Pharmaceutical composition for treating, preventing and / or diagnosing cancer

Abstract

This complex, in which a radioactive isotope and an antibody or a fragment thereof having immunological reactivity with a CAPRIN-1 protein having an amino acid sequence represented by any one of the even SEQ ID Nos of 2-30, or an amino acid sequence having at least 80% sequence identity with said amino acid sequence are bound via a chelating agent, has excellent antitumor effects and cancer detection ability and can thus be used as a radiopharmaceutical.

Description

Pharmaceutical composition for treating, preventing and / or diagnosing cancer

[0001] The present invention relates to a complex of an antibody against CAPRIN-1 protein or a fragment thereof and a radioisotope-labeled complex, and a pharmaceutical composition using the same for treating, preventing and / or diagnosing cancer.

[0002] Various antibody drugs targeting specific antigen proteins on the surface of 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 specifically expressed on the cell membrane surface of many solid cancers, and antibodies against this CAPRIN-1 protein are known to be promising pharmaceutical applications for the treatment and / or prevention of cancer (Patent Document 1).

[0003] In recent years, complexes consisting of molecules that target cancer tissues or cancer cells and unsealed radioisotopes have been put to practical use as radiopharmaceuticals for the treatment, prevention, and diagnosis of cancer. When such radiopharmaceuticals are administered to cancer patients, the radiopharmaceuticals accumulate in the cancer tissues or cancer cells, and the radiation emitted from the radioisotopes enables the treatment and detection of cancer (Non-Patent Documents 1 and 2).

[0004] As a molecule that targets cancer tissues or cancer cells, an antibody that has specific immunological reactivity with the surface of cancer tissues or cancer cells can be used. For example, a therapeutic drug (Zevalin (registered trademark)) in which a monoclonal antibody against CD20 is labeled with the radioisotope indium 111 (111-In) or yttrium 90 (Y-90) has been put to practical use for the treatment and diagnosis of CD20-positive relapsed or refractory low-grade B-cell non-Hodgkin's lymphoma and mantle cell lymphoma.

[0005] Furthermore, as a molecule targeting cancer tissue or cancer cells, a ligand of a receptor expressed on the surface of cancer tissue or cancer cells can be used. Examples include a cancer diagnostic agent (Octreoscan (registered trademark)) in which a somatostatin analog is labeled with indium-111 (111-In) and targeted to somatostatin receptor-positive neuroendocrine tumors, a cancer therapeutic agent (Lutathera (registered trademark)) in which a somatostatin analog is labeled with lutetium-177 (Lu-177), and bipibotide tetraxetane (PLUVICTO (registered trademark)) containing the PMSA analog lutetium-177 (Lu-177) and targeted to PMSA-positive metastatic castration-resistant prostate cancer (mCRPC) with a history of treatment with an androgen receptor (AR) pathway blocker and a taxane anticancer drug.

[0006] WO2010 / 016526

[0007] J. Nucl. Med. , 46, 67S-75S (2005) J. Nucl. Med. , 46 (Suppl. 1), 38S-47S (2005)

[0008] As mentioned above, radiopharmaceuticals that utilize molecules that target cancer tissues or cancer cells have already been put to practical use, but their antitumor effects have been limited. Therefore, an object of the present invention is to provide a radiopharmaceutical that has superior antitumor effects and cancer detection capabilities compared to conventional technologies.

[0009] As a result of intensive research, the present inventors have found that a complex in which a radioisotope is bound via a chelating agent to an antibody or a fragment thereof having immunological reactivity with CAPRIN-1 protein has significantly superior effects in treating, preventing and / or diagnosing cancer than existing radiopharmaceuticals, and have thus completed the present invention.

[0010] Specifically, the present invention has the following features (1) to (18).

[0011] (1) A complex 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 radioisotope bound thereto via a chelating agent.

[0012] (2) The complex 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 one of SEQ ID NOs: 31 to 35, 296 to 299, 308, and 309, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence.

[0013] (3) The complex according to (1) or (2), wherein the antibody is a monoclonal antibody or a polyclonal antibody.

[0014] (4) The conjugate according to any one of (1) to (3), wherein the antibody or a fragment thereof is any one of the following (A) to (M): (A) an antibody or a 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 CAPRIN-1 protein; (B) an antibody or a 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 CAPRIN-1 protein. (C) An antibody or fragment thereof comprising 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 having immunological reactivity with 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 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 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 (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 290, 291 and 292 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 293, 294 and 295, and having immunological reactivity with the CAPRIN-1 protein. (L) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 301, 302 and 303 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 305, 306 and 307, 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 (CDR1, CDR2, and CDR3) of SEQ ID NOs: 134, 135, and 136, respectively, and a light chain variable region containing the complementarity determining regions (CDR1, CDR2, and CDR3) of SEQ ID NOs: 137, 138, and 139, respectively, and having immunological reactivity with a CAPRIN-1 protein.

[0015] (5) The complex according to any one of (1) to (4), wherein the antibody or fragment thereof is any one of the following (a) to (a1): (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, 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.(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, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 92 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 93; (r) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 94 and whose 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, 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; (aa) 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.(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, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 120 and whose light chain variable region comprises the amino acid sequence of SEQ ID NO: 121; (af) an antibody or fragment thereof, whose heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 122 and whose 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.

[0016] (6) The conjugate according to any one of (1) to (5), wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0017] (7) The complex according to any one of (1) to (5), wherein the fragment is F(ab')2, Fab', Fab, or scFv.

[0018] (8) The complex according to any one of (1) to (7), wherein the radioisotope is a radioisotope that emits α-rays, β-rays, or γ-rays.

[0019] (9) The complex according to any one of (1) to (8), wherein the radioisotope is Ra (radium), thorium (Th), actinium (Ac), lutetium (Lu), indium (In), gallium (Ga), lead (Pb), copper (Cu), strontium (Sr), yttrium (Y), samarium (Sm), rhenium (Re), bismuth (Bi), technetium (Tc), or zirconium (Zr).

[0020] (10) The complex according to any one of (1) to (9), wherein the radioisotope is 223-Ra, 227-Th, 225-Ac, 177-Lu, 111-In, 67-Ga, 68-Ga, 212-Pb, 64-Cu, 67-Cu, 89-Sr, 90-Y, 153-Sm, 186-Re, 188-Re, 212-Bi, 213-Bi, 99m-Tc, or 89-Zr.

[0021] (11) The complex according to any one of (1) to (10), wherein the chelating agent is a bifunctional chelating agent.

[0022] (12) The complex according to (11), wherein the bifunctional chelating agent is a cyclic or linear chelating agent.

[0023] (13) The cyclic chelating agent is DOTA, NOTA, TETA, TCMC, DEPA, NETA, H ２ The complex according to (12), which is selected from the group consisting of macropa, Crown, PCTA, NODAGA, NODASA, Sar bicyclic chelator, MANOTA, and derivatives thereof.

[0024] (14) The linear chelating agent is HOPO, DTPA, MAG ２ -GABA, Trisuccin, DFO, H ２ dedpa, H ４ The complex according to (12), which is selected from the group consisting of octapa, HYNIC, HBED-CC, THP, and derivatives thereof.

[0025] (15) A pharmaceutical composition for treating, preventing and / or diagnosing cancer, comprising the complex according to any one of (1) to (14) as an active ingredient.

[0026] (16) The pharmaceutical composition according to (15), wherein the cancer expresses CAPRIN-1 protein on the cell membrane surface.

[0027] (17) The pharmaceutical composition according to (15) or (16), wherein the cancer is 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, 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.

[0028] (18) A method for treating, preventing, and / or diagnosing cancer, comprising administering to a subject the complex according to any one of (1) to (14) or the pharmaceutical composition according to any one of (15) to (17).

[0029] The conjugate of the present invention not only exhibits a significantly stronger antitumor effect than an antibody against the CAPRIN-1 protein or a fragment thereof alone, but also has a significantly superior antitumor effect and / or cancer detectability than existing radiopharmaceuticals. Therefore, the conjugate of the present invention can be used as an active ingredient in a pharmaceutical composition for the treatment, prevention, and / or diagnosis of cancer.

[0030] The antitumor activity of a conjugate of an antibody against the CAPRIN-1 protein or a fragment thereof (hereinafter referred to as an "anti-CAPRIN-1 antibody") used in the present invention and a radioisotope can be evaluated by examining the inhibition of tumor growth in a cancer-bearing animal in vivo, as described below. Furthermore, the detection of cancer by the conjugate can be evaluated by administering the conjugate to a cancer-bearing animal in vivo, as described below, and measuring the radiation dose, including positional information, from a conjugate in which an antibody against the CAPRIN-1 protein or a fragment thereof is labeled with a radioisotope.

[0031] In the present invention, the term "conjugate" refers to a complex in which a chelating agent bound to an antibody and a radioisotope are linked by a coordinate bond. The antibody and the chelating agent may be linked via a linker.

[0032] The anti-CAPRIN-1 antibody forming the above-described complex of the present invention may be a monoclonal or polyclonal antibody, preferably a monoclonal antibody. Furthermore, the complex 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 or cancer detection ability.

[0033] The radioisotope forming the complex according to the present invention is an element having the ability to emit radiation (radioactivity), also called a radioisotope or radionuclide. The radioisotope used in the present invention is not particularly limited as long as it is a radioisotope having the ability to emit radiation useful for the treatment, prevention, and / or diagnosis of cancer.

[0034] Furthermore, subjects for cancer treatment and / or prevention and cancer detection in the present invention include mammals such as humans, pet animals, livestock, and sport animals, with humans being the preferred subject.

[0035] The following describes the anti-CAPRIN-1 antibody, radioisotope, conjugate of anti-CAPRIN-1 antibody and radioisotope, pharmaceutical composition using the conjugate, and method for treating and / or preventing cancer and method for detecting cancer, all of which relate to the present invention.

[0036] <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.

[0037] 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.

[0038] In the present invention, the anti-CAPRIN-1 antibody used to prepare a complex refers to an antibody or a fragment thereof that is immunologically reactive with the full-length CAPRIN-1 protein or a fragment thereof. Here, "immunological reactivity" refers to the property of an antibody binding to the CAPRIN-1 protein or a partial polypeptide thereof in vivo, and is also referred to as "antigen-binding ability."

[0039] The anti-CAPRIN-1 antibody used in the present invention may be a monoclonal or polyclonal antibody. Furthermore, the anti-CAPRIN-1 antibody may be of any class of immunoglobulin molecule, such as IgG, IgE, IgM, IgA, IgD, or IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The antibodies used in the present invention include human antibodies, humanized antibodies, chimeric antibodies, and non-human animal antibodies.

[0044] 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.

[0045] 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).

[0046] Specific examples of the anti-CAPRIN-1 antibody fragment include F(ab')2, Fab', Fab, and scFv, which are structural fragments of the anti-CAPRIN-1 antibody full body that have antigen-binding ability, and these can be prepared by known methods.

[0047] 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).

[0048] 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.

[0049] 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.

[0050] 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).

[0051] 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).

[0052] The anti-CAPRIN-1 antibody used to obtain the conjugate of the present invention may have amino acids in its 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.

[0053] 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:

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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:

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] In addition, the following anti-CAPRIN-1 antibodies are also preferably used.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] <Radioactive Isotope> The radioactive isotope used in the present invention is not particularly limited as long as it has the ability to emit radiation useful for the treatment, prevention, and / or diagnosis of cancer. However, radioactive isotopes that emit particle radiation such as α (alpha) rays and β (beta) rays (β+ or β−), or electromagnetic radiation such as γ (gamma) rays are preferably used.

[0108] Specific examples of preferred radioisotopes include radium (Ra), thorium (Th), actinium (Ac), lutetium (Lu), indium (In), gallium (Ga), lead (Pb), copper (Cu), strontium (Sr), yttrium (Y), samarium (Sm), rhenium (Re), bismuth (Bi), technetium (Tc), and zirconium (Zr). More specific examples of preferred radioisotopes include 227-Th, 225-Ac, 177-Lu, 111-In, 67-Ga, 68-Ga, 212-Pb, 64-Cu, 67-Cu, 89-Sr, 90-Y, 153-Sm, 186-Re, 188-Re, 212-Bi, 213-Bi, 99m-Tc, and 89-Zr.

[0109] Specific examples of radioisotopes that emit α-rays include 223-Ra, 227-Th, 225-Ac, 212-Pb, 186-Re, 188-Re, 212-Bi, and 213-Bi. The energy of the α-rays emitted by the radioisotopes that emit α-rays is preferably 5 to 8 MeV.

[0110] Among the radioisotopes that emit β-rays, specific examples of radioisotopes that emit β- rays include 177-Lu, 64-Cu, 67-Cu, 89-Sr, 90-Y, and 153-Sm. Specific examples of radioisotopes that emit β+ rays include 64-Cu, 68-Ga, and 89-Zr. The energy of the radioisotopes that emit β+ rays is preferably 0.5 to 2.3 MeV.

[0111] Specific examples of gamma-ray-emitting radioisotopes include 111-In, 67-Ga, 177-Lu, and 99m-Tc. The energy of the gamma-ray-emitting radioisotope is preferably 0.1 to 0.5 MeV.

[0112] <Chelating Agent> The chelating agent used in the present invention is preferably one that forms a complex with a radioisotope through a coordination bond and that can bind to the anti-CAPRIN-1 antibody directly or via a linker, and specifically is a bifunctional chelating agent that has both a protein-binding site and a site for complex formation with a radioisotope. More preferably, the site for complex formation is a cyclic or linear chelating agent.

[0113] Specific examples of bifunctional cyclic chelating agents include, but are not limited to, DOTA, NOTA, TETA, TCMC, DEPA, NETA, H2 macropa, Crown, PCTA, NODAGA, NODASA, Sar bicyclic chelator, MANOTA, and derivatives thereof.

[0114] Specific examples of bifunctional linear chelating agents include HOPO, DTPA, MAG2-GABA, Trisuccin, DFO, and H ２ dedpa, H ４ Examples of such compounds include, but are not limited to, octapa, HYNIC, HBED-CC, THP, and derivatives thereof.

[0115] It is preferable to use a chelating agent that easily forms a complex with the radioisotope. For example, when using 223-Ra, H ２Macropa is preferred, and when 225-Ac is used, H ２ When 227-Th is used, DOTA or Me-3,2-HOPO is preferred. When 212-Pb is used, DOTA or TCMC is preferred. When 212-Bi or 213-Bi is used, 3p-C-DEPA, NETA or CHX-A-DTPA is preferred. When 177-Lu is used, DOTA, DOTATATE, DOTANOC, NETA, NODAGA, PCTA, DTPA, CHX-A-DTPA, 1B4M-DTPA or H ４ When 90-Y is used, DOTA, DOTATATE, DOTANOC, NETA, CHX-A-DTPA, DTPA, or 1B4M-DTPA is preferred; when 67-Cu is used, DOTA or NOTA is preferred; when 64-Cu is used, TETA, DOTA, NOTA, p-SCN-Bn-Oxo-DO3A, p-SCN-Bn-Oxo-PCTA, NODAGA, Sar Bicyclic chelators, or MANOTA is preferred; when 188-Re is used, MAG ２ -GABA, Trisuccin or NYNIC is preferred, and when 67-Ga is used, DFO, NOTA, DOTA, p-SCN-Bn-Oxo-DO3A, DFO, H ２ CHXdedpa or H4CXHoctapa is preferred, and when 68-Ga is used, DOTA, DOTATATE, NOTA, NODASA, DTPA, H ２ dedpa or HBED-CCTHP is preferred, and when 111-In is used, DOTA, DOTATATE, DOTANOC, NOTA, NODAGA, NODASA, CHX-A-DTPA, H ４ octapa, DTPA, 1B4M-DTPA, H ２ CHXdedpa,H ４ CXHoctapa or EDDA / HYNIC-TOC is preferred, and when 99m-Tc is used, HYNIC, EDDA / HYNIC-TOC or HBEC-CC is preferred, and when 89-Zr is used, DFO, DFO* (an optical isomer of DFO) or DFOcyclo is preferred.

[0116] The complex of the radioisotope and the chelating agent is described in Theranostics 2021, Vol. 11, Issue 13, Molecules 2022, 27,3062, US2022 / 363768, US2019 / 091354, US2017 / 319721, US2015 / 110817, US2010 / 129315, US2008 / 138899, US2023 / 072421, US2022 / 125960, US2022 / 118123, US2017 / 340759, US2019 / 298865, US2011 / 250137, US2011 / 123444, EP1590005 A, EP2239274A, WO2008 / 046463A, WO2008 / 028530A, EP1861127A, EP1700608A, WO2022 / 162210A, WO2022 / 157094A, WO20 21 / 160708A, WO2012 / 104225A, WO2012 / 069410A, WO2011 / 110490A, US6183721, US5776894, US5753627, US5686410, US56 50134, US2022 / 401593, US2022 / 378954, EP0515313A, WO2022 / 043557A, WO2022 / 043558A, EP4034176A, WO2023 / 152671 A, WO2022 / 219569A, WO2022 / 136317A, WO2022 / 043556A, US2023 / 321285, US2023 / 338587, US2024 / 050597, US2023 / 3573 81, US2024 / 197926, US2024 / 197715, US2024 / 252693, WO2024 / 116053A, WO2024 / 121722A, EP4279092A, US2024 / 108766, US2024 / 156999, US2024 / 245582, WO2023 / 222557A, and WO2023 / 159229A, and these complexes can also be used as components of the complex of the present invention.

[0117] <Method for Producing Conjugate> The conjugate of the present invention may be in a form in which a complex of a radioisotope and a chelating agent is directly bound to an anti-CAPRIN-1 antibody, or in a form in which a complex of an anti-CAPRIN-1 antibody and a chelating agent is bound via a linker. Details of the linker-mediated binding method can be performed in accordance with known methods (see, for example, Greg T. Hermanson, Bioconjugate Techniques, Third Edition, WO2004 / 010957, WO2014 / 012479).

[0118] Reactive groups attached to the anti-CAPRIN-1 antibody and chelator and linker to form the conjugates of the present invention include the following:

[0119] Unless specifically chemically modified, reactive groups present in the amino acid sequence of an anti-CAPRIN-1 antibody or 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 can be generated in glycoproteins by oxidizing glycosyl groups with sodium metaperiodate.

[0120] Specific examples of the reactive groups attached to the chelating agent and linker include the following:

[0121] 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.

[0122] Carbodiimides, diazoalkanes, diazoacetyl compounds, carbonyldiimidazoles are reactive groups that can react with carboxyls and amines.

[0123] Reactive groups that can react with thiols include maleimide, haloacetamide, pyridyl disulfide, thiosulfone, vinyl sulfone, haloacetyl, aziridine, acryloyl, and aryl.

[0124] 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.

[0125] Isocyanate is a reactive group that can react to hydroxyl.

[0126] Photoreactive reactive groups include diazirine, aryl azide, aryl, benzophenol, and diazo compounds.

[0127] Specific examples of the linker having the reactive group include the following:

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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, Propyl-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.

[0132] In another embodiment, by using polyethylene glycol (PEG) as a linker, as described in WO2015 / 057699 and WO2017 / 165851, a conjugate that is expected to have better in vivo kinetics can be obtained.

[0133] The linker used in the present invention may be cleavable under intracellular conditions, and may release a substance having antitumor activity containing a radioisotope and a chelating agent, or a radioisotope, a chelating agent, and a portion of the linker, within the cell. For example, the linker may be 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 polypeptides (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 may be used.

[0134] The linker mediated between the anti-CAPRIN-1 antibody and the radioisotope may be composed of a single type or multiple types.

[0135] A preferred method for preparing the conjugate of the present invention is to bind the functional group of a chelating agent to an ε-amino group or a thiol group that the anti-CAPRIN-1 antibody originally has or that is formed by chemical treatment.

[0136] When an ε-amino group is used, the ε-amino group of a lysine residue in an anti-CAPRIN-1 antibody can be used. In this case, since the anti-CAPRIN-1 antibody has many lysine residues, binding with a chelating agent occurs nonspecifically.

[0137] When using thiol groups, disulfide bonds in cysteine ​​residues of anti-CAPRIN-1 antibodies can be converted to thiol groups using a reducing agent such as mercaptoethanol or dithiothreitol (DTT). Alternatively, thiols can be formed on antibodies by adding thiol groups to primary amines in lysine residues of the antibody using Traut's reagent (2-Iminothiolane or N-Succinimidyl S-Acetylthioacetate (SATA)). Alternatively, THIOMAB (registered trademark) technology (see Nature Biotechnology 26, 925-932 (2008)) or ThioBridge (registered trademark) technology can be used to introduce a predetermined number of thiol groups into specific parts of an antibody (see Methods Mol. Biol. 2078:113-129 (2020)), or RESPECT® technology (see MAbs. 9(6):907-915 (2017)).

[0138] 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. For example, N-ethylmaleimide (NEM) or 2-iodoacetamide (IAA) can be used for capping.

[0139] Forming a conjugate using a thiol group added to an antibody can be accomplished 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 chelating agent allows the formation of an amide bond with the N-succinimide group of SMCC, thereby obtaining a conjugate.

[0140] In another embodiment, an amide bond is first formed with SMCC at the amino group present in the chelating agent, and then a thiol group added to the antibody side is reacted with the maleimide group of the SMCC to which the chelating agent is bound, thereby obtaining a conjugate.

[0141] In another embodiment, an antibody can be conjugated to a chelating agent using two linkers. For example, a primary amino group present in a lysine residue on the antibody is amide-bonded to the N-succinimide group of SATA (N-succinimidyl-S-acetylthioacetate), and a thiol group is added to the antibody. A chelating agent having an amino group or one to which an amino group has been added according to a standard method is reacted with SMCC to form an amide bond with the N-succinimide group in SMCC, and a conjugate can be obtained by reacting the maleimide group in the chelating agent-bound SMCC with the thiol group in the antibody-bound SATA.

[0142] In another embodiment, the antibody and chelating agent can be linked 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 chelating agent having an amino group is reacted with the benzyloxycarbonyl (PAB) in mc-Val-Cit-PAB to prepare a chelating agent bound to mc-val-Cit-PAB, which can then be reacted with the aforementioned thiol-attached antibody to obtain a conjugate.

[0143] In yet another embodiment, SATA is attached to a primary amino group in a lysine residue of an antibody to add a thiol group, and succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is reacted with a chelating agent having an amino group to form an amide bond with the N-succinimide group in SPDP.

[0144] Furthermore, methods for stabilizing thiol-mediated bonds include the use of sulfonephenyloxadiazoles and 4-cyanoethynyloxy derivatives. These bonds are more stable for longer periods than bonds formed by the conjugation reaction of cysteine ​​with maleimide. Furthermore, since stability improves 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 cysteine ​​thiols in antibodies form disulfide bonds, and a radioisotope can be attached between the two thiols. For example, cross-linking can be achieved 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.

[0145] 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)).

[0146] In another embodiment, a linker having glucuronic acid (preferably β-D-glucuronide) as described in WO 2007 / 0711968 can be used 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 chelating agent to the anti-CAPRIN-1 antibody. Alternatively, the means described in WO 2013 / 173337, WO 2015 / 095755, WO 2015 / 123679, and WO 2018 / 031690 can be used.

[0147] In another embodiment, a chelating agent can be site-specifically bound to an anti-CAPRIN-1 antibody using, for example, the means described in WO2006 / 65533 and WO2018 / 160683.

[0148] As a method for producing the conjugate of the present invention, the anti-CAPRIN-1 antibody may be bound to a chelating agent and then a complex of the radioisotope may be formed, or a complex of the radioisotope and a chelating agent may be formed and then bound to the anti-CAPRIN-1 antibody. However, it is preferable to bind the anti-CAPRIN-1 antibody to a chelating agent and then form a complex of the radioisotope.

[0149] In a method for forming a complex of a radioisotope after conjugating an anti-CAPRIN-1 antibody with a chelating agent, first, the reaction product obtained by conjugating an anti-CAPRIN-1 antibody with a chelating agent by the above-described method is subjected to, for example, gel filtration chromatography to separate out antibodies with higher molecular weights than the antibody before conjugation with the chelating agent, thereby obtaining a conjugate of an anti-CAPRIN-1 antibody with a chelating agent.The conjugate of the present invention can then be obtained by mixing and reacting the conjugate of an anti-CAPRIN-1 antibody with a chelating agent, a radioisotope, and a buffer.

[0150] Examples of buffers that can be used include, but are not limited to, sodium acetate buffer, ammonium acetate buffer, HEPES buffer, MES buffer, ascorbic acid buffer, gentisic acid buffer, and mixtures thereof. The pH of the buffer is, for example, 1 to 11, preferably 3 to 9, and more preferably 4 to 7. The reaction temperature is, for example, 5 to 95°C, preferably 20 to 80°C, and more preferably 35 to 45°C. The reaction time can be, for example, 5 minutes to 48 hours, and more preferably 30 minutes to 24 hours, but is not limited thereto.

[0151] <Physical Properties of the Conjugate> The number of chelating agent molecules bound per anti-CAPRIN-1 antibody molecule in the conjugate of the present invention can be quantified by a method such as mass spectrometry. When the conjugate of the present invention is used as an active ingredient in a pharmaceutical composition for treating, preventing, and / or diagnosing cancer, the number of chelating agent molecules per anti-CAPRIN-1 antibody molecule is preferably 0.5 to 20, more preferably 1 to 10.

[0152] The radiochemical purity (%) of the complex of the present invention can be determined by known methods such as thin layer chromatography, radio TLC, and radio HPLC. When thin layer chromatography is used, specifically, 1) the complex after the reaction is spotted at the origin of a thin layer plate, 2) the thin layer plate is developed using a developing solvent up to the solvent front position, 3) after development, the thin layer plate is cut at the cutting position, and the bottom and top of the thin layer plate are placed in measurement vials, and the radiation dose is measured using a radiation measurement device such as an ionization chamber, 4) the radiochemical purity is measured using the following formula: Radiochemical purity (%) = ((radiation dose at the bottom of the thin layer plate - background) / ((radiation dose at the bottom of the thin layer plate - background) + (radiation dose at the top of the thin layer plate - background)) x 100).

[0153] When the complex of the present invention is used as an active ingredient in a pharmaceutical composition for treating, preventing and / or diagnosing cancer, the radiochemical purity is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and most preferably 90% or more.

[0154] <Pharmaceutical Composition for Treating, Preventing, and / or Diagnosing Cancer> The complex of the present invention can be used as an active ingredient of a pharmaceutical composition, particularly a pharmaceutical composition for treating, preventing, and / or diagnosing cancer.

[0155] As used herein, the terms "tumor" and "cancer" refer to malignant neoplasms and are used interchangeably.

[0156] 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.

[0157] 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.

[0158] Furthermore, preferred subjects (patients) are mammals, including, for example, primates, pet animals, livestock, sport animals, etc., with humans, dogs, and cats being particularly preferred.

[0159] The conjugate of the present invention has antitumor activity in vitro and / or in vivo. Antitumor activity refers to the reduction, elimination, inhibition of growth, apoptosis, necrosis, or killing of targeted cancer cells. Therefore, the therapeutic and / or preventive effect of a pharmaceutical composition containing the conjugate of the present invention as an active ingredient on cancer can be evaluated by examining its antitumor activity against cancer.

[0160] To determine the antitumor effect in vivo, the complex is administered to a living body having cancer, and the size of the tumor is measured after administration. The antitumor effect can be evaluated by examining the size of the cancer over time. The antitumor effect can also be evaluated by examining the survival rate. The antitumor effect can also be determined by examining the prevention of cancer, specifically, the prevention of cancer metastasis or the prevention of cancer recurrence.

[0161] The conjugate of the present invention is expected to have a stronger antitumor effect or cancer detection 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:

[0162] The ability of the complex 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.

[0163] As described above, the conjugate of the present invention has a stronger anti-tumor effect than the anti-CAPRIN-1 antibody alone. By utilizing the functions of the anti-CAPRIN-1 antibody and the radioisotope, the conjugate of the present invention can also be used as an active ingredient in a pharmaceutical composition for diagnosing whether a subject (patient) is suffering from the aforementioned cancer. Specifically, by administering the conjugate of the present invention to the body of a subject (patient) and detecting radiation from the conjugate using a PET / CT or SPECT / CT device, it is possible to detect not only cancer tissue or cancer cells in the subject (patient), but also the location and size of the cancer tissue or cancer cells in the subject (patient). Furthermore, cancer diagnosis is preferably performed in combination with immunohistochemical staining or diagnostic imaging using X-rays or the like.

[0164] Specific examples of subjects (patients) to whom the conjugate of the present invention is administered for the diagnosis of cancer include healthy individuals, subjects (patients) suspected of having cancer, and subjects (patients) who are already known to have cancer but are suspected of having a recurrence of cancer after treatment.

[0165] In cancer diagnosis using the conjugate of the present invention, a subject (patient) in whom cancer tissue or cancer cells have been detected in the body may be a target for treatment by surgical treatment, radiotherapy, and / or chemotherapy. There are no particular limitations on the surgical treatment and / or chemotherapy that can be applied, but since the conjugate of the present invention contains an anti-CAPRIN-1 antibody as a constituent component, treatment using a pharmaceutical composition containing an anti-CAPRIN-1 antibody as an active ingredient is preferably applied.

[0166] Pharmaceutical compositions containing the complex of the present invention as an active ingredient can be formulated by methods known to those skilled in the art, and can be used parenterally, for example, in the form of an injection of a sterile solution or suspension in water or other pharmaceutically acceptable liquid.

[0167] Furthermore, when preparing a formulation, the conjugate of the present invention may be appropriately combined with a pharmacologically acceptable carrier, medium, or additive, specifically, sterilized water, physiological saline, isotonic solution, buffer (buffer, etc.), vegetable oil, oily liquid, antioxidant, solubilizer, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, binder, etc., and preferably, the conjugate may be formulated by mixing with 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.

[0168] For example, a freeze-dried preparation can be prepared by blending the complex of the present invention with any salt, surfactant, buffer, sugar, or cryoprotectant (including some sugars), and freeze-drying the mixture.

[0169] 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 benzyl benzoate or benzyl alcohol as a solubilizer. Buffers such as phosphate buffers and sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants may also be added. The prepared injection solutions are typically filled into appropriate ampoules.

[0170] The administration is preferably parenteral administration. Specific examples include injections, intranasal administrations, pulmonary administrations, transdermal administrations, etc. Examples of injections include intravenous injections, intramuscular injections, intraperitoneal injections, subcutaneous injections, and intratumoral injections, which can be used for systemic or local administration.

[0171] Furthermore, the dosage and administration method can be selected appropriately 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 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 0.001 to 100,000 mg / body per patient, although it is not necessarily limited to these values. Alternatively, the dosage of the pharmaceutical composition can be determined based on the radiation dose. For example, it can be selected from 10 KBq to 10 GBq per patient per administration, but it is not necessarily limited to these values.

[0172] 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.

[0173] Example 1: Anti-CAPRIN-1 Polyclonal Antibody The anti-CAPRIN-1 polyclonal antibody immunologically reactive with the CAPRIN-1 protein used in the complex of the present invention was prepared by mixing 1 mg of human CAPRIN-1 recombinant protein shown in SEQ ID NO: 2, 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). Furthermore, 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.

[0174] 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. Anti-CAPRIN-1 polyclonal antibody #2 against the partial polypeptide represented by SEQ ID NO: 37 (SEQ ID NO: 31 herein) described in WO2011 / 096528; Anti-CAPRIN-1 polyclonal antibody #3 against the partial polypeptide represented by SEQ ID NO: 5 (SEQ ID NO: 32 herein) described in WO2013 / 018894; Anti-CAPRIN-1 polyclonal antibody #4 against the partial polypeptide represented by SEQ ID NO: 5 (SEQ ID NO: 33 herein) described in WO2013 / 125654; ​​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; Anti-CAPRIN-1 polyclonal antibody #6 against the partial polypeptide represented by SEQ ID NO: 37 (SEQ ID NO: 35 herein) described in WO2011 / 096534.

[0175] Example 2 Anti-CAPRIN-1 Monoclonal Antibody The following anti-CAPRIN-1 monoclonal antibody was used in the complex of the present invention.

[0176] First, the following anti-CAPRIN-1 monoclonal antibodies were prepared.

[0177] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0178] A monoclonal antibody against CAPRIN-1 obtained by the method described in WO2015 / 020212, wherein CDR1 to CDR3 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 CDR1 to CDR3 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.

[0179] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0180] A monoclonal antibody against CAPRIN-1 obtained by the method described in WO2013 / 125654, wherein CDR1 to CDR3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and CDR1 to CDR3 of the light chain variable region consist of the amino acid sequences of SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively.

[0181] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0182] The following monoclonal antibodies against CAPRIN-1 were obtained by the method described in WO2011 / 096528: 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. 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.

[0183] The following monoclonal antibodies against CAPRIN-1 were obtained by the method described in WO2011 / 096533: An antibody 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. An antibody 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.

[0184] The following monoclonal antibodies against CAPRIN-1 were obtained by the method described in WO2011 / 096534: An antibody 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. An antibody 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.

[0185] The following monoclonal antibodies against CAPRIN-1 obtained by the method described in WO2010 / 016526: 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 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 95; 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; 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.

[0186] The following monoclonal antibodies against CAPRIN-1 were obtained by the method described in WO 2013 / 018894: An antibody 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. 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.

[0187] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0188] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0189] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0190] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0191] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0192] The following monoclonal antibodies against CAPRIN-1 obtained by the method described in WO2013 / 125654: an antibody 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; and an antibody 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.

[0193] A monoclonal antibody against CAPRIN-1 obtained by the method 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.

[0194] The following monoclonal antibodies against CAPRIN-1 were obtained by the method described in WO 2015 / 020212: 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; 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.

[0195] Next, a humanized antibody was prepared from the above anti-CAPRIN-1 monoclonal antibody.

[0196] A nucleotide sequence was designed to enable expression of a heavy chain variable region in which CDR1 to CDR3 of the heavy chain variable region consisted of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and in which the framework regions contained a human antibody sequence, and this was 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 to enable expression of a light chain variable region in which CDR1 to CDR3 of the light chain variable region consisted of SEQ ID NOs: 40, 41, and 42, respectively, and in which the framework regions contained a human antibody sequence, and this was 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 transfected into mammalian cells according to standard methods to obtain a culture supernatant containing humanized monoclonal antibody #1 against CAPRIN-1 (humanized antibody #1), in which CDR1 to CDR3 of the heavy chain variable region consisted of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and CDR1 to CDR3 of the light chain variable region consisted of SEQ ID NOs: 40, 41, and 42, respectively.

[0197] Similarly, culture supernatants containing the following humanized anti-CAPRIN-1 monoclonal antibodies #2 to #4 (humanized antibodies #2 to #4) were obtained.

[0198] Humanized anti-CAPRIN-1 monoclonal antibody #2 (humanized antibody #2), in which CDRs 1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NOs: 44, 45, and 46, and CDRs 1 to 3 of the light chain variable region consist of the amino acid sequences of SEQ ID NOs: 48, 49, and 50, respectively.

[0199] Humanized anti-CAPRIN-1 monoclonal antibody #3 (humanized antibody #3), in which CDRs 1 to 3 of the heavy chain variable region consist 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 consist of the amino acid sequences of SEQ ID NOs: 56, 57, and 58, respectively.

[0200] Humanized anti-CAPRIN-1 monoclonal antibody #4 (humanized antibody #4), in which CDRs 1 to 3 of the heavy chain variable region consist 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 consist of the amino acid sequences of SEQ ID NOs: 64, 65, and 66, respectively.

[0201] Similarly, culture supernatants containing the following humanized anti-CAPRIN-1 monoclonal antibodies #9 to #41 (humanized antibodies #9 to #41) were obtained.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] Furthermore, by using a mammalian expression vector into which the heavy chain constant region of human IgG1 has been inserted, in which the amino acid serine (Ser) at EU numbering position 239 has been substituted with aspartic acid (Asp) and the amino acid isoleucine (Ile) at EU numbering position 332 has been substituted with glutamic acid (Glu), culture supernatants containing humanized anti-CAPRIN-1 monoclonal antibodies #5 to #8 (humanized antibodies #5 to #8) each having a full-length heavy chain amino acid sequence consisting of a human IgG1 heavy chain constant region in which the amino acid serine (Ser) at EU numbering position 239 has been substituted with aspartic acid (Asp) and the amino acid isoleucine (Ile) at EU numbering position 332 has been substituted with glutamic acid (Glu), and the same full-length light chain amino acid sequence as humanized antibodies #1 to #4 were obtained.

[0236] Similarly, culture supernatants containing humanized anti-CAPRIN-1 monoclonal antibodies #42 to #74 (humanized antibodies #42 to #74) were obtained, in which the amino acid serine (Ser) at EU numbering position 239 in the heavy chain variable region of each of the humanized antibodies #9 to #41 prepared above was substituted with aspartic acid (Asp) and the amino acid isoleucine (Ile) at EU numbering position 332 was substituted with glutamic acid (Glu).

[0237] The culture supernatants containing the obtained humanized antibodies #1 to #74 were purified using Hitrap Protein A Sepharose FF (GE Healthcare) according to standard methods, and then the medium was replaced with PBS(-) and filtered through a 0.22 μm filter (Millipore) to prepare samples.

[0238] (Example 3) Conjugation of anti-CAPRIN-1 antibodies with a chelating agent (DOTA) Conjugates of each of the anti-CAPRIN-1 polyclonal antibodies #1 to #6 described in Example 1 and the humanized antibodies #1 to #74 described in Example 2 with DOTA were prepared according to the following method.

[0239] The solution containing each antibody was substituted with 0.1 M phosphate buffer (pH 8.0) that had been treated with Chelex (registered trademark) 100 in advance to remove metal ions in the solution, using an AmiconUltra-50K according to a standard method, and the antibody-containing solution was adjusted to 7.5 mg / ml.

[0240] DOTA-NHS-ester (DOTA-NHS-ester / CFCOOH / HPF6 (C084, CheMatech)) was adjusted to 10 mg / ml using distilled water that had been treated with Chelex (registered trademark) 100 in advance to remove metal ions from the solution, to obtain a DOTA-NHS-ester solution. The DOTA-NHS-ester solution was added to each antibody so that the molar ratio was 10, and the mixture was reacted for 4 hours at room temperature with shaking. The mixture was then reacted for approximately 18 hours at 4°C to obtain a solution containing a conjugate of the antibody and DOTA. The solution containing the conjugates was replaced with DPBS(-) that had been treated with Chelex 100 (registered trademark) in advance to remove metal ions from the solution using an AmiconUltra-50K, and the solution was adjusted to 10 mg / ml. After that, the solution was filtered using a 0.22 μm sterilization membrane filter to obtain solutions containing conjugates of each of anti-CAPRIN-1 polyclonal antibodies #1 to #6 with DOTA (polyclonal antibody conjugates #1 to #6) and conjugates of each of humanized antibodies #1 to #74 with DOTA (humanized antibody conjugates #1 to #74).

[0241] The number of DOTA bound to each anti-CAPRIN-1 antibody molecule in the conjugate prepared above was measured by mass spectrometry, and it was found that approximately 2 to 4 DOTA were bound to each antibody molecule.

[0242] Example 4 Specific reactivity of polyclonal antibody conjugate and humanized antibody conjugate to CAPRIN-1 and CAPRIN-1-expressing cancer cells The conjugates prepared in Example 3 were evaluated for their specific reactivity to CAPRIN-1 protein and their reactivity to the cell membrane surface of human cancer cells and mouse cancer cells in which CAPRIN-1 protein is expressed on the cell membrane surface.

[0243] Specific reactivity to CAPRIN-1 protein was confirmed using ELISA. 100 μL of 2 μg / mL CAPRIN-1 protein solution was added per well of a 96-well plate and incubated at 4°C for approximately 18 hours. After washing each well three times with PBS-T, 200 μL of DPBS solution containing 2% bovine serum albumin (BSA) was added per well and incubated at room temperature for 1 hour. The solution was removed, and the wells were washed three times with 300 μL of PBS-T per well. Then, 100 μL of solutions containing polyclonal antibody conjugates #1-#6, humanized antibody conjugates #1-#74, and their respective positive control antibodies (polyclonal antibodies #1-#6 and humanized antibodies #1-#74) were added per well and incubated at room temperature for 1 hour. After washing each well three times with PBS-T, 50 μL of HRP-labeled anti-rabbit IgG antibody or HRP-labeled anti-human IgG antibody (Abcam) diluted 5000-fold with DPBS was added per well and allowed to stand at room temperature for 30 minutes. After washing the wells three times with PBS-T, 50 μL of TMB substrate solution was added per well and allowed to stand for 3-5 minutes to allow the color reaction to occur. After color development, 50 μL of Stop Solution (Thermo Scientific) was added per well to stop the reaction, and the absorbance at 450 nm and 630 nm was measured using an absorbance meter. As a result, polyclonal antibody conjugates #1 to #6 and humanized antibody conjugates #1 to #74 showed absorbance values ​​equivalent to those of their respective positive control antibodies (polyclonal antibodies #2 to #6 and humanized antibodies #1 to #74), confirming that they react specifically with the CAPRIN-1 protein.

[0244] Next, the reactivity of cancer cells expressing CAPRIN-1 on the cell membrane surface was confirmed by flow cytometry. ４50 μL of a solution containing each conjugate or control antibody was added to each of HT29 (ATCC) human colon cancer cells and 4T1 (ATCC) mouse breast cancer cells, and the cells were allowed to stand for 30 minutes at 4° C. After washing with DPBS, Alexa488-labeled anti-rabbit IgG antibody or Alexa488-labeled anti-human IgG antibody diluted to 10 μg / mL with DPBS(-) containing 1% FBS (1% FBS-DPBS(-)) was added, and the cells were allowed to stand for 30 minutes at 4° C. After washing with 1% FBS-DPBS(-), the cells were reacted with BD Horizon Fixable Viability Stain 450 (FVS450, BD Biosciences) to stain dead cells, and then fluorescence intensity was measured using LSRFortessa™ (BD Biosciences). As a result, polyclonal antibody conjugates #1 to #6 and humanized antibody conjugates #1 to #74 each had higher fluorescence intensity than the negative control antibody (rabbit IgG antibody or human IgG antibody), confirming that they strongly react with the cell surface of human cancer cell HT29 and mouse cancer cell 4T1, which express CAPRIN-1.

[0245] Similarly, the reactivity of the conjugate prepared in Example 3 with the following various human cancer cells and mouse cancer cells was confirmed: breast cancer cells (BT-474), colon cancer cells (HT29, HCT116, and 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, and TOV112D), renal cancer cells (Caki-2), brain cancer cells (U-87MG), bladder cancer cells (T24, UMUC3), and cholangiocarcinoma cells (KKU213), which are human cancer cells in which expression of the CAPRIN-1 gene has been confirmed and in which expression of CAPRIN-1 has been confirmed on the cell membrane surface of cancer cells. ), 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 and in which expression of CAPRIN-1 has been confirmed on the cell membrane surface of cancer cells. The conjugates prepared in Example 3 all had stronger fluorescence intensity than the negative control antibody, and were confirmed to react strongly with the cell membrane surface of cancer cells in which CAPRIN-1 is expressed.

[0246] Example 5 Labeling of Polyclonal Antibody Conjugates and Humanized Antibody Conjugates with Radioisotope (111-In) The polyclonal antibody conjugates and humanized antibody conjugates prepared in Example 3 were labeled with the metallic radioisotope 111-In to prepare conjugates. Each solution containing polyclonal antibody conjugates #1 to #6 and humanized antibody conjugates #1 to #74 obtained in Example 3 was adjusted to approximately 10 mg / ml, mixed with 0.5 M HEPES buffer (pH 5.3±0.3), and then 111-InCl was added. ３(Curium Pharma) was added (mixing volume ratio = 5:10:8) and reacted for approximately 30 minutes at approximately 38° C. A portion of the solution after the reaction was collected, and the radiochemical purity was calculated by thin layer chromatography using 0.1 M citrate buffer as a developing solvent.

[0247] Thereafter, purification was carried out using AmiconUltra-50K to obtain 111-In-labeled polyclonal antibody conjugates #1 to #6 and 111-In-labeled humanized antibody conjugates #1 to #74 with radiochemical purity of 95% or more.

[0248] (Example 6) Detection of Cancer Using 111-In-Labeled Polyclonal Antibody Conjugate and 111-In-Labeled Humanized Antibody Conjugate The 111-In-labeled humanized antibody conjugate #1 prepared in Example 5 was evaluated for its in vivo cancer detection ability using cancer-bearing mice in which human-derived cancer cells (human colon cancer cells HT29) expressing CAPRIN-1 protein on the cell membrane surface were transplanted into NMRI-Nude mice. ７ Human colon cancer cells HT29 were mixed with ECM gel (SIGMA-Aldrich) and subcutaneously implanted to form tumors, producing tumor-bearing mice. Under isoflurane anesthesia, approximately 6.5 MBq of the 111-In-labeled humanized antibody conjugate #1 was administered as a single dose via the tail vein of the HT29 tumor-bearing mice.

[0249] Seventy-two hours after administration of In-111-labeled humanized antibody conjugate #1 to the tumor-bearing mice, the mice were anesthetized with isoflurane and gamma rays emitted from the In-111-labeled humanized antibody conjugate #1 were detected using a NanoSPECT / CT (Bioscan) imaging device according to standard methods, and the tumors transplanted into the mice were detected. HiSPECT NG and InVivoScope (IVS) were used for image reconstruction. Evaluation of cancer detection demonstrated that images of In-111-labeled humanized antibody conjugate #1 strongly accumulated in tumors, demonstrating that cancer could be detected. Similarly, cancers were detected with In-111-labeled polyclonal antibody conjugates #1 to #6 and In-111-labeled humanized antibody conjugates #2 to #74.

[0250] Furthermore, as described above, organs of HT29 cancer-bearing mice administered with 111-In-labeled humanized antibody conjugate #1 were collected 72 hours after administration of the conjugate, including blood, bone, brain, digestive tract, gonads, uterus and fallopian tubes, heart, kidney, liver, lung, muscle, skin, spleen, and tumor. They were then weighed, and the radiation dose emitted from each organ was measured using a γ-counter Wizard2 (PerkinElmer). The percentage of radiation accumulated in each organ was calculated from the administered radiation dose and the radiation dose accumulated in each organ and tumor.

[0251] As a result, assuming the radiation dose emitted by the administered conjugate to be 100%, the percentage of accumulation in the HT29 tumor 72 hours after administration was approximately 19% per tumor weight. Meanwhile, the average percentage of radiation dose in various mouse organs other than the tumor was approximately 6% per organ weight (5.5% excluding the liver, which is the main metabolic pathway). These results demonstrate that conjugates in which anti-CAPRIN-1 antibodies are labeled with 111-In, a radioisotope, can detect cancer. Similar results were obtained for 111-In-labeled polyclonal antibody conjugates #1 to #6 and 111-In-labeled humanized antibody conjugates #2 to #74.

[0252] Example 7 Labeling of Polyclonal Antibody Conjugates and Humanized Antibody Conjugates with Radioisotope (177-Lu) The polyclonal antibody conjugates and humanized antibody conjugates prepared in Example 3 were labeled with the metallic radioisotope 177-Lu to prepare conjugates. Each solution containing polyclonal antibody conjugates #1 to #6 and humanized antibody conjugates #1 to #74 obtained in Example 3 was adjusted to approximately 10 mg / ml, mixed with 0.2 M sodium acetate buffer (pH 5.3 to 5.6), and then 177-LuCl was added. ３A solution (ITM Medical Isotopes) was added (mixing volume ratio = 20:25:2), and the mixture was allowed to react at approximately 38°C for approximately 18 hours. A portion of the solution after the reaction was collected, and the radiochemical purity was calculated by the following standard method using thin-layer chromatography with 0.1 M citrate buffer as the developing solvent. Subsequently, purification was carried out using an AmiconUltra-50K to obtain 177-Lu-labeled polyclonal antibody conjugates #1 to #6 and 177-Lu-labeled humanized antibody conjugates #1 to #74 with radiochemical purity of 95% or more.

[0253] Example 8: Antitumor Effects of 177-Lu-Labeled Polyclonal Antibody Conjugate and 177-Lu-Labeled Humanized Antibody Conjugate in Tumor-Bearing Mice (HT29) The 177-Lu-labeled humanized antibody conjugate #1 prepared in Example 7 was evaluated for its antitumor effect in vivo in mice bearing human colon cancer cell HT29 tumors prepared in the same manner as in Example 6. Approximately 10 MBq of 177-Lu-labeled humanized antibody conjugate #1 was administered as a single dose via the tail vein to mice bearing HT29 tumors. Comparative controls included a saline-administered group (untreated group), a humanized antibody #1-administered group, a non-radioisotope-labeled humanized antibody conjugate #1-administered group, and a 177-Lu-labeled DOTATATE (equivalent to Lutathera®, a marketed therapeutic agent for somatostatin receptor-positive cancers)-administered group. HT29 expresses the somatostatin receptor, which is the target of 177-Lu-labeled DOTATATE (Biomedicines 2021, 9, 1743).

[0254] 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 31 after the start of administration, when the tumor volume of the group administered with humanized antibody conjugate #1 not labeled with a radioisotope was taken as 100%, the tumor volume in the mice administered with 177-Lu-labeled humanized antibody conjugate #1 was approximately 45% or less. The tumor volume in the mice administered with the conjugate not labeled with a radioisotope was comparable to that of the untreated group and the group administered with humanized antibody #1. Furthermore, when the tumor volume in the mice administered with DOTATATE not labeled with a radioisotope was taken as 100%, the tumor volume in the mice administered with DOTATATE labeled with a radioisotope was approximately 80% or more. The tumor volume in the mice administered with DOTATATE not labeled with a radioisotope was comparable to that of the untreated group. The results of this evaluation showed that 177-Lu-labeled humanized antibody conjugate #1 exhibited significantly stronger antitumor effects than humanized antibody #1, humanized antibody conjugate #1 not labeled with a radioisotope, and 177-Lu-labeled DOTATATE. Similar results to those of 177-Lu-labeled humanized antibody conjugate #1 were obtained for 177-Lu-labeled polyclonal antibody conjugates #1 to #6 and 177-Lu-labeled humanized antibody conjugates #2 to #74.

[0255] Example 9: Preparation of anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2 The solvent for each of humanized antibodies #1 to #74 was replaced with Digestion buffer (Thermo Scientific) using Zeba Spin Desalting Columns 40K MWCO according to standard procedures. Papain solution (1 / 60 of the antibody amount) was added and reacted at 37°C for 240 minutes. After centrifugation of the reaction solution, the supernatant was collected and passed through a 0.22 μm filter to remove Papain. Subsequently, unreacted antibody and Fc fragments were removed using AKTA pure25 (GE Healthcare), yielding anti-CAPRIN-1 Fab #1 to #74.

[0256] The solvent for each of humanized antibodies #1 to #74 was replaced with 0.1 M citrate buffer (pH 3.5) using Zeba Spin Desalting Columns 40K MWCO according to standard procedures. Pepsin solution (1 / 90 of the antibody amount) was added, and the mixture was incubated at 37°C for 60 minutes. The reaction was then terminated by adding 1 M Tris-HCl (pH 9.0). Subsequently, the solvent was replaced with DPSB(-) according to standard procedures using an AmiconUltra-10K, and the mixture was fractionated using an AKTA pure25 (GE Healthcare) to obtain anti-CAPRIN-1 F(ab')2 #1 to #74.

[0257] Example 10: Conjugation of anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2 with a chelating agent (DOTA) Conjugates of each of anti-CAPRIN-1 Fab #1 to #74 and anti-CAPRIN-1 F(ab')2 #1 to #74 described in Example 9 with DOTA were prepared according to the following method.

[0258] The solutions containing each anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2 were replaced with 0.1 M phosphate buffer (pH 8.0) using an Amicon Ultra-10K according to a standard method, and the solutions containing the anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2 were adjusted to 2.5 mg / ml and 5 mg / ml, respectively.

[0259] DOTA-NHS-ester (DOTA-NHS-ester / CFCOOH / HPF6 (C084, CheMatech)) was adjusted to 10 mg / ml using distilled water to obtain a DOTA-NHS-ester solution. The DOTA-NHS-ester solution was added to each anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2 so that the molar ratio was 10, and the mixture was reacted at room temperature for 4 hours with shaking. The mixture was then reacted at 4°C for approximately 18 hours to obtain a solution containing conjugates of the anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2 with DOTA. The solution containing the conjugates was replaced with DPBS(-) using an AmiconUltra-10K, adjusted to 10 mg / ml, and then filtered using a 0.22 μm sterilizing filter membrane to obtain solutions containing conjugates of each of anti-CAPRIN-1 Fabs #1 to #74 with DOTA (Fab conjugates #1 to #74) and conjugates of each of anti-CAPRIN-1 F(ab')2 #1 to #74 with DOTA (F(ab')2 conjugates #1 to #74).

[0260] The conjugates prepared above were measured by mass spectrometry to determine the number of DOTA bound per molecule of anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2. As a result, it was found that approximately 1 to 4 DOTA were bound per molecule of anti-CAPRIN-1 Fab and anti-CAPRIN-1 F(ab')2.

[0261] Example 11 Specific Reactivity of Fab Conjugates and F(ab')2 Conjugates to CAPRIN-1 and CAPRIN-1-Expressing Cancer Cells The Fab conjugates and F(ab')2 conjugates prepared in Example 10 were evaluated for their specific reactivity to CAPRIN-1 protein, and for their reactivity to the cell membrane surface of human cancer cells and mouse cancer cells in which CAPRIN-1 protein is expressed on the cell membrane surface.

[0262] Specific reactivity to the CAPRIN-1 protein was confirmed using ELISA. 100 μL of a 2 μg / mL CAPRIN-1 protein solution was added per well of a 96-well plate and incubated at 4°C for approximately 18 hours. After washing each well three times with PBS-T, 200 μL of 2% bovine serum albumin (BSA) PBS solution was added per well and incubated at room temperature for 1 hour. The solution was removed, and the wells were washed three times with 300 μL of PBS-T. 100 μL of solutions containing Fab complexes #1 to #74, F(ab')2 complexes #1 to #74, and each positive control was added per well and incubated at room temperature for 1 hour. After washing each well three times with PBS-T, 50 μL of HRP-labeled anti-human IgG (F(ab')2) antibody (Abcam) diluted 5,000-fold with DPBS was added per well and allowed to stand at room temperature for 30 minutes. After washing the wells three times with PBS-T, 50 μL of TMB substrate solution was added per well and allowed to stand for 3-5 minutes to allow the color reaction to occur. After color development, 50 μL of Stop Solution (Thermo Scientific) was added per well to stop the reaction, and the absorbance values ​​at 450 nm and 630 nm were measured using an absorbance meter. As a result, Fab complexes #1 to #74 and F(ab')2 complexes #1 to #74 showed absorbance values ​​equivalent to their respective positive controls, confirming that they react specifically with CAPRIN-1 protein.

[0263] Next, the reactivity of cancer cells expressing CAPRIN-1 on the cell membrane surface was confirmed by flow cytometry. ４50 μL of a solution containing the Fab conjugate, the F(ab')2 conjugate, and the control antibody was added to each of HT29 human colon cancer cells (obtained from ATCC) and 4T1 mouse breast cancer cells (obtained from ATCC), and the cells were incubated at 4°C for 30 minutes. After washing with DPBS, FITC-labeled anti-IgG (F(ab')2) antibody (Jackson ImmunoResearch) diluted to 10 μg / mL with DPBS(-) containing 1% FBS (1% FBS-DPBS(-)) was added, and the cells were incubated at 4°C for 30 minutes. After washing with 1% FBS-DPBS(-), the cells were reacted with BD Horizon Fixable Viability Stain 450 (FVS450, BD Biosciences) to stain dead cells, and then the fluorescence intensity was measured using an LSRFortessa (BD Biosciences). As a result, Fab complexes #1 to #74 and F(ab')2 complexes #1 to #74 each had higher fluorescence intensity than the negative control antibody, confirming that they strongly react with the cell surface of human cancer cell HT29 and mouse cancer cell 4T1, which express CAPRIN-1.

[0264] Similarly, the reactivity of the Fab complex and F(ab')2 complex prepared in Example 10 with the following various human cancer cells and mouse cancer cells was confirmed: human cancer cells in which expression of the CAPRIN-1 gene has been confirmed and in which expression of CAPRIN-1 on the cell membrane surface of cancer cells has been confirmed: breast cancer cells (BT-474), colon cancer cells (HT29, HCT116, and 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, and TOV112D), renal cancer cells (Caki-2), brain cancer cells (U-87MG), bladder cancer cells (T24, UMUC3), and cholangiocarcinoma cells (KKU213). It was confirmed that the Fab conjugate and F(ab')2 conjugate both had stronger fluorescence intensity than the negative control and reacted strongly with the cell membrane surface of cancer cells expressing CAPRIN-1 against 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), and mouse renal cancer cells (Renca) and mouse breast cancer cells (4T1), in which expression of the CAPRIN-1 gene has been confirmed and in which expression of CAPRIN-1 has been confirmed on the cell membrane surface of cancer cells.

[0265] (Example 12) Labeling of Fab conjugate and F(ab')2 conjugate with radioactive isotope (111-In) The Fab conjugate and F(ab')2 conjugate prepared in Example 10 were labeled with the metal radioactive isotope 111-In to prepare conjugates. Each solution containing the conjugate obtained in Example 10 was buffer-substituted with ammonium acetate buffer, adjusted to approximately 10 mg / ml, and mixed with a reaction buffer (sodium acetate solution + sodium ascorbate solution + gentisic acid solution, pH 7.4±0.3) to prepare 111-InCl ３A solution (Curium Pharma) was added (mixing volume ratio = 4.3:1:10) and reacted for 60 minutes at approximately 37° C. A portion of the solution after the reaction was collected, and the radiochemical purity was calculated by thin layer chromatography using 0.1 M citrate buffer as a developing solvent.

[0266] Thereafter, purification was carried out using AmiconUltra-10K to obtain 111-In-labeled Fab conjugates #1 to #74 and 111-In-labeled F(ab')2 conjugates #1 to #74 with radiochemical purity of 95% or more.

[0267] (Example 13) Detection of Cancer Using 111-In-Labeled Fab Conjugate and 111-In-Labeled F(ab')2 Conjugate The in vivo cancer detection ability of the 111-In-labeled F(ab')2 conjugate #1 prepared in Example 12 was evaluated using tumor-bearing Balb / c mice transplanted with mouse-derived cancer cells (mouse breast cancer cells 4T1) expressing CAPRIN-1 protein on the cell membrane surface. ５ Mouse breast cancer cells (4T1) were subcutaneously transplanted to form tumors, generating tumor-bearing mice. Under isoflurane anesthesia, approximately 10 MBq of the In-111-labeled F(ab')2 conjugate #1 was administered as a single dose via the tail vein of the 4T1 tumor-bearing mice.

[0268] Twenty-four hours after administration to the tumor-bearing mice, gamma rays emitted from the 111-In-labeled F(ab')2 conjugate #1 were detected under isoflurane anesthesia using a NanoSPECT / CT (Bioscan) imaging device according to standard methods, and the tumors transplanted into the mice were detected. HiSPECT NG and InVivoScope (IVS) were used for image reconstruction. Evaluation of cancer detection demonstrated that images of the 111-In-labeled F(ab')2 conjugate #1 strongly accumulated in the tumor, demonstrating that cancer could be detected. Similarly, cancers were detected with the 111-In-labeled Fab conjugates #1 to #74 and F(ab')2 conjugates #2 to #74.

[0269] Furthermore, as described above, the organs of 4T1 cancer-bearing mice administered with In-labeled F(ab')2 conjugate #1 were collected 24 hours after administration of the conjugate, including blood, bone, brain, digestive tract, gonads, uterus and oviduct, heart, kidney, liver, lung, muscle, skin, spleen, and tumor. After measuring their weights, the radiation dose emitted from each organ was measured using a γ counter Wizard2 (PerkinElmer), and the percentage of radiation accumulated in each organ was calculated from the administered radiation dose and the radiation dose accumulated in each organ and tumor.

[0270] As a result, assuming the radiation dose emitted by the administered conjugate to be 100%, the percentage of radiation accumulated in the 4T1 tumor 24 hours after administration was approximately 13% per tumor weight. Meanwhile, the average radiation dose in various organs of the mice other than the tumor was 9% per organ weight (approximately 4% excluding the kidney, which is the main metabolic pathway). Similar results were obtained for 111-In-labeled Fab conjugates #1 to #74 and F(ab')2 conjugates #2 to #74. These results demonstrate that conjugates in which Fab conjugates and F(ab')2 conjugates are labeled with 111-In, a radioisotope, can detect cancer. (Example 14) Labeling of Fab conjugates and F(ab')2 conjugates with a radioisotope (177-Lu) The Fab conjugates and F(ab')2 conjugates prepared in Example 10 were labeled with the metallic radioisotope 177-Lu to prepare conjugates. The Fab complex and F(ab')2 complex solutions were adjusted to approximately 10 mg / ml, mixed with 0.2 M sodium acetate buffer (pH 5.3±0.3), and then added with 177-LuCl ３ A solution (ITM Medical Isotopes) was added (mixing volume ratio = 20:25:2.8), and the mixture was allowed to react at approximately 38°C for approximately 18 hours. A portion of the solution after the reaction was collected, and the radiochemical purity was calculated by the following standard method using thin-layer chromatography with 0.1 M citrate buffer as the developing solvent. Subsequently, the mixture was purified using an AmiconUltra-10K to obtain 177-Lu-labeled Fab conjugates #1 to #74 and 177-Lu-labeled F(ab')2 conjugates #1 to #74 with radiochemical purity of 95% or higher.

[0271] Example 15 Antitumor Effect of 177-Lu-Labeled Fab Conjugate and 177-Lu-Labeled F(ab')2 Conjugate in Tumor-Bearing Mice (4T1) The 177-Lu-labeled Fab conjugate #1 and 177-Lu-labeled F(ab')2 conjugate #1 prepared in Example 13 were evaluated for their antitumor effect in vivo in mice bearing 4T1 tumors prepared in the same manner as in Example 12. Approximately 15 MBq of the 177-Lu-labeled Fab conjugate #1 and 177-Lu-labeled F(ab')2 conjugate #1 were administered as a single dose via the tail vein to mice bearing 4T1 tumors. Controls included a saline-administered group (untreated group), a Fab#1-administered group, an F(ab')2#1-administered group, a non-radioisotope-labeled Fab conjugate #1-administered group, and a non-radioisotope-labeled F(ab')2 conjugate #1-administered group.

[0272] 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). ２The tumor volume was calculated using a ρ = 0.5 × 0.5 ratio. As a result of the evaluation, on day 21 after the start of administration, when the tumor volume in the group administered with non-radioisotope-labeled Fab conjugate #1 was taken as 100%, the tumor volume in the mice administered with 177-Lu-labeled Fab conjugate #1 was approximately 56% or less. Similarly, when the tumor volume in the group administered with non-radioisotope-labeled F(ab')2 conjugate #1 was taken as 100%, the tumor volume in the mice administered with 177-Lu-labeled F(ab')2 conjugate #1 was approximately 36% or less. The tumor volumes in the mice administered with Fab, F(ab')2, and non-radioisotope-labeled conjugates were comparable to those in the untreated group. The results of this evaluation demonstrated that 177-Lu-labeled Fab conjugate #1 and 177-Lu-labeled F(ab')2 conjugate #1 exhibited significantly strong antitumor effects. Similar results were obtained for 177-Lu-labeled Fab conjugates #2 to #74 and 177-Lu-labeled F(ab')2 conjugates #2 to #74 as for 177-Lu-labeled Fab conjugate #1 and 177-Lu-labeled F(ab')2 conjugate #1. Note that 4T1 expresses somatostatin receptors (Anti-Cancer Drugs 28(5): p. 489-502, June 2017). Based on the results of this Example and Example 8, it was estimated that the 177-Lu-labeled Fab conjugates and 177-Lu-labeled F(ab')2 conjugates also have significantly superior efficacy compared to 177-Lu-labeled DOTATATE (equivalent to Lutathera (registered trademark) which is marketed as a therapeutic agent for somatostatin receptor-positive cancers).

Claims

1. A complex comprising an antibody or a fragment thereof having immunological reactivity with 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 to said amino acid sequence, and a radioisotope bound thereto via a chelating agent.

2. The complex according to claim 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 one of SEQ ID NOs: 31 to 35, 296 to 299, 308, and 309, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence.

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

4. The conjugate according to claim 1, wherein the antibody or a fragment thereof is any one of the following (A) to (M): (A) an antibody or a 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 CAPRIN-1 protein; (B) an antibody or a 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 CAPRIN-1 protein. (C) An antibody or a fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 52, 53 and 54 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 56, 57 and 58, and having immunological reactivity with CAPRIN-1 protein. (D) An antibody or a fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 60, 61 and 62 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 64, 65 and 66, and having immunological reactivity with CAPRIN-1 protein. (E) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 170, 171 and 172 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 173, 174 and 175, and having immunological reactivity with CAPRIN-1 protein. (F) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 176, 177 and 178 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 179, 180 and 181, 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 (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 182, 183 and 184 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 185, 186 and 187, 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 (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 188, 189 and 190 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 191, 192 and 193, 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 (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 146, 147 and 148 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 149, 150 and 151, 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 (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 272, 273 and 274 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 275, 276 and 277, 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 (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 290, 291 and 292 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 293, 294 and 295, 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 (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 301, 302 and 303 and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3, respectively) of SEQ ID NOs: 305, 306 and 307, 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 (CDR1, CDR2 and CDR3) of SEQ ID NOs: 134, 135 and 136, and a light chain variable region comprising the complementarity determining regions (CDR1, CDR2 and CDR3) of SEQ ID NOs: 137, 138 and 139, and having immunological reactivity with CAPRIN-1 protein.

5. The conjugate according to claim 1, wherein the antibody or a fragment thereof is any one of the following (a) to (al): (a) an antibody or a fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 39 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 43; (b) an antibody or a fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 47 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 51; (c) an antibody or a fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 55 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 59; (d) an antibody or a fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 63 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 67; (e) an antibody or a fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 68 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 69; (f) an antibody or a fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 70 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO:

71. (g) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 72 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 73; (h) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 74 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 75; (i) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 76 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 77; (j) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 78 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 79; (k) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 80 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 81; (l) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 82 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO:

83. (m) 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.(n) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 86 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 87; (o) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 88 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 89; (p) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 90 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 91; (q) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 92 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 93; (r) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 94 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 95; (s) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 96 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO:

97. (t) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 98 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 99; (u) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 100 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 101; (v) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 102 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 103; (w) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 104 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 105; (x) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 106 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 107; (y) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 108 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO:

109. (z) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 110 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 111; (aa) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 112 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 113.(ab) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 114 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 115; (ac) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 116 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 117; (ad) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 118 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 119; (ae) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 120 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 121; (af) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 122 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 123; (ag) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 124 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO:

125. (ah) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 126 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 127; (ai) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 128 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 129; (aj) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 130 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 131; (ak) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 132 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO: 133; (al) an antibody or fragment thereof, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO: 300 and the light chain variable region of which comprises the amino acid sequence of SEQ ID NO:

304.

6. The conjugate of claim 1, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

7. The complex of claim 1, wherein the fragment is F(ab')2, Fab', Fab or scFv.

8. The complex of claim 1, wherein the radioisotope is an alpha-, beta- or gamma-emitting radioisotope.

9. The complex of claim 1, wherein the radioisotope is Ra (radium), thorium (Th), actinium (Ac), lutetium (Lu), indium (In), gallium (Ga), lead (Pb), copper (Cu), strontium (Sr), yttrium (Y), samarium (Sm), rhenium (Re), bismuth (Bi), technetium (Tc), or zirconium (Zr).

10. The complex of claim 1, wherein the radioisotope is 223-Ra, 227-Th, 225-Ac, 177-Lu, 111-In, 67-Ga, 68-Ga, 212-Pb, 64-Cu, 67-Cu, 89-Sr, 90-Y, 153-Sm, 186-Re, 188-Re, 212-Bi, 213-Bi, 99m-Tc, or 89-Zr.

11. The complex of claim 1, wherein the chelating agent is a bifunctional chelating agent.

12. The complex of claim 11, wherein the bifunctional chelator is a cyclic or linear chelator.

13. The cyclic chelating agent is DOTA, NOTA, TETA, TCMC, DEPA, NETA, H ２ The complex of claim 12, which is selected from the group consisting of macropa, Crown, PCTA, NODAGA, NODASA, Sar bicyclic chelator, MANOTA and derivatives thereof.

14. The linear chelating agent is HOPO, DTPA, MAG ２ -GABA, Trisuccin, DFO, H ２ Dedpa, H ４ The complex of claim 12, which is selected from the group consisting of octapa, HYNIC, HBED-CC, THP and derivatives thereof.

15. A pharmaceutical composition for treating, preventing and / or diagnosing cancer, comprising as an active ingredient the complex according to any one of claims 1 to 14.

16. The pharmaceutical composition according to claim 15, wherein the cancer is a cancer that expresses CAPRIN-1 protein on the cell membrane surface.

17. The pharmaceutical composition of claim 15, wherein the cancer is breast cancer, renal cancer, pancreatic cancer, colon 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.

18. A method for treating, preventing and / or diagnosing cancer, comprising administering to a subject a conjugate according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15.