Pharmaceutical composition for treating and / or preventing cancer

Abstract

Provided is a conjugate obtained by binding a thiol-containing maytansinoid and an antibody or a fragment thereof having immunological reactivity with an amino acid sequence represented by any one of even SEQ ID NOs: 2 to 30 or a CAPRIN -1 protein having an amino acid sequence having 80% or more sequence identity with the antibody. The conjugate can be used as an antibody-drug complex (ADC) for treating and / or preventing cancer.

Description

Pharmaceutical composition for treating and / or preventing cancer

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

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

[0003] In recent years, studies have been conducted to enhance the efficacy of antibody drugs against cancer, and in particular, the development of antibody-drug conjugates (ADCs), which are conjugated between antitumor drugs and antibodies, has progressed. As an example of the practical application of an ADC containing a thiol-containing maytansinoid, an antitumor compound that inhibits cell mitosis by inhibiting microtubule polymerization, ELAHERE (registered trademark) (mirvetuximab soravtansine-gynx), in which DM4, a thiol-containing maytansinoid, is linked to mirvetuximab, an antibody against the folate receptor α (FRα receptor), was observed to have an objective response rate (ORR) of 31.7% and a median duration of response (DOR) of 6.9 months in a single-arm study in patients with FRα-positive platinum-resistant epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer who had previously received bevacizumab, and is currently used to treat some of the above cancer patients (Non-Patent Document 1). Furthermore, KADCYLA (registered trademark) (ado-trastuzumab emtansine (TDM-1)), in which trastuzumab, an antibody against HER2, is conjugated to DM1, a thiol-containing maytansinoid, has been shown to be effective in patients with HER2-positive advanced breast cancer who have previously been treated with taxane anticancer drugs. In a comparison of lapatinib and capecitabine combination therapy with TDM-1 monotherapy, the median progression-free survival was 6.4 months for lapatinib and capecitabine combination therapy, while it was 9.6 months for TDM-1. Furthermore, the median overall survival was 25.1 months for lapatinib and capecitabine combination therapy, while it was 30.9 months for TDM-1 (Non-Patent Document 2).

[0004] WO2010 / 016526

[0005] HIGHLIGHTS OF PRESCRIBING INFORMATION (Reference ID: 5077370) (https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2022 / 761310s000lbl.pdf) The New England Journal of Medicine 2012 Nov 8;367(19):1783-91

[0006] As mentioned above, ADCs using thiol-containing maytansinoids for the purpose of enhancing the efficacy of antibody drugs against cancer have already been put to practical use, but their success has been limited to the targeting of some cancers, and their antitumor effects have also been limited. Furthermore, as shown in the Examples of the present specification, the efficacy of a conjugate of trastuzumab and a thiol-containing maytansinoid, which is commercially available as a cancer antibody drug, is only slightly enhanced in antitumor effect compared to trastuzumab alone, and it has been newly discovered that ADC technology using thiol-containing maytansinoids is not a technology that significantly enhances the antitumor effects of all cancer antibody drugs.

[0007] Therefore, an object of the present invention is to create an ADC that utilizes a thiol-containing maytansinoid and has excellent antitumor effects.

[0008] As a result of intensive research, the present inventors have found that a conjugate of an antibody against CAPRIN-1 protein or a fragment thereof with a thiol-containing maytansinoid exhibits an extremely strong antitumor effect compared to an antibody against CAPRIN-1 protein or a fragment thereof alone, and further that the antitumor effect enhancement effect when an antibody against CAPRIN-1 protein or a fragment thereof is conjugated with a thiol-containing maytansinoid is significantly superior to the antitumor effect enhancement effect when an existing cancer antibody pharmaceutical is conjugated with a thiol-containing maytansinoid, thereby completing the present invention.

[0009] Specifically, the present invention has the following features (1) to (12).

[0010] (1) A conjugate comprising an antibody or a fragment thereof immunologically reactive with a CAPRIN-1 protein having an amino acid sequence represented by any of the even-numbered SEQ ID NOS: 2 to 30, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence, and a thiol-containing maytansinoid bound thereto.

[0011] (2) The conjugate according to (1), wherein the antibody or a fragment thereof is immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having an amino acid sequence represented by any 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 to said amino acid sequence.

[0012] (3) The conjugate according to (1) or (2), wherein the antibody is a monoclonal antibody or a polyclonal antibody.

[0013] (4) The conjugate according to any one of (1) to (3), wherein the antibody or fragment thereof is any one of the following (A) to (M): (A) an antibody or fragment 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 fragment 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 the CAPRIN-1 protein. (D) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 60, 61, and 62 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 64, 65, and 66 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (E) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 170, 171, and 172 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 173, 174, and 175 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with 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.

[0014] (5) The conjugate 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.

[0015] (6) The conjugate according to any one of (1) to (5), wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, or a single-chain antibody.

[0016] (7) The conjugate according to any one of (1) to (6), wherein the antibody or fragment thereof and the thiol-containing maytansinoid are bound via a linker.

[0017] (8) The conjugate according to any one of (1) to (7), wherein the thiol-containing maytansinoid is DM1, DM4, or a derivative thereof.

[0018] (9) A pharmaceutical composition for treating and / or preventing cancer, comprising the conjugate according to any one of (1) to (8) as an active ingredient.

[0019] (10) The pharmaceutical composition according to (9), wherein the cancer is a cancer that expresses CAPRIN-1 protein on the cell membrane surface.

[0020] (11) The pharmaceutical composition according to (9) or (10), 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.

[0021] (12) A method for treating and / or preventing cancer, comprising administering to a subject the conjugate according to any one of (1) to (8) or the pharmaceutical composition according to any one of (9) to (11).

[0022] The conjugate of the present invention not only exhibits an extremely strong antitumor effect compared to an antibody against CAPRIN-1 protein or a fragment thereof alone, but also has a significantly superior antitumor effect compared to previously known conjugates of cancer antibody drugs and thiol-containing maytansinoids. Furthermore, the antitumor effect enhancement achieved by conjugating an antibody against CAPRIN-1 protein or a fragment thereof with a thiol-containing maytansinoid is significantly superior to the antitumor effect enhancement achieved by conjugating an existing cancer antibody drug with a thiol-containing maytansinoid. Therefore, the conjugate of the present invention is effective in treating or preventing cancer.

[0023] The antitumor activity of the conjugate of the present invention between an antibody against CAPRIN-1 protein or a fragment thereof (hereinafter referred to as "anti-CAPRIN-1 antibody") and a thiol-containing maytansinoid can be evaluated by examining the inhibition of tumor growth in vitro or in vivo (specifically, in tumor-bearing animals), as described below.

[0024] In the present invention, the term "conjugate" refers to a compound in which an antibody or a fragment thereof and a drug are covalently linked. A linker may be used between the antibody and the drug.

[0025] The anti-CAPRIN-1 antibody forming the conjugate of the present invention may be a monoclonal or polyclonal antibody, preferably a monoclonal antibody. The conjugate of the present invention may be any type of antibody, including a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, or a non-human animal antibody, as long as it can exhibit antitumor activity.

[0026] The thiol-containing maytansinoid forming the conjugate of the present invention is a microtubule-targeted maytansine derivative that inhibits cell proliferation during mitosis.

[0027] Subjects for cancer treatment and / or prevention in the present invention include mammals such as humans, pet animals, livestock, and sport animals, with humans being the preferred subject.

[0028] The following describes the anti-CAPRIN-1 antibody, the thiol-containing maytansinoid, the conjugate of the anti-CAPRIN-1 antibody and the thiol-containing maytansinoid, the pharmaceutical composition using the conjugate, and the method for treating and / or preventing cancer according to the present invention.

[0029] <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.

[0030] The anti-CAPRIN-1 antibody used in the present invention refers to an antibody or antigen-binding fragment thereof that is immunologically reactive with the full-length CAPRIN-1 protein, which is the antigen, or a fragment thereof. Here, "immunological reactivity" refers to the property of the antibody specifically binding to the CAPRIN-1 protein or a partial polypeptide thereof in vivo. Specific examples include antibodies that are immunologically reactive with a CAPRIN-1 protein variant that has 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity with any of the amino acid sequences represented by the even-numbered SEQ ID NOS: 2 to 30. The term "% sequence identity" used herein 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 (arranged) to maximize similarity, with or without gaps.

[0031] The anti-CAPRIN-1 antibody used in the present invention may be a monoclonal antibody or a polyclonal antibody.

[0032] 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, or the like 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 a partial peptide is bound, or the like.

[0033] 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). Methods for producing the CAPRIN-1 protein can be obtained by referring to WO2014 / 012479, or cells expressing the CAPRIN-1 protein can also be used.

[0034] 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 the CAPRIN-1 protein 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.

[0035] The antibodies used in the present invention include human antibodies, humanized antibodies, chimeric antibodies, and non-human animal antibodies.

[0036] 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.

[0037] 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).

[0038] 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).

[0039] 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.

[0040] 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.

[0041] 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, as described above, purified by ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange column, affinity column bound to CAPRIN-1 protein or a partial peptide, or the like. Furthermore, monoclonal antibodies can be obtained from non-human animals by collecting immune cells from the immunized animal and subjecting them to cell fusion 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).

[0042] The antibodies used in the present invention can also be obtained as recombinant antibodies produced by cloning antibody genes from hybridomas, incorporating them into appropriate vectors, and introducing them into hosts 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).

[0043] The anti-CAPRIN-1 antibody used in the present invention may have amino acids in the variable region (e.g., FR) or constant region substituted with other amino acids. The amino acid substitutions are single or multiple, for example, fewer than 15, fewer than 10, 8 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids, preferably 1 to 9 amino acids. The substituted antibody preferably has the same or higher antigen-specific binding properties and antigen-binding affinity as the unsubstituted antibody, and further preferably does not cause a rejection reaction when administered to humans.

[0044] 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 is.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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, WO2011 / 096535, WO2011 / 096536, WO2011 / 096537, WO2011 / 096538, WO2011 / 096539, WO2011 / 096540, WO2011 / 096541, WO2011 / 096542, WO2011 / 096543, WO2011 / 096544, WO2011 / 096545, WO2011 / 096546, WO2011 / 096547, WO2011 / 096548, WO2011 / 096549, WO2011 / 096550, WO2011 / 096551, WO2011 / 096552, WO2011 / 096553, WO2011 / 096554, WO2011 / 096555, WO2011 / 096556, WO2011 / 096557, WO2011 / 096558, WO2011 / 096559, WO2011 / 096560, WO2 These can be obtained by reference to 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.

[0049] Specific examples of the anti-CAPRIN-1 antibody used in the present invention include those described in the aforementioned WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, WO2013 / 018892, WO2013 / 0188 91, 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:

[0050] 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.

[0051] 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.

[0052] An antibody or fragment thereof immunologically reactive with a partial polypeptide of a CAPRIN-1 protein having the amino acid sequence set forth in SEQ ID NO: 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[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: 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.

[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: 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.

[0063] In addition, the following anti-CAPRIN-1 antibodies are also preferably used.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] In the Examples described below, conjugates of thiol-containing maytansinoids were prepared using the above-mentioned polyclonal and monoclonal antibodies against the full-length CAPRIN-1 protein and a portion of the polypeptide region expressed on the cell membrane surface of cancer cells, and their strong antitumor effects were confirmed.

[0099] <Thiol-Containing Maytansinoid> Thiol-containing maytansinoid is a type of maytansine derivative that targets microtubules and inhibits cell proliferation during mitosis. Specific examples include DM1 (N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine, CAS No. 139504-50-0), DM4 (N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine, CAS No. 799840-96-3), and derivatives thereof. In the present invention, DM1, DM4, and their derivatives are preferably used.

[0100] Specific examples of DM1 derivatives (including specific examples of conjugate compounds containing a linker) include N(2')-deacetyl-N2'-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine and compounds described in U.S. Pat. No. 5,208,020.

[0101] Specific examples of DM4 derivatives (including specific examples of complex compounds containing a linker) include Ravtansine (also known as IUPAC name: (1S,2R,3S,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8, 23-dioxo-4, 24-dioxa-9, 22-diazatetracyclo [19.3.1.110, 14.03,5] hexacosa-10, 12, 14 (26),16,18-pentaen-6-yl)(2S)-2-[methyl-(4-methyl-4-sulfanylpentanoyl)amino]pr opanoate, CAS:1194029-15-6, WOB38VS2NI, UNII-WOB38VS2NI, N2'-Deacetyl-N2'-(4-mer capto-4-methyl-1-oxopentyl)-maytansine (14S, 16S, 32S, 33S, 2R, 4S, 10E, 12E, 14R) -86- chloro-14-hydroxy-85, 14-dimethoxy-33, 2, 7, 10-tetramethyl-12, 6-dioxo-7-aza-1 (6, 4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(4-mercapto-4-methylpentanoyl)-N-methyl-L-alaninate), Maytansinoid DM4 (IUPAC name as alias: [(1S, 3S, 5S, 6S, 16E, 18E, 20R, 21S)-11-chloro-21-hydroxy-12, 20-dimethoxy-2, 2, 5, 9, 16-pe ntamethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,[(16Z,18Z)-11-chloro-21-hydroxy-12,20-dimethoxy-2,2,5,9,16-pentamethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] (2S)-2-[methyl-(4-methyl-4-sulfanylpentanoyl)amino]propanoate, 799840-96-3, SCHEMBL21954026, HY-100503, CS-0019630), [(16Z,18Z)-11-chloro-21-hydroxy-12,20-dimethoxy-2,2,5,9,16-pentamethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] 2-[methyl-(4-methyl-4-sulfanylpentanoyl)amino]propanoate, [(16E,18Z)-11-chloro-21-hydroxy-12,20-dimethoxy-2,2,5,9,16-pentamethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] 2-[methyl-(4-methyl-4-sulfanylpentanoyl)amino]propanoate, Compound CID: 131859751, Compound CID: 134693255, DM4-SPDB, DM4-SPDB-L-Lysine, Soravtansine (DM4-Sulfo-TBA), S-Me-DM4, Slfo-SPDB-DM4, SPDB-DM4, DM4-Sme, Soravatan sine-lysine, D-Lin-K-DM4, N2'-(4-((3-Carboxypropyl)dithio)-4-methyl-1-oxopentyl)-N2'-deacetylmaytansine (also known as OX9S087G5M or DBA-DM4), DM4-SMCC, DBA-DM4, SSP-DM4, Maytansinoid DM4 impurity 2-d6, Sulfo-PDBA-DM4, DM4-SPDP (Carlsson et al., Biochem. J.,173:723-737 (1978)), DM4 (hydrate), Maytansinoid DM4 impurity 5-d6, Maytansinoid DM4 impurity 3-d6, Maytansinoid DM4-d6, Sulfo-PDBA-DM4, DM4-d6, or derivatives thereof. Other examples include compounds described in U.S. Patent Nos. 5,208,020 and 7,276,497, and DM4 containing a non-cleavable linker described in WO 2005 / 037992. Non-cleavable linkers include those that are cleaved by acid induction, light induction, peptidase induction, esterase induction, and those whose disulfide bond is cleaved by a reducing compound.

[0102] <Conjugate of anti-CAPRIN-1 antibody and thiol-containing maytansinoid> In the present invention, the bond between the anti-CAPRIN-1 antibody and the thiol-containing maytansinoid in the conjugate of the anti-CAPRIN-1 antibody and the thiol-containing maytansinoid is not particularly limited as long as it is a bond that can maintain anti-cancer activity, but a bond that forms a linker structure between the anti-CAPRIN-1 antibody and the thiol-containing maytansinoid is preferred.

[0103] Here, the term "linker" refers to a compound capable of binding an antibody or a fragment thereof to a drug. Various known linkers may be used, or the drug may be chemically modified in its structure to be equivalent to a linker and then bound to the antibody (including the compounds described above in the section "Thiol-containing maytansinoids").

[0104] The type of linker and the details of the binding method can be determined in accordance with known methods (see, for example, Greg T. Hermanson, Bioconjugate Techniques, Third Edition, WO2004 / 010957 and WO2014 / 012479).

[0105] In embodiments of the invention, reactive groups attached to the anti-CAPRIN-1 antibody, thiol-containing maytansinoid, and linker include:

[0106] Unless specifically chemically modified, reactive groups present in the amino acid sequence of an antibody or in glycoproteins modified with amino acids include primary amines (ε-amino), carboxyls, thiols (sulfhydryls), carbonyls (ketones or aldehydes), and hydroxyls. Primary amines are found at the N-terminus of polypeptides and in the side chains of lysine residues. They are positively charged under physiological conditions and are usually located on the outside of proteins, allowing them to be used for conjugation without altering the protein structure. Carboxyls are found at the C-terminus of polypeptides and in the side chains of aspartic acid and glutamic acid. Sulfhydryls are found in the side chains of cysteine ​​and form disulfide bonds that maintain the higher-order structure of proteins. Ketones or aldehydes are generated in glycoproteins by oxidizing glycosyl groups with sodium metaperiodate.

[0107] The conjugates of the invention are prepared by attaching a drug to a linker attached to the reactive group on the antibody, by attaching a thiol-containing maytansinoid to a linker attached to the drug, or by attaching a drug directly to the antibody.

[0108] Specific examples of the reactive groups attached to the thiol-containing maytansinoid and linker include:

[0109] Reactive groups that can react with amines include N-hydroxysuccinimide (NHS) esters, N-sulfosuccinimidyl 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.

[0110] Carbodiimides, diazoalkanes, diazoacetyl compounds, carbonyldiimidazoles are reactive groups that can react with carboxyls and amines.

[0111] Reactive groups that can react with thiols include maleimide, haloacetamide, pyridyl disulfide, thiosulfone, vinyl sulfone, haloacetyl, aziridine, acryloyl, and aryl.

[0112] 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.

[0113] Isocyanate is a reactive group that can react to hydroxyl.

[0114] Photoreactive reactive groups include diazirine, aryl azide, aryl, benzophenol, and diazo compounds.

[0115] A compound containing a dithiopyridyl group capable of forming a disulfide bond.

[0116] Specific examples of the linker having the reactive group include the following:

[0117] 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.

[0118] 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 ), a linker having an NHS ester and a pyridyldithiol as a reactive group (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, SBA, 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 that have an imide as a reactive group (e.g., DCC, EDC, EDAC, NHS, Sulfo-NHS), linkers that have a maleimide and a hydrazide as a reactive group (e.g., BMPH, EMCH, MPBH, KMUH), linkers that have a pyridyldithiol and a hydrazide as a reactive group (e.g., PDPH), linkers that have an isocyanate and a maleimide as a reactive group (e.g., PMPI), and linkers that have an NHS ester and a psoralen as a reactive group (e.g., SPB) are used.

[0119] 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.

[0120] 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-aminooxy-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-PEG ６ -Tos, N-Tos-N-(t-butoxycarbonyl)-aminoxy-PEG ４ -Tos, Ms-PEG-Ms, Ms-PEG-t-butyl ester, PEG-Ms, Propargyl-PEG-Ms), Boc-PEG (e.g., Amino-PEG-t-Boc-Hydrazide, Azido-PEG-t-Boc-Hydrazide, Boc-NH-PEG-NH-Boc, Bromoacetamido-PEG-Boc-amine, m-PEG-ONHBoc, Mal-Alkyl-t-Boc-amine, N-Boc-PEG-alcohol, N-Boc-PEG-bromide, N-methyl-N-(t-Boc)-PEG-acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH ２ CO ２H, t-Boc-N-Amido-PEG-amine, t-Boc-N-amido-PEG-azide, t-Boc-N-amido-PEG-NHS ester, t-Boc-N-amido-PEG-sulfonic acid), PEG NHS ester (e.g., Acid-PEG-NHS ester, Azido-PEG-NHS ester, Bis-PEG-NHS, Fmoc-PEG-NHS ester, m-PEG-NHS ester, m-PEG-NHS Carbonate, Mal-PEG-NHS ester, Propargyl-PEG-NHS ester, t-Boc-N-amido-PEG-NHS ester, t-Butoxycarbonyl-PEG-NHS ester), Fmoc-PEG (e.g., Fmoc-N-amido-PEG-acid, Fmoc-NH-PEG-CH ２ CO ２ H, Fmoc-PEG-NHS ester), Biotin PEG (e.g., Biotin PEG-acid, Biotin PEG-alcohol, Biotin PEG-alkyne, Biotin PEG-amine, Biotin PEG-azide, Biotin PEG-DBCO, Biotin PEG-hydrazide, Biotin-PEG-Mal, Biotin-PEG-NHS, Biotin-EDA-PEG-NHS, Biotin-PEG-oxyamine, Biotin-PEG-PFP, Biotin-EDA-PEG-PFP, Biotin-PEG-Tetrazine, Biotin-PEG-TFP, Azide-SS-biotin, Biotin-PEG ３ -SS-azide, DBCO-S-S-PEG ３ -Biotin, Dde Biotin-PEG4-Alkyne, Dde Biotin-PEG ４ -Azide, Dde Biotin-PEG ４ -DBCO, Diazo Biotin-PEG ３ -Alkyne, Diazo Biotin-PEG ３ -Azide, Diazo Biotin-PEG ３-DBCO、Diol Biotin-PEG ３ -Alkyne、Diol Biotin-PEG ３ -Azide、PC Biotin-PEG ３ -Alkyne、PC-Biotin-PEG ４ -PEG ４ -Alkyne、PC-Biotin-PEG ４ -PEG4-Alkyne、PC Biotin-PEG ３ -Azide、PC-Biotin-PEG4-PEG ３ -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.

[0121] In another embodiment, polyethylene glycol (PEG) as described in WO2015 / 057699 and WO2017 / 165851 can be used to obtain a conjugate that is expected to have better in vivo kinetics.

[0122] The linker mediated between the anti-CAPRIN-1 antibody and the thiol-containing maytansinoid may be composed of a single type or multiple types.

[0123] In another embodiment, the charged linkers described in WO2014 / 186403 are used.

[0124] Methods for preparing the conjugate of the present invention include a method using the ε-amino group of a lysine side chain of an antibody or a fragment thereof, and a method in which cysteine ​​residues forming disulfide bonds in an antibody are reduced to form a thiol bond to conjugate the antibody.

[0125] When the ε-amino group of a lysine residue of an antibody is used, for example, an amide bond is formed by reacting it with an active ester (e.g., N-hydroxysuccinimide ester). In this case, since an antibody or a fragment thereof contains many lysine residues, the binding reaction proceeds nonspecifically.

[0126] When using a thiol that forms a disulfide bond present in the side chain of a cysteine ​​of an antibody or its fragment, a method is used in which the disulfide bond in the antibody or its fragment is converted to a thiol using a reducing agent such as mercaptoethanol, followed by reaction with a maleimide or α-haloamide. Furthermore, methods using, for example, sulfonephenyloxadiazole or 4-cyanoethynyloxy derivatives can be used to stabilize thiol-mediated bonds. These bonds are more stable for longer periods than bonds formed by the conjugation reaction of cysteine ​​to maleimide. Furthermore, since stability is improved when the imide ring formed by the thiol group attached to the maleimide is opened by hydrolysis to form an amide bond, a linker having an amino group near the imide group can also be used. Another method involves conjugating a drug between two thiols formed by the cysteine ​​thiol in an antibody or its fragment via a disulfide bond. For example, a cross-linked bond can be formed using a linker with two disulfide bond sites, which can be generated from an amide group with two sulfones at the β-position, or dibromomaleimide.

[0127] The conjugates of the present invention can be produced, for example, using THIOMAB™ technology, which is a method for introducing a fixed number of thiol groups into a specific portion of an antibody or a fragment thereof (see Nature Biotechnology 26, 925-932 (2008)). Alternatively, a method using ThioBridge™ technology (see Methods Mol Biol. 2078: 113-129 (2020)). Alternatively, a method using RESPECT™ technology (see MAbs. 9 (6): 907-915 (2017)).

[0128] The conjugates of the present invention can be prepared, for example, by reducing an antibody or a fragment thereof with the reducing agent dithiothreitol (DTT) in a phosphate buffer solution to obtain an antibody having a thiol-reactive group, followed by formation of a conjugate with a thiol-containing maytansinoid. In addition to the method using a reducing agent, the conjugates can also be obtained by adding a thiol group to the primary amine of a lysine residue in the antibody by introducing Traut's reagent (2-Iminothiolane or N-Succinimidyl S-Acetylthioacetate (SATA)).

[0129] The amount of thiol added to an antibody or a fragment thereof 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)).

[0130] The thiol groups added by cleaving disulfide bonds in the antibody through reduction treatment are preferably subjected to a treatment (capping) to prevent re-formation of disulfide bonds, such as with N-ethylmaleimide (NEM) or 2-iodoacetamide (IAA).

[0131] Formation of a conjugate using a thiol group added to an antibody or a fragment thereof can be carried out 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) can be used as a linker having a maleimide group. In this case, if an amino group is present on the drug side, a conjugate can be obtained by forming an amide bond with the N-succinimide group of SMCC.

[0132] In another embodiment, an amide bond is first formed with SMCC at the amino group present on the drug side, and then a thiol group added to the antibody or its fragment side is reacted with the maleimide group of SMCC on the drug side to obtain a conjugate.

[0133] In another embodiment, a conjugate can be formed using two linkers. For example, a conjugate can be prepared by amide-bonding a primary amino group present in a lysine residue on an antibody or a fragment thereof with an N-succinimide group on SATA (N-succinimidyl-S-acetylthioacetate), adding a thiol group to the antibody or fragment thereof, and then reacting SMCC with an amino group present on the drug side or one to which an amino group has been added according to a standard method to form an amide bond with the N-succinimide group on SMCC, and then reacting the maleimide group on the SMCC on the drug side with the thiol group on SATA on the antibody or fragment thereof to obtain a conjugate.

[0134] In another embodiment, conjugates can be prepared using, for example, maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PAB) as a linker. mc-val-Cit-PAB (mc-vc-PAB) is a linker that can be cleaved by intracellular proteases (e.g., cathepsin B). A thiol group is attached to an antibody or a fragment thereof dissolved in phosphate buffer using DTT or the like. Meanwhile, a thiol-containing maytansinoid having an amino group is reacted with the benzyloxycarbonyl (PAB) in mc-Val-Cit-PAB to prepare a thiol-containing maytansinoid bound to mc-val-Cit-PAB, which can then be reacted with the thiol-added antibody or a fragment thereof to obtain a conjugate.

[0135] In yet another embodiment, SATA is attached to a primary amino group of a lysine residue of an antibody or fragment thereof to add a thiol group, while succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is reacted with an amino group on the drug to form an amide bond with the N-succinimide group of SPDP.

[0136] The linker used in the present invention can be one that is cleavable under intracellular conditions. When such a linker is used, a substance having antitumor activity containing a drug or a drug and a portion of the linker is released intracellularly. For example, the linker is cleaved by intracellular peptidases or proteases. Preferred linkers are those that are 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 can be used.

[0137] Furthermore, in another embodiment, as a means for improving the stability, solubility, and metabolism of the conjugate of the present invention in blood and the binding ability of a drug to an antibody or a fragment thereof, a linker having glucuronic acid (preferably β-D-glucuronide) as described in WO 2007 / 011968 can be used. Alternatively, means as described in WO 2013 / 173337, WO 2015 / 095755, WO 2015 / 123679, and WO 2018 / 031690 can be used.

[0138] Furthermore, in another embodiment, site-specific binding of a drug to an antibody or a fragment thereof can be achieved using, for example, the means described in WO2006 / 65533 and WO2018 / 160683.

[0139] In yet another embodiment, a conjugate of an anti-CAPRIN-1 antibody to which two or more drugs, including a thiol-containing maytansinoid, are bound can be obtained by the method described in WO2018 / 112253.

[0140] The conjugate of the present invention can be obtained by, for example, subjecting the antibody to gel filtration chromatography or the like, and isolating a peak that is higher in molecular weight than the antibody before linker attachment. To detect the mass of the conjugate while maintaining the intact bivalent antibody, for example, the method described in WO 2013 / 049410 can be used.

[0141] The number of thiol-containing maytansinoids bound per antibody molecule of the conjugate of the present invention can be quantified according to known methods such as mass spectrometry, ELISA, electrophoresis, and chromatography such as HPLC.

[0142] <Anti-tumor effect of conjugate> The conjugate of the present invention has anti-tumor activity in vitro or in vivo. Anti-tumor activity means reduction, elimination, inhibition of growth, apoptosis, necrosis, or killing of targeted cancer cells. Therefore, the anti-tumor effect of the conjugate of the present invention can be determined by examining the anti-tumor activity against cancer.

[0143] The in vivo antitumor effect can be assessed by administering the conjugate to a cancer-bearing organism, measuring the size of the tumor after administration, and examining the size of the cancer over time. The antitumor effect of the present invention can also be assessed by examining the survival rate. It can also be assessed by examining the ability to produce cytokines or chemokines. The antitumor effect of the conjugate of the present invention can be further assessed by examining the prevention of cancer, metastasis, or recurrence.

[0144] The conjugate of the present invention is expected to have a stronger antitumor effect if it has a higher binding affinity with the CAPRIN-1 protein on the surface of cancer cells. ７ M －１ , at least 10 ８ M －１ , at least 5 × 10 ８ M －１ , at least 10 ９ M －１ , at least 5 × 10 ９ M －１ , at least 10 １０ M －１ , at least 5 × 10 １０ M －１ , at least 10 １１ M －１ , at least 5 × 10 １１ M －１ , at least 10 １２ M －１ , or at least 10 １３ M －１ is.

[0145] The ability of the conjugate of the present invention to bind to the CAPRIN-1 protein can be determined by using a binding assay using, for example, surface plasmon resonance (SPR), ELISA, Western blotting, immunofluorescence, flow cytometry, or the like.

[0146] As described above, the conjugates of the present invention have an enhanced anti-tumor effect compared to an anti-CAPRIN-1 antibody alone, and the enhancement rate is preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 65% ​​or more, and most preferably 70% or more. The enhancement rate of the anti-tumor effect of the conjugates of the present invention compared to an anti-CAPRIN-1 antibody alone can be calculated by administering an effective amount of each to tumor-bearing mice under the same conditions and comparing the tumor volumes on or after day 10 after the start of administration.

[0147] <Pharmaceutical Composition, Method for Treating and / or Preventing Cancer> The target of the pharmaceutical composition of the present invention for treating and / or preventing cancer is not particularly limited, as long as it is a cancer (cell) that expresses the CAPRIN-1 protein.

[0148] As used herein, the terms "tumor" and "cancer" refer to malignant neoplasms and are used interchangeably.

[0149] 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.

[0150] 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.

[0151] Furthermore, preferred subjects (patients) are mammals, including, for example, primates, pet animals, livestock, sport animals, etc., with humans, dogs, and cats being particularly preferred.

[0152] When the conjugates used in the present invention are used as pharmaceutical compositions, they can be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of a sterile solution or suspension injection in water or other pharmaceutically acceptable liquid. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterilized water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, binders, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is such that an appropriate dose within the indicated range can be obtained.

[0153] When the conjugate of the present invention is used as a pharmaceutical composition, it can be formulated in a lyophilized state containing any salt, surfactant, buffer, sugar, and cryoprotectant (including some sugars). For example, a pharmaceutical composition solution described in WO2007 / 019232 containing excipients including sucrose, polysorbate 20, polysorbate 80, cyclodextrin, glucose, glycerol, polyethylene glycol, mannitol, sodium chloride, and an amino acid, and having a pH of 4 to 8, preferably 4.5 to 7.6, can be used.

[0154] 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 isotonic solutions containing physiological saline, glucose, or other adjuvants (specifically, D-sorbitol, D-mannose, D-mannitol, and sodium chloride), and suitable solubilizers, such as alcohols (specifically, ethanol, polyalcohols, propylene glycol, and polyethylene glycol), and nonionic surfactants (specifically, 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 (specifically, phosphate buffer, sodium acetate buffer), soothing agents (specifically, procaine hydrochloride), stabilizers (specifically, benzyl alcohol and phenol), and antioxidants may also be added. The prepared injection solutions are usually filled into appropriate ampoules. Examples of oily liquids include sesame oil and soybean oil, and they may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Buffers (specifically, phosphate buffer, sodium acetate buffer), soothing agents (specifically, procaine hydrochloride), stabilizers (specifically, benzyl alcohol, phenol), and antioxidants may also be added. The prepared injection solution is usually filled into appropriate ampoules.

[0155] Administration may be oral or parenteral, preferably parenteral, and specific examples include injections, intranasal administrations, pulmonary administrations, transdermal administrations, etc. Examples of injections include intravenous injections, intramuscular injections, intraperitoneal injections, subcutaneous injections, intratumoral injections, etc., which can be used for systemic or local administration.

[0156] Furthermore, an appropriate administration method can be selected depending on the patient's age, body weight, sex, symptoms, etc. The dosage of a pharmaceutical composition containing an antibody or a polynucleotide encoding an antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration, such as 0.5 mg, 1 mg, 2 mg, 3 mg, 5 mg, 10 mg, 20 mg, 50 mg, 75 mg, 100 mg, 200 mg, 500 mg, or 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg / body per patient, although it is not necessarily limited to these values.

[0157] The dosage and administration method will vary depending on the patient's weight, age, sex, symptoms, etc., but can be appropriately selected by those skilled in the art.

[0158] By administering to a subject a pharmaceutical composition for treating and / or preventing cancer, which contains the conjugate of the present invention as an active ingredient, cancers that express CAPRIN-1 on the cell membrane surface, preferably breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, liver cancer, gallbladder cancer, sarcoma, mast cell tumor, melanoma, adrenocortical carcinoma, Ewing's tumor, Hodgkin's lymphoma, mesothelioma, multiple myeloma, testicular cancer, thyroid cancer, head and neck cancer, and urothelial cancer, can be treated and / or prevented.

[0159] 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.

[0160] Example 1: Anti-CAPRIN-1 Polyclonal Antibody The anti-CAPRIN-1 polyclonal antibody immunologically reactive with the CAPRIN-1 protein used in the conjugate of the present invention was prepared by mixing 1 mg of human CAPRIN-1 recombinant protein represented by SEQ ID NO: 2 and SEQ ID NO: 4, prepared according to Example 3 of WO 2010 / 016526, with an equal volume of incomplete Freund's adjuvant (IFA) solution. This mixture was subcutaneously administered to rabbits four times every two weeks. Blood was then collected to obtain antisera containing the polyclonal antibody. The obtained antisera was purified using a protein G carrier (GE Healthcare Biosciences) to obtain a polyclonal antibody against the CAPRIN-1 protein (anti-CAPRIN-1 polyclonal antibody #1). 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] Anti-CAPRIN-1 polyclonal antibody #6 against a partial polypeptide represented by SEQ ID NO: 37 (SEQ ID NO: 35 herein) described in WO2011 / 096534.

[0167] Example 2 Anti-CAPRIN-1 Monoclonal Antibody The following anti-CAPRIN-1 monoclonal antibody was used in the conjugate of the present invention.

[0168] An anti-CAPRIN-1 monoclonal antibody described in WO2011 / 096528, comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO:39 and the amino acid sequence of a light chain variable region represented by SEQ ID NO:43, 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.

[0169] An anti-CAPRIN-1 monoclonal antibody described in WO2015 / 020212, comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO:47 and the amino acid sequence of a light chain variable region represented by SEQ ID NO:51, 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.

[0170] An anti-CAPRIN-1 monoclonal antibody described in WO2011 / 096519, comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO:55 and the amino acid sequence of a light chain variable region represented by SEQ ID NO:59, 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.

[0171] An anti-CAPRIN-1 monoclonal antibody described in WO2013 / 125654, comprising the amino acid sequence of a heavy chain variable region represented by SEQ ID NO: 63 and the amino acid sequence of a light chain variable region represented by SEQ ID NO: 67, 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.

[0172] An anti-CAPRIN-1 monoclonal antibody 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.

[0173] Anti-CAPRIN-1 monoclonal antibodies are disclosed in WO2011 / 096528, including an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 70 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 71; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 72 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 73; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 74 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 75; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 76 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 77; and an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 78 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 79.

[0174] An anti-CAPRIN-1 monoclonal antibody described in WO2011 / 096533, comprising an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 80 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 81; and an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 82 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 83.

[0175] An anti-CAPRIN-1 monoclonal antibody described in WO2011 / 096534, comprising an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 84 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 85; and an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 86 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 87.

[0176] Anti-CAPRIN-1 monoclonal antibodies described in WO2010 / 016526 include an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 88 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 89; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 90 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 91; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 92 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 93; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 94; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 96 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 97; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 98 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 99; and an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 100 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 101.

[0177] An anti-CAPRIN-1 monoclonal antibody described in WO2013 / 018894, comprising an antibody having a heavy chain variable region with the amino acid sequence of SEQ ID NO: 102 and a light chain variable region with the amino acid sequence of SEQ ID NO: 103; and an antibody having a heavy chain variable region with the amino acid sequence of SEQ ID NO: 104 and a light chain variable region with the amino acid sequence of SEQ ID NO: 105.

[0178] An anti-CAPRIN-1 monoclonal antibody 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.

[0179] An anti-CAPRIN-1 monoclonal antibody 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.

[0180] An anti-CAPRIN-1 monoclonal antibody 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.

[0181] An anti-CAPRIN-1 monoclonal antibody 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.

[0182] An anti-CAPRIN-1 monoclonal antibody 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.

[0183] An anti-CAPRIN-1 monoclonal antibody described in WO2013 / 125654, comprising an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 116 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 117; and an antibody having a heavy chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 118 and a light chain variable region with an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 119.

[0184] An anti-CAPRIN-1 monoclonal antibody 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.

[0185] an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 122 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 123; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 124 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 125; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 126 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 127; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 128 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 129; an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 130 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 131; and an antibody comprising the amino acid sequence of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 132 and the amino acid sequence of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 133.

[0186] For the above-mentioned anti-CAPRIN-1 monoclonal antibodies, a nucleotide sequence was designed so that a heavy chain variable region in which CDR1 to CDR3 consist of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and in which the framework regions comprise a human antibody sequence, could be expressed, and inserted into a mammalian expression vector into which the heavy chain constant region of human IgG1 had been inserted. Similarly, a nucleotide sequence was designed so that a light chain variable region in which CDR1 to CDR3 consist of SEQ ID NOs: 40, 41, and 42, respectively, and in which the framework regions comprise a human antibody sequence, could be expressed, and inserted into a mammalian expression vector into which the light chain constant region of human IgG1 had been inserted. The above two recombinant expression vectors were introduced into mammalian cells according to a standard method to obtain a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #1 (humanized antibody #1), in which CDRs 1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and CDRs 1 to 3 of the light chain variable region consist of the amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively.

[0187] Similarly, a nucleotide sequence was designed to enable expression of a heavy chain variable region represented by SEQ ID NO: 47, in which CDRs 1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively, and in which the framework regions comprise the sequence of a human antibody, and this was inserted into a mammalian expression vector into which the heavy chain constant region of human IgG1 had been inserted. Similarly, a base sequence was designed to enable expression of a heavy chain variable region represented by SEQ ID NO:51, in which CDR1 to 3 of the light chain variable region consist of the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively, and the framework regions comprise the sequence of a human antibody. This base sequence was inserted into a mammalian expression vector into which the heavy chain constant region of human IgG1 had been inserted, and the two recombinant expression vectors were introduced into mammalian cells according to standard methods to obtain a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #2 (humanized antibody #2), in which CDR1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, and CDR1 to 3 of the light chain variable region consist of the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively.

[0188] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #3 (humanized antibody #3) was obtained, in which CDRs 1 to 3 of the heavy chain variable region consisted of the amino acid sequences of SEQ ID NOs: 52, 53, and 54, respectively, and CDRs 1 to 3 of the light chain variable region consisted of the amino acid sequences of SEQ ID NOs: 56, 57, and 58, respectively.

[0189] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #4 (humanized antibody #4) was obtained, in which CDRs 1 to 3 of the heavy chain variable region consisted of the amino acid sequences of SEQ ID NOs: 60, 61, and 62, respectively, and CDRs 1 to 3 of the light chain variable region consisted of the amino acid sequences of SEQ ID NOs: 64, 65, and 66, respectively.

[0190] Similarly, culture supernatants containing the following humanized anti-CAPRIN-1 monoclonal antibodies #9 to #41 (humanized antibodies #9 to #41) were obtained.

[0191] Humanized anti-CAPRIN-1 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.

[0192] Humanized anti-CAPRIN-1 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.

[0193] Humanized anti-CAPRIN-1 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.

[0194] Humanized anti-CAPRIN-1 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.

[0195] Humanized anti-CAPRIN-1 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.

[0196] Humanized anti-CAPRIN-1 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.

[0197] Humanized anti-CAPRIN-1 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.

[0198] Humanized anti-CAPRIN-1 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.

[0199] Humanized anti-CAPRIN-1 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.

[0200] Humanized anti-CAPRIN-1 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.

[0201] Humanized anti-CAPRIN-1 monoclonal antibody #19 (humanized antibody #19) 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.

[0202] Humanized anti-CAPRIN-1 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.

[0203] Humanized anti-CAPRIN-1 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.

[0204] Humanized anti-CAPRIN-1 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.

[0205] Humanized anti-CAPRIN-1 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.

[0206] Humanized anti-CAPRIN-1 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.

[0207] Humanized anti-CAPRIN-1 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.

[0208] Humanized anti-CAPRIN-1 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.

[0209] Humanized anti-CAPRIN-1 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.

[0210] Humanized anti-CAPRIN-1 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.

[0211] Humanized anti-CAPRIN-1 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.

[0212] Humanized anti-CAPRIN-1 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.

[0213] Humanized anti-CAPRIN-1 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.

[0214] Humanized anti-CAPRIN-1 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.

[0215] Humanized anti-CAPRIN-1 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.

[0216] Humanized anti-CAPRIN-1 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.

[0217] Humanized anti-CAPRIN-1 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.

[0218] Humanized anti-CAPRIN-1 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.

[0219] Humanized anti-CAPRIN-1 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.

[0220] Humanized anti-CAPRIN-1 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.

[0221] Humanized anti-CAPRIN-1 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.

[0222] Humanized anti-CAPRIN-1 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.

[0223] Humanized anti-CAPRIN-1 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.

[0224] Furthermore, for humanized antibody #1 of the above anti-CAPRIN-1 monoclonal antibodies, a nucleotide sequence was designed to enable expression of a heavy chain variable region in which CDRs 1 to 3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively, and in which the framework regions comprise the sequence of a human antibody, and this was inserted into a mammalian expression vector containing the heavy chain constant region of human IgG1 in which serine (Ser), amino acid 239 (EU numbering), has been substituted with aspartic acid (Asp) and isoleucine (Ile), amino acid 332 (EU numbering), has been substituted with glutamic acid (Glu). Furthermore, a nucleotide sequence was designed to enable expression of a light chain variable region in which CDRs 1 to 3 of the light chain variable region consist of SEQ ID NOs: 40, 41, and 42, respectively, and in which the framework regions comprise the sequence of a human antibody, and this was inserted into a mammalian expression vector containing the light chain constant region of human IgG1. The two recombinant expression vectors were introduced into mammalian cells according to standard methods to obtain a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #5 (humanized antibody #5), which consisted of the heavy chain full-length amino acid sequence consisting of the heavy chain variable region prepared above and a heavy chain constant region of human IgG1 in which serine (Ser), amino acid 239 (EU numbering), has been substituted with aspartic acid (Asp) and isoleucine (Ile), amino acid 332 (EU numbering), has been substituted with glutamic acid (Glu), and the light chain full-length amino acid sequence consisting of the light chain variable region and human light chain constant region prepared above.

[0225] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #6 (humanized antibody #6) consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of humanized antibody #2 prepared above was obtained.

[0226] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #7 (humanized antibody #7) consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the humanized antibody #3 prepared above was obtained.

[0227] Similarly, a culture supernatant containing humanized anti-CAPRIN-1 monoclonal antibody #8 (humanized antibody #8) consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the humanized antibody #4 prepared above was obtained.

[0228] Similarly, culture supernatants containing humanized anti-CAPRIN-1 antibodies #42 to #74 (humanized antibodies #42 to #74) each consisting of the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of each of the humanized antibodies #9 to #41 prepared above were obtained.

[0229] The culture supernatants containing the obtained humanized anti-CAPRIN-1 monoclonal antibodies #1 to #74 were purified using Hitrap Protein A Sepharose FF (GE Healthcare) according to standard methods, and then substituted with PBS(-) and filtered through a 0.22 μm filter (Millipore) to prepare samples.

[0230] Example 3 Preparation of Conjugates of Anti-CAPRIN-1 Antibodies and Thiol-Containing Maytansinoid DM4 Conjugates of the anti-CAPRIN-1 polyclonal antibodies #1 to #6 described in Example 1 and the thiol-containing maytansinoid DM4 were prepared. Anti-CAPRIN-1 polyclonal antibody #1 was dissolved in PBS(-) (pH 8.5) at a concentration of 4 mg / ml, and DMA was added to prepare a solution containing 10% DMA. A solution of SPDB-DM4 (CAS No. 1626359-62-3, MedChemExpress) dissolved in DMA to a concentration of 10 mM was added to the solution containing anti-CAPRIN-1 polyclonal antibody #1 in an amount of 6 molar equivalents, and the mixture was allowed to react for 3 hours at room temperature. After the reaction, the mixture was replaced with PBS (pH 7.4) using a Zeba™ Spin Desalting Column (MWCO 40 k) to remove free SPDB-DM4, thereby obtaining a solution containing the anti-CAPRIN-1 polyclonal antibody #1-SPDB-DM4 (conjugate 1) of the present invention.

[0231] Similarly, conjugates were prepared using SPDB-DM4 for anti-CAPRIN-1 polyclonal antibodies #2 to #6, and solutions containing the conjugates were obtained (the conjugate using anti-CAPRIN-1 polyclonal antibody #2: Conjugate 2, the conjugate using anti-CAPRIN-1 polyclonal antibody #3: Conjugate 3, the conjugate using anti-CAPRIN-1 polyclonal antibody #4: Conjugate 4, the conjugate using anti-CAPRIN-1 polyclonal antibody #5: Conjugate 5, and the conjugate using anti-CAPRIN-1 polyclonal antibody #6: Conjugate 6). Furthermore, using the same procedure as above, a solution containing a conjugate with SPDB-DM4 (control conjugate 1) was obtained in the same manner for the rabbit control antibody described in Example 1 that does not react with the CAPRIN-1 protein.

[0232] Furthermore, using the same method as above, a solution containing a conjugate with SPDB-DM4 (conjugate 10) was obtained using humanized antibody #4, which is an anti-CAPRIN-1 monoclonal antibody described in Example 2.

[0233] Furthermore, a conjugate (conjugate 7) of SPDB-DM4 with humanized antibody #1, the anti-CAPRIN-1 monoclonal antibody described in Example 2, was prepared according to a standard method. Solutions containing conjugates using SPDB-DM4 were also prepared for humanized antibodies #2, #3, and #5 to #74 (conjugate 8, conjugate 9, conjugate 11 to conjugate 80) in the same manner.

[0234] The number of DM4 molecules bound to each antibody molecule of the above conjugate was measured by mass spectrometry according to a standard method, and the average number was approximately 4 to 7.

[0235] Furthermore, a solution containing a conjugate of a human IgG control antibody that does not react with the CAPRIN-1 protein with SPDB-DM4 (control conjugate 2) was obtained in the same manner as above.

[0236] Example 4 Specific Reactivity of Conjugate with CAPRIN-1 Protein and CAPRIN-1-Expressing Cancer Cells The conjugate (anti-CAPRIN-1 antibody-SPDB-DM4 conjugate) prepared in Example 3 was evaluated for its specific reactivity with CAPRIN-1 protein and its reactivity on the cell membrane surface of human cancer cells and mouse cancer cells expressing CAPRIN-1 protein.

[0237] Specific reactivity to the CAPRIN-1 protein was confirmed using ELISA. 100 μL of a 1 μg / mL CAPRIN-1 protein solution was added per well of a 96-well plate and incubated at 4°C for 18 hours. After washing each well three times with PBS-T, 400 μL of 0.5% bovine serum albumin (BSA) solution was added per well and incubated at room temperature for 3 hours. The solution was removed, and the wells were washed three times with 400 μL of PBS-T. Then, 100 μL of each solution containing conjugates 1 to 6 and control conjugate 1 was added per well and incubated at room temperature for 2 hours. After washing each well three times with PBS-T, 100 μL of HRP-labeled anti-rabbit IgG antibody diluted 5000-fold with PBS was added per well and incubated at room temperature for 1 hour. After washing the wells three times with PBS-T, 100 μL of TMB substrate solution was added per well and the plate was left to stand for 15 to 30 minutes to allow the color reaction to occur. After color development, 100 μL of 1 N sulfuric acid was added per well to stop the reaction, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorbance meter. As a result, conjugates 1 to 6 had higher absorbance values ​​than the negative control, control conjugate 1, confirming that they react specifically with the CAPRIN-1 protein.

[0238] Similarly, specific reactions to the CAPRIN-1 protein were detected by ELISA using HRP-labeled anti-human antibodies for anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates 7 to 80) using humanized antibodies #1 to #74 prepared in Example 3. As a result, the absorbance values ​​were higher than those of human IgG (SIGMA) used as a negative control and control conjugate 2, confirming that the conjugates react specifically with the CAPRIN-1 protein.

[0239] Next, the reactivity of the CAPRIN-1 protein to the cell membrane surface of cancer cells expressing the protein on the cell surface was confirmed by flow cytometry. ５Human breast cancer cells BT-474 (obtained from ATCC) and mouse breast cancer cells 4T1 (obtained from ATCC) were centrifuged in 1.5 ml microcentrifuge tubes, and 100 μL of a solution containing conjugates 1 to 6 and control conjugate 1 was added to each tube and allowed to stand at 4°C for 1 hour. After washing with PBS, Alexa488-labeled anti-rabbit IgG (H+L) diluted 100-fold with PBS(-) containing 0.5% FBS (0.5% FBS-PBS(-)) was added and allowed to stand at 4°C for 1 hour. After washing with 0.5% FBS-PBS(-), the cells were reacted with BD Horizon Fixable Viability Stain (FVS) Reagents (FVS450, Becton, Dickinson and Company) to stain dead cells, and then the fluorescence intensity was measured using a FACSFortessa™ (Becton, Dickinson and Company). As a result, the anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates 1 to 6) had higher fluorescence intensity than the negative control, control conjugate 1, confirming that they strongly react with the cell surface of human breast cancer cell BT474 and mouse breast cancer cell 4T1, which express CAPRIN-1 protein on their cell surface.

[0240] Similarly, for anti-CAPRIN-1 antibody-SPDB-DM4 conjugates using humanized antibodies #1 to #74, specific reactivity with human cancer cell line BT474 and mouse cancer cell line 4T1, which express CAPRIN-1 protein on the cell membrane surface, was detected using Alexa488-labeled anti-human IgG (H+L) antibody. As a result, it was confirmed that anti-CAPRIN-1 antibody-SPDB-DM4 conjugates using humanized antibodies #1 to #74 (conjugates 7 to 80) had higher fluorescence intensity than the negative control control conjugate 2, i.e., they strongly reacted with the cell surface of human breast cancer cell line BT474 and mouse breast cancer cell line 4T1, which express CAPRIN-1 protein on the cell surface.

[0241] Similarly, the reactivity of anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates 7 to 80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 with various human cancer cells and mouse cancer cells was confirmed. These human cancer cells are confirmed to express the CAPRIN-1 gene and to express the CAPRIN-1 protein on the cell membrane surface of cancer cells, including breast cancer cells (BT-474, MDA-MB-453, HCC70, MCF7, MDA-MB-231, SKBR3, T47D), colon cancer cells (HT-29, HCT116, Lovo, SW480, DLD-1), and lung cancer cells. cells (A549, NCI-H322, NCI-H2228), gastric cancer cells (NCI-N87, NUGC3), uterine cancer cells (HEC-1-A), prostate cancer cells (22Rv1), pancreatic cancer cells (Panc10.5), liver cancer cells (HepG2), ovarian cancer cells (MCAS, SKOV3, TOV112D), kidney cancer cells (Caki-2), brain tumor cells (U-87MG), bladder cancer cells ( The anti-CAPRIN-1 antibody-SPDB-DM4 conjugate prepared in Example 3 was used on mouse ovarian cancer cells (T24, UMUC3, RT4, 5637), bile duct cancer cells (KKU213), fibrosarcoma cells (HT-1080), esophageal cancer cells (OE33), leukemia cells (OCI-AML5), lymphoma cells (Ramos), gallbladder cancer cells (TGBC14TKB), melanoma cells (Malme-3M), head and neck cancer cells (FaDu), mouse renal cancer cells (Renca) and mouse breast cancer cells (4T1) in which expression of the CAPRIN-1 gene has been confirmed. The fluorescence intensity of the anti-CAPRIN-1 antibody-SPDB-DM4 conjugate prepared in Example 3 was stronger than that of the negative controls, Control Conjugate 1 and Control Conjugate 2, and it was confirmed that it reacts strongly with the cell membrane surface of cancer cells in which CAPRIN-1 is expressed.

[0242] Example 5 Internalization of DM4 Conjugates The internalization of anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates 1 to 80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 into cancer cells was confirmed using a confocal laser microscope. The anti-CAPRIN-1 antibody-SPDB-DM4 conjugates were reacted at 100 μg / mL with cancer cells HT29 that had been cultured overnight in a culture plate on ice for 1 hour, and then incubated at 37°C and 5% CO for 1 to 6 hours. ２ The cells were reacted under the same conditions. After the reaction, the cells were washed with PBS and fixed at room temperature using PBS containing 4% paraformaldehyde. The fixed cells were then washed with PBS and then subjected to a cell membrane permeabilization treatment using a PBS solution containing 0.3% Triton-X. After permeabilization, the cells were washed with PBS and subjected to a blocking treatment using a PBS solution containing 5% BSA. After blocking, the cells were reacted with an Alexa488-labeled anti-rabbit IgG secondary antibody or anti-human IgG secondary antibody (A11013, Invitrogen) at a concentration of 10 μg / ml for 1 hour at room temperature. After the secondary antibody reaction, the above samples were washed with PBS and then internalization of the conjugate of the present invention into cancer cells was observed using a confocal laser microscope (Nicon A1R inverted microscopy). As a result of the observation, it was shown that the anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates #1 to #80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 were clearly present intracellularly, and that the conjugates of the present invention were internalized. On the other hand, no internalization into cancer cells was observed for the control conjugates 1 and 2 prepared in Example 3.

[0243] (Example 6) Antitumor Activity of DM4 Conjugates The antitumor activity of anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates #1 to #80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 against cancer cells was evaluated in vitro. In Example 4, human cancer cells in which CAPRIN-1 expression on the cell membrane surface of cancer cells was confirmed, including breast cancer cells (BT-474), colon cancer cells (HT-29), lung cancer cells (NCI-H322, NCI-H2228), uterine cancer cells (Ishikawa, HEC-1-A), pancreatic cancer cells (Panc10.5), ovarian cancer cells (MCAS, TOV112D), and head and neck cancer cells, were evaluated. The antitumor activity against FaDu, gastric cancer (NU-GC-3), bladder cancer (UMUC3), prostate cancer cells (22Rv1), liver cancer cells (HepG2), renal cancer cells (Caki-2), brain tumor cells (U-87MG), cholangiocarcinoma cells (KKU213), fibrosarcoma cells (HT-1080), gallbladder cancer cells (TGBC14TKB), and melanoma cells (Malme-3M) was evaluated.

[0244] The cancer cells were cultured by a standard method and seeded into a black 96-well plate suitable for cell culture (96 well optical Black Plate with Lid, Cell Culture Sterile, PS 165305, Thermo Corp.) at 1,000 to 5,000 cells per well, at 90 μL / well. The cells were then incubated at 5% CO ２ The cells were cultured in an incubator at 37°C for 18 to 24 hours. －２ μg / mL to 10 ２ The anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates #1 to #80) prepared in Example 3 using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 were added at 10 μL per well, and the plates were incubated in 5% CO ２The plates were incubated in an incubator at 37°C. Three to four days after the start of incubation, cell viability was measured using a Cell Titer Glow Luminescent Cell Viability Assay (#7573, Promega). 100 μL of substrate was added per well, and the plates were shaken for 2 minutes using a plate shaker. After allowing to stand for 10 minutes, the luminescence signal intensity was measured using a SpectraMax® iD3 (MOLECULAR DEVICE) to calculate the ATP content of surviving cells.

[0245] As a result, when the anti-CAPRIN-1 antibody-SPDB-DM4 conjugates (conjugates #1 to #80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 described in the present invention were used, the number of surviving cancer cells in a well to which no conjugate was added was taken as 100%, and at a concentration range of 1 μg / mL or more, the number of surviving cancer cells was 70% or less. These results demonstrated that the anti-CAPRIN-1 antibody-SPDB-DM4 conjugates exhibit antitumor activity against cancer cells.

[0246] Example 7 Antitumor Effect of DM4 Conjugate in Tumor-Bearing Mice 1 Conjugate 1 and Conjugate 7 (both of which have approximately four DM4 molecules bound per antibody molecule), which are the anti-CAPRIN-1 antibody-SPDB-DM4 conjugates prepared in Example 3, were evaluated for their in vivo antitumor effect in tumor-bearing mice. The antitumor effect of the conjugate of the present invention was examined using NOD-SCID mice transplanted with human-derived cancer cells expressing the CAPRIN-1 protein.

[0247] Approximately 10 per fish ７ Human head and neck cancer cells, FaDu, were mixed with Matrigel (SIGMA) and subcutaneously implanted, and the average tumor volume was 250 mm ３Conjugate 7 and Conjugate 10 were each administered once at 10 mg / kg via the tail vein to tumor-bearing mice grown to ≥ 100 mg / kg. FaDu expresses CAPRIN-1 protein on the cell membrane surface, and as shown in Example 4, Conjugate 1 and Conjugate 7 each bind to FaDu.

[0248] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor). ２ As a result of the evaluation, on day 14 after the start of administration, when the tumor volume of the negative control was taken as 100%, the tumor volumes of the mice administered with Conjugate 1 and Conjugate 7 were 7 to less than 50%.

[0249] Similar antitumor effects were also obtained with the other anti-CAPRIN-1 antibody-SPDB-DM4 conjugates prepared in Example 3.

[0250] These results demonstrate that the anti-CAPRIN-1 antibody-SPDB-DM4 conjugate prepared in Example 3 exhibits a significantly stronger antitumor effect than the negative control (untreated control group).

[0251] Example 8 Antitumor Effect of DM4 Conjugate in Tumor-Bearing Mice 2 The antitumor effect of Conjugate 1 and Conjugate 7 (both of which have approximately four DM4 molecules bound per antibody molecule), which are the anti-CAPRIN-1 antibody-SPDB-DM4 conjugates prepared in Example 3, was evaluated in vivo in tumor-bearing mice. The antitumor effect of the conjugate of the present invention was examined using Balb / c nude mice transplanted with human-derived cancer cells expressing the CAPRIN-1 protein.

[0252] Human gastric cancer cells NUGC3 were mixed with Matrigel (SIGMA) and implanted subcutaneously, and the average tumor volume was 100 mm ３Conjugate 1 and Conjugate 7 were each administered at 10 mg / kg via the tail vein to tumor-bearing mice grown to or larger than 10 mg / kg. NUGC3 expresses CAPRIN-1 protein on the cell membrane surface, and as shown in Example 4, Conjugate 1 and Conjugate 7 each bound to NUGC3.

[0253] Furthermore, a conjugate of trastuzumab, an antibody other than an anti-CAPRIN-1 antibody, and SPDB-DM4 (a conjugate in which approximately four molecules of DM4 are bound per antibody molecule) was prepared according to the method described in Example 3, and the same amount was administered to the above-mentioned tumor-bearing mice as a comparative control. Note that HER2 protein, the target antigen of trastuzumab, is expressed on the cell membrane surface of NUGC3, and the trastuzumab-SPDB-DM4 conjugate showed reactivity with NUGC3.

[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 The evaluation results showed that, on and after day 20 after administration, when the tumor volume of the anti-CAPRIN-1 antibody not conjugated with DM4 (control) was taken as 100%, the tumor volumes of the mice administered with Conjugate 1 and Conjugate 7 showed complete regression (0%) to less than 50%.

[0255] On the other hand, the tumor volume of the mice administered with the trastuzumab-SPDB-DM4 conjugate was 80% or more, assuming that the tumor volume of the mice administered with trastuzumab not conjugated with DM4 was 100%.

[0256] The results of this evaluation showed that the anti-CAPRIN-1 antibody-SPDB-DM4 conjugate had a significantly enhanced antitumor effect compared to the anti-CAPRIN-1 antibody not conjugated with DM4 (control), but the enhancement of the efficacy of the trastuzumab-DM4 conjugate over trastuzumab not conjugated with DM4 was limited.

[0257] Example 9 Preparation of Conjugates of Anti-CAPRIN-1 Antibodies and Thiol-Containing Maytansinoid DM1 Conjugates of the anti-CAPRIN-1 polyclonal antibodies #1 to #6 described in Example 1 and the thiol-containing maytansinoid DM1 were prepared. Anti-CAPRIN-1 polyclonal antibody #1 was dissolved in PBS(-) (pH 7.2) at a concentration of 2.5 mg / ml. SMCC-DM1 (CAS No. 1228105-51-8, MedChemExpress) solution dissolved in DMSO to a concentration of 1 mM was added to the above solution containing anti-CAPRIN-1 polyclonal antibody #1 and 19% (v / v) DMSO in an amount of 7.5 to 12 molar equivalents, and the mixture was allowed to react for 1 hour at 33°C. After the reaction, the solution was replaced with PBS (pH 7.2) using Illustra NAP-10 Columns (GE Healthcare, Cat. No. 17085402) to remove free SMCC-DM1, thereby obtaining a solution containing the anti-CAPRIN-1 polyclonal antibody #1-SMCC-DM1 (DM1 conjugate 1) of the present invention.

[0258] Similarly, conjugates were prepared using SMCC-DM1 for anti-CAPRIN-1 polyclonal antibodies #2 to #6, and solutions containing DM1 conjugates were obtained (the conjugate using anti-CAPRIN-1 polyclonal antibody #2: DM1 conjugate 2, the conjugate using anti-CAPRIN-1 polyclonal antibody #3: DM1 conjugate 3, the conjugate using anti-CAPRIN-1 polyclonal antibody #4: DM1 conjugate 4, the conjugate using anti-CAPRIN-1 polyclonal antibody #5: DM1 conjugate 5, and the conjugate using anti-CAPRIN-1 polyclonal antibody #6: DM1 conjugate 6). Furthermore, using the same procedure as above, a solution containing a conjugate of SMCC-DM1 with the rabbit control antibody described in Example 1 that does not react with the CAPRIN-1 protein (DM1 control conjugate 1) was obtained in the same manner.

[0259] Furthermore, using a method similar to that described above, a solution containing a conjugate with SMCC-DM1 (DM1 conjugate 10) was obtained using humanized antibody #4, an anti-CAPRIN-1 monoclonal antibody described in Example 2.

[0260] Furthermore, a conjugate (DM1 conjugate 7) of SMCC-DM1 with humanized antibody #1, the anti-CAPRIN-1 monoclonal antibody described in Example 2, was prepared according to a standard method. Solutions containing conjugates using SMCC-DM1 were also prepared for humanized antibodies #2, #3, and #5 to #74 in the same manner (DM1 conjugate 8, DM1 conjugate 9, DM1 conjugate 11 to DM1 conjugate 80).

[0261] The number of DM1 molecules bound to each antibody molecule of the above conjugate was measured by mass spectrometry according to a standard method, and the average number was approximately 4 to 6.

[0262] Furthermore, a solution containing a conjugate of a human IgG control antibody that does not react with the CAPRIN-1 protein with SMCC-DM1 (DM1 control conjugate 2) was obtained in the same manner as above.

[0263] (Example 10) Specific reactivity of DM1 conjugate with CAPRIN-1 protein and CAPRIN-1-expressing cancer cells The DM1 conjugate (anti-CAPRIN-1 antibody-SMCC-DM1 conjugate) prepared in Example 9 was evaluated for its specific reactivity with CAPRIN-1 protein and its reactivity on the cell membrane surface of human cancer cells and mouse cancer cells expressing CAPRIN-1 protein.

[0264] Specific reactivity to the CAPRIN-1 protein was confirmed using ELISA. 100 μL of a 1 μg / mL CAPRIN-1 protein solution was added per well of a 96-well plate and incubated at 4°C for 18 hours. After washing each well three times with PBS-T, 400 μL of 0.5% bovine serum albumin (BSA) solution was added per well and incubated at room temperature for 3 hours. The solution was removed, and the wells were washed three times with 400 μL of PBS-T. Then, 100 μL of each solution containing conjugates 1 to 6 and control conjugate 1 was added per well and incubated at room temperature for 2 hours. After washing each well three times with PBS-T, 100 μL of HRP-labeled anti-rabbit IgG antibody diluted 5000-fold with PBS was added per well and incubated at room temperature for 1 hour. After washing the wells three times with PBS-T, 100 μL of TMB substrate solution was added per well and the plate was left to stand for 15 to 30 minutes to allow the color reaction to occur. After color development, 100 μL of 1 N sulfuric acid was added per well to stop the reaction, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorbance meter. As a result, conjugates 1 to 6 had higher absorbance values ​​than the negative control, control conjugate 1, confirming that they react specifically with the CAPRIN-1 protein.

[0265] Similarly, specific reaction with the CAPRIN-1 protein was detected by ELISA using an HRP-labeled anti-human antibody for anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (DM1 conjugates 7 to DM1 conjugates 80) prepared using humanized antibodies #1 to #74 prepared in Example 3. As a result, the absorbance values ​​were higher than those of human IgG (SIGMA) and DM1 control conjugate 2 used as negative controls, confirming that the conjugates react specifically with the CAPRIN-1 protein.

[0266] Next, the reactivity of the CAPRIN-1 protein to the cell membrane surface of cancer cells expressing the protein on the cell surface was confirmed by flow cytometry. ５Human breast cancer cells BT-474 (obtained from ATCC) and mouse breast cancer cells 4T1 (obtained from ATCC) were centrifuged in 1.5 ml microcentrifuge tubes, and 100 μL of a solution containing DM1 conjugates 1 to 6 and DM1 control conjugate 1 was added to each tube and allowed to stand at 4°C for 1 hour. After washing with PBS, Alexa488-labeled anti-rabbit IgG (H+L) diluted 100-fold with PBS(-) containing 0.5% FBS (0.5% FBS-PBS(-)) was added and allowed to stand at 4°C for 1 hour. After washing with 0.5% FBS-PBS(-), the cells were reacted with BD Horizon Fixable Viability Stain (FVS) Reagents (FVS450, Becton, Dickinson and Company) to stain dead cells, and then the fluorescence intensity was measured using a FACSFortessa™ (Becton, Dickinson and Company). As a result, the anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (conjugates 1 to 6) had higher fluorescence intensity than the negative control, control conjugate 1, confirming that they strongly react with the cell surface of human breast cancer cell BT474 and mouse breast cancer cell 4T1, which express CAPRIN-1 protein on their cell surface.

[0267] Similarly, anti-CAPRIN-1 antibody-SMCC-DM1 conjugates using humanized antibodies #1 to #74 were used to detect their specific reactivity with human cancer cell line BT474 and mouse cancer cell line 4T1, which express CAPRIN-1 protein on their cell membrane surface, using Alexa488-labeled anti-human IgG (H+L) antibody. As a result, the anti-CAPRIN-1 antibody-SMCC-DM1 conjugates using humanized antibodies #1 to #74 (DM1 Conjugate 7 to DM1 Conjugate 80) showed higher fluorescence intensity than the negative control DM1 Control Conjugate 2, confirming that they strongly react with the cell surface of human breast cancer cell line BT474 and mouse breast cancer cell line 4T1, which express CAPRIN-1 protein on their cell surface.

[0268] Similarly, the reactivity of anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (DM1 conjugate 7 to DM1 conjugate 80) prepared using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 with various human and mouse cancer cells was confirmed. These human cancer cells are confirmed to express the CAPRIN-1 gene and to express the CAPRIN-1 protein on the cell membrane surface of cancer cells, including breast cancer cells (BT-474, MDA-MB-453, HCC70, MCF7, MDA-MB-231, SKBR3, T47D), colon cancer cells (HT-29, HCT116, Lovo, SW480, DLD-1), and lung cancer cells. (A549, NCI-H322, NCI-H2228), gastric cancer cells (NCI-N87, NUGC3), uterine cancer cells (HEC-1-A), prostate cancer cells (22Rv1), pancreatic cancer cells (Pa nc10.5), liver cancer cells (HepG2), ovarian cancer cells (MCAS, SKOV3, TOV112D), kidney cancer cells (Caki-2), brain tumor cells (U-87MG), bladder cancer cells (T24) , UMUC3, RT4, 5637), bile duct cancer cells (KKU213), fibrosarcoma cells (HT-1080), esophageal cancer cells (OE33), leukemia cells (OCI-AML5), lymphoma cells (Ramos), gallbladder cancer cells (TGBC14TKB), melanoma cells (Malme-3M), head and neck cancer cells (FaDu), mouse renal cancer cells (Renca), and mouse breast cancer cells (4T1) in which expression of the CAPRIN-1 gene has been confirmed. The anti-CAPRIN-1 antibody-SMCC-DM1 conjugate prepared in Example 3 had stronger fluorescence intensity than the negative controls DM1 control conjugate 1 and DM1 control conjugate 2, and was confirmed to react strongly with the cell membrane surface of cancer cells in which CAPRIN-1 is expressed.

[0269] Example 5 Internalization of DM1 Conjugates The internalization of anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (DM1 conjugate 1 to DM1 conjugate 80) into cancer cells using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 was confirmed using a confocal laser microscope. The anti-CAPRIN-1 antibody-SMCC-DM1 conjugates were reacted at 100 μg / mL with cancer cells HT29 that had been cultured overnight in a culture plate on ice for 1 hour, and then incubated at 37°C and 5% CO for 1 to 6 hours. ２ The cells were reacted under the same conditions. After the reaction, the cells were washed with PBS and fixed at room temperature using PBS containing 4% paraformaldehyde. The fixed cells were then washed with PBS and then subjected to a cell membrane permeabilization treatment using a PBS solution containing 0.3% Triton-X. After permeabilization, the cells were washed with PBS and subjected to a blocking treatment using a PBS solution containing 5% BSA. After blocking, the cells were reacted with an Alexa488-labeled anti-rabbit IgG secondary antibody or anti-human IgG secondary antibody (A11013, Invitrogen) at a concentration of 10 μg / ml for 1 hour at room temperature. After the secondary antibody reaction, the above samples were washed with PBS and then internalization of the conjugate of the present invention into cancer cells was observed using a confocal laser microscope (Nicon A1R inverted microscopy). As a result of the observation, it was shown that the anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (DM1 conjugates #1 to #80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 were clearly present intracellularly, indicating that the conjugates of the present invention were internalized. On the other hand, no internalization into cancer cells was observed for DM1 control conjugate 1 and DM1 control conjugate 2 prepared in Example 3.

[0270] (Example 6) Antitumor Activity of DM1 Conjugates The antitumor activity of anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (conjugates #1 to #80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 against cancer cells was evaluated in vitro. In Example 4, human cancer cells in which CAPRIN-1 expression on the cell membrane surface was confirmed were breast cancer cells (BT-474), colon cancer cells (HT-29), lung cancer cells (NCI-H322, NCI-H2228), uterine cancer cells (Ishikawa, HEC-1-A), pancreatic cancer cells (Panc10.5), ovarian cancer cells (MCAS, TOV112D), and head and neck cancer cells. The antitumor activity against FaDu, gastric cancer (NU-GC-3), bladder cancer (UMUC3), prostate cancer cells (22Rv1), liver cancer cells (HepG2), renal cancer cells (Caki-2), brain tumor cells (U-87MG), cholangiocarcinoma cells (KKU213), fibrosarcoma cells (HT-1080), gallbladder cancer cells (TGBC14TKB), and melanoma cells (Malme-3M) was evaluated.

[0271] The cancer cells were cultured by a standard method and seeded into a black 96-well plate suitable for cell culture (96 well optical Black Plate with Lid, Cell Culture Sterile, PS 165305, Thermo Corp.) at 1,000 to 5,000 cells per well, at 90 μL / well. The cells were then incubated at 5% CO ２ The cells were cultured in an incubator at 37°C for 18 to 24 hours. －２ μg / mL to 10 ２ The anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (DM1 conjugates #1 to #80) prepared in Example 3 were added at 10 μL per well, and the conjugates were incubated in a 5% CO atmosphere for 1 hour. ２The plates were incubated in an incubator at 37°C. Three to four days after the start of incubation, cell viability was measured using a Cell Titer Glow Luminescent Cell Viability Assay (#7573, Promega). 100 μL of substrate was added per well, and the plates were shaken for 2 minutes using a plate shaker. After allowing to stand for 10 minutes, the luminescence signal intensity was measured using a SpectraMax® iD3 (MOLECULAR DEVICE) to calculate the ATP content of surviving cells.

[0272] As a result, for anti-CAPRIN-1 antibody-SMCC-DM1 conjugates (DM1 conjugates #1 to #80) using the anti-CAPRIN-1 polyclonal antibody and humanized antibodies #1 to #74 prepared in Example 3 described in the present invention, the number of surviving cancer cells in a well to which no conjugate was added was 80% or less at a concentration range of 5 μg / mL or higher, assuming that the number of surviving cancer cells in the well to which no conjugate was added was 100%. These results demonstrate that the anti-CAPRIN-1 antibody-SMCC-DM1 conjugates exhibit antitumor activity against cancer cells.

[0273] Example 7 Antitumor Effect of DM1 Conjugate in Cancer-Bearing Mice The antitumor effect of DM1 Conjugate 1 and DM1 Conjugate 7 (both of which have approximately four DM1 molecules bound per antibody molecule), which are the anti-CAPRIN-1 antibody-SMCC-DM1 conjugates prepared in Example 3, was evaluated in vivo in cancer-bearing mice. The antitumor effect of the conjugate of the present invention was examined using NOD-SCID mice transplanted with human-derived cancer cells expressing the CAPRIN-1 protein.

[0274] Human colon cancer cells HT29 were mixed with Matrigel (SIGMA) and implanted subcutaneously, and the average tumor volume was 50 mm ３DM1 Conjugate 1 and DM1 Conjugate 7 were each administered at 10 mg / kg via the tail vein to tumor-bearing mice grown to HT29 or larger. HT29 expresses CAPRIN-1 protein on the cell membrane surface, and as shown in Example 4, DM1 Conjugate 1 and DM1 Conjugate 7 each bound to HT29.

[0275] Furthermore, a conjugate of trastuzumab, an antibody other than an anti-CAPRIN-1 antibody, with SMCC-DM1 (a conjugate in which approximately four molecules of DM1 are bound per antibody molecule) was prepared according to the method described in Example 3, and the same amount was administered to the above-mentioned tumor-bearing mice as a control. Note that HER2 protein, the target antigen of trastuzumab, is expressed on the cell membrane surface of HT29, and the trastuzumab-SMCC-DM1 conjugate showed reactivity with HT29.

[0276] After administration, the size of the tumor in the tumor-bearing mice was measured over time using a vernier caliper, and the tumor volume was calculated according to the standard method using the formula: (length of the longest axis of the tumor) x (length of the shortest axis of the tumor). ２ The evaluation results showed that on and after day 14 after administration, when the tumor volume of the anti-CAPRIN-1 antibody not conjugated with DM1 (control) was taken as 100%, the tumor volumes of the mice administered with DM1 Conjugate 1 and DM1 Conjugate 7 were less than 65%.

[0277] On the other hand, the tumor volume of the mice administered with the trastuzumab-SMCC-DM1 conjugate was 80% or more, assuming that the tumor volume of the mice administered with trastuzumab not conjugated with DM1 was 100%.

[0278] The results of this evaluation showed that the anti-CAPRIN-1 antibody-SMCC-DM1 conjugate had a significantly enhanced anti-tumor effect compared to an anti-CAPRIN-1 antibody not conjugated with DM1 (control), but that the enhancement of the efficacy of the trastuzumab-DM1 conjugate over trastuzumab not conjugated with DM1 was limited.

Claims

1. A conjugate comprising a thiol-containing maytansinoid bound to an antibody or fragment thereof that is 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 that has 80% or more sequence identity with said amino acid sequence.

2. The conjugate according to claim 1, wherein the antibody or 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 fragment thereof is any one of the following (A) to (M): (A) an antibody or fragment 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; or (B) an antibody or fragment 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 the CAPRIN-1 protein. (D) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 60, 61, and 62 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 64, 65, and 66 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with the CAPRIN-1 protein. (E) An antibody or fragment thereof comprising a heavy chain variable region comprising the complementarity determining regions of SEQ ID NOs: 170, 171, and 172 (CDR1, CDR2, and CDR3, respectively) and a light chain variable region comprising the complementarity determining regions of SEQ ID NOs: 173, 174, and 175 (CDR1, CDR2, and CDR3, respectively), and having immunological reactivity with 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, and a light chain variable region containing the complementarity determining regions (CDR1, CDR2 and CDR3) of SEQ ID NOs: 137, 138 and 139, and having immunological reactivity with a CAPRIN-1 protein.

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

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

7. The conjugate of claim 1, wherein the antibody or fragment thereof and the thiol-containing maytansinoid are linked via a linker.

8. The conjugate of claim 1, wherein the thiol-containing maytansinoid is DM1, DM4, or a derivative thereof.

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

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

11. The pharmaceutical composition of claim 10, 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.

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