Pharmaceutical composition for treating and / or preventing cancer

Abstract

A conjugate, in which an antibody or a fragment thereof having immunological reactivity with a CAPRIN-1 protein that has an amino acid sequence represented by any one of the even numbered SEQ ID NOS: 2 to 30 or an amino acid sequence having 80% or more sequence identity with the aforesaid amino acid sequence is linked to a benzodiazepine, is useful as an antibody-drug conjugate (ADC).

Description

Pharmaceutical composition for treating and / or preventing cancer

[0001] The present invention relates to a conjugate of an antibody against CAPRIN-1 protein or a fragment thereof with a benzodiazepine, and to a pharmaceutical use thereof as a therapeutic and / or preventive agent for cancer, etc.

[0002] Various antibody drugs targeting specific antigen proteins on cancer cells have been applied to cancer treatment as cancer therapeutic drugs with few side effects due to their cancer specificity. For example, cytopasmic-activation and proliferation-associated protein 1 (CAPRIN-1) is expressed on the cell membrane surface of many solid cancers, and antibodies against this CAPRIN-1 protein are known to be promising pharmaceuticals for the treatment and / or prevention of cancer (Patent Document 1).

[0003] In addition, some benzodiazepines are known to exhibit antitumor activity by recognizing specific DNA sequences and binding to the minor groove (DNA minor groove), thereby interfering with DNA synthesis and inhibiting cell proliferation; specific examples include pyrrolobenzodiazepines (PBDs), indolinobenzodiazepines (IGNs), and pyridinobenzodiazepines (PDDs).

[0004] In recent years, studies have been made to enhance the efficacy of antibody drugs against cancer, and in particular, the development of antibody-drug conjugates (ADCs), which conjugate antibodies with drugs that have strong cell killing ability directly, has been actively pursued (Non-Patent Documents 1 and 2). There are few successful examples of ADCs using benzodiazepines, but ZYNLONTA is an ADC in which Tesirine (a derivative of SG3249), a PBD derivative, is linked to an antibody against CD19. ＴＭ(loncastuximab tesirine-lpyl) has been approved for use in large B-cell lymphoma, achieving a complete or partial response rate of 48% or more in relapsed / refractory diffuse large B-cell lymphoma (DLBCL). Other drugs that have been developed include robalpituzumab tesirine, in which tesirine is linked to an antibody against DLL-3; vadastuximab talirine (SGN-CD33A), in which talirine is linked to an antibody against CD33; camidanlumab tesirine (Cami), in which tesirine is linked to an antibody against CD25; and SGN-CD70a, in which SGD-1882 is linked to an antibody against CD70a.

[0005] WO2010 / 016526

[0006] Lancet Oncol. 2016;17:e256-62Pharm Res. 2015 Nov;32(11):3526-40

[0007] As mentioned above, ADCs that use benzodiazepines to enhance the efficacy of antibody drugs against cancer have already been put to practical use, but their success has been limited to targeting only a few types of cancer, and their antitumor effects have also been limited.

[0008] Therefore, an object of the present invention is to enhance the antitumor effect of benzodiazepine-based ADCs compared to conventional techniques.

[0009] As a result of intensive research, the present inventors have found that a conjugate of an antibody against the CAPRIN-1 protein and a benzodiazepine exhibits an extremely strong antitumor effect compared to an antibody against the CAPRIN-1 protein or a fragment thereof alone, and further that the antitumor effect enhancement effect when an antibody against the CAPRIN-1 protein or a fragment thereof is conjugated with a benzodiazepine is significantly superior to the antitumor effect enhancement effect when an existing cancer antibody pharmaceutical is conjugated with a benzodiazepine, thereby completing the present invention.

[0010] Specifically, the present invention has the following features (1) to (15): (1) A conjugate comprising an antibody or a fragment thereof immunologically reactive with a CAPRIN-1 protein having an amino acid sequence represented by any of the even-numbered SEQ ID NOs: 2 to 30, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence, and a benzodiazepine. (2) The conjugate according to (1), wherein the antibody or a fragment thereof is immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having an amino acid sequence represented by any of SEQ ID NOs: 31 to 35, 296 to 299, 308, or 309, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence. (3) The conjugate according to claim 1 or 2, wherein the antibody is a monoclonal antibody or a polyclonal antibody. (4) The conjugate according to any one of (1) to (3), wherein the antibody or a fragment thereof is any of the following (A) to (M): (A) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 36, 37, and 38 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 40, 41, and 42 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (B) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 44, 45, and 46 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 48, 49, and 50 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (C) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions (CDR1, CDR2, and CDR3) of SEQ ID NOs: 52, 53, and 54, respectively, and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2, and CDR3) of SEQ ID NOs: 56, 57, and 58, respectively, and having immunological reactivity with a CAPRIN-1 protein.(D) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 60, 61, and 62 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 64, 65, and 66 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (E) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 170, 171, and 172 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 173, 174, and 175 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (F) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 176, 177, and 178 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 179, 180, and 181 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein. (G) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 182, 183, and 184 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 185, 186, and 187 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein. (H) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 188, 189, and 190 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 191, 192, and 193 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein. (I) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 146, 147, and 148 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 149, 150, and 151 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein.(J) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 272, 273, and 274 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 275, 276, and 277 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein. (K) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 290, 291, and 292 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 293, 294, and 295 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein. (L) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 301, 302, and 303 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 305, 306, and 307 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein. (M) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 134, 135, and 136 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 137, 138, and 139 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with CAPRIN-1 protein. (5) The conjugate according to any one of (1) to (4), wherein the antibody or fragment thereof is any of the following (a) to (al): (a) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 43; (b) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 47 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 51; (c) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 55 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 59; (d) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 67.(e) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 68 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 69; (f) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 70 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 71; (g) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 72 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 73; (h) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 74 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 75; (i) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 77; (j) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 78 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 79. (k) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 80 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 81; (l) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 82 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 83; (m) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 84 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 85; (n) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 86 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 87; (o) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 88 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 89; (p) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 91. (q) an antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 92 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 93; (r) an antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 94 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95.(s) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 96 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 97; (t) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 98 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 99; (u) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 100 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 101; (v) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 102 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 103; (w) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 104 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 105; (x) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 106 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 107. (y) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 108 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 109; (z) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 110 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 111; (aa) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 112 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 113; (ab) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 114 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 115; (ac) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 116 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 117; (ad) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 118 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 119. (ae) An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 120 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 121. (af) An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 122 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 123.(ag) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 124 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 125; (ah) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 126 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 127; (ai) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 128 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 129; (aj) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 130 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 131; (ak) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 132 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 133; (al) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 300 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 304. (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. (7) The conjugate according to any one of (1) to (6), wherein the antibody or a fragment thereof and a benzodiazepine are bound via a linker. (8) The conjugate according to any one of (1) to (7), wherein the benzodiazepine is a benzodiazepine having antitumor activity. (9) The conjugate according to any one of (1) to (8), wherein the benzodiazepine having antitumor activity is a pyrrolobenzodiazepine (PBD), an indolinobenzodiazepine (IGN), a pyridinobenzodiazepine (PDD), or an isoquinolizinobenzodiazepine (IQB).(10) The benzodiazepine is selected from the group consisting of DSB-120, SJG-136 (SG2000), DC-81, DSB-120, SJG-136, SG2057, SG2202, SG2285, SGD-1882, SGD-1910, SG3199, SG3249, SG2219, IMGN779, IMGN632, (S)—N-(4-aminophenyl)-4-(4-(4-((2-methoxy-12-oxo-6a ,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide)phenyl)-1-methyl-1H-pyrrole-2-carboxamide, D211, D221, D231, GWL-78, KMR-28-39, or a derivative thereof. (11) The conjugate according to (10), wherein the derivative is Tesirine (a derivative of SG3249), Talirine (a derivative of SGD-1910), SG3364, SG3227, SG3140 (MC-Phe-Lys-PAB-SG2057), SG3170, SG3203 (MC-Phe-Lys-PAB-SG2057), SG3231, SG3400, SG3376, DGN642, DGN549, FGX5-67, FGX-2-62, or FGX11-38. (12) A pharmaceutical composition for treating and / or preventing cancer, comprising the conjugate according to any one of (1) to (11) as an active ingredient. (13) The pharmaceutical composition according to (12), wherein the cancer is a cancer that expresses CAPRIN-1 protein on the cell membrane surface. (14) The pharmaceutical composition according to (12) or (13), wherein the cancer is 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, 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 cancer. (15) A method for treating and / or preventing cancer, comprising administering to a subject the conjugate according to any one of (1) to (11), or the pharmaceutical composition according to any one of (12) to (14).

[0011] The conjugate of the present invention not only exhibits an extremely strong antitumor effect compared to an antibody against the CAPRIN-1 protein or a fragment thereof alone, but also has a significantly superior antitumor effect compared to previously known conjugates of cancer antibody drugs and benzodiazepines. Furthermore, the enhancement of the antitumor effect achieved by conjugating an antibody against the CAPRIN-1 protein or a fragment thereof with a benzodiazepine is significantly superior to the enhancement of the antitumor effect achieved by conjugating an existing cancer antibody drug with a benzodiazepine. Therefore, the conjugate of the present invention is effective in treating and preventing cancer.

[0012] The antitumor activity of a conjugate of an antibody against the CAPRIN-1 protein or a fragment thereof (hereinafter referred to as "anti-CAPRIN-1 antibody") used in the present invention with a benzodiazepine can be evaluated by examining the inhibition of tumor growth in tumor-bearing animals in vivo, as described below.

[0013] In the present invention, the term "conjugate" refers to a compound in which an antibody and a benzodiazepine are linked by a covalent bond. The bond between the antibody and the benzodiazepine may be mediated by a linker.

[0014] The anti-CAPRIN-1 antibody forming the conjugate of the present invention may be a monoclonal or polyclonal antibody, preferably a monoclonal antibody. The conjugate of the present invention may be any type of antibody, including a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, or a non-human animal antibody, as long as it can exhibit antitumor activity.

[0015] The benzodiazepine forming the conjugate according to the present invention is known as one of the substances that is toxic to cancer cells by recognizing a specific DNA sequence and binding to the minor groove (DNA minor groove), thereby interfering with DNA synthesis and inhibiting cell proliferation.

[0016] Furthermore, subjects for cancer treatment and / or prevention in the present invention include mammals such as humans, pet animals, livestock, and sport animals, with humans being the preferred subject.

[0017] The following describes the anti-CAPRIN-1 antibody, benzodiazepine, conjugate of anti-CAPRIN-1 antibody and benzodiazepine, pharmaceutical composition using the conjugate, and method for treating and / or preventing cancer according to the present invention.

[0018] <Anti-CAPRIN-1 Antibody> Among CAPRIN-1 proteins having an amino acid sequence represented by any of the even-numbered SEQ ID NOS: 2 to 30, which are immunologically reactive with the anti-CAPRIN-1 antibody used in the present invention, the amino acid sequences represented by SEQ ID NOS: 6, 8, 10, 12, and 14 are the amino acid sequences of canine CAPRIN-1 protein, the amino acid sequences represented by SEQ ID NOS: 2 and 4 are the amino acid sequences of human CAPRIN-1 protein, the amino acid sequence represented by SEQ ID NOS: 16 is the amino acid sequence of bovine CAPRIN-1 protein, the amino acid sequence represented by SEQ ID NOS: 18 is the amino acid sequence of equine CAPRIN-1 protein, the amino acid sequences represented by SEQ ID NOS: 20 to 28 are the amino acid sequences of mouse CAPRIN-1 protein, and the amino acid sequence represented by SEQ ID NOS: 30 is the amino acid sequence of chicken CAPRIN-1 protein.

[0019] Furthermore, the anti-CAPRIN-1 antibody used in the present invention may be immunologically reactive with a variant of the CAPRIN-1 protein having 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity with the amino acid sequence represented by any of the even-numbered SEQ ID NOs: 2 to 30. As used herein, "% sequence identity" refers to the percentage (%) of identical amino acids (or bases) relative to the total number of amino acids (or bases) when the two sequences are aligned to maximize similarity, with or without introducing gaps.

[0020] In the present invention, the anti-CAPRIN-1 antibody used to prepare a conjugate refers to an antibody or an antigen-binding fragment thereof that is immunologically reactive with the full-length CAPRIN-1 protein, which is the antigen, or a fragment thereof. Here, "immunological reactivity" refers to the property of the antibody binding to the CAPRIN-1 protein or a partial polypeptide thereof in vivo.

[0021] The anti-CAPRIN-1 antibody used in the present invention may be a monoclonal antibody or a polyclonal antibody.

[0022] Polyclonal antibodies immunologically reactive with the full-length CAPRIN-1 protein or a fragment thereof (anti-CAPRIN-1 polyclonal antibodies) can be obtained by immunizing mice, human antibody-producing mice, rats, rabbits, chickens, etc. with, for example, the natural CAPRIN-1 protein, a fusion protein with GST or the like, or a partial peptide thereof, and then obtaining serum. The obtained serum can be subjected to ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange column, affinity column to which the CAPRIN-1 protein or partial peptide is bound, or the like.

[0023] The full-length CAPRIN-1 protein or a fragment thereof used in the immunization, the nucleotide sequence and amino acid sequence of CAPRIN-1 and its homologs, can be obtained, for example, by accessing 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). In addition, a method for producing the CAPRIN-1 protein can be obtained by referring to WO2014 / 012479, and cells expressing the CAPRIN-1 protein can also be used.

[0024] A monoclonal antibody immunologically reactive with the full-length CAPRIN-1 protein or a fragment thereof (anti-CAPRIN-1 monoclonal antibody) can be obtained, for example, by immunizing a mouse with SK-BR-3 breast cancer cells expressing CAPRIN-1 or the full-length CAPRIN-1 protein or a fragment thereof, fusing spleen cells isolated from the mouse with myeloma cells, and selecting a clone producing an anti-CAPRIN-1 monoclonal antibody from the resulting fused cells (hybridoma). The antibody produced by the selected hybridoma can be obtained by a method similar to the method for purifying polyclonal antibodies described above.

[0025] The antibodies used in the present invention include human antibodies, humanized antibodies, chimeric antibodies, and non-human animal antibodies.

[0026] Human antibodies can be obtained by sensitizing human lymphocytes infected with EB virus with a protein, protein-expressing cells, or a lysate thereof, fusing the sensitized lymphocytes with myeloma cells such as human-derived U266 cells, and then obtaining antibodies immunologically reactive with the full-length CAPRIN-1 protein or a fragment thereof from the resulting fused cells.

[0027] A humanized antibody is a modified antibody, also known as a reshaped human antibody. Humanized antibodies are constructed by grafting the complementarity-determining regions (CDRs) of an antibody derived from an immunized animal onto the CDRs of a human antibody. Genetic recombination, a common technique for this purpose, is well known. Specifically, for example, a DNA sequence designed to link the CDRs of a mouse or rabbit antibody with the framework regions of a human antibody is synthesized by PCR from several oligonucleotides engineered to have overlapping ends. The resulting DNA is ligated to DNA encoding the constant regions of a human antibody, incorporated into an expression vector, and then introduced into a host for production (see EP 239400 and WO 96 / 02576). The framework regions of the human antibody linked via the CDRs are selected so that the CDRs form a good antigen-binding site. If necessary, amino acids in the framework regions of the variable regions of the antibody may be substituted so that the complementarity-determining regions of the reshaped human antibody form an appropriate antigen-binding site (Sato K. et al., Cancer Research 1993, 53:851-856). Alternatively, they may be substituted with framework regions derived from various human antibodies (see WO99 / 51743).

[0028] Antibodies are typically heteromeric glycoproteins containing at least two heavy chains and two light chains. Antibodies consist of two identical light chains and two identical heavy chains. Heavy chains have a heavy chain variable region at one end, followed by several constant regions. Light chains have a light chain variable region at one end, followed by several constant regions. The variable regions exhibit specific variable regions called complementarity-determining regions (CDRs) that confer binding specificity to the antibody. Portions of the variable regions that are relatively conserved are called framework regions (FRs). Complete heavy and light chain variable regions each contain four FRs connected by three CDRs (CDR1 to CDR3).

[0029] The sequences of the constant and variable regions of human-derived heavy and light chains are available from NCBI (USA: GenBank, UniGene, etc.). For example, reference can be made to the sequences of the human IgG1 heavy chain constant region under accession number J00228, the human IgG2 heavy chain constant region under accession number J00230, the human light chain κ constant region under accession numbers V00557, X64135, X64133, etc., and the human light chain λ constant region under accession numbers X64132, X64134, etc.

[0030] A chimeric antibody is an antibody produced by combining sequences derived from different animals, such as an antibody consisting of the heavy chain variable region and light chain variable region of a mouse antibody and the heavy chain variable region and light chain variable region constant region of a human antibody. Chimeric antibodies can be produced using known methods, for example, by linking DNA encoding an antibody V region with DNA encoding a human antibody C region, incorporating the resultant into an expression vector, and introducing the vector into a host for production.

[0031] Non-human animal antibodies can be obtained by immunizing an animal with a sensitizing antigen according to known methods. A typical method is to inject the sensitizing antigen intraperitoneally, intradermally, or subcutaneously into an animal such as a mouse. When injecting the sensitizing antigen, the antigen is mixed with an appropriate amount of various adjuvants, such as Freund's complete adjuvant (CFA), and administered to the animal multiple times. After immunizing an animal and confirming that the serum contains anti-CAPRIN-1 antibodies, the serum can be obtained and purified, as described above, by ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange columns, affinity columns coupled with CAPRIN-1 protein or partial peptides, or the like. Furthermore, monoclonal antibodies can be obtained from non-human animals by collecting immune cells from the immunized animal and fusing them with myeloma cells. The fusion of the immune cells with myeloma cells can be carried out according to known methods (see Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).

[0032] The antibodies used in the present invention can also be obtained as recombinant antibodies produced by cloning an antibody gene from a hybridoma, incorporating it into a suitable vector, and introducing it into a host using genetic engineering techniques (see Carl, A.K., Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0033] The anti-CAPRIN-1 antibody used to obtain the conjugate of the present invention may have amino acids in the variable region (e.g., FR) or constant region substituted with other amino acids. The amino acid substitutions are single or multiple, for example, fewer than 15, fewer than 10, 8 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids, preferably 1 to 9 amino acids. The substituted antibody should have the same or higher antigen-specific binding properties and antigen-binding affinity as the unsubstituted antibody, and should not cause rejection reactions when administered to humans.

[0034] The anti-CAPRIN-1 antibody used in the present invention is expected to have a stronger antitumor effect if it has a higher binding affinity with the CAPRIN-1 protein on the surface of cancer cells. 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 It is desirable that:

[0035] The binding ability of the anti-CAPRIN-1 antibody used in the present invention to effector cells can be improved by substituting one, two, or several amino acids in the heavy chain constant region of the antibody, or by removing fucose bound to N-acetylglucosamine in the N-glycoside-linked sugar chain bound to the heavy chain constant region. The above may be achieved by amino acid substitution alone, or may be a composition with an antibody bound to fucose.

[0036] Antibodies in which one, two, or several amino acids in the heavy chain constant region have been substituted can be produced by referring to, for example, WO2004 / 063351, WO2011 / 120135, U.S. Patent No. 8,388,955, WO2011 / 005481, U.S. Patent No. 6,737,056, and WO2005 / 063351.

[0037] An antibody from which fucose bound to N-acetylglucosamine in the N-glycoside-linked sugar chain in the heavy chain constant region has been removed, or a cell producing such an antibody, 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. An antibody from which fucose bound to N-acetylglucosamine in the N-glycoside-linked sugar chain bound to the heavy chain constant region has been removed, or a composition of an antibody to which fucose has been bound, or a cell producing such an antibody, can be prepared with reference to, for example, U.S. Patent No. 8,642,292.

[0038] The anti-CAPRIN-1 polyclonal antibody, anti-CAPRIN-1 monoclonal antibody, antibody production method, purification method, and method for producing the CAPRIN-1 protein or its partial polypeptide used in immunization used in the present invention are described in WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, and WO2011 / 09653 5, WO2013 / 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.

[0039] Specific examples of the anti-CAPRIN-1 antibody of the present invention include those described in WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, WO2013 / 018892, and WO2013 / 01889 1, WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176, and WO2015 / 020212, but preferred anti-CAPRIN-1 antibodies include the following:

[0040] An antibody or fragment thereof that is immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence that has 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and still more preferably 99% or more) sequence identity with said amino acid sequence.

[0041] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence represented by SEQ ID NO: 31 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 said amino acid sequence. An antibody or fragment thereof immunologically reactive with a CAPRIN-1 protein, preferably comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 36, 37, and 38 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 40, 41, and 42 (CDR1, CDR2, and CDR3, respectively), or an antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 140, 141, and 142 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 143, 144, and 145. or an antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 164, 165, and 166 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 167, 168, and 169 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 43, or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 71, or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79.

[0042] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 33 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 60, 61, and 62 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 64, 65, and 66 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67.

[0043] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 32 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 52, 53, and 54 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 56, 57, and 58 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59.

[0044] An antibody or fragment thereof that is immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence represented by SEQ ID NO: 34 or an amino acid sequence that has 80% or more (preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more) sequence identity with said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 170, 171, and 172 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 173, 174, and 175 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with the CAPRIN-1 protein; or an antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 176, 177, and 178 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 179, 180, and 181 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with the CAPRIN-1 protein. More preferably, the antibody or fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 80 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 81, or an antibody or fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 83.

[0045] An antibody or fragment thereof that is immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 35 or an amino acid sequence that has 80% or more (preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more) sequence identity with said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 182, 183, and 184 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 185, 186, and 187 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with the CAPRIN-1 protein; or an antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 188, 189, and 190 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 191, 192, and 193 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with the CAPRIN-1 protein. More preferably, the antibody or fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85, or an antibody or fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 87.

[0046] An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions (CDR1, CDR2, and CDR3) of SEQ ID NOs: 44, 45, and 46, and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2, and CDR3) of SEQ ID NOs: 48, 49, and 50, and having immunological reactivity with a CAPRIN-1 protein. Preferably, the antibody or fragment thereof comprises the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 51.

[0047] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 296 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 146, 147, and 148 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 149, 150, and 151 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73.

[0048] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 297 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 272, 273, and 274 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 275, 276, and 277 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 114 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 115.

[0049] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 298 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 290, 291, and 292 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 293, 294, and 295 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 120 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 121.

[0050] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 299 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 301, 302, and 303 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 305, 306, and 307 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 300 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 304.

[0051] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 308 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 134, 135, and 136 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 137, 138, and 139 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 69.

[0052] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 309 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 said amino acid sequence. Preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 134, 135, and 136 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity-determining regions of SEQ ID NOs: 137, 138, and 139 (CDR1, CDR2, and CDR3, respectively), and is immunologically reactive with a CAPRIN-1 protein. More preferably, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 69.

[0053] In addition, the following anti-CAPRIN-1 antibodies are also preferably used.

[0054] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 69.

[0055] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 70 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 71.

[0056] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 73.

[0057] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 74 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 75.

[0058] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 77.

[0059] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 78 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79.

[0060] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 80 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 81.

[0061] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83.

[0062] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 84 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 85.

[0063] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 86 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 87.

[0064] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 89.

[0065] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 91.

[0066] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 92 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 93.

[0067] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 94 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 95.

[0068] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 96 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 97.

[0069] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:98 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:99.

[0070] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 100 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 101.

[0071] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 102 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 103.

[0072] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 104 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 105.

[0073] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 106 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 107.

[0074] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 108 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 109.

[0075] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 110 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 111.

[0076] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 112 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 113.

[0077] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 114 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 115.

[0078] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 116 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 117.

[0079] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 118 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 119.

[0080] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 120 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 121.

[0081] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 122 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 123.

[0082] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 124 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 125.

[0083] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 126 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 127.

[0084] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 128 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 129.

[0085] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 130 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 131.

[0086] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 132 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 133.

[0087] An antibody or fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 300 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 304.

[0088] In the Examples described below, conjugates of microtubule-regulating agents were prepared using the above-mentioned polyclonal and monoclonal antibodies against the full-length CAPRIN-1 protein and a portion of the polypeptide region expressed on the cell membrane surface of cancer cells, and their strong antitumor effects were confirmed.

[0089] <Benzodiazepines> Some benzodiazepines are known to exhibit antitumor effects by recognizing specific DNA sequences and binding to the minor groove (DNA minor groove), thereby interfering with DNA synthesis and inhibiting cell proliferation, and benzodiazepines having such antitumor activity are preferably used in the present invention.

[0090] Specific examples of benzodiazepines having antitumor activity include pyrrolobenzodiazepines (PBDs), indolinobenzodiazepines (IGNs), pyridinobenzodiazepines (PDDs) and isoquinolidinobenzodiazepines (IQBs).

[0091] PBD has an aromatic ring A (A ring), ring B (B ring), and pyrrolo ring C (C ring), and in ring B, the N10-N11 positions are either imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine methyl ether (NH-CH(OMe)). PBD can also take the form of a C2-unsaturated PBD dimer, a PBD dimer with C2 aryl substitution, or polypyrrole-PBD. PBD has the ability to recognize and bind to a specific sequence in DNA, with the preferred sequence being 5'PuGPu3'.

[0092] Specific examples of PBDs include the antitumor antibiotic anthramycin and its analogs, including abbeymycin, chicamycin, mazethramycin, neothramycin A, meothramycin B, porothramicin, prothracarcin, sibanomycin (DC-102), sibiromycin, tomamycin, didehydroanhydroanthramicin, spadicomycin, and DC-81.

[0093] Specific examples of PBD include "Mantaj, J et al. 2016 Angew. Chem. Int Ed. 55, 2-29; D. Antonow and D. Thurston, Chem. Rev. 2011", "Leanna et al. J. Med. Chem. 2020, 63, 9603-9622", "Jhon et al. Expert Opinion on Biological Therapy Vol. 21, 2021, 931-943", and "Julia et al. Angew Chem Int Ed Engl. 2017 Jan 9;56(2):462-488" are included.

[0094] Specific examples of PBD and linker composite compounds include 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, WO2015 / 052532, WO2015 / 181559, and WO2017 / 020972. , WO2017 / 059289, WO2017 / 137555, WO2017 / 137556, WO2017 / 186894, WO20 18 / 031662, WO2018 / 069490, WO2018 / 091646, WO2018 / 146188, WO2018 / 146 189, WO2018 / 18234, WO2018 / 192944, WO2019 / 034764, WO2019 / 065964, WO2 019 / 096788, WO2019 / 104289, WO2019 / 126691, WO2019 / 224340, WO2020 / 00 6722, WO2020 / 079229, WO2020 / 079239, WO2020 / 100954, WO2020 / 141923, WO2020 / 152462, WO2020 / 1964474, WO2020 / 196712, WO2021 / 137646, WO202 2 / 063853, WO2016 / 083468, WO2014 / 057073, WO2014 / 057113, WO2014 / 0571 14, WO2014 / 057115, WO2014 / 057117, WO2014 / 057118, WO2014 / 057119, WO2 014 / 057120, WO2014 / 057122, U.S. Patent 4,316,900, WO2020 / 245283, WO2019 / 197602, WO2004 / 043963, WO2005 / 085260, WO2006 / 111759, WO2010 / 010347, WO 2010 / 043877, WO2011 / 128650, WO2011 / 130598, WO2011 / 130613, WO2011 / 1 30616, WO2013 / 041606, WO2013 / 053871, WO2013 / 053873, WO2013 / 055987,WO2013 / 053872, WO2013 / 164593, WO2013 / 164592, WO2014 / 096368, WO2014 / 140862 , WO2014 / 140174, WO2015 / 052322, WO2015 / 052532, WO2015 / 052533, WO2015 / 052534 , WO2015 / 052535, WO2016 / 038383, WO2016 / 037644, WO2016 / 044560, WO2017 / 137553, WO2017 / 129652, WO2017 / 223275, WO2018 / 146199, and WO2018 / 182341.

[0095] Examples of IGN and IGN-linker composite compounds include those disclosed in WO2017 / 015495, WO2017 / 015496, WO2012 / 128868, WO2012 / 112687, WO2012 / 112708, WO2016 / 036801, WO2017 / 015502, WO2012 / 112708, Examples include compounds described in WO2018 / 140435, WO2018 / 195245, WO2018 / 098258, WO2010 / 091150, WO2016 / 036804, WO2019 / 133652, WO2020102051, WO2020 / 102053, and WO2020 / 205564.

[0096] Examples of PDD and PDD-linker composite compounds include compounds described in WO2012 / 152915, WO2015 / 166289, WO2017 / 032983, WO2016 / 198869, WO2017 / 194960, WO2019 / 043417, WO2020 / 049286, WO2020 / 157491, and WO2022 / 023735.

[0097] Examples of IQB and conjugate compounds of IQB and a linker include compounds described in WO2016 / 149546, WO2018 / 071455, WO2018 / 053552, WO2017 / 011803, WO2017 / 091615, U.S. Patent No. 9974864, U.S. Patent No. 9504694, and U.S. Patent No. 10350218.

[0098] The benzodiazepine used in the present invention may be a monomer or a dimer. In the case of a dimer, it may be a homodimer or a heterodimer with another antitumor compound. The dimer may be a dimer bonded to each other via the 8th, 7th, or 2nd carbon of the benzodiazepine.

[0099] Benzodiazepines preferably used in the present invention include DSB-120, SJG-136 (SG2000), DC-81, DSB-120, SJG-136, SG2057, SG2202, SG2285, SGD-1882, SGD-1910, SG3199, SG3249, SG2219, IMGN779, IMGN632, (S)—N-(4-aminophenyl)-4-(4-(4- ((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide)phenyl)-1-methyl-1H-pyrrole-2-carboxamide, D211, D221, D231, GWL-78, KMR-28-39, or a derivative thereof.

[0100] Furthermore, the derivative is preferably a derivative in which a linker is bound to the compound, and specific examples include Tesirine (a derivative of SG3249), Talirine (a derivative of SGD-1910), SG3364, SG3227, SG3140 (MC-Phe-Lys-PAB-SG2057), SG3170, SG3203 (MC-Phe-Lys-PAB-SG2057), SG3231, SG3400, SG3376, DGN642, DGN549, FGX5-67, FGX-2-62, and FGX11-38.

[0101] <Conjugate of anti-CAPRIN-1 antibody and benzodiazepine> In the present invention, the bond between the anti-CAPRIN-1 antibody and the benzodiazepine in the conjugate of the anti-CAPRIN-1 antibody and the benzodiazepine is not particularly limited as long as it is a bond that can maintain anti-cancer activity, but a bond in which a linker structure is formed between the anti-CAPRIN-1 antibody and the benzodiazepine is preferred.

[0102] Here, the linker refers to a compound capable of binding an anti-CAPRIN-1 antibody to a benzodiazepine. Various known linkers may be used, or the activator may be directly bound after appropriate chemical modification of the structure on the activator side.

[0103] The type of linker and the details of the binding method can be determined in accordance with known methods (see, for example, Greg T. Hermanson, Bioconjugate Techniques, Third Edition, WO2004 / 010957, WO2014 / 012479).

[0104] In embodiments of the invention, reactive groups attached to the anti-CAPRIN-1 antibody, benzodiazepine, and linker include:

[0105] Unless specifically chemically modified, reactive groups present in the amino acid sequence of an antibody or in glycoproteins modified with amino acids include primary amines (ε-amino), carboxyls, thiols (sulfhydryls), carbonyls (ketones or aldehydes), and hydroxyls. Primary amines are found at the N-terminus of polypeptides and in the side chains of lysine residues. They are positively charged under physiological conditions and are usually located on the outside of proteins, allowing them to be used for conjugation without altering the protein structure. Carboxyls are found at the C-terminus of polypeptides and in the side chains of aspartic acid and glutamic acid. Sulfhydryls are found in the side chains of cysteine ​​and form disulfide bonds that maintain the higher-order structure of proteins. Ketones or aldehydes are generated in glycoproteins by oxidizing glycosyl groups with sodium metaperiodate.

[0106] The conjugates of the present invention are prepared by attaching the benzodiazepine to a linker attached to the reactive group on the antibody, by attaching the benzodiazepine to a linker attached to the benzodiazepine, or by attaching the benzodiazepine directly to the antibody.

[0107] Specific examples of the reactive groups attached to the PBD and linker include the following:

[0108] Reactive groups that can react with amines include N-hydroxysuccinimide (NHS) esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphine, isothiocyanates, isocyanates, acyl azides, N-hydroxyl esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryls, carbodiimides, and carboxylic acid anhydrides.

[0109] Carbodiimides, diazoalkanes, diazoacetyl compounds, carbonyldiimidazoles are reactive groups that can react with carboxyls and amines.

[0110] Reactive groups that can react with thiols include maleimide, haloacetamide, pyridyl disulfide, thiosulfone, vinyl sulfone, haloacetyl, aziridine, acryloyl, and aryl.

[0111] Reactive groups that can react with aldehydes include hydrazide and alkoxyamine, and reactive groups that can react with hydroxyl include epoxy, oxirane, carbonyldiimidazole, N,N'-disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, and isocyanate.

[0112] Isocyanate is a reactive group that can react to hydroxyl.

[0113] Photoreactive reactive groups include diazirine, aryl azide, aryl, benzophenol, and diazo compounds.

[0114] Specific examples of the linker having the reactive group include the following:

[0115] Examples of linkers having the same reactive group terminal include linkers having N-hydroxysuccinimide ester as the reactive group (e.g., Disuccinimidyl Glutarate (DSG), Disuccinimidyl Suberate (DSS), Bis(sulfosuccinimidyl)Suberate (BS3), Tris-(Succinimidyl)Aminotriacetate (TSAT), PEGylated Bis(Sulfosuccinimidyl)Suberate (BS(PEG))). ５ , BS(PEG) ９ ), Dithiobis (Succinimidyl Propionate) (DSP), 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), Sulfo-ethylene glycol bis(succinimidyl succinate (Sulfo-EGS), Dimethyl adipimidate・2HCl (DMA), Dimethyl pimelimidate・2HCl (DMP), Dimethyl suberimidate・2HCl (DMS), Dimethyl3,3'-dithiobispropionimidate・2H Cl (DTBP), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), Disuccinimidyl tartrate (DST), Bis[2-(Succinimidooxycarbonyloxy)ethyl]Sulfone (BSOCOES), and linkers with maleimide as the reactive group (e.g., Bismaleimidoethane (BMOE), 1,4-bismaleimidobutane (BMB), Bismaleimidohexane (BMH), Tris(2-maleimidothyl)amine (TMEA), 1,8-bismaleimido-(PEG)). ２ (BM (PEG) ２ ), 1,8-bismaleimido-(PEG) ３ (BM (PEG) ３), Dithiobismaleimidoethane (DTME) is used.

[0116] The main linkers having different reactive group ends include linkers having NHS ester and maleimide reactive groups (e.g., AMAS, BMPS, GMBS, Sulfo-MBS, MBS, Sulfo-MBS, SMCC, Sulfo-SMCC, EMCS, Sulfo-EMCS, SMPB, Sulfo-SMPB, SMPH, LC-SMCC, Sulfo-KMUS, SM(PEG) ２ , SM(PEG) ４ , SM(PEG) ６ , SM(PEG) ８ , SM(PEG) １２ , SM(PEG) ２４ ), linkers with NHS ester and pyridyldithiol as reactive groups (e.g., SPDP, LC-SPDP, Sulfo-LC-SPDP, SMPT, (PEG) ４ SPDP, PEG12-SPDP), linkers with NHS ester and haloacetyl as reactive groups (e.g., SIA, SBAP, SIAB, Sulfo-SIAB), linkers with NHS ester and aryl azide as reactive groups (e.g., ANB-NOS, Sulfo-SANPAH, ATFB), linkers with NHS ester and diazirine as reactive groups (e.g., SDA, Sulfo-SDA, LC-SDA, SDAD, Sulfo-SDAD), carbodiimide Linkers having a hydrazide as a reactive group (e.g., DCC, EDC, EDAC, NHS, Sulfo-NHS), linkers having a maleimide and a hydrazide as a reactive group (e.g., BMPH, EMCH, MPBH, KMUH), linkers having a pyridyldithiol and a hydrazide as a reactive group (e.g., PDPH), linkers having an isocyanate and a maleimide as a reactive group (e.g., PMPI), and linkers having an NHS ester and a psoralen as a reactive group (e.g., SPB) are used.

[0117] Other linkers include polypeptide-containing linkers, such as Fmoc-Ala-Ala-Asn-PAB, Fmoc-Ala-Ala-Asn(Trt)-PAB, and Fmoc-PEG. ３-Ala-Ala-Asn(Trt)-PAB, Fmoc-PEG ４ -Ala-Ala-Asn(Trt)-PAB, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Ala-Ala-Asn(Trt)-PAB-PNP, Fmoc-PEG ３ -Ala-Ala-Asn(Trt)-PAB-PNP, Azide-PEG ４ -Ala-Ala-Asn(Trt)-PAB-PNP, Mal-PEG ４ -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. are used.

[0118] Also, Bis-PEG-acid, PEG Acid (e.g., Acid-PEG-TEMPO, Amino-PEG-acid, Amino-PEG-CH ２ CO ２ H, Aminoxy-PEG-acid, Azido-PEG-acid, Carboxy-PEG-sulfonic acid, Fmoc-N-amido-PEG-acid, Fmoc-N-amido-PEG-CH ２ CO ２ H, Fmoc-aminoxy-PEG-acid, Hydroxy-PEG-acid, Hydroxy-PEG-CH ２ CO ２ H, m-PEG-acid, m-PEG-(CH ２ ) ３ -acid, Methoxytrityl-N-PEG-acid, N-methyl-N-(t-Boc)-PEG-acid, Propargyl-PEG-acid, Propargyl-PEG-CH ２ CO ２ H, Propargyl-PEG-(CH ２ ) ３ -acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH ２ CO ２H, t-Boc-Aminoxy-PEG-acid, Acid-PEG-PFP ester, Miscellaneous PEG acid, ), PEG PFP ester (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, Hydroxy-PEG-Tos, m-PEG-Tos, t-Boc-Aminoxy-PEG-Tos, Trifluoroethyl-PEG-Tos, Tos-PEG-acid, Tos-PEG-CH ２ CO ２ H, Tos-PEG-alkyne, Tos-PEG-t-butyl ester, Tos-PEG-CH ２ CO ２ tBu, Tos-PEG-Tos, S-acetyl-PEG6-Tos, N-Tos-N-(t-butoxycarbonyl)-aminoxy-PEG ４ -Tos, Ms-PEG-Ms, Ms-PEG-t-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, N-Boc-PEG-bromide, N-methyl-N-(t-Boc)-PEG-acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH ２ CO ２H, 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), PEG NHS 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-CH ２ CO ２ H, 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-PEG ３ -SS-azide, DBCO-S-S-PEG ３ -Biotin, Dde Biotin-PEG4-Alkyne, Dde Biotin-PEG ４ -Azide, Dde Biotin-PEG ４ -DBCO, Diazo Biotin-PEG ３ -Alkyne, Diazo Biotin-PEG ３ -Azide, Diazo Biotin-PEG ３-DBCO、Diol Biotin-PEG ３ -Alkyne、Diol Biotin-PEG ３ -Azide、PC Biotin-PEG ３ -Alkyne、PC-Biotin-PEG ４ -PEG ４ -Alkyne、PC-Biotin-PEG ４ -PEG4-Alkyne、PC Biotin-PEG ３ -Azide、PC-Biotin-PEG4-PEG3-Azide、PC-Biotin-PEG ４ -NHS carbonate、PC DBCO-PEG ３ -Biotin、WSPC Biotin-PEG ３-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 (e.g., endo-BCN-PEG, exo-BCN-PEG, Propargyl-PEG-acid, Propargyl-PEG-CH ２ CO ２ H, Propargyl-PEG-(CH ２ ３ -acid, Propargyl-PEG-(CH ２ ３ ​​-methyl ester、Prropargyl-･Gmcrrylat e、Propargglll･･･alcohol、rrop argylmm･･mamine、.. G-methylamiine、| Propargylla. de、Propargyllm･･bromide、rro pargyl-･･omMaleimide、rropa gylmPEy-ｭs、Propargyllmm･om｢ｳester、PrropargylllEGsulffoiic I ester、Propargyl-･ym｣ｨ ２ 39 ２ tBu、Propargylll･･thiol、ｰcbd gummies carbonaate、 nooxy-EEorropargyl、 BismPropargyl-･1、mm PEGPropargyll) . ２ 39 ２ 2、2zido-E((3ｨ ２ ) ) ３ -methyl ester、｡zidomE･m｡crylatee｡ do-PEGalcoholl ２ ) ) ３ OH、｡zido-EGamine、｡zid o-PEmazide、｡zido-EMM I ylamine、｡zido-･mmethyll ester、｡zido-EE1mｮｳ esterr ２ 39 ２-NHS, Azido-PEG-oxazolidin-2-one, Azido-PEG-PFP ester, Azido-PEG-phosphonic acid, Azido-PEG-phosphonic acid ethyl ester, Azido-PEG-sulfonic acid, Azido-PEG-t-Boc-Hydrazide, Azido-PEG-t-butyl ester, Azido-PEG-CH ２ CO ２ -t-butyl ester, Azido-PEG-TFP ester, Azido-PEG-Tos, Aminoxy-PEG-azide, Bromo-PEG-azide, Bromoacetamide-PEG-azide, Carboxyrhodamine 110-PEG-Azide, Isothiocyanato-PEG-Azide, Isothiocyanato- PEG-Azide, m-PEG-azide, Propargyl-PEG-azide, TAMRA-PEG-Az ide, t-Boc-N-Amido-PEG-Azide, t-Boc-Aminooxy-PEG-Azide, T hiol-PEG-Azide, Trifluoroethyl-PEG-Azide, Azido-PEG-amino acid, Azido-PEG ４ -4-nitrophenyl carbonate, S-acetyl-PEG ３ -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 is used.

[0119] In another embodiment, polyethylene glycol (PEG) described in WO2015 / 057699 and WO2017 / 165851 can be used to obtain a conjugate that is expected to have better in vivo kinetics.

[0120] The linker between the anti-CAPRIN-1 antibody and the benzodiazepine may be composed of a single type or multiple types.

[0121] Methods for preparing a conjugate of an anti-CAPRIN-1 antibody and a benzodiazepine include a method in which a benzodiazepine is conjugated using the ε-amino group of a lysine side chain of the antibody, and a method in which a cysteine ​​residue forming a disulfide bond of the antibody is reduced to form a thiol.

[0122] When the ε-amino group of a lysine residue of an antibody is used, for example, an amide bond is formed by reacting it with an active ester (e.g., N-hydroxysuccinimide ester). In this case, since there are many lysine residues in an antibody, the binding reaction proceeds nonspecifically.

[0123] When using thiols forming disulfide bonds in the side chains of cysteine ​​residues of antibodies, a method is used in which the disulfide bonds on the antibody are converted to thiols using a reducing agent such as mercaptoethanol, followed by reaction with maleimide or α-haloamide. Furthermore, methods using sulfonephenyloxadiazole, 4-cyanoethynyloxy derivatives, etc., can be used to stabilize thiol-mediated bonds. These bonds are more stable for longer periods than bonds formed by the conjugation reaction of cysteine ​​to maleimide. Furthermore, since stability is improved when the imide ring formed by the thiol group attached to the maleimide is opened by hydrolysis to form an amide bond, a linker with an amino group near the imide group can also be used. Furthermore, the thiols of cysteine ​​residues in antibodies form disulfide bonds, and a benzodiazepine can be attached between them via two thiols. For example, cross-linking can be formed using a linker with two disulfide bond sites, which can be generated from an amide group with two sulfones at the β-position, or dibromomaleimide.

[0124] The conjugates of the present invention can be produced, for example, using THIOMAB™ technology (see Nature Biotechnology 26, 925-932 (2008)), a method that allows a fixed number of thiol groups to be introduced into a specific portion of an antibody, or a method that uses ThioBridge™ technology (see Methods Mol Biol. 2078: 113-129 (2020)), or RESPECT™ technology (see MAbs. 9 (6): 907-915 (2017)).

[0125] The conjugate of the present invention can be prepared, for example, by reducing an antibody with the reducing agent dithiothreitol (DTT) in a phosphate buffer solution to obtain an antibody having a thiol-reactive group, followed by formation of a conjugate with a benzodiazepine. In addition to the method using a reducing agent, the conjugate can also be obtained by adding a thiol group to the primary amine of a lysine residue in the antibody by introducing Traut's reagent (2-Iminothiolane or N-Succinimidyl S-Acetylthioacetate (SATA)).

[0126] The amount of thiol added to the antibody can be quantified, for example, by mixing a sample solution containing 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) and an SH group with phosphate buffer (pH 8.0) and distilled water, adding a 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 G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959)).

[0127] The thiol groups added by cleaving disulfide bonds in the antibody through reduction treatment are preferably subjected to a treatment (capping) to prevent re-formation of disulfide bonds, such as with N-ethylmaleimide (NEM) or 2-iodoacetamide (IAA).

[0128] Forming a conjugate by binding a benzodiazepine to a thiol group added to an antibody can be achieved by known methods. Specifically, for example, linker reagents having a maleimide group or a bromoacetamide group can be used as linker reagents that specifically bind to the thiol group of a reduced antibody. For example, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) is used as a linker having a maleimide group. In this case, the presence of an amino group in the benzodiazepine allows the formation of an amide bond with the N-succinimide group of SMCC, thereby obtaining a conjugate.

[0129] In another embodiment, an amide bond is first formed with SMCC at the amino group present in the activator, and then a thiol group added to the antibody side is reacted with the maleimide group of the benzodiazepine-bound SMCC to obtain a conjugate.

[0130] In another embodiment, a conjugate of an antibody and a benzodiazepine can be formed by using two linkers. For example, a conjugate can be prepared by forming an amide bond between a primary amino group present in a lysine residue on the antibody and the N-succinimide group of SATA (N-succinimidyl-S-acetylthioacetate), adding a thiol group to the antibody, and then synthesizing a benzodiazepine containing an amino group or one to which an amino group has been added according to a standard method, reacting it with SMCC to form an amide bond with the N-succinimide group in SMCC. A conjugate can then be obtained by reacting the maleimide group in the benzodiazepine-bound SMCC with the thiol group in the antibody-bound SATA.

[0131] In another embodiment, conjugates of antibodies and benzodiazepines can be prepared using, for example, maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PAB) as a linker. mc-val-Cit-PAB (mc-vc-PAB) is a linker that can be cleaved by intracellular proteases (e.g., cathepsin B). A thiol group is attached to an antibody dissolved in phosphate buffer using DTT or the like. Meanwhile, a benzodiazepine having an amino group is reacted with the benzyloxycarbonyl (PAB) in mc-Val-Cit-PAB to prepare a benzodiazepine bound to mc-val-Cit-PAB, which can then be reacted with the aforementioned thiol-attached antibody to obtain a conjugate.

[0132] In yet another embodiment, SATA is attached to a primary amino group in a lysine residue of an antibody to add a thiol group, while succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is reacted with a benzodiazepine having an amino group to form an amide bond with the N-succinimide group in SPDP.

[0133] The linker used in the present invention is cleavable under intracellular conditions, liberating a substance having antitumor activity comprising a benzodiazepine or a benzodiazepine and a portion of the linker within the cell. For example, the linker is cleaved by intracellular peptidases or proteases. Preferred linkers are those cleaved by lysosomal or endosomal proteases, cathepsin B, cathepsin D, or plasmin. Examples include linkers containing a polypeptide (Val-Cit, Phe-Leu, or Gly-Phe-Leu-Gly) that can be cleaved by cathepsin B. More specifically, linkers described in U.S. Patent No. 6,214,345 can be used.

[0134] Furthermore, in another embodiment, as a means for improving the stability, solubility, and metabolism of the conjugate of the present invention in blood and the binding affinity of the benzodiazepine to the anti-CAPRIN-1 antibody, for example, a linker having glucuronic acid (preferably β-D-glucuronide) described in WO 2007 / 0711968 can be used. Alternatively, means described in WO 2013 / 173337, WO 2015 / 095755, WO 2015 / 123679, and WO 2018 / 031690 can be used.

[0135] Furthermore, in another embodiment, benzodiazepines can be site-specifically bound to anti-CAPRIN-1 antibodies using the methods described in, for example, WO2006 / 65533 and WO2018 / 160683.

[0136] In yet another embodiment, a conjugate of an anti-CAPRIN-1 antibody bound to two or more drugs including a benzodiazepine can be obtained by the method described in WO2018 / 112253.

[0137] To obtain a composition containing a conjugate of an anti-CAPRIN-1 antibody of the present invention and a benzodiazepine, the composition can be subjected to, for example, gel filtration chromatography, and then the peak with a higher molecular weight than the antibody before linker attachment can be isolated. To detect the mass of the conjugate while maintaining the intact bivalent antibody, for example, the method described in WO2013 / 049410 can be used.

[0138] The number of benzodiazepines bound per antibody of the conjugate of the anti-CAPRIN-1 antibody and benzodiazepine of the present invention can be quantified according to known methods such as mass spectrometry, ELISA, electrophoresis, and chromatography such as HPLC.

[0139] <Anti-tumor effect of the conjugate> The conjugate of the present invention between an anti-CAPRIN-1 antibody and a benzodiazepine has anti-tumor activity in vitro or in vivo. Anti-tumor activity means reduction, elimination, inhibition of growth, apoptosis, necrosis, or killing of targeted cancer cells. Therefore, the anti-tumor effect of the conjugate of the present invention can be determined by examining the anti-tumor activity against cancer.

[0140] The in vivo antitumor effect can be assessed by administering the conjugate to a cancer-bearing organism, measuring the size of the tumor after administration, and examining the size of the cancer over time. The antitumor effect of the present invention can also be assessed by examining the survival rate. It can also be assessed by examining the ability to produce cytokines or chemokines. The antitumor effect of the conjugate of the present invention can be further assessed by examining the prevention of cancer, metastasis, or recurrence.

[0141] The conjugate of the present invention is expected to have a stronger antitumor effect if it has a higher binding affinity with the CAPRIN-1 protein on the surface of cancer cells. 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 It is desirable that:

[0142] The ability of the conjugate of the present invention to bind to CAPRIN-1 can be determined using binding assays such as surface plasmon resonance (SPR), ELISA, Western blotting, immunofluorescence, and flow cytometry.

[0143] As described above, the conjugates of the present invention have an enhanced anti-tumor effect compared to an anti-CAPRIN-1 antibody alone, and the enhancement rate is preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 65% ​​or more, and most preferably 70% or more. The enhancement rate of the anti-tumor effect of the conjugates of the present invention compared to an anti-CAPRIN-1 antibody alone can be calculated by administering an effective amount of each to tumor-bearing mice under the same conditions and comparing the tumor volumes on or after day 10 after the start of administration.

[0144] <Pharmaceutical Composition, Method for Treating and / or Preventing Cancer> The target of the pharmaceutical composition of the present invention for treating and / or preventing cancer is not particularly limited, as long as it is a cancer (cell) that expresses the CAPRIN-1 protein.

[0145] As used herein, the terms "tumor" and "cancer" refer to malignant neoplasms and are used interchangeably.

[0146] The cancers targeted by the present invention may be any cancers that express CAPRIN-1 protein on the cell membrane surface. Preferred cancers include breast cancer, kidney cancer, pancreatic cancer, colon 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, and multiple myeloma, as well as testicular cancer, thyroid cancer, head and neck cancer, and urothelial cancer.

[0147] More specifically, the cancers include, for example, breast adenocarcinoma, hybrid breast adenocarcinoma, malignant mixed breast tumor, intraductal papillary adenocarcinoma, recurrent metastatic breast cancer, lung adenocarcinoma, non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tissue tumors such as glioma, glioblastoma, neuroblastoma, ependymoma, neuronal tumor, embryonal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, digestive lymphoma, small to medium cell lymphoma, cecum 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 ductal carcinoma, invasive pancreatic ductal carcinoma, adenocarcinoma of pancreatic cancer, metastatic adenocarcinoma, Acinar cell carcinoma, adenosquamous carcinoma, giant cell tumor, intraductal papillary mucinous neoplasm, mucinous cystadenocarcinoma, pancreatoblastoma, pancreatic head cell tumor, Frants' tumor, serous cystadenocarcinoma, solid papillary carcinoma, gastrinoma, glucagonoma, insulinoma, multiple endocrine neoplasia 1 (Wermer's syndrome), nonfunctioning islet cell tumor, somatostatinoma, VIP-secreting tumor, cervical cancer, endometrial cancer, fibrosarcoma, bone and joint sarcoma, Ewing's sarcoma, Wilms' tumor, hepatoblastoma, soft tissue sarcoma, acute leukemia, chronic leukemia, spinal cord tumor, malignant soft tissue tumor, teratoma group tumor, head and neck cancer includes, but is not limited to, hypopharyngeal cancer, oropharynx cancer, tongue cancer, nasopharyngeal cancer, oral cancer, lip cancer, paranasal sinus cancer, laryngeal cancer, recurrent brain tumor, etc.

[0148] Furthermore, preferred subjects (patients) are mammals, including, for example, primates, pet animals, livestock, sport animals, etc., with humans, dogs, and cats being particularly preferred.

[0149] When the conjugates used in the present invention are used as pharmaceutical compositions, they can be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of a sterile solution or suspension injection in water or other pharmaceutically acceptable liquid. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterilized water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, binders, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is such that an appropriate dose within the indicated range can be obtained.

[0150] When the conjugate of the present invention is used as a pharmaceutical composition, it can be formulated in a lyophilized state containing any salts, surfactants, buffers, sugars, and cryoprotectants (including some sugars).

[0151] Sterile compositions for injection can be formulated according to standard pharmaceutical practice using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These solutions may be used in combination with appropriate solubilizers, such as alcohols, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and nonionic surfactants such as Polysorbate 80™ and HCO-60. Examples of oily solutions include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. These solutions may also be combined with buffers such as phosphate buffer and sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants. The prepared injection solutions are typically filled into appropriate ampoules. Examples of oily solutions include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution may also be formulated with a buffer such as a phosphate buffer solution or a sodium acetate buffer solution, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, or an antioxidant. The prepared injection solution is usually filled into an appropriate ampule.

[0152] Administration may be oral or parenteral, preferably parenteral, and specific examples include injections, intranasal administrations, pulmonary administrations, transdermal administrations, etc. Examples of injections include intravenous injections, intramuscular injections, intraperitoneal injections, subcutaneous injections, intratumoral injections, etc., which can be used for systemic or local administration.

[0153] Furthermore, an appropriate administration method can be selected depending on the patient's age, body weight, sex, symptoms, etc. The dosage of a pharmaceutical composition containing an antibody or a polynucleotide encoding an antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration, 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 per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg / body per patient, although it is not necessarily limited to these values.

[0154] The dosage and administration method will vary depending on the patient's weight, age, sex, symptoms, etc., but can be appropriately selected by those skilled in the art.

[0155] By administering to a subject a pharmaceutical composition for treating and / or preventing cancer, which comprises the conjugate of the present invention as an active ingredient, it is possible to treat and / or prevent the above-mentioned cancers that express CAPRIN-1 on the cell membrane surface, preferably breast cancer, kidney cancer, pancreatic cancer, colon 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 cancer.

[0156] The present invention will be specifically described below based on examples, but the scope of the present invention is not limited to these specific examples.

[0157] Example 1: Anti-CAPRIN-1 Polyclonal Antibody The anti-CAPRIN-1 polyclonal antibody immunologically reactive with the CAPRIN-1 protein used in the conjugate of the present invention was prepared by mixing 1 mg of human CAPRIN-1 recombinant protein represented by SEQ ID NO: 2 and SEQ ID NO: 4, prepared according to Example 3 of WO 2010 / 016526, with an equal volume of incomplete Freund's adjuvant (IFA) solution. This mixture was subcutaneously administered to rabbits four times every two weeks. Blood was then collected to obtain antisera containing the polyclonal antibody. The obtained antisera was purified using a protein G carrier (GE Healthcare Biosciences) to obtain a polyclonal antibody against the CAPRIN-1 protein (anti-CAPRIN-1 polyclonal antibody #1). Additionally, serum from a rabbit not administered the antigen was purified using a protein G carrier in the same manner as above to serve as a rabbit control antibody.

[0158] Furthermore, the following polyclonal antibodies #2 to #6 against partial polypeptides of CAPRIN-1 were obtained in the same manner as in the preparation of the polyclonal antibodies against the CAPRIN-1 protein.

[0159] Anti-CAPRIN-1 polyclonal antibody #2 against a partial polypeptide represented by SEQ ID NO: 37 (SEQ ID NO: 31 herein) described in CAPRIN-1 protein WO2011 / 096528.

[0160] Anti-CAPRIN-1 polyclonal antibody #3 against a partial polypeptide represented by SEQ ID NO: 5 (SEQ ID NO: 32 herein) described in WO2013 / 018894.

[0161] Anti-CAPRIN-1 polyclonal antibody #4 against a partial polypeptide represented by SEQ ID NO: 5 (SEQ ID NO: 33 herein) described in WO2013 / 125654.

[0162] Anti-CAPRIN-1 polyclonal antibody #5 against the partial polypeptide represented by SEQ ID NO: 37 (SEQ ID NO: 34 herein) described in WO2011 / 096533.

[0163] Anti-CAPRIN-1 polyclonal antibody #6 against a partial polypeptide represented by SEQ ID NO: 37 (SEQ ID NO: 35 herein) described in WO2011 / 096534.

[0164] Example 2 Anti-CAPRIN-1 Monoclonal Antibody The following anti-CAPRIN-1 monoclonal antibody was used in the conjugate of the present invention.

[0165] A monoclonal antibody against CAPRIN-1 described in WO2011 / 096528, wherein CDR1 to CDR3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively, and CDR1 to CDR3 of the light chain variable region consist of the amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively (for example, an antibody comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO: 39, which includes CDR1 to CDR3 of the heavy chain variable region, and the amino acid sequence of a light chain variable region represented by SEQ ID NO: 43, which includes CDR1 to CDR3 of the light chain variable region).

[0166] A monoclonal antibody against CAPRIN-1 described in WO2015 / 020212, wherein CDR1 to CDR3 of a heavy chain variable region consist of the amino acid sequences of SEQ ID NOs: 44, 45, and 46, respectively, and CDR1 to CDR3 of a light chain variable region consist of the amino acid sequences of SEQ ID NOs: 48, 49, and 50, respectively (for example, an antibody comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO: 47, which includes CDR1 to CDR3 of the heavy chain variable region, and the amino acid sequence of a light chain variable region represented by SEQ ID NO: 51, which includes CDR1 to CDR3 of the light chain variable region).

[0167] A monoclonal antibody against CAPRIN-1 described in WO2011 / 096519, wherein CDR1 to CDR3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, respectively, and CDR1 to CDR3 of the light chain variable region consist of the amino acid sequences of SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58, respectively (for example, an antibody comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO: 55, which includes CDR1 to CDR3 of the heavy chain variable region, and the amino acid sequence of a light chain variable region represented by SEQ ID NO: 59, which includes CDR1 to CDR3 of the light chain variable region).

[0168] A monoclonal antibody against CAPRIN-1 described in WO2013 / 125654, wherein CDR1 to CDR3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NOs: 60, 61, and 62, respectively, and CDR1 to CDR3 of the light chain variable region consist of the amino acid sequences of SEQ ID NOs: 64, 65, and 66, respectively (for example, an antibody comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO: 63, which includes CDR1 to CDR3 of the heavy chain variable region, and the amino acid sequence of a light chain variable region represented by SEQ ID NO: 67, which includes CDR1 to CDR3 of the light chain variable region).

[0169] A monoclonal antibody against CAPRIN-1 described in WO2011 / 096517, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 68 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 69.

[0170] Monoclonal antibodies against CAPRIN-1 described in WO2011 / 096528 include an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 70 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 71; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 72 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 73; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 74 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 75; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 76 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 77; and an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 78 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 79.

[0171] A monoclonal antibody against CAPRIN-1 described in WO2011 / 096533, comprising an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 80 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 81; and an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 82 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 83.

[0172] A monoclonal antibody against CAPRIN-1 described in WO2011 / 096534, comprising an antibody having an amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 84 and an amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 85; and an antibody having an amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 86 and an amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 87.

[0173] Monoclonal antibodies against CAPRIN-1 described in WO2010 / 016526, which comprise an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 88 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 89; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 90 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 91; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 92 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 93; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 94; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 96 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 97; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 98 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 99; and an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 100 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 101.

[0174] A monoclonal antibody against CAPRIN-1 described in WO2013 / 018894, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 102 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 103; and an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 104 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 105.

[0175] A monoclonal antibody against CAPRIN-1 described in WO2013 / 018892, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 106 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 107.

[0176] A monoclonal antibody against CAPRIN-1 described in WO2013 / 018891, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 108 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 109.

[0177] A monoclonal antibody against CAPRIN-1 described in WO2013 / 018889, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 110 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 111.

[0178] A monoclonal antibody against CAPRIN-1 described in WO2013 / 018883, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 112 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 113.

[0179] A monoclonal antibody against CAPRIN-1 described in WO2013 / 125636, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 114 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 115.

[0180] A monoclonal antibody against CAPRIN-1 described in WO2013 / 125654, comprising an antibody having an amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 116 and an amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 117; and an antibody having an amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 118 and an amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 119.

[0181] A monoclonal antibody against CAPRIN-1 described in WO2013 / 125630, comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 120 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 121.

[0182] an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 123; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 124 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 125; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 126 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 127; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 128 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 129; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 130 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 131; and an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 132 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 133.

[0183] For the above-mentioned anti-CAPRIN-1 monoclonal antibodies, a nucleotide sequence was designed so that a heavy chain variable region in which CDR1 to CDR3 consist of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and in which the framework regions comprise a human antibody sequence, could be expressed, and inserted into a mammalian expression vector into which the heavy chain constant region of human IgG1 had been inserted. Similarly, a nucleotide sequence was designed so that a light chain variable region in which CDR1 to CDR3 consist of SEQ ID NOs: 40, 41, and 42, respectively, and in which the framework regions comprise a human antibody sequence, could be expressed, and inserted into a mammalian expression vector into which the light chain constant region of human IgG1 had been inserted. The above two recombinant expression vectors were introduced into mammalian cells according to a standard method to obtain a culture supernatant containing humanized monoclonal antibody #1 against CAPRIN-1 (humanized antibody #1), in which CDRs 1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and CDRs 1 to 3 of the light chain variable region consist of the amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively.

[0184] Similarly, a nucleotide sequence was designed to enable expression of a heavy chain variable region represented by SEQ ID NO: 47, in which CDRs 1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively, and in which the framework regions comprise the sequence of a human antibody, and this was inserted into a mammalian expression vector into which the heavy chain constant region of human IgG1 had been inserted. Similarly, a base sequence was designed to enable expression of a heavy chain variable region represented by SEQ ID NO:51, in which CDR1 to 3 of the light chain variable region consist of the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively, and the framework regions comprise the sequence of a human antibody. This base sequence was inserted into a mammalian expression vector into which the heavy chain constant region of human IgG1 had been inserted, and the two recombinant expression vectors were introduced into mammalian cells according to standard methods to obtain a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #2 (humanized antibody #2), in which CDR1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, and CDR1 to 3 of the light chain variable region consist of the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively.

[0185] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #3 (humanized antibody #3) was obtained, in which CDRs 1 to 3 of the heavy chain variable region consisted of the amino acid sequences of SEQ ID NOs: 52, 53, and 54, respectively, and CDRs 1 to 3 of the light chain variable region consisted of the amino acid sequences of SEQ ID NOs: 56, 57, and 58, respectively.

[0186] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #4 (humanized antibody #4) was obtained, in which CDRs 1 to 3 of the heavy chain variable region consisted of the amino acid sequences of SEQ ID NOs: 60, 61, and 62, respectively, and CDRs 1 to 3 of the light chain variable region consisted of the amino acid sequences of SEQ ID NOs: 64, 65, and 66, respectively.

[0187] Similarly, culture supernatants containing the following humanized anti-CAPRIN-1 monoclonal antibodies #9 to #41 (humanized antibodies #9 to #41) were obtained.

[0188] Humanized monoclonal antibody #9 (humanized antibody #9) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 68 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 69.

[0189] Humanized monoclonal antibody #10 (humanized antibody #10) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 70 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 71.

[0190] Humanized monoclonal antibody #11 (humanized antibody #11) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 72 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 73.

[0191] Humanized monoclonal antibody #12 (humanized antibody #12) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 74 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 75.

[0192] Humanized monoclonal antibody #13 (humanized antibody #13) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 76 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 77.

[0193] Humanized monoclonal antibody #14 (humanized antibody #14) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 78 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 79.

[0194] Humanized monoclonal antibody #15 (humanized antibody #15) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 80 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 81.

[0195] Humanized monoclonal antibody #16 (humanized antibody #16) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 82 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 83.

[0196] Humanized monoclonal antibody #17 (humanized antibody #17) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 84 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 85.

[0197] Humanized monoclonal antibody #18 (humanized antibody #18) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 86 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 87.

[0198] Humanized monoclonal antibody #19 (humanized antibody #19) comprising the amino acid sequence of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 88 and the amino acid sequence of the light chain variable region represented by the amino acid sequence of SEQ ID NO: 89.

[0199] Humanized monoclonal antibody #20 (humanized antibody #20) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 90 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 91.

[0200] Humanized monoclonal antibody #21 (humanized antibody #21) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 92 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 93.

[0201] Humanized monoclonal antibody #22 (humanized antibody #22) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 94 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 95.

[0202] Humanized monoclonal antibody #23 (humanized antibody #23) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 96 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 97.

[0203] Humanized monoclonal antibody #24 (humanized antibody #24) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 98 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 99.

[0204] Humanized monoclonal antibody #25 (humanized antibody #25) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 100 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 101.

[0205] Humanized monoclonal antibody #26 (humanized antibody #26) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 102 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 103.

[0206] Humanized monoclonal antibody #27 (humanized antibody #27) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 104 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 105.

[0207] Humanized monoclonal antibody #28 (humanized antibody #28) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 106 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 107.

[0208] Humanized monoclonal antibody #29 (humanized antibody #29) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 108 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 109.

[0209] Humanized monoclonal antibody #30 (humanized antibody #30) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 110 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 111.

[0210] Humanized monoclonal antibody #31 (humanized antibody #31) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 112 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 113.

[0211] Humanized monoclonal antibody #32 (humanized antibody #32) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 114 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 115.

[0212] Humanized monoclonal antibody #33 (humanized antibody #33) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 116 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 117.

[0213] Humanized monoclonal antibody #34 (humanized antibody #34) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 118 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 119.

[0214] Humanized monoclonal antibody #35 (humanized antibody #35) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 120 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 121.

[0215] Humanized monoclonal antibody #36 (humanized antibody #36) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 123.

[0216] Humanized monoclonal antibody #37 (humanized antibody #37) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 124 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 125.

[0217] Humanized monoclonal antibody #38 (humanized antibody #38) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 126 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 127.

[0218] Humanized monoclonal antibody #39 (humanized antibody #39) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 128 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 129.

[0219] Humanized monoclonal antibody #40 (humanized antibody #40) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 130 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 131.

[0220] Humanized monoclonal antibody #41 (humanized antibody #41) comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 132 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 133.

[0221] Furthermore, for humanized antibody #1 of the above anti-CAPRIN-1 monoclonal antibodies, a nucleotide sequence was designed to enable expression of a heavy chain variable region in which CDRs 1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and in which the framework regions comprise the sequence of a human antibody, and this was inserted into a mammalian expression vector containing the heavy chain constant region of human IgG1 in which serine (Ser), amino acid 239 (EU numbering), has been substituted with aspartic acid (Asp) and isoleucine (Ile), amino acid 332 (EU numbering), has been substituted with glutamic acid (Glu). Furthermore, a nucleotide sequence was designed to enable expression of a light chain variable region in which CDRs 1 to 3 of the light chain variable region consist of SEQ ID NOs: 40, 41, and 42, respectively, and in which the framework regions comprise the amino acid sequence of a human antibody, and this was 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 according to standard methods to obtain a culture supernatant containing monoclonal antibody #5 against humanized CAPRIN-1 (humanized antibody #5), which consisted of the heavy chain full-length amino acid sequence consisting of the heavy chain variable region prepared above and a heavy chain constant region of human IgG1 in which serine (Ser), amino acid 239 (EU numbering), has been substituted with aspartic acid (Asp) and isoleucine (Ile), amino acid 332 (EU numbering), has been substituted with glutamic acid (Glu), and the light chain full-length amino acid sequence consisting of the light chain variable region and human light chain constant region prepared above.

[0222] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #6 (humanized antibody #6) consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the humanized antibody #2 prepared above was obtained.

[0223] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #7 (humanized antibody #7) consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the humanized antibody #3 prepared above was obtained.

[0224] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #8 (humanized antibody #8) consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the humanized antibody #4 prepared above was obtained.

[0225] Similarly, culture supernatants containing humanized anti-CAPRIN-1 antibodies #42 to #74 (humanized antibodies #42 to #74) each consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of each of the humanized antibodies #9 to #41 prepared above were obtained.

[0226] The culture supernatants containing the obtained humanized anti-CAPRIN-1 monoclonal antibodies #1 to #74 were purified using Hitrap Protein A Sepharose FF (GE Healthcare) according to standard methods, and then substituted with PBS(-) and filtered through a 0.22 μm filter (Millipore) to prepare samples.

[0227] Example 3: Preparation of conjugates of anti-CAPRIN-1 antibodies and PBD derivative (SGD-1910) Conjugates of the anti-CAPRIN-1 polyclonal antibodies #1 to #6 described in Example 1 and SGD-1910 (CAS #1342820-51-2) were prepared according to standard methods. Purified anti-CAPRIN-1 polyclonal antibody #1 was dissolved in PBS(-) at a concentration of 10 mg / ml. A PBS(-) solution containing 100 mM EDTA and 50 mM L-cysteine ​​was added to the solution containing polyclonal antibody #1 in a 1 / 10 ratio, followed by a reduction reaction at 4°C for 1 hour. After reduction, the solution was replaced with PBS(-) containing 1 mM EDTA using a Zeba™ Spin Desalting Column (MWCO 70k or 40k) and allowed to stand at room temperature for 18 to 72 hours to reoxidize the polyclonal antibody. Propylene glycol was added to the reoxidized polyclonal antibody #1 to a concentration of 50%. 10 mM SGD-1910 dissolved in DMSO was added to the solution containing polyclonal antibody #1 at a molar ratio of antibody to SDG-1910 of 1:5, and the mixture was allowed to react at room temperature for at least 1 hour. After the reaction, SGD-1910 that did not bind to the antibody was removed using a Zeba™ Spin Desalting Column (MWCO 70k or 40k), and the solvent was replaced with PBS(-) (repeated twice). After replacing the buffer with PBS(-), the solution was filtered using a 0.22 μm sterilizing filter membrane to obtain a solution containing the anti-CAPRIN-1 polyclonal antibody #1-SDG-1910 (conjugate 1) of the present invention. Similarly, solutions containing conjugates using SDG-1910 were obtained for anti-CAPRIN-1 polyclonal antibodies #2 to #6 (the conjugate using anti-CAPRIN-1 polyclonal antibody #2: conjugate 2, the conjugate using anti-CAPRIN-1 polyclonal antibody #3: conjugate 3, the conjugate using anti-CAPRIN-1 polyclonal antibody #4: conjugate 4, the conjugate using anti-CAPRIN-1 polyclonal antibody #5: conjugate 5, and the conjugate using anti-CAPRIN-1 polyclonal antibody #6: conjugate 6).Furthermore, using the same method as above, a solution containing a conjugate with SDG-1910 (conjugate 10) was obtained using humanized antibody #4, an anti-CAPRIN-1 monoclonal antibody described in Example 2. Using the same procedure as above, a solution containing a conjugate with the SDG-1910 linker (control conjugate 1) was also obtained using the same method as above, but for the rabbit control antibody described in Example 1, which does not react with the CAPRIN-1 protein.

[0228] Furthermore, a conjugate of humanized antibody #1, an anti-CAPRIN-1 monoclonal antibody described in Example 2, and SGD-1910 (CAS# 1342820-51-2) was prepared according to a standard method. Purified humanized antibody #1 was dissolved in PBS(-) at a concentration of 10 mg / mL to prepare a solution. A PBS(-) solution containing TCEP at a final concentration of 10 mM was added to the solution containing humanized antibody #1, followed by a reduction reaction at room temperature for 1 hour. After reduction, the solution was replaced with PBS(-) containing 1 mM EDTA using a Zeba™ Spin Desalting Column (MWCO 70k or 40k), and then an equal amount of propylene glycol was added. To the solution containing humanized antibody #1, 10 mM SGD-1910 dissolved in DMSO was added so that the molar ratio of antibody to SDG-1910 was 1:3, and the mixture was allowed to react at room temperature for at least 1 hour. After the reaction, SGD-1910 that had not bound to the antibody was removed using a Zeba™ Spin Desalting Column (MWCO 70k or 40k), and the solvent was replaced with PBS(-) (repeated twice). After replacing the buffer with PBS(-), the mixture was filtered using a 0.22 μm sterilizing filter membrane to obtain a solution containing humanized antibody #1-SDG-1910 (conjugate 7) of the present invention. Similarly, solutions containing conjugates using SDG-1910 were obtained for anti-CAPRIN-1 polyclonal antibodies #2 to #6 and humanized antibodies #2, #3, and #5 to #74 (conjugates 8, 9, 11 to 53). Using the same procedure as above, a solution containing a conjugate with the SDG-1910 linker (control conjugate 2) was also obtained using a similar method for a human IgG control antibody that does not react with the CAPRIN-1 protein.

[0229] Example 4 Specific Reactivity of Conjugate with CAPRIN-1 Protein and CAPRIN-1-Expressing Cancer Cells The conjugate (anti-CAPRIN-1 antibody-SDG-1910 conjugate) prepared in Example 3 was evaluated for its specific reactivity with CAPRIN-1 protein and its reactivity on the cell membrane surface of human cancer cells and mouse cancer cells expressing CAPRIN-1 protein.

[0230] Specific reactivity to the CAPRIN-1 protein was confirmed using ELISA. 100 μL of a 1 μg / mL CAPRIN-1 protein solution was added per 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 per well and incubated at room temperature for 3 hours. The solution was removed, and the wells were washed three times with 400 μL of PBS-T. Then, 100 μL of each solution containing conjugates 1 to 6 and control conjugate 1 was added per 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-fold with PBS was added per 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 per well and the plate was left to stand for 15 to 30 minutes to allow the color reaction to occur. After color development, 100 μL of 1 N sulfuric acid was added per well to stop the reaction, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorbance meter. As a result, conjugates 1 to 6 had higher absorbance values ​​than the negative control, control conjugate 1, confirming that they react specifically with the CAPRIN-1 protein.

[0231] Similarly, a specific reaction to the CAPRIN-1 protein was detected by ELISA using an HRP-labeled anti-human antibody for the anti-CAPRIN-1 antibody-SDG-1910 conjugates using humanized antibodies #1 to #74 prepared in Example 3. As a result, the absorbance value was higher than that of human IgG (SIGMA) used as a negative control and control conjugate 2, confirming that the conjugates react specifically with the CAPRIN-1 protein.

[0232] Next, the reactivity of CAPRIN-1-expressing cancer cells to the cell membrane surface was confirmed by flow cytometry. ５ Human breast cancer cells BT-474 (obtained from ATCC) and mouse breast cancer cells 4T1 (obtained from ATCC) were centrifuged in 1.5 ml microcentrifuge tubes, and 100 μL of a solution containing conjugates 1 to 6 and control conjugate 1 was added to each tube and allowed to stand 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 allowed to stand at 4°C for 1 hour. After washing with 0.5% FBS-PBS(-), the cells were reacted with BD Horizon Fixable Viability Stain (FVS) Reagents (FVS450, Becton, Dickinson and Company) to stain dead cells, and then the fluorescence intensity was measured using a FACSFortessa™ (Becton, Dickinson and Company). As a result, the anti-CAPRIN-1 antibody-SDG-1910 conjugates (conjugates 1 to 6) had higher fluorescence intensity than the negative control, control conjugate 1, confirming that they strongly react with the cell surface of human cancer cell line BT474, which expresses CAPRIN-1.

[0233] Similarly, for anti-CAPRIN-1 antibody-SDG-1910 conjugates using humanized antibodies #1 to #74, specific reactivity to CAPRIN-1-expressing cancer cells, human cancer cell BT474 and mouse cancer cell 4T1, was detected using Alexa488-labeled anti-human IgG (H+L) antibody. As a result, it was confirmed that anti-CAPRIN-1 antibody-SDG-1910 conjugates using humanized antibodies #1 to #74 had higher fluorescence intensity than the negative control control conjugate 2, i.e., they strongly reacted with the cell surface of human cancer cell BT474 and 4T1, which express CAPRIN-1.

[0234] Similarly, the reactivity of the anti-CAPRIN-1 polyclonal antibody prepared in Example 3 and the anti-CAPRIN-1 antibody-SDG-1910 conjugates using humanized antibodies #1 to #74 with various human cancer cells and mouse cancer cells was confirmed. Human cancer cells in which CAPRIN-1 gene expression has been confirmed and in which CAPRIN-1 expression on the cell membrane surface of cancer cells has been confirmed include breast cancer cells (BT-474), colon cancer cells (HT-29, HCT116, Lovo), lung cancer cells (A549), gastric cancer cells (NCI-N87), uterine cancer cells (HEC-1-A), prostate cancer cells (22Rv1), pancreatic cancer cells (Panc10.5), liver cancer cells (HepG2), ovarian cancer cells (MCAS, SKOV3, TOV112D), renal cancer cells (Caki-2), brain cancer cells (U-87MG), bladder cancer cells (T24, UMUC3), cholangiocarcinoma cells (KKU213), and fibrosarcoma 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) in which expression of the CAPRIN-1 gene has been confirmed. The anti-CAPRIN-1 antibody-SDG-1910 conjugate prepared in Example 3 showed stronger fluorescence intensity than the negative controls, Control Conjugate 1 and Control Conjugate 2, and was confirmed to react strongly with the cell membrane surface of cancer cells in which CAPRIN-1 is expressed.

[0235] Example 5 Internalization The internalization of anti-CAPRIN-1 antibody-SDG-1910 conjugates into cancer cells using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 was evaluated in vitro. The anti-CAPRIN-1 antibody-SDG-1910 conjugate (4 μg / mL) and Incucyte™ Human Fabfluor-pH Antibody Labeling Dye (2 μg / mL) were mixed in equal amounts and left to stand at 37°C for 1 hour to conjugate the anti-CAPRIN-1 antibody-SDG-1910 conjugate with Human Fabfluor-pH Antibody Labeling Dye. 100 μL of this conjugate was diluted to 2 × 10 ５ The conjugate was added to HT29 cancer cells and incubated at 37°C for 6 hours. After the incubation, internalization was evaluated by measuring fluorescence intensity using flow cytometry according to a standard method. As a result of the evaluation, the anti-CAPRIN-1 antibody-SDG-1910 conjugate exhibited a stronger fluorescence intensity than the negative control (HT29 cells incubated with only the anti-CAPRIN-1 antibody-SDG-1910 conjugate). These results demonstrated that the anti-CAPRIN-1 antibody-SDG-1910 conjugate was internalized into human cancer cells. Similarly, when the internalization into HT29 of Trastuzumab, an antibody against HER2, Cetuximab, an antibody against EGFR, Enfortumab vedotin, an ADC targeting Nectin-4, and Sacituzumab govitecan, an ADC targeting TROP2, was evaluated by flow cytometry, the anti-CAPRIN-1 antibody-SDG-1910 conjugate had a stronger fluorescence intensity than any of the antibodies.

[0236] (Example 6) Antitumor Activity of Conjugates The antitumor activity of anti-CAPRIN-1 antibody-SDG-1910 conjugates against cancer cells was evaluated in vitro using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3. The antitumor activity was evaluated against human cancer cells in which CAPRIN-1 expression on the cell membrane surface has been confirmed in Example 4: breast cancer cells (BT-474), colon cancer cells (HT-29, HCT116), gastric cancer cells (NCI-N87), uterine cancer cells (Ishikawa), pancreatic cancer cells (Panc10.5), ovarian cancer cells (MCAS, TOV112D), and head and neck cancer cells (FaDu).

[0237] The cancer cells were cultured by a standard method and plated in a black 96-well plate suitable for cell culture (96 well optical Black Plate Polymer Base Black with Lid Cell Culture Sterile PS 165305, Thermo) at 1 to 4 x 10 cells per well. ４ The cells were seeded at 90 μL / well and incubated in 5% CO ２ The cells were cultured in an incubator at 37°C for 18 to 24 hours. －２ μg / mL to 10 ２ The anti-CAPRIN-1 antibody-SDG-1910 conjugates prepared in Example 3 using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 were added at 10 μL per well, and the plates were incubated in 5% CO ２ The plates were incubated in an incubator at 37°C. Three to four days after the start of incubation, cell viability was measured using a Cell Titer Glow Luminescent Cell Viability Assay (#7573, Promega). 100 μL of substrate was added per well, and the plates were shaken for 2 minutes using a plate shaker. After allowing to stand for 10 minutes, the luminescence signal intensity was measured using a SpectraMax® iD3 (MOLECULAR DEVICE) to calculate the ATP content of surviving cells.

[0238] As a result, when the anti-CAPRIN-1 antibody-SDG-1910 conjugates using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 described in the present invention were used, the number of surviving cancer cells in a well without conjugate added was 100%, and 70% or less of the cancer cells survived at concentrations of 1 μg / mL or higher. These results demonstrate that conjugates of antibodies against CAPRIN-1 and PBD exhibit antitumor activity against cancer cells.

[0239] (Example 7) Antitumor Effect of Conjugates in Cancer-Bearing Mice Conjugate 7 and conjugate 10, which are the anti-CAPRIN-1 antibody-SDG-1910 conjugates prepared in Example 3, were evaluated for their antitumor effect in vivo in cancer-bearing mice. The antitumor effect of the conjugate of the present invention was examined using NOD-SCID mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. 10 per mouse ７ Human colon cancer cells HT29 were mixed with Matrigel (SIGMA) and subcutaneously implanted, and the tumors grew to 50 mm ３ Tumor-bearing mice were prepared by growing the tumor to a size of 100 or larger. HT29 expresses CAPRIN-1 protein on the cell membrane surface, and as shown in Example 4, Conjugates 7 and 10 bind to HT29. Conjugates 7 and 10 were administered twice a week via the tail vein at 0.3 mg / kg to HT29 tumor-bearing mice. Furthermore, according to the method described in Example 3, a solution containing a conjugate of trastuzumab (Chugai Pharmaceutical Co., Ltd.) and SDG-1910 was prepared so that the drug bound to the same extent as the conjugate using an antibody against CAPRIN-1. The same amount was administered to the tumor-bearing mice as a comparative control. HT29 expresses HER2 protein, the target antigen of trastuzumab, on the cell membrane surface, to which the conjugate of trastuzumab and PBD specifically binds. PBS(-) was administered to tumor-bearing mice as a negative control.

[0240] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor).２ × 0.5. As a result of the evaluation, on day 7 after the start of administration, when the tumor volume of the negative control was taken as 100%, the tumor volumes of both the mice administered with Conjugate 7 and Conjugate 10 were less than 50%. When the tumor volume of the cancer-bearing mice administered only the antibody against CAPRIN-1 used in the above conjugate was taken as 100%, the tumor volume of the cancer-bearing mice administered with the above conjugate was less than 50%. On the other hand, the tumor volume of the mice administered with a solution containing a conjugate of trastuzumab and PBD, used as a comparison control, was 80% or more of the negative control. Furthermore, when the tumor volume of the cancer-bearing mice administered with trastuzumab only was taken as 100%, the tumor volume of the cancer-bearing mice administered with the above conjugate was less than 75%.

[0241] The results of this evaluation showed that the conjugate of SDG-1910 prepared in Example 3 using an antibody against CAPRIN-1 exhibited a significantly stronger antitumor effect than the negative control (untreated control group).Furthermore, it was shown to have a significantly stronger antitumor effect than the conjugate of trastuzumab and SDG-1910 prepared as a comparative control.

[0242] Example 8 Preparation of Conjugates of Anti-CAPRIN-1 Antibody and PBD Derivative (Tesirine) Similar to Chemical Example 3 below, SATA was attached as a spacer to the lysine residue of anti-CAPRIN-1 polyclonal antibody #1 described in Example 1 according to a standard method, and then tesirine, a PBD derivative represented by the following chemical structure, was attached to the antibody via a maleimide group to obtain a solution containing the conjugate (conjugate 54). Similarly, a conjugate of humanized antibody #1, the anti-CAPRIN-1 monoclonal antibody described in Example 2, with tesirine was prepared, and a solution containing the conjugate (conjugate 60) was obtained. Similarly, conjugates of tesirine derivatives were prepared for anti-CAPRIN-1 polyclonal antibodies #2 to #6 and humanized anti-CAPRIN-1 monoclonal antibodies #2 to #47. Using the same procedures as above, solutions containing conjugates with the above-mentioned tesirine were obtained in the same manner for the rabbit control antibody and human IgG control antibody described in Example 1, which do not react with the CAPRIN-1 protein (control conjugate 3, control conjugate 4). The prepared conjugates were evaluated in the same manner as in Examples 4 to 6, and it was confirmed that the conjugates exhibited specific reactivity with the CAPRIN-1 protein and CAPRIN-1-expressing cancer cells, were internalized, and exhibited antitumor activity.

[0243]

[0244] (Example 9) Antitumor effect of conjugates of anti-CAPRIN-1 antibody and PBD derivative (tesirine) in tumor-bearing mice The antitumor effect of conjugates 60 and 63 prepared in Example 8 was evaluated in vivo in tumor-bearing mice. The antitumor effect of the conjugate of the present invention was examined using NOD-SCID mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. 10 per mouse ７ Human colon cancer cells HT29 were mixed with Matrigel (SIGMA) and subcutaneously implanted, and tumors were grown to 100-200 mm ３Tumor-bearing mice were prepared by growing the tumor to a tumor size of 100 or more. HT29 expresses the CAPRIN-1 protein on the cell membrane surface, and conjugates 60 and 63 bind to HT29. Conjugates 60 and 63 were administered once via the tail vein to HT29 tumor-bearing mice at 2 mg / kg and 6 mg / kg, respectively. PBS(-) was administered to negative control tumor-bearing mice.

[0245] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor). ２ The calculation was performed using a ratio of 0.5 to 1. As a result of the evaluation, on and after the seventh day after the start of administration, when the tumor volume of the negative control was taken as 100%, the tumor volumes of the mice administered 2 mg / kg of conjugate 60 and conjugate 63 were each less than 50% of the tumor volume of the negative control. On the other hand, tumors regressed in cancer-bearing mice administered 6 mg / kg of conjugate 60 and conjugate 63.

[0246] The results of this evaluation showed that the conjugate of Tesirine prepared in Example 8 using an antibody against CAPRIN-1 exhibited a significantly stronger antitumor effect than the negative control (untreated control group).

[0247] Example 10: Preparation of conjugates of anti-CAPRIN-1 antibodies and IGN derivatives (DGN549) As in Example 3, SATA was attached as a spacer to the lysine residue of anti-CAPRIN-1 polyclonal antibody #1 described in Example 1 according to a standard method, and then DGN549, an IGN derivative represented by the following chemical structure, was attached to the antibody via a maleimide group to obtain a solution containing the conjugate (conjugate 107). Similarly, a conjugate of humanized antibody #1, the anti-CAPRIN-1 monoclonal antibody described in Example 2, and DGN549 was prepared, and a solution containing the conjugate (conjugate 113) was obtained. Similarly, conjugates of anti-CAPRIN-1 polyclonal antibodies #2 to #6 and humanized anti-CAPRIN-1 monoclonal antibodies #2 to #47 with DGN549 were prepared. Using the same procedures as above, solutions containing conjugates with DGN549 were obtained in the same manner for the rabbit control antibody and human IgG control antibody described in Example 1, which do not react with CAPRIN-1 protein (control conjugate 5, control conjugate 6). The conjugates prepared above were evaluated in the same manner as in Examples 4 to 6, and it was confirmed that the conjugates exhibited specific reactivity with CAPRIN-1 protein and CAPRIN-1-expressing cancer cells, were internalized, and exhibited antitumor activity.

[0248]

[0249] (Example 11) Antitumor Effect of Conjugates of Anti-CAPRIN-1 Antibody and IGN (DGN549) in Cancer-Bearing Mice 1 Conjugate 107 and conjugate 113 prepared in Example 10 were evaluated for their antitumor effects in vivo in cancer-bearing mice. The antitumor effect of the conjugate of the present invention was examined using NOD-SCID mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. 10 per mouse ７ Human head and neck cancer cells, FaDu, were mixed with Matrigel (SIGMA) and implanted subcutaneously, and tumors were grown to 100-200 mm ３Tumor-bearing mice were generated and allowed to grow to a size of 100 or larger. FaDu expresses CAPRIN-1 protein on the cell membrane surface, and conjugates 107 and 113 bind to FaDu. Conjugates 107 and 113 were administered once via the tail vein to the FaDu-bearing mice at 2 mg / kg and 6 mg / kg, respectively. PBS(-) was administered to the negative control tumor-bearing mice.

[0250] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor). ２ The calculation was performed using a ratio of 0.5 to 1. As a result of the evaluation, on and after the seventh day after the start of administration, when the tumor volume of the negative control was taken as 100%, the tumor volumes of the mice administered 2 mg / kg of conjugate 103 and conjugate 117 were less than 50% of the tumor volume of the negative control. On the other hand, tumors regressed in cancer-bearing mice administered 6 mg / kg of conjugate 103 and conjugate 117.

[0251] The results of this evaluation showed that the conjugate of DGN549 prepared in Example 10 using an antibody against CAPRIN-1 exhibited a significantly stronger antitumor effect than the negative control (untreated control group).

[0252] (Example 12) Antitumor Effect of Conjugates of Anti-CAPRIN-1 Antibody and IGN (DGN549) in Tumor-Bearing Mice 2 The antitumor effects of conjugates 107 and 113 prepared in Example 10 were evaluated in vivo in tumor-bearing mice. The antitumor effect of the conjugate of the present invention was examined using C.B-17 SCID mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. 5 × 10 per mouse 6 Human ovarian cancer cells TOV112D were mixed with Matrigel (SIGMA) and implanted subcutaneously, and tumors grew to an average of 150-200 mm. ３Tumor-bearing mice were generated and allowed to grow to a size of 100 or larger. TOV112D expresses the CAPRIN-1 protein on the cell membrane surface, and conjugates 107 and 113 bind to TOV112D. Conjugates 107 and 113 were each administered once at 0.5 mg / kg via the tail vein to TOV112D-bearing mice. PBS(-) was administered to negative control tumor-bearing mice.

[0253] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor). ２ As a result of the evaluation, 15 days after the start of administration, the tumor volumes of the cancer-bearing mice administered Conjugate 103 and Conjugate 117 were 13% or less when the tumor volume of the negative control was taken as 100%.

[0254] The results of this evaluation showed that the conjugate of DGN549 prepared in Example 10 using an antibody against CAPRIN-1 exhibited a significantly stronger antitumor effect than the negative control (untreated control group).

[0255] (Example 13) Antitumor Effect of Conjugates of Anti-CAPRIN-1 Antibody and IGN (DGN549) in Tumor-Bearing Mice 3 The antitumor effects of conjugates 107 and 113 prepared in Example 10 were evaluated in vivo in tumor-bearing mice. The antitumor effect of the conjugate of the present invention was examined using Balb / c-nu / nu (Balb / c nude) mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. 5 × 10 per mouse 6 Human bladder cancer cells UMUC3 were mixed with Matrigel (SIGMA) and implanted subcutaneously, and tumors grew to an average of 120-150 mm. ３Tumor-bearing mice were prepared by growing the tumor until the tumor size reached 100 μg / mL. UMUC3 expresses the CAPRIN-1 protein on the cell membrane surface, and conjugates 107 and 113 bind to UMUC3. Conjugates 107 and 113 were each administered once at 0.5 mg / kg via the tail vein to UMUC3 tumor-bearing mice. PBS(-) was administered to negative control tumor-bearing mice.

[0256] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor). ２ As a result of the evaluation, 15 days after the start of administration, the tumor volumes of the cancer-bearing mice administered Conjugate 103 and Conjugate 117 were 11% or less when the tumor volume of the negative control was taken as 100%.

[0257] The results of this evaluation showed that the conjugate of DGN549 prepared in Example 10 using an antibody against CAPRIN-1 exhibited a significantly stronger antitumor effect than the negative control (untreated control group).

[0258] (Example 14) Preparation of a conjugate of an anti-CAPRIN-1 antibody and a PDD derivative As in Example 3, SATA was conjugated as a spacer to the lysine residue of the anti-CAPRIN-1 polyclonal antibody #1 described in Example 1 according to a standard method, and then a compound having the following chemical structure, in which a linker having a maleimide group added to the compound described in Example 41 of U.S. Patent No. 10,975,072 B2 ((S)—N-(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide) was conjugated to the antibody via the maleimide group, to obtain a solution containing the conjugate (conjugate 160). Similarly, a conjugate of the humanized antibody #1, an anti-CAPRIN-1 monoclonal antibody described in Example 2, with the compound was prepared, and a solution containing the conjugate (conjugate 166) was obtained. Similarly, conjugates of the anti-CAPRIN-1 polyclonal antibodies #2 to #6 and the humanized anti-CAPRIN-1 monoclonal antibodies #2 to #47 with the compound were prepared. Using the same procedures as above, solutions containing conjugates of the rabbit control antibody and human IgG control antibody described in Example 1, which do not react with CAPRIN-1 protein, with the PDD derivative were also obtained in the same manner (control conjugate 7, control conjugate 8). The prepared conjugates were evaluated in the same manner as in Examples 4 to 6, and it was confirmed that the conjugates exhibited specific reactivity to CAPRIN-1 protein and CAPRIN-1-expressing cancer cells, internalization, and antitumor activity.

[0259]

[0260] (Example 15) Antitumor effect of conjugates of anti-CAPRIN-1 antibody and PDD derivative in tumor-bearing mice The antitumor effect of conjugates 160 and 166 prepared in Example 12 was evaluated in vivo in tumor-bearing mice. The antitumor effect of the conjugate of the present invention was examined using NOD-SCID mice transplanted with human-derived cancer cells expressing CAPRIN-1 protein. ７ Human head and neck cancer cells, FaDu, were mixed with Matrigel (SIGMA) and implanted subcutaneously, and tumors were grown to 100-200 mm ３ Tumor-bearing mice were generated and allowed to grow to a size of 100 or larger. FaDu expresses CAPRIN-1 protein on the cell membrane surface, and conjugates 160 and 166 bind to FaDu. Conjugates 160 and 166 were administered once via the tail vein to the FaDu-bearing mice at 6 mg / kg and 12 mg / kg, respectively. PBS(-) was administered to negative control tumor-bearing mice.

[0261] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor). ２ The results were calculated using a ratio of 0.5 to 1.0. As a result of the evaluation, on and after the 30th day after the start of administration, when the tumor volume of the negative control was taken as 100%, the tumor volumes of the mice administered 6 mg / kg of conjugate 160 and conjugate 166 were less than 50% of the tumor volume. On the other hand, the tumor volumes of the cancer-bearing mice administered 12 mg / kg of conjugate 160 and conjugate 166 were less than 30%.

[0262] The results of this evaluation showed that the conjugate of the PDD derivative prepared in Example 12 using an antibody against the CAPRIN-1 protein exhibited a significantly stronger antitumor effect than the negative control (untreated control group).

Claims

1. A conjugate comprising a benzodiazepine bound to an antibody or fragment thereof that is immunoreactive with the CAPRIN-1 protein having an amino acid sequence represented by any even-numbered sequence number among sequence numbers 2 to 30, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence.

2. The conjugate according to claim 1, wherein the antibody or fragment thereof has immunological reactivity with a partial polypeptide of the CAPRIN-1 protein having an amino acid sequence represented by any of SEQ ID NOs. 31-35, 296-299, 308, or 309, or an amino acid sequence having 80% or more sequence identity with said 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 its fragment is any of the following (A) to (M). (A) An antibody or fragment having immunological reactivity with the CAPRIN-1 protein, comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 36, 37, and 38 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 40, 41, and 42 (CDR1, CDR2, and CDR3, respectively). (B) An antibody or fragment having immunological reactivity with the CAPRIN-1 protein, comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 44, 45, and 46 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 48, 49, and 50 (CDR1, CDR2, and CDR3, respectively). (C) An antibody or fragment having immunological reactivity with the CAPRIN-1 protein, comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 52, 53, and 54 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 56, 57, and 58 (CDR1, CDR2, and CDR3, respectively). (D) An antibody or fragment having immunological reactivity with the CAPRIN-1 protein, comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 60, 61, and 62 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 64, 65, and 66 (CDR1, CDR2, and CDR3, respectively). (E) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs: 170, 171, and 172 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs: 173, 174, and 175 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (F) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs: 176, 177, and 178 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs: 179, 180, and 181 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (G) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 182, 183, and 184 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 185, 186, and 187 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (H) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs: 188, 189, and 190 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs: 191, 192, and 193 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (I) An antibody or fragment thereof that comprises a heavy chain variable region including the complementarity-determining regions of SEQ ID NOs. 146, 147, and 148 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region including the complementarity-determining regions of SEQ ID NOs. 149, 150, and 223 (CDR1, CDR2, and CDR3, respectively), and which has immunological reactivity with the CAPRIN-1 protein. (J) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 272, 273, and 274 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 275, 276, and 277 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (K) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 290, 291, and 292 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 293, 294, and 295 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (L) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs. 300, 301, and 302 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs. 304, 305, and 306 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (M) An antibody or fragment thereof comprising a heavy chain variable region containing the complementarity-determining regions of SEQ ID NOs: 134, 135, and 136 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region containing the complementarity-determining regions of SEQ ID NOs: 137, 138, and 139 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein.

5. The conjugate according to claim 1, wherein the antibody or its fragment is any of the following (a) to (al). (a) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 39 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

43. (b) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 47 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

51. (c) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 55 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

59. (d) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 63 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

67. (e) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 68 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

69. (f) An antibody or fragment thereof having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 70 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

71. (g) An antibody or fragment thereof having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 72 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

73. (h) An antibody or fragment thereof having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 74 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 75 (i) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 76 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

77. (j) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 78 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

79. (k) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 80 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

81. (l) An antibody or fragment thereof having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 82 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

83. (m) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 84 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

85. (n) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 86 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

87. (o) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 88 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

89. (p) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

91. (q) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 92 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

93. (r) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 94 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

95. (s) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 96 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

97. (t) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 98 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

99. (u) An antibody or fragment thereof having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 100 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 101 (v) An antibody or fragment thereof having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 102 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

103. (w) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 104 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

105. (x) An antibody or fragment thereof having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 106 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

107. (y) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 108 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

109. (z) An antibody or fragment thereof in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 110 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 111 (aa) An antibody or fragment thereof in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 112 and the light chain variable region contains the amino acid sequence of SEQ ID NO:

113. (ab) An antibody or fragment thereof in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 114 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 115 (ac) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 116 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

117. (ad) An antibody or fragment thereof in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 118 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 119 (ae) An antibody or fragment thereof in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 120 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 121 (af) An antibody or fragment thereof in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 122 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 123 (ag) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 124 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

125. (ah) An antibody or fragment thereof in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 126 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 127 (ai) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 128 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

129. (aj) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 130 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

131. (ak) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 132 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

133. (al) An antibody or fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 303 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

307.

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 claim 1, wherein the antibody or a fragment thereof is bound to a benzodiazepine via a linker.

8. The conjugate according to claim 1, wherein the benzodiazepine is a benzodiazepine having antitumor activity.

9. The conjugate according to claim 1, wherein the benzodiazepine is pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IGN), pyridinobenzodiazepine (PDD), or isoquinolidinobenzodiazepine (IQB).

10. The aforementioned benzodiazepines are DSB-120, SJG-136 (SG2000), DC-81, SJG-136, SG2057, SG2202, SG2285, SGD-1882, SGD-1910, SG3199, SG3249, SG2219, IMGN779, IMGN632, (S)-N-(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a, The conjugate according to claim 1, wherein the conjugate is 7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-3-yl)oxy)butanamide)-1-methyl-1H-pyrrole-2-carboxamide)phenyl)-1-methyl-1H-pyrrole-2-carboxamide, D211, D221, D231, GWL-78, KMR-28-39 or a derivative thereof.

11. The conjugate according to claim 10, wherein the derivative is Tesirine (a derivative of SG3249), Talirine (a derivative of SGD-1910), SG3364, SG3227, SG3140 (MC-Phe-Lys-PAB-SG2057), SG3170, SG3203 (MC-Phe-Lys-PAB-SG2057), SG3231, SG3400, SG3376, DGN642, DGN549, FGX5-67 or FGX-2-62, or FGX11-38.

12. A pharmaceutical composition for the treatment and / or prevention of cancer, comprising the conjugate described in any one of claims 1 to 11 as an active ingredient.

13. The pharmaceutical composition according to claim 12, wherein the cancer is a cancer that expresses the CAPRIN-1 protein on the cell membrane surface.

14. The pharmaceutical composition according to claim 12, 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 lymphoma, mesothelioma, multiple myeloma, testicular cancer, thyroid cancer, head and neck cancer, or urothelial carcinoma.