocarcinoma cells (KKU213), fibrosarcoma cells (HT-1080), gallbladder cancer cells (TGBC14TKB), and melanoma cells (Malme-3M), which were confirmed to express CAPRIN-1 protein on the cell membrane surface of cancer cells in Example 4, was evaluated.

[0300] The cancer cells obtained by standard methods were seeded at a rate of 90 μL / well (1–4 × 10⁶ cells per well) in black 96-well plates (96well optical Btm Plt PolymerBase Black w / Lid Cell Culture Sterile PS 165305, Thermo Scientific) capable of cell culture, at a rate of 1–4 × 10⁶ cells per well. 4 Individual samples were incubated for 18–24 hours in a 5% CO2 incubator at 37°C. Then, they were cultured to become 10... -3 μg / mL~10 2Using a range of μg / mL, 10 μL of the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-eciletane conjugates (eciletane conjugate 1 to eciletane conjugate 80) prepared in Example 8, containing anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74, was added to each well and incubated in a 5% CO2 incubator at 37°C. Cell viability was measured from 3 to 4 days after the start of incubation using the Cell Titer Glow Luminescent Cell Viability Assay (#7573, Promega). 100 μL of substrate was added to each well, and the cells were shaken for 2 minutes using a microplate shaker. After incubation for 10 minutes, the luminescence signal intensity was measured using a SpectraMax (registered trademark) iD3 (MOLECULARDEVICE) to calculate the ATP content of surviving cells.

[0301] As a result, when the number of cancer cells surviving in the wells without the conjugate was taken as 100%, the number of cancer cells surviving at concentrations above 1 μg / mL using the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ecithecan conjugates (ecithecan conjugate 1 to ecithecan conjugate 80) described in this invention, which use anti-CAPRIN-1 polyclonal antibodies and humanized antibodies #1 to #74, was less than 70%. These results indicate that the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ecithecan conjugates exhibit antitumor activity against cancer cells.

[0302] (Example 13) Antitumor effect of eczema conjugate in cancer-bearing mice

[0303] The antitumor effects of ixenocarbazin-ixenocarbazin conjugates 1 and 10 (both conjugates in which approximately 4 molecules of ixenocarbazin are bound per antibody molecule) prepared in Example 8, as part of the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ixenocarbazin conjugate, were evaluated in cancer-bearing mice. The antitumor effects of the conjugates of the present invention were studied using SCID mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. For each mouse, 10... 7 Personal colorectal cancer cells HT-29 were mixed with Matrigel (Corning Corporation) and transplanted subcutaneously, creating a tumor that grew to 120mm. 3The above-mentioned cancer-bearing mice. HT-29 expresses CAPRIN-1 protein on its cell membrane surface. As shown in Example 4, eczetidine conjugate 1 and eczetidine conjugate 10 bind to HT-29. Ecitidine conjugate 1 and eczetidine conjugate 10 were administered to HT-29-bearing mice via tail vein at a dose of 10 mg / kg, twice a week, for a total of 4 administrations.

[0304] In addition, following the method described in Example 8, a conjugate of trastuzumab (Selleck) as an anti-HER2 antibody with PEG9-EVA-PAB-eciletane was prepared, with the same number of drug binding molecules per antibody molecule (4-5) as eciletane conjugate 1 and eciletane conjugate 10. This conjugate was administered to the aforementioned cancer-bearing mice in the same amount as a comparative control. HT-29 cells express the HER2 protein, which is the target antigen of trastuzumab, on their cell membrane surface and specifically binds to the aforementioned conjugate of trastuzumab and topoisomerase I inhibitor.

[0305] The size of the tumor in the cancer-bearing mice was measured over time using vernier calipers. The tumor volume was calculated using the formula: (length of the major axis of the tumor) × (length of the minor axis of the tumor) according to standard methods. 2 The result was calculated as ×0.5. The evaluation results showed that, on day 10 of the initial administration, with the tumor volume of cancer-bearing mice administered the unconjugated eczema anti-CAPRIN-1 antibody (comparative control) as 100%, the tumor volume of cancer-bearing mice administered the above conjugate was less than 55%.

[0306] On the other hand, the tumor volume of mice given the trastuzumab-PEG9-EVA-PAB-ecienta conjugate was approximately 65% ​​of that of cancer-bearing mice given the unconjugated ecienta conjugate trastuzumab, compared to 100% of the tumor volume of mice given the unconjugated ecienta conjugate trastuzumab.

[0307] The results of this evaluation indicate that the anti-CAPRIN-1 antibody-PEG9-EVA-PAB-ethicane conjugate showed enhanced anti-tumor efficacy compared to the anti-CAPRIN-1 antibody without ethicane (comparative control), but the enhancement of anti-tumor efficacy of the trastuzumab-PEG9-EVA-PAB-ethicane conjugate was weaker compared to the trastuzumab without ethicane.

Claims

1. An antibody or fragment thereof conjugate with a topoisomerase I inhibitor, wherein the antibody or fragment thereof is immunoreactive with CAPRIN-1 protein, wherein the CAPRIN-1 protein has an amino acid sequence represented by any one of the even-numbered sequence numbers from Serial Number 2 to 30, or an amino acid sequence having more than 80% sequence identity with such amino acid sequence.

2. The conjugate according to claim 1, wherein the antibody or its fragment is immunoreactive with a portion of the CAPRIN-1 protein polypeptide, wherein the portion of the CAPRIN-1 protein polypeptide has an amino acid sequence represented by any one of sequence numbers 31-35, 296-299, 308, 309, or an amino acid sequence having more than 80% sequence identity with the amino acid sequence.

3. The conjugate according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

4. The conjugate according to claim 1, wherein the antibody or a fragment thereof is any one of (A) to (M) below. (A) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 36, 37, and 38, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 40, 41, and 42, designated as CDR1, CDR2, and CDR3, respectively. (B) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 44, 45, and 46, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 48, 49, and 50, designated as CDR1, CDR2, and CDR3, respectively. (C) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 52, 53, and 54, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 56, 57, and 58, designated as CDR1, CDR2, and CDR3, respectively. (D) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 60, 61, and 62, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 64, 65, and 66, designated as CDR1, CDR2, and CDR3, respectively. (E) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 170, 171, and 172, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 173, 174, and 175, designated as CDR1, CDR2, and CDR3, respectively. (F) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 176, 177, and 178, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 179, 180, and 181, designated as CDR1, CDR2, and CDR3, respectively. (G) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 182, 183, and 184, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 185, 186, and 187, designated as CDR1, CDR2, and CDR3, respectively. (H) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 188, 189, and 190, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 191, 192, and 193, designated as CDR1, CDR2, and CDR3, respectively. (I) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 146, 147, and 148, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 149, 150, and 151, designated as CDR1, CDR2, and CDR3, respectively. (J) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 272, 273, and 274, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 275, 276, and 277, designated as CDR1, CDR2, and CDR3, respectively. (K) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 290, 291, and 292, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 293, 294, and 295, designated as CDR1, CDR2, and CDR3, respectively. (L) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 301, 302, and 303, designated as CDR1, CDR2, and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 305, 306, and 307, designated as CDR1, CDR2, and CDR3, respectively. (M) An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region and being immunoreactive with CAPRIN-1 protein, wherein the heavy chain variable region comprises complementarity-determining regions of sequence numbers 134, 135 and 136 as CDR1, CDR2 and CDR3, respectively, and the light chain variable region comprises complementarity-determining regions of sequence numbers 137, 138 and 139 as CDR1, CDR2 and CDR3, respectively.

5. The conjugate according to claim 1, wherein the antibody or a fragment thereof is any one of (a) to (a1) below, (a) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 39 and whose light chain variable region contains the amino acid sequence of sequence number 43. (b) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 47 and whose light chain variable region contains the amino acid sequence of sequence number 51. (c) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 55 and whose light chain variable region contains the amino acid sequence of sequence number 59. (d) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 63 and whose light chain variable region contains the amino acid sequence of sequence number 67. (e) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 68 and whose light chain variable region contains the amino acid sequence of sequence number 69. (f) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 70 and whose light chain variable region contains the amino acid sequence of sequence number 71. (g) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 72 and whose light chain variable region contains the amino acid sequence of sequence number 73. (h) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 74 and whose light chain variable region contains the amino acid sequence of sequence number 75. (i) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 76 and whose light chain variable region contains the amino acid sequence of sequence number 77. (j) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 78 and whose light chain variable region contains the amino acid sequence of sequence number 79. (k) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 80 and whose light chain variable region contains the amino acid sequence of sequence number 81. (l) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 82 and whose light chain variable region contains the amino acid sequence of sequence number 83. (m) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 84 and whose light chain variable region contains the amino acid sequence of sequence number 85. (n) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 86 and whose light chain variable region contains the amino acid sequence of sequence number 87. (o) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 88 and whose light chain variable region contains the amino acid sequence of sequence number 89. (p) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 90 and whose light chain variable region contains the amino acid sequence of sequence number 91. (q) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 92 and whose light chain variable region contains the amino acid sequence of sequence number 93. (r) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 94 and whose light chain variable region contains the amino acid sequence of sequence number 95. (s) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 96 and whose light chain variable region contains the amino acid sequence of sequence number 97. (t) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 98 and whose light chain variable region contains the amino acid sequence of sequence number 99. (u) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 100 and whose light chain variable region contains the amino acid sequence of sequence number 101. (v) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 102 and whose light chain variable region contains the amino acid sequence of sequence number 103. (w) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 104 and whose light chain variable region contains the amino acid sequence of sequence number 105. (x) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 106 and whose light chain variable region contains the amino acid sequence of sequence number 107. (y) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 108 and whose light chain variable region contains the amino acid sequence of sequence number 109. (z) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 110 and whose light chain variable region contains the amino acid sequence of sequence number 111. (aa) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 112 and whose light chain variable region contains the amino acid sequence of sequence number 113. (ab) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 114 and whose light chain variable region contains the amino acid sequence of sequence number 115. (ac) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 116 and whose light chain variable region contains the amino acid sequence of sequence number 117. (ad) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 118 and whose light chain variable region contains the amino acid sequence of sequence number 119. (ae) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 120 and whose light chain variable region contains the amino acid sequence of sequence number 121. (af) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 122 and whose light chain variable region contains the amino acid sequence of sequence number 123. (ag) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 124 and whose light chain variable region contains the amino acid sequence of sequence number 125. (ah) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 126 and whose light chain variable region contains the amino acid sequence of sequence number 127. (ai) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 128 and whose light chain variable region contains the amino acid sequence of sequence number 129. (aj) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 130 and whose light chain variable region contains the amino acid sequence of sequence number 131. (ak) An antibody or fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 132 and whose light chain variable region contains the amino acid sequence of sequence number 133. (al) An antibody or a fragment thereof whose heavy chain variable region contains the amino acid sequence of sequence number 300 and whose light chain variable region contains the amino acid sequence of sequence number 304.

6. The conjugate according to claim 1, wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, or a single-chain antibody.

7. The conjugate according to any one of claims 1 to 6, wherein the antibody or a fragment thereof binds to the topoisomerase I inhibitor via a linker.

8. The conjugate according to claim 1, wherein the topoisomerase I inhibitor is eczema, SN38, or a derivative thereof.

9. A pharmaceutical composition for the treatment and / or prevention of cancer, comprising the conjugate of any one of claims 1 to 8 as an active ingredient.

10. The pharmaceutical composition according to claim 9, wherein the cancer is a cancer that expresses CAPRIN-1 protein on the cell membrane surface.

11. The pharmaceutical composition according to claim 10, wherein the cancer is breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor, stomach cancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, liver cancer, gallbladder cancer, bile duct cancer, sarcoma, mast cell tumor, melanoma, adrenocortical carcinoma, Ewing's tumor, Hodgkin's lymphoma, mesothelioma, multiple myeloma, testicular cancer, thyroid cancer, head and neck cancer, or urothelial carcinoma.

12. A method of treating and / or preventing cancer, comprising administering to a subject the conjugate of any one of claims 1 to 8, or the pharmaceutical composition of any one of claims 9 to 11.

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