Anti-CADM1 antibodies

Abstract

The purpose of the present invention is to provide an antibody that binds to CADM1 expressed on a cell and that can singly induce intracellular internalization thereof. Specifically, the present invention pertains to an antibody or an antigen-binding fragment thereof that binds to Cell adhesion molecule 1 (CADM1) and that induces, by binding to CADM1 on a cell surface, intracellular internalization thereof. Representative examples of the antibody include an antibody having a heavy chain CDR1 including an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 including an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 including an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 including an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 including an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 including an amino acid sequence represented by SEQ ID NO: 6.

Description

[Technical Field]

[0001] The present invention relates to anti-CADM1 antibodies, more specifically to anti-CADM1 antibodies and fragments thereof that bind to CADM1 on the cell surface and are internalized into cells, and uses thereof. [Background technology]

[0002] CADM1 / TSLC1 (Cell adhesion molecule 1) is a molecule identified as a tumor suppressor gene in lung cancer and belongs to the immunoglobulin superfamily of cell adhesion molecules (Non-patent Document 1). CADM1 expression is downregulated in epithelial cell-derived cancers such as non-small cell lung cancer, breast cancer, liver cancer, and pancreatic cancer. On the other hand, it is known that ectopically high expression of ATLL is observed in adult T-cell leukemia / lymphoma (ATLL) (Non-Patent Documents 2 and 3). ATLL is an intractable peripheral T-cell tumor caused by infection with HTLV-1 (human T-cell leukemia virus type 1), but many details of its onset mechanism remain unknown, and the prognosis is extremely poor.

[0003] CADM1, which is highly expressed in ATLL, is expected to be a target not only for rapid diagnosis of ATLL but also for treatment using drug delivery systems (DDS). Therefore, efforts have been made to develop antibodies that recognize CADM1. For example, Furuno et al. generated a chicken IgY antibody (9D2 clone) against synaptic cell adhesion molecule (SynCAM) (CADM1) and reported that this antibody binds to CADM1 on the surface of mast cells and inhibits CADM1 homotypic binding (Non-Patent Document 4). Patent Document 1 also disclosed antibodies that specifically recognize IgSF4 / TSLC1 / CADM1 expressed on ATLL cells and are thought to be suitable for the diagnosis of ATLL. These antibodies can be used for the diagnosis of ATLL because they can bind to CADM1 on cells, but it is unclear whether they can be used for treatment with DSS, for example, as an antibody-drug conjugate (ADC). Antibodies suitable for use as ADCs must be capable of binding to antigens on the cell surface and subsequently being internalized into the cell.

[0004] Patent Document 2 discloses an antibody (anti-CADM1 human IgG) that binds to CADM1 on cancer cells and induces ADCC (antibody-dependent cellular cytotoxicity). Patent Document 2 shows that cell death is induced when a saporin-conjugated anti-human IgG antibody (a conjugate of human IgG and saporin) is bound to the anti-CADM1 human IgG bound to CADM1 on cells. However, there is no mention of whether internalization can be induced by anti-CADM1 human IgG alone. As described above, none of the anti-CADM1 antibodies reported to date can induce intracellular internalization by themselves. Therefore, the development of antibodies that can be used for disease treatment using ADCs remains a challenge in this field. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2015-7030 [Patent Document 2] WO2010102175 [Non-patent literature]

[0006] [Non-Patent Document 1] Kuromachi et al., Nat Genet. 27:427-430 2001. [Non-patent document 2] Sasaki et al., Blood. 105:1204-1213 2005. [Non-patent document 3] Nakahata et al., Leukemia. 26:1238-1246 2012. [Non-patent document 4] Furuno et al., J. Immunol. 174:6934-6942 2005. Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above circumstances, an object of the present invention is to develop an antibody that binds to CADM1 expressed on cells and that can induce internalization into cells by itself. [Means for solving the problem]

[0008] The inventors attempted to prepare monoclonal antibodies using four types of immunizing antigens, a mixture of monomeric CADM1 and Fc-fused CADM1 capable of forming dimers, and succeeded in preparing antibodies that exhibit the desired functions. That is, the present invention provides the following (1) to (11). (1) An antibody or an antigen-binding fragment thereof that binds to CADM1 (cell adhesion molecule 1), which binds to CADM1 on the cell surface and induces its internalization into the cell. (2) The antibody or antigen-binding fragment thereof according to (1) above, wherein the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (A) or (B) below: (A) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5; and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6; (B) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5; and It has a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:6. (3) The antibody or antigen-binding fragment thereof according to (2) above, which satisfies either (a) or (b) below: (a) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19; (b) A heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19. (4) The antibody or antigen-binding fragment thereof according to (2) above, wherein the CADM1 is dimerized on the cell surface. (5) An antibody or its antigen-binding fragment that binds to CADM1, characterized by binding to CADM1 on the cell surface and inducing its internalization into the cell, and competitively inhibits the binding of the antibody described in (2) above to CADM1. (6) The antibody or antigen-binding fragment thereof according to (2) above, which is a humanized or chimeric antibody. (7) The antibody or antigen-binding fragment thereof according to (2) above, characterized in that it is a human antibody. (8) The antibody or antigen-binding fragment thereof according to (2) above, characterized in that it is bound to a substance having antitumor activity. (9) The antigen-binding fragment according to (2) above, which is Fab, Fab', F(ab')2, Fv, a single-chain antibody, scFv, an scFv dimer, or a dsFv. (10) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of (1) to (9) above. (11) The pharmaceutical composition according to (10) above, wherein the disease to be treated is adult T-cell leukemia / lymphoma. In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of it. [Effects of the Invention]

[0009] The antibodies of the present invention have ADCC activity. Furthermore, since the antibodies of the present invention can bind to CADM1 on cells and induce its internalization into the cells, the antibodies are expected to function as ADC antibodies. For these reasons, the antibodies of the present invention may be effective as therapeutic agents for ATLL and other diseases. [Brief explanation of the drawings]

[0010] [Figure 1] Analysis of CADM1 variants in ATLL. a) Primer locations used to amplify the CADM1 gene region using the 5'RACE method. b) Schematic diagram of the gene structure of CADM1 isoform 3 (full length), exon 10 deletion variant (Δ10), and exon 9-10 deletion variant (Δ9-10). TM: transmembrane domain. [Figure 2] Four types of immunizing antigens used to generate anti-CADM1 antibodies. The structures of the CADM1 extracellular domain Δ10 variant (ecΔ10), Δ9-10 variant (ecΔ9-10), Δ10 variant dimerized via human Fc, and Δ9-10 variant dimerized via human Fc are shown schematically. [Figure 3] Cloning of the variable region of the rat anti-CADM1 antibody (YTH-W-2C2). (a) shows the positions of the primers used to amplify the coding regions of the heavy and light chains of the YTH-W-2C2 antibody by 5' RACE. (b) shows the results of agarose gel electrophoresis of the PCR amplification products. [Figure 4] Expression and purification of recombinant antibodies. (a) Schematic diagram of the antibody light chain and heavy chain expression construct structures. (b) SDS-PAGE profiles of affinity-purified YTH-W-2C2 rat antibody and YTH-W-2C2 chimeric antibody. (c) Absorbance (left) and SDS-PAGE (right) profiles of size-exclusion purified YTH-W-2C2 rat antibody and YTH-W-2C2 chimeric antibody. [Figure 5] 1 shows the results of flow cytometric analysis of the binding of an antibody according to an embodiment of the present invention to a human T cell line. [Figure 6] 1 shows the results of analyzing the binding ability of an antibody according to an embodiment of the present invention to cells using a flow cytometer after labeling the antibody with biotin. [Figure 7] 1 shows the results of directly labeling an antibody according to an embodiment of the present invention with Alexa488 and then analyzing its binding to cells using a flow cytometer. [Figure 8] 1 shows the results of examining the internalization of a complex between an antibody according to an embodiment of the present invention (Rat-IgG; YTH-W-2C2 rat antibody) and CADM1 into cells, including the results of flow cytometry analysis and fluorescence microscopic observation of cells. [Figure 9]1 shows the results of examining the internalization of a complex between an antibody according to an embodiment of the present invention (human-Fc-Rat-IgG; YTH-W-2C2 chimeric antibody) and CADM1 into cells, including the results of flow cytometry analysis and fluorescence microscopic observation of cells. [Figure 10] The results of observing the cells subjected to the fluorescence microscope observation in FIG. 9 with a confocal laser microscope are shown. [Figure 11] 1 shows the results of investigation into the production of ADCs using an antibody according to an embodiment of the present invention (Human-Fc-Rat-IgG; YTH-W-2C2 chimeric antibody). [Figure 12] FIG. 1 shows the results of flow cytometric analysis of the effect of an ADC using an antibody according to an embodiment of the present invention (Human-Fc-Rat-IgG; YTH-W-2C2 chimeric antibody) on ATL cells derived from chronic ATL patients. [Figure 13] 1 shows the results of examining the ADCC effect using an antibody according to an embodiment of the present invention (YTH-W-2C2 rat antibody). [Figure 14] 1 shows the results of examining the ADCC effect of an antibody according to an embodiment of the present invention (YTH-W-2C2 chimeric antibody). DETAILED DESCRIPTION OF THE INVENTION

[0011] A first embodiment of the present invention is an antibody that binds to CADM1 (cell adhesion molecule 1), particularly human CADM1, and that binds to CADM1 on the cell surface (particularly dimerized CADM1 on the cell surface) and induces its internalization into the cell (hereinafter also referred to as the "anti-CADM1 antibody according to this embodiment") or an antigen-binding fragment thereof. The anti-CADM1 antibody according to this embodiment can be prepared, for example, as follows, without any particular limitation. A mixture of four types of immunogens, consisting of monomeric CADM1 and Fc-fused CADM1 capable of forming a dimer, can be used. Specifically, the immunogen can be a mixture of four proteins: the Δ9-10 variant of the CADM1 extracellular domain, the Δ10 variant of the CADM1 extracellular domain, the Δ9-10 Fc variant in which the CADM1 extracellular domain is fused with human Fc, and the Δ10 Fc variant in which the CADM1 extracellular domain is fused with human Fc. For details, see the Examples.

[0012] The term "antibody" as used herein is not particularly limited in terms of its preparation method or structure, and includes all "antibodies" that bind to a desired antigen with desired properties, such as monoclonal antibodies, polyclonal antibodies, or nanoantibodies. When the anti-CADM1 antibody according to this embodiment is a polyclonal antibody, it can be prepared, for example, by injecting a mixture of antigen and adjuvant into an animal to be immunized (e.g., but not limited to, rabbit, goat, sheep, chicken, guinea pig, mouse, rat, or pig). Typically, the antigen and / or adjuvant are injected subcutaneously or intraperitoneally into the animal multiple times. Adjuvants include, but are not limited to, Freund's complete and monophosphoryl lipid A synthetic-trehalose dicorynomycolate (MPL-TMD). After immunization with the antigen, the anti-CADM1 antibody can be purified from the serum derived from the immunized animal by a standard method (e.g., a method using Sepharose loaded with Protein A).

[0013] Furthermore, when the anti-CADM1 antibody according to this embodiment is a monoclonal antibody, it can be produced, for example, as follows: In this specification, the term "monoclonal" refers to the characteristics of an antibody obtained from a substantially homogeneous antibody population (a population of antibodies in which the heavy and light chain amino acid sequences constituting the antibody are identical), and is not to be interpreted restrictively as meaning that the antibody is produced by a specific method (e.g., hybridoma method, etc.). Examples of methods for producing monoclonal antibodies include the hybridoma method (Kohler and Milstein, Nature 256:495-497, 1975) and recombinant methods (U.S. Patent No. 4,816,567). Alternatively, the anti-CADM1 antibody of this embodiment may be isolated from a phage antibody library (e.g., Clackson et al., Nature 352:624-628, 1991; Marks et al., J. Mol. Biol. 222:581-597, 1991). More specifically, when preparing the antibody using the hybridoma method, the preparation method includes, for example, the following four steps: (i) immunizing an animal with an antigen, (ii) recovering lymphocytes secreting (or potentially secreting) a monoclonal antibody, (iii) fusing the lymphocytes with immortalized cells, and (iv) selecting cells secreting the desired monoclonal antibody. Immunized animals can include, for example, mice, rats, guinea pigs, hamsters, and rabbits. After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusing agent such as polyethylene glycol or electrofusion to establish hybridoma cells. For example, rat or mouse myeloma cell lines are used as fused cells. After cell fusion, the cells are grown in an appropriate medium containing a substrate that inhibits the growth or survival of unfused lymphocytes and the immortalized cell line. A common technique involves using parent cells lacking the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, aminopterin is added to the medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows hybridoma growth. Hybridomas producing the desired antibody are selected from the hybridomas thus obtained, and the desired monoclonal antibody can be isolated from the medium in which the selected hybridomas grow, using standard methods. The hybridomas thus prepared are cultured in vitro or in vivo in ascites of mice, rats, guinea pigs, hamsters, etc., and the desired antibody can be prepared from the culture supernatant or ascites.

[0014] Nanobodies are polypeptides consisting of the variable domain of the heavy chain of an antibody (VHH). While antibodies in humans and other animals are typically composed of heavy and light chains, camelids, such as llamas, alpacas, and camels, produce single-chain antibodies (heavy-chain antibodies) consisting only of heavy chains. Heavy-chain antibodies, like conventional antibodies composed of heavy and light chains, can recognize and bind to target antigens. The variable domain of a heavy-chain antibody is the smallest unit that has binding affinity to an antigen, and this variable domain fragment is called a "nanobody." Nanobodies are highly heat-resistant, digestion-resistant, and stable at room temperature, and can be easily produced in large quantities using genetic engineering techniques. Nanobodies can be produced, for example, as follows: Camelids are immunized with an antigen, and the presence or absence of the desired antibody is detected from the collected serum. cDNA is produced from RNA derived from peripheral blood lymphocytes of immunized animals in which the desired antibody titer is detected. DNA fragments encoding VHHs are amplified from the obtained cDNA and inserted into phagemids to prepare a VHH phagemid library. The VHH phagemid library thus produced can be subjected to several rounds of screening to produce the desired nanobodies.

[0015] The anti-CADM1 antibody according to this embodiment may be a recombinant antibody. Examples of recombinant antibodies include, but are not limited to, humanized antibodies and chimeric antibodies with a human antibody. Chimeric antibodies are antibodies in which variable and constant regions derived from different animal species are linked (e.g., antibodies in which the variable region of a rat-derived antibody is linked to a human-derived constant region) (e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6851-6855 1984), and can be easily constructed using recombinant techniques.

[0016] A humanized antibody is an antibody whose framework regions (FR) contain sequences derived from humans and whose complementarity-determining regions (CDRs) are derived from other animal species (e.g., mouse). Humanized antibodies can be produced by first grafting the CDRs from the variable regions of an antibody derived from another animal species (e.g., mouse) into the variable regions of a human antibody, reconstituting the heavy and light chain variable regions, and then linking these humanized reconstituted human antibody variable regions to a human antibody constant region. Methods for producing such humanized antibodies are well known in the art (e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86, 10029-10033, 1989).

[0017] An antigen-binding fragment of an antibody of the present invention is a partial region of the antibody of the present invention and refers to an antibody fragment that binds to human CADM1. Examples of the fragment include Fab, Fab', F(ab')2, Fv (variable fragment of antibody), single-chain antibodies (heavy chain, light chain, heavy chain variable region, light chain variable region, nanobody, etc.), scFv (single chain Fv), diabody (scFv dimer), dsFv (disulfide-stabilized Fv), and peptides that contain at least a portion of the CDR of the antibody of the present invention.

[0018] Fab is an antibody fragment that has antigen-binding activity and is obtained by treating an antibody molecule with the protease papain, in which approximately the N-terminal half of the heavy chain and the entire light chain are linked by a disulfide bond. Fab can be produced by treating an antibody molecule with papain to obtain a fragment, or by constructing an appropriate expression vector into which DNA encoding Fab is inserted, introducing this into appropriate host cells (e.g., mammalian cells such as CHO cells, yeast cells, insect cells, etc.), and then expressing Fab in the cells.

[0019] F(ab')2 is an antibody fragment that retains antigen-binding activity and is slightly larger than the fragments obtained by treating an antibody molecule with the protease pepsin, in which Fab is linked via a disulfide bond in the hinge region. In addition to obtaining fragments by treating an antibody molecule with pepsin, F(ab')2 can also be produced by bonding Fab via a thioether bond or disulfide bond, and can also be produced by genetic engineering techniques, similar to Fab.

[0020] Fab' is an antibody fragment having antigen-binding activity, obtained by cleaving the disulfide bond in the hinge region of the F(ab')2. Fab' can also be prepared by genetic engineering techniques, similar to Fab.

[0021] An scFv is an antibody fragment that has antigen-binding activity and is a VH-linker-VL or VL-linker-VH polypeptide in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker. scFv can be produced by genetic engineering techniques using cDNA encoding the heavy and light chain variable regions of an antibody.

[0022] A diabody is an antibody fragment formed by dimerization of scFv and has bivalent antigen-binding activity. The bivalent antigen-binding activity may be the same antigen-binding activity or may be different antigen-binding activities. Diabodies can be produced by genetic engineering techniques by obtaining cDNA encoding the heavy and light chain variable regions of an antibody and constructing cDNA encoding an scFv in which the heavy and light chain variable regions are linked via a peptide linker.

[0023] A dsFv is a polypeptide in which one amino acid residue in each of the heavy chain variable region and the light chain variable region is substituted with a cysteine ​​residue, and the cysteine ​​residues are linked via a disulfide bond. The amino acid residue to be substituted with a cysteine ​​residue can be selected based on the predicted three-dimensional structure of the antibody. A dsFv can be produced by genetic engineering techniques by obtaining cDNAs encoding the heavy chain variable region and the light chain variable region of an antibody and constructing DNA encoding the dsFv.

[0024] CDR-containing peptides are constructed to contain at least one region of the heavy or light chain CDRs (CDRs 1 to 3). Peptides containing multiple CDRs can be linked directly or via an appropriate peptide linker. CDR-containing peptides are prepared by constructing DNA encoding the CDRs of the heavy or light chain of an antibody and inserting it into an expression vector. The type of vector is not particularly limited and may be selected appropriately depending on the type of host cell into which it will be subsequently introduced. These vectors can be introduced into appropriate host cells (e.g., mammalian cells such as CHO cells, yeast cells, insect cells, etc.) to express them as antibodies and produce them. CDR-containing peptides can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

[0025] Human antibodies (fully human antibodies) generally have the same structures as human antibodies in the hypervariable region, which is the antigen-binding site of the V region, the remaining parts of the V region, and the constant region. Human antibodies can be easily produced by those skilled in the art using known techniques. Human antibodies can be obtained, for example, by a method using a human antibody-producing mouse carrying a human chromosome fragment containing human antibody H-chain and L-chain genes (see, for example, Tomizuka et al., Proc. Natl. Acad. Sci. USA, 97, 722-727, 2000), or by a method of obtaining human antibodies derived from phage display selected from a human antibody library (see, for example, Siriwardena et al., Ophthalmology, 109, 427-431, 2002).

[0026] Antigen-binding fragments of antibodies of the present invention can be used to construct multispecific antibodies. Multispecificity refers to having binding specificity for two or more antigens, and examples include monoclonal antibodies or proteins containing antigen-binding fragments that have binding specificity for two or more antigens. This can be achieved by those skilled in the art using known techniques. Several methods for constructing multispecific antibodies have been developed, including the construction of asymmetric IgGs in which two different antibody heavy chain molecules are engineered to form heterodimers, and the linking of low-molecular-weight antigen-binding fragments obtained from antibodies to each other or to other antibody molecules. Specific construction methods can be found in, for example, the following literature: Kontermann et al., Drug Discovery Today, 20, 838-847, 2015.

[0027] Examples of anti-CADM1 antibodies and antigen-binding fragments thereof according to this embodiment include antibodies and antigen-binding fragments thereof characterized in that the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (A) or (B) below. (A) The heavy chain CDR1 amino acid sequence is NYDIS (SEQ ID NO: 1), The heavy chain CDR2 amino acid sequence is YIHTGSGGTYYNEKFKG (SEQ ID NO: 2). The heavy chain CDR3 amino acid sequence is TPYVYYGSGYFDF (SEQ ID NO: 3). The light chain CDR1 amino acid sequence is KSSQSLLYSGNQKNYLA (SEQ ID NO: 4). the light chain CDR2 amino acid sequence is WASTRQS (SEQ ID NO: 5), and The light chain CDR3 amino acid sequence has QQYYDTPDT (SEQ ID NO: 6). (B) The heavy chain CDR1 amino acid sequence is GYTFSNY (SEQ ID NO: 7). The heavy chain CDR2 amino acid sequence is HTGSGG (SEQ ID NO: 8), The heavy chain CDR3 amino acid sequence is TPYVYYGSGYFDF (SEQ ID NO: 3). The light chain CDR1 amino acid sequence is KSSQSLLYSGNQKNYLA (SEQ ID NO: 4). the light chain CDR2 amino acid sequence is WASTRQS (SEQ ID NO: 5), and The light chain CDR3 amino acid sequence has QQYYDTPDT (SEQ ID NO: 6).

[0028] Furthermore, the anti-CADM1 antibodies and antigen-binding fragments thereof according to this embodiment include antibodies having either a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19, and antibodies comprising an amino acid sequence that shares at least about 70%, preferably at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, more preferably at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and most preferably at least about 99% amino acid sequence identity with the amino acid sequences of the heavy chain variable regions and / or light chain variable regions constituting these antibodies, which antibodies or antigen-binding fragments thereof bind to CADM1 on the cell surface and induce its internalization into cells.

[0029] A second embodiment of the present invention relates to an antibody that binds to CADM1, characterized by binding to CADM1 on the cell surface and inducing its internalization into cells. The antibody competitively inhibits the binding of the antibody according to the first embodiment (i.e., the anti-CADM1 antibody according to this embodiment) to CADM1 (hereinafter also referred to as the "competitive antibody according to this embodiment") or an antigen-binding fragment thereof. The competitive antibody according to this embodiment can be prepared and obtained by competition experiments well known to those skilled in the art. Specifically, when the binding of a first anti-CADM1 antibody (the antibody according to the first embodiment) to CADM1 is competitively inhibited by a second anti-CADM1 antibody, the first anti-CADM1 antibody and the second anti-CADM1 antibody are determined to bind to substantially the same or closely adjacent antigenic sites. Furthermore, when the second anti-CADM1 antibody binds to CADM1 on the cell surface and has the function of inducing its internalization into cells, the second anti-CADM1 antibody is a competitive antibody according to this embodiment. For example, methods using Fab fragments or the like are commonly used in the art for such competition experiments. See, for example, WO95 / 11317, WO94 / 07922, WO2003 / 064473, WO2008 / 118356 and WO2004 / 046733.

[0030] A third embodiment of the present invention is the antibody according to the first embodiment or the antibody according to the second embodiment, or an antigen-binding fragment thereof, to which a substance having antitumor activity, particularly preferably a substance having antitumor activity against adult T-cell leukemia-lymphoma, is bound. Antibody-targeted cancer therapy can be performed by conjugating an antibody with a substance having antitumor activity, such as a drug (hereinafter, such a conjugate is also referred to as an "antibody-drug conjugate"). In this case, the substance having antitumor activity includes, but is not limited to, cytotoxic drugs such as anticancer drugs, radioisotopes, and substances that indirectly induce antitumor activity by manipulating the immune system.

[0031] In the third embodiment, a drug exhibiting antitumor activity can be used, and such a conjugate is called an antibody-drug conjugate. Drugs with known antitumor activity include, but are not limited to, tubulin inhibitors and microtubule polymerization inhibitors such as auristatins (e.g., MMAE, MMAF), maytansines (e.g., DM1, DM4), tubulysins, cryptophycins, and rhizoxin; antibiotics such as calicheamicins, doxorubicin, and anthracyclines; DNA synthesis inhibitors such as duocarmycins, PBDs (benzodiazepines), and IGNs (indolinobenzodiazepines); topoisomerase I inhibitors such as camptothecin analogs (e.g., SN-38 and DXd); RNA polymerase II inhibitors such as amanitins; RNA spliceosome inhibitors and apoptosis-related protein inhibitors such as spliceostatins and thailanstatins (for details, see, for example, Yaghoubi et al., J Cell Physiol. 235:31-64 2020). doi:10.1002 / jcp.28967). Furthermore, compounds that become toxic when excited by light energy can also be used as drugs exhibiting antitumor activity. Such antibody-drug conjugates can be administered into the body, allowed to bind to tumor cells, and then used in a treatment called photoimmunotherapy (PIT) (Kobayashi et al., Int Immunol. 33:7-15 2021.), in which tumor cells are killed by external application of light energy such as near-infrared light. The anti-CADM1 antibody according to this embodiment may also be used as an antibody for photoimmunotherapy. Known compounds that can be used include, but are not limited to, IRDye 700DX.

[0032] Numerous chemical modification methods for attaching antitumor agents to antibodies have been proposed, including covalent bonding to the side chains of lysine residues and cysteine ​​residues, site-specific chemical modification of unnatural amino acids introduced into antibody peptide chains, site-specific enzymatic modification of specific amino acid sequences or modified sugar chains in antibodies, and enzyme-based modification for peptide linkage. Another common approach is to chemically modify drugs and use them as linkers for protein conjugation. Many types of chemical linkers are known, and their properties significantly affect the pharmacological activity of antibody-drug conjugates in vivo. For example, hydrazone linkers, valine-citrulline linkers, disulfide bond linkers, and pyrophosphate linkers can be cleaved by endogenous enzymes, allowing the drug to be separated from the antibody, resulting in the preparation of antibody-drug conjugates with high antitumor efficacy. However, chemical structures that cannot be cleaved in vivo are also commonly used as chemical linkers. An overview of the above methods is described in, for example, the following literature: Tsuchikama and An, Protein and Cell, 9, 33-46 2018.

[0033] A fourth embodiment of the present invention is a pharmaceutical composition for preventing or treating cancer, particularly preferably adult T-cell leukemia-lymphoma, comprising an antibody-drug conjugate or an antigen-binding fragment thereof according to the third embodiment (hereinafter also referred to as the "pharmaceutical composition according to this embodiment"). The pharmaceutical composition according to this embodiment may be administered in the form of a pharmaceutical composition containing one or more formulation additives in addition to the antibody-drug conjugate or its antigen-binding fragment as the active ingredient. Furthermore, the pharmaceutical composition according to this embodiment may also contain other known drugs.

[0034] The pharmaceutical composition according to this embodiment may be in an oral or parenteral dosage form, and examples thereof include, but are not limited to, tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, and injections. These preparations are prepared according to conventional methods. Liquid preparations may be dissolved or suspended in water or other suitable solvents before use. Tablets and granules may also be coated by known methods. Injections are prepared by dissolving the antibody or functional fragment thereof according to this embodiment in water, but may also be dissolved in physiological saline or glucose solution as needed, and buffers and preservatives may also be added.

[0035] The type of formulation additive used in the production of the pharmaceutical composition according to this embodiment, the ratio of the formulation additive to the active ingredient, or the method of producing the pharmaceutical composition can be appropriately selected by those skilled in the art depending on the form of the pharmaceutical composition. The formulation additive can be an inorganic or organic substance, or a solid or liquid substance, and can generally be blended in an amount of, for example, 0.1% to 99.9% by weight, 1% to 95.0% by weight, or 1% to 90.0% by weight relative to the weight of the active ingredient. Specific examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0036] To prepare solid dosage forms for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, or anhydrous silicic acid to form a powder, or, if necessary, with binders such as sucrose, hydroxypropyl cellulose, or polyvinylpyrrolidone, and disintegrants such as carboxymethylcellulose or calcium carboxymethylcellulose, followed by wet or dry granulation to form granules. To prepare tablets, these powders and granules can be compressed directly or with the addition of lubricants such as magnesium stearate or talc. These granules or tablets can also be coated with enteric-coated bases such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer to form enteric-coated formulations, or coated with ethylcellulose, carnauba wax, or hydrogenated oil to form sustained-release formulations. To prepare capsules, the powder or granules can be filled into hard capsules, or the active ingredient can be dissolved directly or in glycerin, polyethylene glycol, sesame oil, olive oil, or the like, and then coated with gelatin to form soft capsules.

[0037] To prepare an injection, the active ingredient is dissolved in distilled water for injection, if necessary, along with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate or sodium dihydrogen phosphate, and an isotonic agent such as sodium chloride or glucose, and the solution is sterile filtered and filled into ampoules. Alternatively, mannitol, dextrin, cyclodextrin, gelatin, or the like may be added and the mixture is vacuum freeze-dried to produce an injection that can be dissolved before use. Alternatively, the active ingredient can be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, or the like to produce an emulsion for injection.

[0038] To prepare a rectal preparation, the active ingredient may be dissolved by wetting together with a suppository base such as cacao butter, tri-, di-, or monoglycerides of fatty acids, or polyethylene glycol, and then poured into a mold and cooled; alternatively, the active ingredient may be dissolved in polyethylene glycol or soybean oil, etc., and then coated with a gelatin film or the like.

[0039] The dosage and frequency of administration of the pharmaceutical composition of this embodiment are not particularly limited, and can be appropriately selected at the discretion of a physician or pharmacist depending on conditions such as the purpose of preventing the worsening or progression of the disease to be treated and / or treating it, the type of disease, and the weight and age of the patient. Generally, the daily oral dose for adults is about 0.01 to 1,000 mg (weight of active ingredient), and can be administered once a day, in divided doses, or every few days. When used as an injection, it is desirable to administer 0.001 to 100 mg (weight of active ingredient) per day to adults continuously or intermittently.

[0040] Another example of the pharmaceutical composition according to this embodiment is a cytotoxic cell, such as a T cell, that expresses an antibody according to this embodiment or an antigen-binding fragment thereof on its cell surface. Chimeric antigen receptor-expressing T cell (CAR-T) therapy is a treatment method in which a fusion gene (chimeric antigen receptor gene) consisting of the antigen-binding site of an antibody and a portion of a T cell receptor is expressed in T cells, which are then transferred into the body of a cancer patient. The transferred T cells then specifically attack cancer cells, resulting in anti-tumor activity. A gene encoding the antibody according to this embodiment or its antigen-binding fragment is used as a component of the chimeric antigen receptor gene to construct expressing T cells, thereby enabling the construction of a CAR-T therapy that specifically attacks tumors expressing CADM1 molecules, such as adult T-cell leukemia-lymphoma. Additionally, the antibody according to this embodiment can also be used as a ligand for nanoparticles such as gold nanoparticles, or DDS carriers such as micelles and liposomes.

[0041] A fifth embodiment of the present invention is a method for preventing and / or treating cancer (e.g., adult T-cell leukemia / lymphoma, etc.), which comprises administering a pharmaceutical composition according to this embodiment to a patient (hereinafter also referred to as the "prophylactic or therapeutic method according to this embodiment"). Here, "treatment" means preventing or alleviating the progression and worsening of the disease in patients who already have cancer such as adult T-cell leukemia / lymphoma, and is a procedure aimed at preventing or alleviating the progression and worsening of cancer. "Prevention" refers to the prevention of cancer, such as adult T-cell leukemia / lymphoma, in those at risk of developing cancer that requires treatment, and is a treatment aimed at preventing the onset of cancer. Furthermore, "prevention" also includes treatment to prevent recurrence of cancer after cancer treatment. Furthermore, the subjects of treatment and prevention are not limited to humans, but may also include mammals other than humans, such as mice, rats, dogs, and cats, as well as livestock such as cows, horses, and sheep, and primates such as monkeys, chimpanzees, and gorillas, with humans being particularly preferred.

[0042] A sixth embodiment of the present invention relates to a method for diagnosing or assisting in the diagnosis of adult T-cell leukemia / lymphoma using an anti-CADM1 antibody according to this embodiment. The anti-CADM1 antibody according to this embodiment can specifically bind to CADM1 molecules, and by labeling with a fluorescent substance, a radioisotope, an enzyme, or the like, adult T-cell leukemia / lymphoma cells expressing CADM1 molecules can be detected. Detection methods include, for example, immunostaining, flow cytometry, Western blotting, ELISA, RIA, CLIA, and PET. This method can directly detect cancer cells in vivo or observe the expression level of CADM1 in patient samples. Furthermore, by estimating the expression level of CADM1 in a patient in advance using a method using an anti-CADM1 antibody according to this embodiment, it is possible to predict (or assist in predicting) the therapeutic effect of administering a pharmaceutical composition according to this embodiment.

[0043] The disclosures of all documents cited herein are incorporated by reference in their entirety. Also, throughout this specification, when the words "a," "an," and "the" appear in the singular, they include the plural as well as the singular, unless the context clearly indicates otherwise. The present invention will be further explained below by showing examples. However, the examples are merely illustrative of embodiments of the present invention and are not intended to limit the scope of the present invention. [Example]

[0044] 1. Identification of CADM1 variants expressed in adult T-cell leukemia / lymphoma (ATLL) cells To identify CADM1 variants expressed in ATLL cells, total RNA was extracted from ATLL cell lines (ATN1, TL-Om1) and HTLV-1 (human T-cell leukemia virus type 1) cell lines (MT-2, HUT102). ATN1 and HUT102 were donated by the Japanese Foundation for Cancer Research. TL-Om1 was provided by Professor Kazuo Sugamura (currently Professor Emeritus) of Tohoku University. MT-2 was provided by Professor Hiroo Hoshino (currently Professor Emeritus) of Gunma University. For total RNA extraction, cell pellets were immediately suspended in TRIzol RNA Isolation Reagents (Invitrogen, Thermo Fisher Scientific), treated with phenol and chloroform, and then precipitated with propanol. The CADM1 gene region was amplified using 5' and 3' RACE using the SMARTer RACE 5' / 3' Kit (TaKaRa). The primers used for 5' RACE / 3' RACE are as follows: 5'RACE GATTACGCCAAGCTTCTAGATGAAGTACTCTTTCTTTTCTTCGGAGTTGTTCTGTCCTCCTTCTGC (SEQ ID NO: 21) 3'RACE GATTACGCCAAGCTTATGGCGAGTGTAGTGCTGCCGAGCGG (SEQ ID NO: 22) The amplified DNA was subjected to agarose gel electrophoresis, followed by gel excision and DNA extraction. CADM1 was cloned using the In-Fusion HD cloning kit (TaKaRa), and its amino acid sequence was determined by sequence analysis. The determined amino acid sequence of CADM1 revealed that the full-length extracellular domain of CADM1 was not expressed in ATLL cell lines and HTLV-1 cell lines, and only the exon 9-10 deletion variant (Δ9-10) and exon 10 deletion variant (Δ10) were expressed (Figure 1). Similar results were reported by Nakahata et al. (Nakahata et al., Haematologica. 2020 Feb 13:haematol.2019.234096.)

[0045] 2. Preparation of Immunizing Antigen We designed the antigens based on the following criteria: they must be variants confirmed to be expressed in ATLL, must contain antigens that mimic the cellular conformation, and must contain multiple variants. Consequently, we decided to use a mixture of four proteins, including monomeric CADM1 and dimeric Fc-fused CADM1 (Figure 2). Specifically, DNA sequences encoding the amino acid sequences of the CADM1 extracellular domain Δ9-10 variant (CADM1ecΔ9-10), the CADM1 extracellular domain Δ10 variant (CADM1ecΔ10), the CADM1 extracellular domain Δ9-10 Fc variant (CADM1FcΔ9-10) fused to human Fc, and the CADM1 extracellular domain Δ10 Fc variant (CADM1FcΔ10) fused to human Fc were amplified by PCR and inserted into the pcDNA3.4 TOPO vector (ThermoFisher) using NEBuilder HiFi DNA assembly.

[0046] CADM1ecΔ9-10 QNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYDPPTTIPPPTTTTTTTTTTTTTILTIITDSRAGEEGSIRAAAAEQKLISEEDLNSAVDHHHHHH (SEQ ID NO: 9)

[0047] CADM1ecΔ10 QNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCESNIVGKAHSDYMLYVYDPPTTIPPPTTTTTTTTTTTTTILTIITDTTATTEPAVHDSRAGEEGSIRAHHHHHH (SEQ ID NO: 10)

[0048] CADM1FcΔ9-10 (SEQ ID NO: 11)

[0049] CADM1FcΔ10 (SEQ ID NO: 12)

[0050] The constructed vectors were expressed in the culture supernatant of human expi293 cells using the Expi293 Expression system (ThermoFisher). Four CADM1 variants were purified from the culture supernatant by metal affinity chromatography, followed by final purification by size exclusion chromatography. The purified CADM1ecΔ9-10, CADM1ecΔ10, CADM1FcΔ9-10, and CADM1FcΔ10 were mixed and concentrated to 0.7 mg / ml (1 ml) using an Amicon Ultra 30K (Merck) filter. This was used as the immunization antigen.

[0051] 3. Generation of monoclonal antibodies that recognize CADM1 3-1. Cloning of hybridomas producing anti-CADM1 antibodies Monoclonal antibodies were produced according to standard methods (Kohler and Milstein, Nature, 256, 495-497, 1975). The immunizing antigen prepared in step 2 above was mixed with Freund's adjuvant and immunized into WKAH / Hkm Slc rats (Japan SLC) three times at 2-week intervals. Splenocytes were then prepared from the rats and fused with myeloma cells to produce hybridomas. Culture supernatants of these hybridomas were used to select those producing anti-CADM1 monoclonal antibodies (clone name: YTH-W-2C2) using flow cytometry.

[0052] 3-2. Cloning of the variable region of anti-CADM1 antibody Immediately after harvesting, the YTH-W-2C2 cell pellet was suspended in TRIzol RNA Isolation Reagents (1 ml), treated with phenol and chloroform, and precipitated with propanol to obtain total RNA. Using the SMARTer RACE 5' / 3' Kit (TaKaRa), 5'RACE was performed to amplify the heavy chain variable region to the rat IgG2a heavy chain constant region and the light chain variable region to the kappa light chain constant region of the YTH-W-2C2 antibody (Figure 3a). The amplified DNA was subjected to agarose gel electrophoresis, excised, and DNA was extracted. The heavy and light chain DNA was cloned using the In-Fusion HD cloning kit (TaKaRa) (Figure 3b). Subsequently, the heavy chain amino acid sequence (SEQ ID NO: 13; signal sequence is SEQ ID NO: 14, constant region is SEQ ID NO: 16) and light chain amino acid sequence (SEQ ID NO: 17; signal sequence is SEQ ID NO: 18, constant region is SEQ ID NO: 20) were determined by sequence analysis. The amino acid sequences of the heavy and light chain variable regions are shown below.

[0053] Amino acid sequence of the heavy chain variable region of YTH-W-2C2 antibody QVQLQQSGAELAKPGSSVKISCKASGYTFSNYDISWIKQTTGQGLDYIGYIHTGSGGTYYNEKFKGKATLTVDKSSSTAFMQLSSLTPEDTAVYYCARTPYVYYGSGYFDFWGPGTMVTVSS (SEQ ID NO: 15)

[0054] Amino acid sequence of the light chain variable region of YTH-W-2C2 antibody DIVMTQSPSSLAVSAGETVTINCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFIGSGSGTDFTLTISSVQAEDLAIYYCQQYYDTPDTFGAGTKLELK (SEQ ID NO: 19)

[0055] The above analysis of the amino acid sequences of the heavy and light chains of the YTH-W-2C2 antibody revealed that, with regard to the amino acid sequences of the CDR regions defined by Kabat et al., heavy chain CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively. Furthermore, with regard to the amino acid sequences of the CDR regions defined by Chothia et al., heavy chain CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NOs: 7, 8, and 3, respectively, and light chain CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively. CDR sequence analysis according to the definition of Kabat and Chothia was performed using abYsis (http: / / www.abysis.org / abysis / index.html).

[0056] 3-3. Preparation of YTH-W-2C2 rat antibody and chimeric antibody The cloned heavy and light chain signal sequences and variable region amino acid sequences of the YTH-W-2C2 antibody were inserted into the pcDNA3.4topo expression plasmid. To generate the YTH-W-2C2 rat antibody, the cloned full-length heavy and light chain DNA sequences from the signal sequence to the constant region were inserted into the pcDNA3.4topo plasmid. To generate a chimeric antibody (a chimeric antibody consisting of the variable region of the YTH-W-2C2 antibody and a human IgG constant region, hereafter referred to as the YTH-W-2C2 chimeric antibody), the DNA sequences of the variable region DNA grafted to the constant regions of human IGH1 and IGCκ were inserted into the pcDNA3.4topo plasmid (Fig. 4a). The expression plasmids were introduced into CHO cells, and expression was achieved in the culture supernatant. After 10 to 14 days of culture, the supernatant was separated into cellular components and the supernatant by centrifugation, then filtered. The YTH-W-2C2 rat antibody was purified using a Protein G column, and the chimeric antibody was purified using a Protein A column (Fig. 4b). Thereafter, the YTH-W-2C2 rat antibody and the YTH-W-2C2 chimeric antibody were dialyzed against phosphate buffer and finally purified by size exclusion chromatography (FIG. 4c).

[0057] 4. Examination of the binding of anti-CADM1 antibody (YTH-W-2C2 chimeric antibody) to CADM1 monomer and CADM1 dimer CADM1 is known to form dimers on cells (Shingai et al., J Biol Chem. 278:35421-35427 2003). Therefore, to evaluate the binding affinity of the YTH-W-2C2 chimeric antibody to the antigens (ecΔ9-10, ecΔ10, dimerized FcΔ9-10, and dimerized FcΔ10; see Figure 2) used in immunization of rats for antibody production, we performed surface plasmon resonance (SPR) analysis using Biacore8K. The antibodies were diluted to a concentration of 1 mg / ml in phosphate buffered saline (PBS) and immobilized on a CM5 sensor chip as ligands using the amine coupling method. The analytes were serially diluted from 1.56 nM to 50 nM for Fc full, FcΔ10, and FcΔ9-10, and from 6.25 nM to 200 nM for ec full, ecΔ10, and ecΔ9-10. The binding rate constants (k a ), dissociation rate coefficient (k d ), dissociation constant (K D The results of the analysis are summarized in Table 1. [Table 1] The binding affinity (dissociation constant K D ) and kinetic parameters (binding rate constant k a , dissociation rate constant k d ) was not significantly different from the dimers (Fc full, FcΔ10, FcΔ9-10) but the dimers (ec full, ecΔ10, ecΔ9-10) showed significantly slower dissociation (k d ) and showed higher binding affinity to the dimer. These results suggest that YTH-W-2C2 binds to the full-length, Δ10, and Δ9-10 variants with equal affinity and binds to the dimeric CADM1 with higher affinity than to the monomeric form.

[0058] It has been reported that CADM1 can be expressed as a soluble form of the extracellular domain of CADM1 (sCADM1) through alternative splicing or cleavage of membrane-associated CADM1. This sCADM1 has been reported to be elevated in the plasma of ATL patients from smoldering to acute ATL. In acute ATL, the most aggressive type of lymphoma, blood levels have been shown to rise to a maximum of 1-10 μg / ml (Nakahata et al., Haematologica. 2020 Feb 13:haematol.2019.234096.). In addition, to analyze the biological functions and adhesion affinities of CADM1 and other nectin-like molecules (Necl) as availability proteins, analyses have been conducted using CADM1 fused with an antibody Fc domain to dimerize the extracellular domain (Shingai et al., J Biol Chem. 278:35421-35427 2003; Arase et al., Int Immunol. 17:1227-1237 2005; Ito et al., Front Cell Dev Biol. 6:86 2018). These findings suggest that CADM1 must dimerize to remain active in solution. As mentioned above, the soluble form of sCADM1 is present in blood at concentrations of only 1–10 μg / mL (a few hundred nanomolar order), suggesting that it exists as a monomer, given the need for dimerization to achieve function in solution. YTH-W-2C2 exhibits higher binding affinity for dimerized antigens, suggesting that it may bind with higher affinity to dimeric cellular CADM1 than to circulating sCADM1. This suggests that antibody-based therapeutics may be able to effectively bind to target cells even when the soluble form is present in blood.

[0059] 5. Biological evaluation of anti-CADM1 antibodies (YTH-W-2C2 rat antibody and YTH-W-2C2 chimeric antibody) 5-1. Examination of binding to CADM1 on the cell surface CEM (acute T lymphoblastic leukemia patient-derived T cell line, CADM1 -), TL-Om1 (ATLL patient-derived T cell line, CADM1 ++ ) and MT-2 (HTLV-1-infected immortalized T cell line, CADM1 ++ ) and count 5 x 10 cells. 5 Three 1.5 mL tubes containing cells were prepared. The following three antibody solutions were prepared in advance, each diluted with FACS buffer (PBS + 2% FBS): (a) Anti-CADM1 antibody (Rat-IgG; YTH-W-2C2 rat antibody) 10 μg / mL (b) Anti-CADM1 antibody (Human-Fc-Rat-IgG; YTH-W-2C2 chimeric antibody) 10 μg / mL (c) Isotype control: normal mouse IgG-PE / normal mouse IgG-FITC (10 μg / mL each) 100μL of antibody solution (a), (b), and (c) was added to three tubes for each cell line, mixed well by pipetting, and then incubated in the dark at room temperature for 20 minutes. 500μL of FACS buffer was added to each tube, and the tubes were gently vortexed for approximately 2 seconds (to wash the cells), centrifuged at 1500 rpm for 1 minute at 25℃, and the supernatant was removed.

[0060] The following secondary antibody solutions (d) and (e) were prepared: (d) Anti-rat IgG-Alexa546 (ThermoFisher Scientific) diluted 500-fold in FACS buffer. (e) Anti-human IgG-Alexa546 (ThermoFisher Scientific) diluted 500-fold in FACS buffer. 100 μL of (d) was added to the tube containing antibody (a) for each cell line, and 100 μL of (e) was added to the tube containing antibody (b) for each cell line. After mixing thoroughly by pipetting, the tubes were incubated in the dark at room temperature for 20 minutes. 500 μL of FACS buffer was added to each cell line tube, and the tubes were gently vortexed for approximately 2 seconds (to wash the cells). After centrifugation at 1500 rpm for 1 minute at 25°C, the supernatant was removed. 300 μL of fresh FACS buffer was added to each cell line tube, mixed thoroughly by pipetting, and transferred to a flow cytometer tube.

[0061] The Novocyte flow cytometer (ACEA Biosciences, Inc. / Agilent Technologies, Inc.) was used to detect Alexa488 / FITC or Alexa546 / PE. The data were analyzed using NovoExpress (ACEA Biosciences, Inc. / Agilent Technologies, Inc.) and FlowJo (FlowJo, LLC / Becton, Dickinson and Company (BD)). The results are shown in Figure 5. CADM1-CEM cells, CADM1-HTLV-1-infected cell line MT-2, and ATL patient-derived cell line TL-Om1 were immunostained with anti-CADM1-Rat-IgG (YTH-W-2C2 rat antibody) or anti-CADM1-Human-Fc-Rat-IgG-IgG (YTH-W-2C2 chimeric antibody) as the primary antibody, and anti-Rat-IgG-Alexa546 or anti-Human-IgG-Alexa546 as the secondary antibody, respectively. As a result, both anti-CADM1 IgGs were able to detect CADM1 on the MT-2 cell membrane (Fig. 5, center) and TL-Om1 cell membrane (Fig. 5, right), but did not show nonspecific binding to CEM cells (Fig. 5, left).

[0062] 5-2. Consideration of antibody labeling conditions (biotinylation) 5-2-1. Antibody biotinylation Three types of anti-CADM1 antibodies (YTH-W-2C2 rat antibody, YTH-W-2C2 chimeric antibody, and IgY (MBL)) were biotinylated using the following method. First, 50 μL of antibody (1 μg / μL) and 350 μL of reaction buffer (100 mM NaHCO3, pH 8.4) were added to a Centricut Ultra Mini (W=50: 50 kDa cutoff) and centrifuged (10,000 g, ~20 min, 4°C) until approximately 50 μL remained in the upper chamber. 350 μL of reaction buffer was added again and centrifuged (10,000 g, ~20 min, 4°C) until approximately 50 μL remained. This process was repeated twice, and 50 μL of antibody solution was transferred to a new 1.5 nM tube. A biotin solution was prepared by dissolving biotin in DMSO (dimethyl sulfoxide) to a concentration of 1 mg / mL. 2.5 μL of this biotin solution was added to each antibody solution, mixed by pipetting, and then left in the dark at room temperature for 2 hours. 350 μL of PBS was added to each antibody solution containing biotin, and the mixture was centrifuged (10,000 g, 20 minutes, 4°C) until approximately 50 μL of liquid remained. This process was repeated twice. 50 μL of PBS was added to the biotinylated antibody (50 μL) remaining in the upper chamber to obtain biotinylated antibody (final concentration 500 ng / μL).

[0063] 5-2-2. Verification of biotinylation by flow cytometry analysis CEM cells, TL-Om1 cells, MT-2 cells, and PBMC cells (cell lines derived from chronic ATLL patients) were counted and 5 × 10 5 Four 1.5 mL tubes containing cells were prepared. The following three antibody solutions were prepared in advance, each diluted with FACS buffer (PBS + 2% FBS): (a) Biotinylated anti-CADM1 antibody (Rat-IgG; YTH-W-2C2 rat antibody) 10 μg / mL (b) Biotinylated anti-CADM1 antibody (Human-Fc-Rat-IgG; YTH-W-2C2 chimeric antibody) 10 μg / mL (c) Biotinylated anti-CADM1 antibody (IgY) 10μg / mL (d) No primary antibody (FACS buffer only) 100 μL of antibody solution (a), (b), (c), or (d) or FACS buffer was added to four tubes for each cell line, mixed thoroughly by pipetting, and then incubated in the dark at room temperature for 20 minutes. 500 μL of FACS buffer was added to each tube, and the tubes were gently vortexed for approximately 2 seconds (cell washing). Then, the tubes were centrifuged at 1500 rpm for 1 minute at 25°C, and the supernatant was removed. 100 μL of streptavidin-PE solution (100x diluted in FACS buffer, BioLegend Inc.) was added to each tube, mixed thoroughly by pipetting, and then incubated in the dark at room temperature for 20 minutes. 500 μL of FACS buffer was added to each tube, and the tubes were gently vortexed for approximately 2 seconds (cell washing). Then, the tubes were centrifuged at 1500 rpm for 1 minute at 25°C, and the supernatant was removed. Again, 300 μL of fresh FACS buffer was added to each cell line tube, and after mixing well by pipetting, the cells were transferred to a tube for the flow cytometer.

[0064] PE wavelengths were detected using a Novocyte flow cytometer (ACEA Biosciences, Inc. / Agilent Technologies, Inc.). The data obtained were analyzed using NovoExpress (ACEA Biosciences, Inc. / Agilent Technologies, Inc.) and FlowJo (FlowJo, LLC / Becton, Dickinson and Company (BD)). The analysis results are shown in Figure 6. When the YTH-W-2C2 rat antibody and YTH-W-2C2 chimeric antibody of the present invention were biotinylated, their binding affinity to CADM1-expressing cells was not as strong as that of biotinylated IgY (MBL). However, clear binding was confirmed with two-step labeling (Figure 5) and direct labeling (Figure 7), suggesting that labeling and detection methods other than biotinylation are suitable for these antibodies.

[0065] 5-3. Examination of antibody labeling conditions (Alexa488 direct labeling) Two anti-CADM1 antibodies (YTH-W-2C2 rat antibody and YTH-W-2C2 chimeric antibody) were labeled with Alexa488 (ThermoFisher Scientific). Next, CEM cells, TL-Om1 cells, MT-2 cells, and PBMCs derived from chronic ATL patients were counted and 5 × 10 cells were collected. 5 Four 1.5 mL tubes containing cells were prepared. The following three antibody solutions were prepared in advance, each diluted with FACS buffer (PBS + 2% FBS): (a) Alexa488-anti-CADM1 antibody (Rat-IgG; YTH-W-2C2 rat antibody) 10μg / mL (b) Alexa488-anti-CADM1 antibody (Human-Fc-Rat-IgG; YTH-W-2C2 chimeric antibody) 10μg / mL (c) PE-anti-CADM1 antibody (IgY) 10μg / mL (d) Isotype control: normal mouse IgG-PE / normal mouse IgG-FITC (10 μg / mL each) 100 μL of antibody solution (a), (b), (c), and (d) was added to four tubes for each cell line, mixed thoroughly by pipetting, and then incubated in the dark at room temperature for 20 minutes. 500 μL of FACS buffer was added to each tube, and the tubes were gently vortexed for approximately 2 seconds (to wash the cells). After centrifugation at 1500 rpm for 1 minute at 25°C, the supernatant was removed. 300 μL of fresh FACS buffer was added to each cell line tube, mixed thoroughly by pipetting, and then transferred to a flow cytometer tube. The Novocyte flow cytometer (ACEA Biosciences, Inc. / Agilent Technologies, Inc.) was used to detect Alexa488 / FITC or PE wavelengths. The data were analyzed using NovoExpress (ACEA Biosciences, Inc. / Agilent Technologies, Inc.) and FlowJo (FlowJo, LLC / Becton, Dickinson and Company (BD)). The analysis results are shown in Figure 7. The YTH-W-2C2 rat antibody and the YTH-W-2C2 chimeric antibody of the present invention showed binding affinity comparable to that of the existing PE-IgY (TL-Om1) or even higher (MT-2 and PBMC) when directly fluorescently labeled. In particular, they showed higher detection ability for CADM1 on the surface of tumor T cells derived from chronic-type ATL patients than PE-IgY.

[0066] 5-4. Confirmation of internalization of CADM1 / CADM1 antibody complex into cells Count MT-2 cells and measure 5 x 10 5 Cells were suspended in RPM1 (10% FBS) at 1 mL per well and seeded into 8 wells of a 12-well plate. Two wells of cells were transferred to two 1.5 mL tubes (1 well per tube), and the remaining cell plate was placed in an incubator (37°C, 5% CO2) to begin culturing. The tubes containing the cells were centrifuged at 1500 rpm for 1 minute at 25°C, and the supernatant was removed. 100 μL of the following antibody solutions diluted with FACS buffer (PBS + 2% FBS) was added to each tube, mixed well by pipetting, and then incubated in the dark at room temperature for 20 minutes: (a) Anti-CADM1 antibody (Rat-IgG; YTH-W-2C2 rat antibody) 10 μg / mL (b) Anti-CADM1 antibody (Human-Fc-Rat-IgG; YTH-W-2C2 chimeric antibody) 10 μg / mL Next, 500 μL of FACS buffer was added to each tube, and the tubes were gently vortexed for about 2 seconds (to wash the cells), centrifuged at 1500 rpm for 1 minute at 25° C., and the supernatant was then removed. The cells from each tube were suspended in 1 mL of RPMI (10% FBS) and transferred back to the 8-well plate where the cells were initially seeded (6-hour internalization sample). The same procedure was repeated 2 hours (4-hour internalization sample), 4 hours (2-hour internalization sample), and 6 hours (0-hour internalization sample). Immediately after the 0-hour internalization sample was prepared, the cells from the other wells were transferred to 1.5 mL tubes and centrifuged at 1500 rpm for 1 minute at 25°C. The supernatant was then removed.

[0067] The following antibody solutions were prepared as secondary antibodies. 100 μL of (c) was added to tube (a) and 100 μL of (d) was added to tube (b). After mixing well by pipetting, the mixture was incubated in the dark at room temperature for 20 minutes. (c) Anti-Rat IgG (YTH-W-2C2 rat antibody)-Alexa546 (ThermoFisher Scientific), diluted 500-fold in FACS buffer. (d) Anti-Human IgG (YTH-W-2C2 chimeric antibody)-Alexa488 (ThermoFisher Scientific), diluted 500-fold in FACS buffer. 500 μL of FACS buffer was added to each tube, which was then gently vortexed for approximately 2 seconds (to wash the cells), centrifuged at 1500 rpm for 1 minute at 25°C, and the supernatant was removed. 300 μL of fresh FACS buffer was added to each cell line tube, mixed well by pipetting, and then transferred to a flow cytometer tube. The Novocyte flow cytometer (ACEA Biosciences, Inc. / Agilent Technologies, Inc.) was used to detect Alexa 488 or Alexa 546 wavelengths. The data were analyzed using NovoExpress (ACEA Biosciences, Inc. / Agilent Technologies, Inc.) and FlowJo (FlowJo, LLC / Becton, Dickinson and Company (BD)). After flow cytometry, the remaining cell suspension was attached to a glass slide using a cytospin (800 rpm, 3 minutes, room temperature) and fixed in 4% PFA for 10 minutes without excessive drying. The slide was washed three times with PBS, mounted in mounting solution (80% glycerol + DAPI + anti-fade agent), and observed under a fluorescence microscope (Olympus IX70, OLYMPUS Corp.). The analysis results are shown in Figure 8. In a flow cytometric study, the YTH-W-2C2 rat antibody and YTH-W-2C2 chimeric antibody of the present invention bound to CADM1 on the surface of MT-2 cells, resulting in a decrease in the fluorescent signal after 2 hours. Furthermore, when the MT-2 cells were observed under a fluorescence microscope, the CADM1-antibody complex, which was present in a granular form on the surface of the MT-2 cells at 0 hours, disappeared after 2 hours, except at the cell-cell adhesion sites. Therefore, it is believed that both the YTH-W-2C2 rat antibody and the YTH-W-2C2 chimeric antibody were internalized into the cells in the form of a complex bound to CADM1.

[0068] Next, we performed an experiment using CEM cells overexpressing CADM1. CEM / hCADM1 cells, in which CADM1 was overexpressed, were incubated with anti-CADM1-Human-Fc-Rat-IgG (YTH-W-2C2 chimeric antibody) for 20 minutes at room temperature. After washing, the cells were cultured in RPMI + 10% FBS medium at 37°C for 0, 2, and 4 hours under 5% CO2 conditions to allow for internalization of the CADM1 antibody (anti-CADM1-Human-Fc-Rat-IgG)-CADM1 complex. The cells were then incubated with a secondary antibody (anti-Human-IgG-Alexa546) for 20 minutes at room temperature to detect the CADM1 antibody-CADM1 complex on the cell surface. The same procedure was performed on CEM / control cells (vector control CEM cells) as a negative control. The analytical results are shown in Figure 9. Flow cytometry analysis (Figure 9, left) showed that the amount of CADM1 antibody-CADM1 complexes in CEM / hCADM1 cells decreased after 2 and 4 hours compared to 0 hours. Furthermore, fluorescence microscopy revealed that the intracellular localization of the CADM1 antibody-CADM1 complexes (red) and lysosomes (green) at each time point revealed that the CADM1 antibody-CADM1 complexes, which were localized on the cell surface at 0 hours, localized as clusters near lysosomes after 2 hours, and co-localized with lysosomes after 4 hours. These results suggest that anti-CADM1-Human-Fc-Rat-IgG is internalized upon binding to CADM1 on the cell surface and then taken up into lysosomes for degradation, demonstrating its usefulness as an antibody-drug conjugate (ADC). No signal was detected with anti-CADM1-Human-Fc-Rat-IgG in CEM / control cells, confirming that anti-CADM1-Human-Fc-Rat-IgG specifically binds to CADM1 on the cell surface.

[0069] Furthermore, these cells (cells used for fluorescence microscopy in Figure 9) were observed using a confocal laser microscope. The results showed that even in two-dimensional images, the CADM1 / CADM1 antibody complex (red) was localized near lysosomes (green) over time, and after 4 hours, the red signal alone had decreased (Figure 10). This result indicated that the CADM1 / CADM1 antibody complex was taken up by lysosomes and rapidly degraded.

[0070] 5-5. Preparation of ADC (Antibody Drug Conjugate) and investigation of its effect on cells We constructed anti-CADM1-human Fc-rat IgG ADCs conjugated with MMAE (monomethyl auristatin E) and examined their specific effects on CADM1(+) cells. CADM1(+) cells included CEM / hCADM1 cells (CADM1-overexpressing CEM cells), ATL patient-derived cell lines (TL-Om1, MT-1, and ATN-1), and an HTLV-1-infected cell line (MT-2). CEM(-) cells included CEM / control cells (vector-control CEM cells) and untreated CEM cells. PBS, MMAE, anti-CADM1-human Fc-rat IgG alone, or anti-CADM1-human Fc-rat IgG + MMAE (ADC) were added to the culture medium of these cells. Cell viability was measured by a WST8 assay after 0, 2, and 4 days of incubation. As a result, compared with PBS (untreated) (○ (open circle) in the graph in Figure 11), treatment with anti-CADM1-Human-Fc-Rat-IgG alone did not affect cell proliferation in either cell line (□ (open square) in the graph in Figure 11), and MMAE treatment nonspecifically induced cell death (● (filled circle) in the graph in Figure 11). On the other hand, treatment with anti-CADM1-IgG + MMAE (ADC) resulted in a significant decrease in cell proliferation only in CADM1(+) cells (■ (gray square) in the graph in Figure 11). These results confirmed that the ADC using anti-CADM1-Human chimera-IgG was specifically taken up by CADM1(+) cells and induced cell death.

[0071] Next, we examined the effects of anti-CADM1-Human-Fc-Rat-IgG+MMAE (ADC) on fresh ATL cells from chronic ATL patients. Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from chronic ATL patients and cultured in RPMI + 10% FBS + IL-2 (100 ng / mL) culture medium with PBS, anti-CADM1-Human-Fc-Rat-IgG antibody alone (150 nM), or anti-CADM1-Human-Fc-Rat-IgG+MMAE (ADC) (150 nM) at 37°C under 5% CO2. After 7 days, PBMCs were stained with anti-CADM1-PE, CD7-FITC, CD4-APC, and PI, and the CD7 and CADM1 expression patterns of PI(-) / CD4+ cells were compared (see, for example, HAS-Flow method, Kobayashi et al., Clin Cancer Res; 20(11): 2851-2861, 2014. DOI: 10.1158 / 1078-0432.CCR-13-3169). The HAS-Flow method separates CD7(+) / CADM1(-): uninfected cells, CD7(+) / CADM1(+): HTLV-1-infected cells, and CD7(-) / CADM1(+): ATL tumor cells. As a result, compared with PBS-treated cells, treatment with anti-CADM1-Human-Fc-Rat-IgG antibody alone did not change the HAS-Flow pattern, whereas treatment with CADM1-Human-Fc-Rat-IgG+MMAE (ADC) specifically reduced the CD7(-) / CADM1(+):ATL tumor cell population and relatively increased the proportion of CD7(+) / CADM1(-):uninfected cells (Figure 12, upper panel).

[0072] In addition, the CD4 + Comparing the proportion of T cells, anti-CADM1-Human-Fc-Rat-IgG+MMAE (ADC) treatment also significantly increased the CD4 +The proportion of T cells was significantly reduced, indicating a decrease in the tumorigenic ATL cell population (Figure 12, bottom panel). These results demonstrate that the ADC using this antibody specifically eliminates only ATL cells that overexpress CADM1 from PBMCs of ATL patients, which contain a mixture of various cells, without affecting uninfected T cells.

[0073] 5-6. Confirmation of ADCC activity by anti-CADM1 antibody Normal human peripheral blood mononuclear cells (PBMCs) were used as effector cells. After separating PBMCs from peripheral blood using lymphocyte separation fluid, 8 × 10 6 The target cells were a green fluorescently labeled CADM1-positive HTLV-1-infected T cell line (MT-2), suspended in RPMI (10% FBS, 20 U / mL IL-2) at a concentration of 4 × 10 cells / mL. 5 The cells were suspended in RPMI (10% FBS, 20 U / mL IL-2) at a concentration of 20 μg / mL. The antibodies used were a negative control antibody (rat IgG, human IgG), a positive control antibody (anti-HTLV-1 gp46 antibody (LAT-27), humanized LAT-27 (hu-LAT-27)), and an anti-CADM1 antibody (YTH-W-2C2 rat antibody, YTH-W-2C2 chimeric antibody). Each antibody was suspended in RPMI (10% FBS, 20 U / mL IL-2) at a concentration of 20 μg / mL. Then, 50 μL of effector cells, 25 μL of target cells, and 25 μL of each antibody were seeded into each well of a 96-well U-bottom plate (effector / target ratio = 40) and cultured for 4 hours at 37°C, 5% CO2. After incubation, the cells were fixed with 1% paraformaldehyde and transferred to a tube for flow cytometry. Furthermore, 5 × 10 flow count standard particles (Beckman Coulter) were added to each tube. 3 The number of surviving target cells was counted by flow cytometry (BD FACSCalibur, BD CELLQuest Pro). The number of injured target cells was calculated from these remaining numbers. As a result, it was revealed that both the YTH-W-2C2 rat antibody and the YTH-W-2C2 chimeric antibody according to this embodiment bind to CADM1 on the surface of MT-2 cells and, in cooperation with effector cells, can kill target MT-2 cells (ADCC effect) (Figures 13 and 14).

[0074] These results demonstrate that the anti-CADM1 antibody of this embodiment binds to various cell lines and fresh tumor T cells derived from ATLL patients. Furthermore, experiments confirming cellular internalization demonstrated that the anti-CADM1 antibody of the present invention binds to CADM1 on the cell surface and is internalized into cells in the form of a CADM1 / CADM1 antibody complex, demonstrating its suitability for disease treatment, such as by ADC conversion. Furthermore, direct labeling with a fluorescent dye demonstrated comparable cell surface CADM1 detection capabilities to commercially available PE-anti-CADM1-IgY, demonstrating its usefulness in a variety of diagnostic and therapeutic applications. [Industrial Applicability]

[0075] The antibodies and antigen-binding fragments thereof provided by the present invention are believed to play an important role in providing treatments for diseases such as ATLL or developing therapeutic agents, and therefore the present invention is expected to be useful in the medical and pharmaceutical fields, etc.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CADM1 (cell adhesion molecule 1), wherein the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy either (A) or (B) below, and that binds to CADM1 on the cell surface and induces its internalization into the cell. (A) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5; and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6; (B) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5; and It has a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that it satisfies either (a) or (b) below. (a) having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19; (b) a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

19.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the CADM1 is dimerized on the cell surface.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that it is a humanized antibody or a chimeric antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that a substance having antitumor activity is bound thereto.

6. Fab, Fab', F(ab') 2 2. The antigen-binding fragment of claim 1, which is an Fv, a single-chain antibody, an scFv, an scFv dimer or a dsFv.

7. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 6.

8. 8. The pharmaceutical composition according to claim 7, wherein the disease to be treated is adult T-cell leukemia / lymphoma.

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