Antibody against cancer cell and pharmaceutical composition containing said antibody

Abstract

The present invention provides: an antibody against a cancer cell; and a pharmaceutical composition containing the antibody. The present invention provides, for example: an antibody or an antigen-binding fragment thereof that binds to a tumor cell, wherein the antibody has a heavy-chain variable region comprising a heavy-chain CDR1, a heavy-chain CDR2, and a heavy-chain CDR3, and a light-chain variable region comprising a light-chain CDR1, a light-chain CDR2, and a light-chain CDR3, the heavy-chains CDR1 to CDR3 and the light-chains CDR1 to CDR3 being heavy-chains CDR1 to CDR3 and light-chains CDR1 to CDR3 that are defined in accordance with the numbering system by Kabat et al. in SEQ ID NOs: 7 and 8; or an antibody or an antigen-binding fragment thereof that competes with the above-mentioned antibody for the binding to the tumor cell.

Description

Antibody against cancer cells and pharmaceutical composition containing said antibody

[0001] The present invention relates to an antibody against cancer cells and a pharmaceutical composition comprising said antibody.

[0002] Malignant lymphoma is a tumor that originates in lymphatic tissues, and its treatment is mainly by radiotherapy and chemotherapy using anticancer drugs. In recent years, an anti-CD20 antibody (rituximab) targeting CD20 has been developed, and it has been shown to have an antitumor effect on B-cell lymphoma (Non-Patent Document 1).

[0003] McLaughlin P. et al. , J Clin Oncol. , (1998), 16, 2825-2833

[0004] The present invention provides an antibody against cancer cells and a pharmaceutical composition comprising the antibody.

[0005] The present inventors screened antibodies obtained by immunizing non-human animals with NK / T lymphoma cells and found an antibody with cytotoxic activity against a wide range of hematopoietic tumor cell lines and solid tumors. They named this monoclonal antibody the ANAP antibody, and the clone producing this antibody ANAP. The present inventors also demonstrated that the cytotoxic activity was neither antibody-dependent cellular cytotoxicity (ADCC) nor complement-dependent cytotoxicity (CDC). The antigen of the ANAP antibody was integrin α4, which is expressed on tumor cells. The present invention was made based on these findings.

[0006] The present invention provides the following inventions. (1) An antibody or antigen-binding fragment thereof that binds to integrin α4, wherein the antibody or antigen-binding fragment thereof: (a) has a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the amino acid sequence of the heavy chain variable region of SEQ ID NO: 7, and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3 in the amino acid sequence of the light chain variable region of SEQ ID NO: 8; (b) an antibody or antigen-binding fragment thereof that has a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8; (c) an antibody or antigen-binding fragment thereof that has a heavy chain variable region comprising heavy chain CDR1 to 3 of the heavy chain constant region of SEQ ID NO: 11 and a light chain variable region comprising light chain CDR1 to 3 of the light chain constant region of SEQ ID NO: 14; (d) an antibody or antigen-binding fragment thereof that has a heavy chain variable region comprising heavy chain CDR1 to 3 of the heavy chain constant region of SEQ ID NO: 12 and a light chain variable region comprising light chain CDR1 to 3 of the light chain constant region of SEQ ID NO: 15; (e) an antibody or antigen-binding fragment thereof having a heavy chain variable region having heavy chain CDR1 to CDR3 of the heavy chain constant region set forth in SEQ ID NO: 13 and a light chain variable region having light chain CDR1 to CDR3 of the light chain constant region set forth in SEQ ID NO: 16, or (f) an antibody or antigen-binding fragment thereof that competes for binding to integrin α4 with the antibody set forth in any of (b) and (c) to (e) above {with the proviso that the antibody is preferably not the 9F10 antibody, nor an antibody having the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 of 9F10, nor an antibody containing mutations of 5 or fewer amino acids in the heavy and light chain CDR1 to CDR3.} (2) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof set forth in (1) above.(3) The pharmaceutical composition according to (2) above, wherein (e) the antibody is an antibody having a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region having a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (h) the antibody is an antibody having a heavy chain constant region described in SEQ ID NO: 11 and a light chain constant region described in SEQ ID NO: 14; (i) an antibody having a heavy chain constant region described in SEQ ID NO: 12 and a light chain constant region described in SEQ ID NO: 15; (j) an antibody having a heavy chain constant region described in SEQ ID NO: 13 and a light chain constant region described in SEQ ID NO: 16; or (k) an antibody that competes with the antibody described in any of (g) to (j) above for binding to integrin α4, but precipitates only α4 in the immunoprecipitation of α4β1. (4) A pharmaceutical composition comprising a conjugate of the antibody defined in any one of (1) to (3) above with an anticancer agent. (5) The pharmaceutical composition according to any one of (2) to (4) above for use in treating cancer. (6) The pharmaceutical composition according to (5) above, wherein the cancer is selected from the group consisting of hematopoietic tumors, malignant melanoma, and mesothelioma. (7) The pharmaceutical composition according to (6) above, wherein the hematopoietic tumor is leukemia, acute leukemia, chronic leukemia, monocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and / or lymphoma. (8) The pharmaceutical composition according to (7) above, wherein the hematopoietic tumor is Hodgkin's lymphoma, hairy cell leukemia, multiple myeloma, B-cell lymphoma, and / or T-cell lymphoma hematopoietic tumor. (9) The pharmaceutical composition according to (7) above, wherein the hematopoietic tumor is NK / T-cell tumor. (10) The pharmaceutical composition according to (5) or (6) above, wherein the cancer is integrin α4 positive.

[0007] Figure 1 shows the cytotoxic activity of antibodies against various NK / T lymphoma cell lines. Figure 2 shows the results of flow cytometric analysis of the cytotoxic activity of ANAP antibodies produced by ANAP clones against NKL cell lines. Figure 3 shows the time-dependent decrease in the viable cell percentage of the NK-92 cell line due to the cytotoxic activity of the ANAP antibody. Figure 3-2 shows the antibody concentration dependence of the cytotoxicity of the ANAP antibody against NKL cells. Figure 4 shows scanning electron microscope images of NKL cells in which cell death was induced by the ANAP antibody. Images A and B show NKL cells in the absence of the ANAP antibody. Images C, D, and E show NKL cell images 20 minutes after the addition of the ANAP antibody; F shows a 20-fold magnification of E; and G and H show images of the cells after an additional 2 hours. Figure 5 shows the results of flow cytometric analysis showing the binding of the ANAP antibody to various cell lines. Figure 6 shows the results of flow cytometric analysis using propidium iodide (PI) to detect cell death in various cell lines induced by ANAP antibody. Figure 7 shows the time-dependent changes in trypan blue staining of human malignant mesothelioma cell lines treated with ANAP antibody. Figure 7-2 shows scanning electron microscope images of a human malignant mesothelioma cell line (MSTO-211H) in which cell death was induced by ANAP antibody. Images A and B show images of the human malignant mesothelioma cell line in the absence of ANAP antibody. Images C and D show images of the malignant mesothelioma cell line 20 minutes after the addition of ANAP antibody. Figure 7-3 shows the results of cell size analysis by flow cytometry. FSC is a measurement of the amount of laser beam passing around the cell, and SSC is a measurement of the amount of laser beam reflected by intracellular particles. Figure 8 shows the lack of cytotoxic activity of ANAP antibody against normal human peripheral blood granulocytes. Figure 9A shows the UV absorbance of each elution fraction in ANAP antibody affinity purification. Figure 9B shows the results of SDS-PAGE electrophoresis of fractions 41 to 43. Figure 9C shows the results of flow cytometry demonstrating the binding of ANAP antibody to ITGA4. Figure 9D shows the results of Western blot analysis using anti-ITGA4 antibody on immunoprecipitates of cell lysates with ANAP antibody. Figure 9E shows the results of a competition assay between ANAP antibody and 9F10 antibody.Figure 10 shows the sequences of (A) the heavy chain variable region and a portion of the constant region adjacent thereto, and (B) the light chain variable region and a portion of the constant region adjacent thereto of the ANAP antibody. Figure 11 shows flow cytograms showing the binding of various humanized antibodies to NKL cells. Ibid. Specific Description of the Invention

[0008] As used herein, a "subject" may be a mammal, preferably a human. The subject may be a subject suffering from a tumor or cancer.

[0009] As used herein, "antibody" refers to immunoglobulin, including polyclonal and monoclonal antibodies. A preferred antibody is a monoclonal antibody. The origin of the antibody is not particularly limited, but examples include antibodies from non-human animals, non-human mammals, and human antibodies. The antibody may also be a chimeric antibody, a humanized antibody, or a human antibody. The antibody may also be a bispecific antibody. An antibody selectively binds to an antigen. Selective binding means that the antibody binds only to a specific protein, but not to other proteins with different sequences. The heavy chain of an antibody consists of a heavy chain variable region (VH), a heavy chain constant region (CH1), a hinge, a CH2, and a CH3 region. The light chain of an antibody consists of a light chain variable region (VL) and a light chain constant region (CL).

[0010] As used herein, the term "antigen-binding fragment" refers to a portion of an antibody that retains its ability to bind to an antigen. The antigen-binding fragment may comprise the heavy chain variable region, the light chain variable region, or both of the antibody of the present invention. The antigen-binding fragment may also be a fragment of a chimerized or humanized antibody. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), diabody, and sc(Fv)2 (single-chain (Fv)2). Such antibody fragments can be obtained, but are not limited to, by treating the antibody with an enzyme. For example, Fab can be obtained by digesting the antibody with papain. Alternatively, F(ab')2 can be obtained by digesting the antibody with pepsin, and Fab' can be obtained by further reducing this. Such antibody antigen-binding fragments can be used in the present invention.

[0011] As used herein, "integrin α4" refers to a member of the integrin superfamily expressed on the plasma membrane of cells. It is involved in cell adhesion, particularly cell-extracellular matrix adhesion and cell-cell adhesion, as a receptor for extracellular matrix. Integrins are heterodimers consisting of two chains, an α chain and a β chain. The first integrin group includes LFA1, Mac1, and p150,95, and is expressed exclusively on leukocytes. The second integrin group is the VLA antigen, which is expressed on all cell types except granulocytes and erythrocytes (not limited to leukocytes). An example of the amino acid sequence of human integrin α4 is registered in GenBank as AAB59613.1. Integrin α4 can form heterodimers (VLA-4) with integrin β1 or β7. α4β1 (VLA-4), a type of VLA antigen, is involved in extracellular matrix adhesion as a fibronectin receptor, but also functions as an intercellular adhesion receptor.

[0012] The present invention provides an antibody or antigen-binding fragment thereof that binds to tumor cells, particularly integrin α4, comprising: (a) a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1-3 and light chain CDR1-3 are defined by the numbering of Kabat et al. in SEQ ID NOs: 7 and 8. Here, the numbering of Kabat et al. refers to the numbering of Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. The CDR numbering system refers to the numbering system described in the National Institutes of Health and Human Services, PHS, NIH. Other CDR numbering systems such as Chothia, AbM, contact, IMGT, Aho, and Martin (Enhanced Chothia) are known and can be used for CDR estimation.

[0013] The antibody of the present invention binds to integrin α4 expressed on tumor cells. The antibody of the present invention preferably selectively binds to integrin α4 expressed on tumor cells (e.g., binds specifically and more strongly to α4 than to integrins other than α4). The binding affinity of an antibody to an antigen is quantified by the binding dissociation constant (KD). The KD of an antibody is, for example, 10 -8 M or less, and the smaller the value, the stronger the bond. For example, -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less, for example, 10 -9 M to 10 -12 It can be M.

[0014] According to the present invention, there is provided an antibody or antigen-binding fragment thereof that binds to tumor cells, particularly integrin α4, and (b) has the following CDRs: a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5. (b) an antibody having at least one CDR selected from the group consisting of a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, or having all CDRs; (b) an antibody having a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8; (c') an antibody that competes with the antibody set forth in (b) for binding to the tumor cells, particularly integrin α4, but does not bind to integrin β1; or (d) an antibody that competes with the antibody set forth in (b) for binding to the tumor cells, particularly integrin α4 (provided that the antibody is not the 9F10 antibody, is not an antibody having the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 of 9F10, and is not an antibody containing mutations of 1 amino acid or less, 2 amino acids or less, 3 amino acids or less, 4 amino acids or less, or 5 amino acids or less in the heavy chain and light chain CDR1 to CDR3). The tumor cells preferably include integrin α4-positive cells. Antibody competition can also be tested using integrin α4-positive cells. The antibody may have one or more (e.g., 1 to 3) modifications selected from amino acid insertion, substitution, deletion, and addition. The modifications may be made in at least one CDR selected from the group consisting of: a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, while maintaining some or all of the ability to bind to integrin α4. These antibodies preferably exhibit ADCC or CDC-independent cytotoxicity against integrin α4-expressing tumor cells.The cytotoxicity of these antibodies does not require effector function and is effector function-independent. Alternatively, these antibodies may disrupt the cytoskeleton of integrin α4-expressing tumor cells, resulting in cell damage.

[0015] The present invention provides an antibody or an antigen-binding fragment thereof that binds to tumor cells, particularly integrin α4, wherein the antibody is: (e) an antibody having a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region having a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; or (f) an antibody that competes with the antibody described in (e) above for binding to the tumor cells, particularly integrin α4.

[0016] The present invention provides an antibody or antigen-binding fragment thereof that binds to tumor cells, particularly integrin α4 expressed by said cells, wherein the antibody: (b) has a heavy chain variable region defined by the amino acid sequence of SEQ ID NO:7 and a light chain variable region defined by the amino acid sequence of SEQ ID NO:8; (c) competes with the antibody of (b) above for binding to said tumor cells, particularly integrin α4; or (c'') competes with the antibody of (b) above for binding to said tumor cells, particularly integrin α4, and is capable of precipitating only integrin α4 by immunoprecipitation of a cell lysate derived from α4β1-expressing cells, or is capable of monomerizing the α4β1 heterodimer.

[0017] In one aspect of the present invention, the antibody of the present invention selectively binds to tumor cells and does not exhibit binding to integrin α4-positive non-tumor cells.

[0018] The antibodies of the present invention may exhibit significantly increased cytotoxicity against tumor cells, particularly integrin α4-positive tumor cells (e.g., the NK / T lymphoma cell line NKL or the NK / T lymphoma cell line NKYS), compared to a control IgG. The antibodies of the present invention may not exhibit significant cytotoxicity against normal cells (e.g., peripheral blood mononuclear cells from healthy individuals), compared to a control IgG. Whether or not a tumor is integrin α4-positive can be determined, without particular limitation, by detecting the presence of integrin α4 in tumor cells obtained from a patient using an antibody, for example, by testing the binding ability of an anti-integrin α4 antibody to tumor cells obtained from a patient.

[0019] The tumor cells to which the antibody binds may be, for example, any one or more or all selected from the group consisting of solid tumor cells, hematopoietic tumor cells, NK / T lymphoma cell lines NK-92, NKL, and NKYS; cutaneous T-cell lymphoma HUT78; acute T-cell leukemia cell line Jurkat; Hodgkin's lymphoma line L428; acute lymphoblastic leukemia cell line MOLT-4; Burkitt's lymphoma cell line RAJI; and malignant mesothelioma cell line MSTO-211H. Most hematopoietic tumor cells are integrin α4 positive.

[0020] The antibody of the present invention is preferably a humanized antibody. A humanized antibody has CDRs in the CDR regions of a human antibody that correspond to each CDR of an animal antibody. The subclass of the heavy chain of the humanized antibody may be, for example, human IgG1, human IgG2, human IgG3, or human IgG4. The light chain of the humanized antibody may be a human κ chain or a human λ chain. Those skilled in the art can prepare humanized antibodies from animal antibodies using conventional methods. The subclass of the heavy chain of the humanized antibody may be, for example, human IgG2 or human IgG4.

[0021] The antibodies of the present invention can exhibit cytotoxic activity against various cancer cells, including integrin α4-positive cancer cells. In particular, they can exhibit cytotoxic activity against various blood cancer cells. This cytotoxic activity is different from ADCC activity or CDC activity. Thus, the present invention provides a pharmaceutical composition comprising the antibody of the present invention, for example, any of the antibodies (a) to (f) above, for use in treating cancer. For example, such cancers include malignant mesothelioma.

[0022] According to the present invention, there is provided a pharmaceutical composition for use in treating hematopoietic tumors, comprising an antibody of the present invention, for example, any of the antibodies (a) to (f) above. In one embodiment of the present invention, the hematopoietic tumor may be NK / T lymphoma. The hematopoietic tumor may also be Hodgkin's lymphoma, hairy cell leukemia, multiple myeloma, and / or B-cell lymphoma. The hematopoietic tumor may also be leukemia, acute leukemia, chronic leukemia, monocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and / or lymphoma. Molecules to which the antibodies of the present invention bind are widely expressed and displayed on the surface of these cancer cells.

[0023] In one aspect of the present invention, there is provided a conjugate of the antibody of the present invention, for example, any of the antibodies (a) to (f) above, or an antigen-binding fragment thereof, with a cytotoxic agent. Examples of the cytotoxic agent used in the present invention include radioisotopes for cancer treatment (e.g., P 32 , Y 90 , I 131 , I 125 , Sm 153 , Re 186 , Re 188 , At 211 , Bi 212 , Pb 212and radioactive isotopes of Lu). Examples of cytotoxic agents used in the present invention include anticancer agents. Examples of cytotoxic agents include chemotherapeutic agents (e.g., anticancer agents such as commercially available anticancer agents, for example, auristatins (auristatin E, auristatin F phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin F, and their derivatives), maytansinoids DM1 and DM4, and their derivatives), camptothecins (SN-38, irinotecan, lutotecan, DB67, BMP1350, ST1481, CKD602, topotecan, exatecan, and deruxtecan, and their derivatives), DNA minor groove binders (enediynes, lexitropsin, duocarmycin, and their derivatives), taxanes (paclitaxel and docetaxel, and their derivatives), polyketides (discodermolide and its derivatives), anthraquinones (mitoxantrone and its derivatives), benzo[a]thiazolinone, benzo[b ... Examples of cytotoxic agents that can be used in the ADCs of the present invention include diazepines (pyrrolobenzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), cardiac glycosides (digitoxin and its derivatives), calechiamycin, epothilones, cryptophycins, cemadotin, rhizoxin, netropsin, combretastatin, eleutherobin, etoposide, T67 (Tularik), and nocodazole), and toxins (e.g., diphtheria toxin A, Pseudomonas endotoxin, ricin, saporin, etc.). The cytotoxic agent may be a pharmaceutically acceptable salt, solvate (eg, hydrate), ester, or prodrug of the above-mentioned cytotoxic agent.

[0024] In one aspect of the present invention, a pharmaceutical composition for use in treating cancer is provided, comprising a conjugate (conjugate) of an antibody of the present invention, for example, any of the antibodies (a) to (f) above, or an antigen-binding fragment thereof, with a cytotoxic agent. In one aspect of the present invention, the antibody or antigen-binding fragment thereof and the cytotoxic agent may be linked via a linker. The linker may be a non-cleavable linker or a cleavable linker (e.g., a linker containing a valine-citrulline dipeptide). Various linker structures are known for use in general ADCs, and these may be used as appropriate.

[0025] In another aspect of the present invention, there is provided a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody of the present invention, for example, any of the antibodies (a) to (f) above.

[0026] Another aspect of the present invention provides use of an antibody of the present invention, for example, any of the antibodies (a) to (f) above, in the manufacture of a pharmaceutical composition for use in treating cancer.

[0027] In one aspect of the present invention, an antibody of the present invention, for example, any of the antibodies (a) to (f) above, itself has cytotoxic activity. Furthermore, in this aspect, the antibody of the present invention may further have cytotoxic activity such as antibody-dependent cellular cytotoxicity (ADCC activity) or complement-dependent cytotoxicity (CDC activity). In one aspect of the present invention, a pharmaceutical composition for use in treating cancer or a cancer therapeutic agent is provided, which comprises an antibody of the present invention, for example, any of the antibodies (a) to (f) above, as an active ingredient. As used herein, "treatment" is used in a sense that includes therapy.

[0028] Pharmaceutical compositions or medicaments containing the antibodies of the present invention as an active ingredient can be formulated using known pharmaceutical methods. For example, pharmaceutical compositions or medicaments of the present invention may contain a pharmaceutically acceptable excipient. The excipient can be any vehicle that can be appropriately administered to provide an effective amount of the antibody of the present invention, which is the active ingredient, to a subject. In certain aspects, pharmaceutical compositions or medicaments of the present invention can be injectable, and the excipient for injection can be a sterile aqueous solution, for example, a pharmaceutically acceptable buffer such as Ringer's solution, Hank's solution, or physiological saline, or an isotonic solution containing glucose or other adjuvants. Examples of adjuvants include alcohols such as ethanol, polyalcohols such as polyethylene glycol, and nonionic surfactants such as polysorbate 80, which can be added during formulation. Sesame oil, coconut oil, and soybean oil can be used as oily solutions for injection, and benzyl benzoate or benzyl alcohol can be used as an adjuvant. Pharmaceutical compositions or medicaments of the present invention can be administered parenterally (e.g., intravenously or intrapleurally) in the form of an injection.

[0029] The cytotoxic activity of the antibody of the present invention can be determined by incubating target cancer cells under physiological conditions in the presence of the antibody of the present invention and counting the number of live and / or dead cancer cells. In one aspect of the present invention, the cytotoxic activity of the antibody is not ADCC activity or CDC activity, and testing the cytotoxic activity of the antibody of the present invention does not require effector cells or complement, unlike measuring ADCC activity or CDC activity. However, the antibody of the present invention may have ADCC activity and / or CDC activity.

[0030] The cytotoxic activity may be enhanced by combining with other drugs, for example, a combination of the antibody of the present invention with an existing drug (such as an anticancer drug), or a form in which the antibody of the present invention is conjugated with an existing drug (such as an anticancer drug) (antibody-drug conjugate; ADC). Methods for conjugating the antibody of the present invention with an existing drug include known methods, such as conjugating the drug to the amino group of the antibody of the present invention via glutaraldehyde, or conjugating the amino group of the drug to the carboxyl group of the antibody of the present invention via a water-soluble carbodiimide. Methods for enhancing cytotoxic activity using these methods are known, and antibodies modified in this way can also be used as the antibodies of the present invention.

[0031] The antibody can be genetically modified, for example, a chimeric antibody, a humanized antibody, or a human antibody, by methods well known to those skilled in the art, for the purpose of reducing the antigenicity of the antibody itself in humans. In certain aspects of the present invention, the antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody. The antibody may also be a bispecific antibody.

[0032] In one aspect of the present invention, a conjugate (conjugate) of an antibody of the present invention, for example, any of the antibodies (a) to (f) above, or an antigen-binding fragment thereof, with an imaging probe is provided. According to the present invention, a conjugate (conjugate) of an antibody of the present invention or an antigen-binding fragment thereof with an imaging probe can be used for in vitro or in vivo cancer diagnosis. Therefore, the present invention provides a cancer diagnostic agent or diagnostic kit comprising a conjugate (conjugate) of an antibody of the present invention or an antigen-binding fragment thereof with an imaging probe. The present invention also provides an in vivo cancer diagnostic agent or kit comprising a conjugate (conjugate) of an antibody of the present invention or an antigen-binding fragment thereof with an imaging probe. Imaging probes that can be used for in vivo cancer diagnosis include, for example, fluorescent imaging probes, enhancing agents (e.g., paramagnetic ions) such as contrast agents for nuclear magnetic resonance imaging (MRI), and radionuclides for imaging such as PET molecular imaging probes. In one aspect of the present invention, the antibody or antigen-binding fragment thereof and a cytotoxic agent can be conjugated via a linker.

[0033] The present invention provides nucleic acids encoding antibodies of the present invention, such as any of the antibodies (a) to (f) above, or antigen-binding fragments thereof. Nucleic acids encoding antibodies of the present invention, such as any of the antibodies (a) to (f) above, or antigen-binding fragments thereof, can be determined by those skilled in the art using well-known methods. For example, nucleic acids encoding antibodies of the present invention, such as any of the antibodies (a) to (f) above, or antigen-binding fragments thereof, can be determined from their amino acid sequences.

[0034] According to the present invention, there is provided, for example, a nucleic acid encoding any one or all of the CDRs selected from the group consisting of the CDRs described below: heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, or a combination of these nucleic acids.

[0035] The present invention provides, for example, a nucleic acid encoding a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3. The present invention provides, for example, a nucleic acid encoding a light chain variable region having a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6. The present invention also provides, for example, a nucleic acid encoding a heavy chain comprising a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3. The present invention also provides, for example, a nucleic acid encoding a light chain comprising a light chain variable region having a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0036] The present invention provides, for example, a nucleic acid encoding a heavy chain variable region specified by the amino acid sequence set forth in SEQ ID NO: 7. The present invention also provides, for example, a nucleic acid encoding a light chain variable region specified by the amino acid sequence set forth in SEQ ID NO: 8. The present invention also provides, for example, a nucleic acid encoding a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7. The present invention also provides, for example, a nucleic acid encoding a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. Note that in SEQ ID NO: 7, amino acid numbers 1 to 19 are signal peptides, and in SEQ ID NO: 8, amino acid numbers 1 to 22 are signal peptides. These signal peptides are typically cleaved when antibodies are secreted outside cells, and therefore functional antibodies do not contain these signal peptides.

[0037] The present invention provides an antibody, particularly a humanized antibody, having CDR1 to CDR3 of a heavy chain constant region set forth in SEQ ID NO: 11 and CDR1 to CDR3 of a light chain variable region set forth in SEQ ID NO: 14. The present invention also provides an antibody, particularly a humanized antibody, having CDR1 to CDR3 of a heavy chain constant region set forth in SEQ ID NO: 12 and CDR1 to CDR3 of a light chain variable region set forth in SEQ ID NO: 15. The present invention further provides an antibody, particularly a humanized antibody, having CDR1 to CDR3 of a heavy chain constant region set forth in SEQ ID NO: 13 and CDR1 to CDR3 of a light chain variable region set forth in SEQ ID NO: 16.

[0038] The present invention also provides a humanized antibody comprising a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 11 to 13 and a light chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 14 to 16. The present invention also provides an antibody, particularly a humanized antibody, having a heavy chain constant region set forth in SEQ ID NO: 11 and a light chain variable region set forth in SEQ ID NO: 14. The present invention also provides an antibody, particularly a humanized antibody, having a heavy chain constant region set forth in SEQ ID NO: 12 and a light chain variable region set forth in SEQ ID NO: 15. The present invention also provides an antibody, particularly a humanized antibody, having a heavy chain constant region set forth in SEQ ID NO: 13 and a light chain variable region set forth in SEQ ID NO: 16.

[0039] In one aspect of the present invention, there is provided a protein expression vector incorporating a nucleic acid of the present invention, which is operably linked to an expression promoter capable of expressing the protein in a host cell. Those skilled in the art can appropriately select and use the protein expression vector and its promoter. Those skilled in the art can also appropriately select and use host cells for protein expression, such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells)) and animal cells (e.g., insect cells).

[0040] Antibody Production Polyclonal antibodies can be obtained by immunizing an animal with an antigen and obtaining the plasma of the immunized animal. Monoclonal antibodies can be obtained by immunizing an animal with an antigen, obtaining B lymphocytes from the immunized animal, fusing them with myeloma cells to form hybridomas, and cloning the hybridomas that produce the desired antibody. The antibodies of the present invention can be obtained, for example, by immunizing an animal (e.g., intraperitoneally administering) a cell suspension containing live tumor cells (e.g., integrin α4-positive tumor cells such as NK / T lymphoma cell lines NKL and NKYS). If an adjuvant is used during immunization, the cells will be destroyed, and intracellular substances will also become immunogens. By not using an adjuvant during immunization, cells can be administered in an undestroyed state, enabling effective immune induction against membrane proteins. Therefore, the present disclosure provides a method for obtaining an antibody against the extracellular domain of a membrane molecule (e.g., a membrane protein), the method comprising administering (e.g., intraperitoneally administering) cells expressing the membrane molecule to a non-human mammal in an undisrupted state. An undisrupted state means that the cell membrane of the cell is not disrupted. A living cell is a cell in an undisrupted state.

[0041] Chimeric antibodies can be produced from animal antibodies by methods well known in the art. For example, they can be produced by substituting the constant region of an antibody with the constant region of a human antibody. Humanized antibodies, for example, contain complementarity-determining regions (CDRs) derived from a non-human animal, framework regions derived from a human antibody, and constant regions derived from a human antibody. Humanized antibodies can be obtained, for example, by grafting the above-mentioned CDRs onto a human antibody. Human antibodies can also be obtained, for example, by immunizing a genetically modified mouse that produces human antibodies with an antigen. Bispecific antibodies are antibodies that can bind to two different epitopes or antigens, and can be prepared by methods well known to those skilled in the art. Bispecific antibodies can be produced, for example, by fusing cells producing two different antibodies to produce hybrid hybridomas, or by V H Area and V LThe V domains are expressed on one polypeptide chain via a short linker that does not allow pairing between the two domains, and the V domains are expressed on another polypeptide chain via a short linker that does not allow pairing between the two domains. H Area and V L Complementary V pairing region H Area and V L Bispecific antibodies can be produced by forming a complex with a polypeptide chain having a specific region. Both arms of a bispecific antibody may bind to integrin α4, or one arm may bind to integrin α4 and the other arm may bind to another antigen. The other antigen may be, for example, the T cell receptor complex or a portion thereof (e.g., CD3ε) or another integrin (e.g., integrin β1). Bispecific antibodies against the T cell receptor complex and a tumor antigen are called T cell recruiting antibodies (TR antibodies) and can induce the destruction of tumor cells by T cells. In some aspects of the present invention, the antibodies of the present invention can be chimeric, humanized, or human antibodies with ADCC activity, such as IgG1 or IgG3.

[0042] Antibodies that compete with a given antibody for binding to an antigen can be obtained by competitive assays well known to those skilled in the art. Antibodies that can block the binding of a desired antibody by, for example, at least 20%, preferably at least 20-50%, and more preferably at least 50% in a competitive assay can be considered to be competing antibodies for binding to the same antigen. Competing antibodies can be confirmed by cross-blocking assays, preferably competitive ELISA assays. In cross-blocking assays, an antigen is coated on, for example, a microtiter plate, and a candidate competing antibody is added and incubated to allow binding between the antigen and the candidate antibody. The desired antibody is then labeled and added to the well, incubated, washed, and the amount of binding of the desired antibody is quantified to determine whether the antibodies compete. If there is competition, the amount of label remaining in the well should be reduced. In the present invention, the antibody can be an antibody that competes for binding to tumor cells, particularly integrin α4, or an antibody that competes for binding to antigen molecules (particularly integrin α4) displayed on the surface of cancer cells. In the present invention, the antibody may also have the same epitope as any of the antibodies (a) to (f) above. Antibodies with the same epitope can be confirmed by a competition assay, as described above. In the competition assay, the competition of the antibody with integrin α4-expressing tumor cells is preferably evaluated.

[0043] The present invention provides antibodies (preferably humanized antibodies) having killing activity (particularly killing activity against NK / T lymphoma cells; for example, NKL cells).

[0044] The present invention provides use of an antibody of the present invention or an antigen-binding fragment thereof for the manufacture of a pharmaceutical composition for treating cancer. The antibody of the present invention may be any of the antibodies (a) to (f) above, or may compete with any of the antibodies (a) to (f) above for binding to tumor cells, particularly integrin α4, or may be an antibody having the same epitope as any of the antibodies (a) to (f). As described above, the antibody may be conjugated to a cytotoxic agent. In one embodiment, the antibody does not include heavy chain CDR1-CDR3 and light chain CDR1-CDR3 of the anti-integrin α4 antibody 9F10 clone, or antibodies that differ from these sequences by only 1 to 5 amino acids. In one embodiment, the antibody does not have the ability to bind to integrin β1. The ability to bind to integrin β1 can be determined by testing whether the antibody exhibits significant binding to integrin β1 compared to an isotype antibody. An antibody that emits a signal within two-fold of the background signal of an isotype antibody does not exhibit significant binding to integrin β1.

[0045] The present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody of the present invention or an antigen-binding fragment thereof. The antibody of the present invention may be any of the antibodies (a) to (f) above, or may be an antibody that competes with any of the antibodies (a) to (f) above for binding to tumor cells, or may be an antibody that shares the same epitope as any of the antibodies (a) to (f) above. As described above, the antibody may be conjugated to a cytotoxic agent.

[0046] The present invention will be described below by way of non-limiting examples. However, the following examples are illustrative examples and are not intended to limit the scope of the present invention.

[0047] Example 1: Preparation of hybridomas producing cell death-inducing antibodies (1) Immunization method Two types of NK / T lymphoma cell lines, NKL and NKYS, were used as immunogens. After washing twice with physiological saline, 3 × 10 7The cells were suspended in 200 μL of physiological saline. Two types of live NK / T lymphoma cell lines were intraperitoneally administered alternately to 8-week-old female BALB / c mice (Charles River Laboratories) every two weeks, three times each, for a total of six doses.

[0048] (2) Hybridoma Production Method by Cell Fusion Three days after the final immunization, 1 x 10 spleen cells from the immunized mice were cultured in a 500-well plate. 8 and P3U1 myeloma cells 1 x 10 8 The cells were fused using PEG 4000. The cells were suspended in HAT selection medium and cultured in 50 96-well flat-bottom microtiter plates. Cell fusion was successful, and growing hybridomas were obtained.

[0049] (3) Screening: NK / T lymphoma cell line NK-92, which was not used for immunization, was added to 2 × 10 6 The solution was adjusted to a concentration of 10 ...

[0050] (4) Results As shown in Figure 1, the screening results allowed us to obtain hybridoma #ANAP, which produces an antibody that induces cell death in NK-92 and also in the NKL and NKYS used in immunization (see Figure 1). As shown in Figure 1, cell death caused by the antibody produced by hybridoma #ANAP was accompanied by a change in cell shape and was clearly evident under a microscope. After two rounds of limiting dilution, we obtained cloned hybridoma #ANAP. We confirmed that the subclass of the antibody produced by hybridoma #ANAP (hereinafter referred to as "ANAP antibody") is mouse IgG1.

[0051] Example 2: Evaluation of in vitro cytotoxic activity of ANAP antibody In Example 1, an ANAP antibody was obtained in the screening using its ability to induce cell death of NK-92, NKL, and NKYS as an indicator. In this example, the cytotoxic activity of the ANAP antibody was evaluated in more detail.

[0052] The cytotoxic activity of the antibody was evaluated using the NKL NK / T lymphoma cell line. The ANAP antibody obtained in Example 1 and an anti-HLA class II binding antibody (4713 antibody) were used as a control, and the results were compared.

[0053] 2 x 10 6 50 μL of each cell line was added to a 96-well flat-bottom microtiter plate, and 50 μL of 4713 antibody or ANAP antibody at 0.002, 0.2, or 2 μg / mL was added to each well. After incubation at 37°C for 30 minutes or 1 hour, 7-amino-actinomycin (7AAD) (BD) was added, and cell death was analyzed using a flow cytometer (FACSCant BD). The results are shown in Figure 2.

[0054] As shown in Figure 2, in the group to which the ANAP antibody was added at a concentration of 1 μg / mL, in addition to one sharp peak, 7AAD-positive cells that had undergone cell death were observed to the right of that peak. On the other hand, in the group to which the 4713 antibody was added at a concentration of 1 μg / mL, although the antibody bound to HLA class II on the cell surface, almost no 7AAD-positive cells that had undergone cell death were observed. Note that the flow cytometer is thought to count only cells that retained their cell morphology among the dead cells, and to count the many other cell fragments that had lost their original shape.

[0055] Next, the time course of cell death induction was examined. 6 50 μL of each cell line (1 μg / mL) was added to a 96-well flat-bottom microtiter plate, and 50 μL of 2 μg / mL ANAP antibody was added to each well. After incubation at 37°C for 15, 30, 60, 90, 120, 180, or 240 minutes, cells were stained with trypan blue and counted. The results are shown in Figure 3.

[0056] As shown in FIG. 3, the ANAP antibody induced cell death in the NKL cell line, and after 240 minutes of incubation, it induced cell death in about 80% of the NKL cells.

[0057] Furthermore, these experiments also revealed that the cytotoxic activity of the ANAP antibody does not correspond to either antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0058] Example 3: Comparison of cytotoxic activity with anti-vimentin antibody In this example, the commercially available anti-vimentin monoclonal antibody V9 was compared with the ANAP antibody in terms of cytotoxic activity.

[0059] Anti-vimentin monoclonal antibody V9 was purchased from Santa Cruz Biotechnology. 6 NKL cells (50 μL / mL) were added to a 96-well flat-bottom microtiter plate, and 50 μL of either ANAP antibody or V9 antibody (50 μL each) at the concentrations indicated in the figure was added to each well. After incubation at 37°C for 30 minutes or 1 hour, 7-amino-actinomycin (7AAD) (BD) was added, and cell death was analyzed using a flow cytometer (FACSCant BD). The results are shown in Figure 3-2.

[0060] As shown in Figure 3-2, the V9 antibody, which binds to a molecule (vimentin) different from the molecule bound by the antibody of the present invention, did not induce cell death at any of the concentrations confirmed, while the ANAP antibody exhibited concentration-dependent cytotoxic activity.

[0061] The above examples also revealed that the ANAP antibody recognizes surface molecules on tumor cells, and when it binds to the surface of tumor cells, it opens holes in the tumor cells and kills the cells. Furthermore, the cytotoxic activity of the ANAP antibody does not correspond to either antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), suggesting that the ANAP antibody induces cell death directly or indirectly. The difference between the V9 antibody and the ANAP antibody is the antigen molecule, and it has been suggested that the presence or absence of cytotoxicity is determined by the difference in the antigen molecule.

[0062] Example 4: Electron microscope observation of NKL cells after treatment with ANAP antibody In this example, the morphology of NKL cells in which cell death was induced by ANAP antibody was observed using an electron microscope.

[0063] 2 x 10 6 50 μL of NKL cell line (1 μg / mL) was added to each well of a 96-well flat-bottom microtiter plate, and 50 μL of 2 μg / mL ANAP antibody was added to each well. The plate was then incubated at 37°C for 20 minutes. The cells were harvested, suspended in 0.1 M bicarbonate buffer (pH 7.3) containing 1% glutaraldehyde, and incubated at 4°C for 2 hours. After washing with 0.1 M bicarbonate buffer, the plate was incubated with 1% osmium tetroxide (OsO4) solution at 4°C for 1 hour. The cells were dehydrated with 50-100% ethyl alcohol and then further dehydrated with t-butyl alcohol using a freeze dryer (ID-2, Giko). The cells were mounted on gold-palladium-coated conductive double-sided tape and analyzed using a scanning electron microscope (S-430, Hitachi Ltd.). The results are shown in Figure 4.

[0064] Figure 4A and B show NKL cells in the absence of ANAP antibody. 20 minutes after the addition of ANAP antibody, cell death was induced in the NKL cells, and it was observed that the cell membrane of the NKL cells was severely damaged (Figures 4C, D, E, and F). With continued incubation, the cells were observed to shrink (Figures 4G and H). These electron microscopic images suggest that cell death induced by ANAP antibody is morphologically distinct from apoptosis and necrosis.

[0065] Example 5: Measurement of cytotoxic activity of ANAP antibody against cells other than NKL cells In this example, the cytotoxic activity of ANAP antibody against tumor cells other than NKL cells was examined.

[0066] The cytotoxic activity of the ANAP antibody against four NK / T lymphoma cell lines (NKL), two T lymphoma cell lines (HUT78 and Jurkat), one Hodgkin's lymphoma cell line (L428), one acute lymphoblastic leukemia T cell line (MOLT-4), and a Burkitt's lymphoma B cell line (Raji) was examined. In addition to the FITC-labeled ANAP antibody, a FITC-labeled mouse IgG1 isotype control antibody was used as a negative control.

[0067] 2 x 10 6 50 μL of each cell line (1 μg / mL) was added to a 96-well flat-bottom microtiter plate, followed by 50 μL of 2 μg / mL ANAP antibody. After incubation at 37°C for 1 hour, dead cells were stained with propidium iodide (PI) and analyzed using a flow cytometer. The binding of the antibodies to each cell line was shown in Figure 5, and the cytotoxic activity of the antibodies was shown in Figure 6.

[0068] As shown in Figure 5, the FITC-labeled ANAP antibody bound to all lymphoma cell lines. Furthermore, as shown in Figure 6, the ANAP antibody was found to induce cell death in lymphoma cells other than NK lymphoma and leukemia cells by PI staining.

[0069] Thus, it has become clear that the antigen of the ANAP antibody is widely expressed on the surface of lymphoma and leukemia cells, and that the ANAP antibody exhibits antitumor activity, particularly cytotoxic activity, against a wide range of lymphoma and leukemia cells. It has also become clear that the ANAP antibody exhibits cytotoxic activity against target cells bound to it via molecules expressed outside the cells, probably through a common mechanism between different cells.

[0070] Next, as in Example 3, a human malignant mesothelioma cell line (MSTO-211H), another cancer type, was treated with ANAP antibody, and optical microscope images of trypan blue-stained cells during the process of inducing cell death are shown in Figure 7, and the results of electron microscope observation after induction of cell death are shown in Figure 7-2.

[0071] As shown in FIG. 7, dead cells stained with trypan blue began to appear 20 minutes after the start of ANAP antibody treatment, and after 60 minutes, almost all cells had become dead cells that stained positively with trypan blue.

[0072] Furthermore, Figure 7-2, A and B, show MSTO-211H cells in the absence of ANAP antibody, but as shown in Figure 7-2, C and D, treatment with ANAP antibody induced cell death in MSTO-211H cells. This indicates that cell death is induced not only in lymphoma cells and leukemia cells, but also in other types of cancer cells. Furthermore, as shown in Figure 7-2, C and D, cells in which cell death was induced had severe damage to the cell membrane. Therefore, it was confirmed that induced cell death in other types of cancer cells also occurs by a mechanism different from apoptosis or necrosis.

[0073] Next, we added ANAP antibody to malignant melanoma cells, a different type of cancer, and observed cell size by flow cytometry. The results are shown in Figure 7-3. In the malignant melanoma cell lines 526mel and 1363mel, no cell debris (small FSC and SSC) was observed after 1 hour of incubation when control IgG1 or 4713 antibody was added. However, in the cell lines to which ANAP antibody was added, cell debris (areas with small FSC and SSC; area enclosed by a square in the figure) significantly increased. The cell debris was thought to be derived from dead cells.

[0074] Example 6: Measurement of cytotoxic activity of ANAP antibody against normal cells In this example, it was examined whether or not the ANAP antibody exhibits cytotoxicity against normal cells.

[0075] Peripheral blood from healthy volunteers was used as normal cells. As antibodies, an FITC-labeled mouse IgG1 isotype control antibody was used as a negative control, and an FITC-labeled ANAP antibody was used as a test group.

[0076] Mononuclear cells were collected from the peripheral blood of healthy volunteers by density gradient centrifugation using Ficoll. 6 50 μL of PBMCs (100 μg / mL) were added to a 96-well flat-bottom microtiter plate, and 50 μL of 2 μg / mL isotype control antibody or ANAP antibody was added to each well. After incubation at 37°C for 1 hour, cell death was detected by staining with propidium iodide (PI) and analyzed by flow cytometry. The results are shown in Figure 8.

[0077] As shown in Figure 8, no induction of cell death was observed in normal human peripheral blood granulocytes. The ANAP antibody did not exhibit cytotoxicity against other normal lymphocytes.

[0078] Example 7: Analysis of target molecule of ANAP antibody In this example, an attempt was made to identify the target molecule of ANAP antibody.

[0079] An ANAP antibody affinity column was prepared by binding ANAP antibody (10 mg) to HiTrap NHS-Activated HP according to the GE Healthcare protocol. 9The analytes (80 ... This fraction was subjected to liquid chromatography mass spectrometry (LC-ms / ms) to extract membrane proteins, and integrin α4 (ITGA4) (CD49d) and integrin β1 (ITGB1) were selected as candidates.

[0080] If the antigen of the ANAP antibody is ITGA4, knockdown of ITGA4 would likely result in reduced antibody binding to cells. Therefore, we analyzed negative control and ITGA4 siRNA-transfected malignant mesothelioma cells MSTO-211H by flow cytometry using a control antibody (clone MOPC-21), mAb ANAP, a commercially available anti-ITGA4 antibody (clone 9F10), and an anti-HLA-C antibody (clone DT-9). The results showed that the reactivity of the ANAP antibody and the commercially available anti-ITGA4 antibody to ITGA4-knockdown cells was reduced, strongly suggesting that the antigen of the ANAP antibody is ITGA4 (see Figure 9C).

[0081] ITGA4 forms a heterodimer with integrin β1 (ITGB1) (CD29). Therefore, it is possible that either ITGB1 is the true antigen for the ANAP antibody. To confirm this possibility, NKL cell lysates biotinylated with ANAP and 9F10 antibodies were subjected to immunoprecipitation and subsequent Western blotting. First, chemiluminescence staining was performed using streptavidin-peroxidase and its substrate (see Figure 9D, left). After destaining, the lysates were reacted with anti-ITGA4, anti-IGTB1, or anti-ITGB7 antibodies, respectively, and then stained with a peroxidase-conjugated secondary antibody (goat anti-rabbit IgG). The results are shown in Figure 9D. As shown in Figure 9D, the immunoprecipitates with ANAP and 9F10 antibodies each showed a band of approximately 150 kDa, which was detected with the anti-ITGA4 antibody. A band that bound to the anti-ITGB1 antibody was observed in the immunoprecipitate using the 9G10 antibody (see FIG. 9D), supporting the idea that the ANAP antibody recognizes ITGA4.

[0082] A competition assay between the ANAP antibody and the 9F10 antibody was performed. NKL cells were incubated with the control antibody MOPC-21 (10 μg), and then Alexa Fluor 488-labeled ANAP antibody was added to the NKL cells (Fig. 9E, upper panel). Because the epitopes of the ANAP and MOPC-21 antibodies reside on two different molecules on the cells, the labeled ANAP antibody bound freely to the cells independently of the control antibody, emitting a strong signal in FCM analysis (Fig. 9E, upper panel).

[0083] On the other hand, when NKL cells were first incubated with a pre-existing anti-ITGA4 antibody (9F10) and then the Alexa Flour 488-labeled ANAP antibody was added to the cells, interference with the ANAP-Alexa Flour 488 binding was clearly observed, as evidenced by a significant downward shift in the signal peak in FCM (see Figure 9E, upper panel). These results indicate that the epitopes of the two antibodies are in close proximity on a single molecule, namely ITGA4. This conclusion was also verified by the reverse experiment, in which NKL cells were first incubated with ANAP and then the labeled anti-ITGA4 (9F10)-FITC antibody was added to the cells (see Figure 9E, lower panel).

[0084] Example 8: Sequencing of ANAP antibody gene 1. Mouse antibody (IgG) sequence-specific RT reaction cDNA was synthesized using a mouse antibody (IgG) heavy chain-specific primer (H-RT1:TCCAKAGTTCCA (SEQ ID NO: 9)) and total RNA prepared according to standard methods from a hybridoma producing the ANAP antibody as a template. Similarly, cDNA was synthesized using a light chain-specific primer (L-RT1:GCTGTCCTGATC (SEQ ID NO: 10)). The RT reaction was carried out according to the instructions for the SMARTer™ RACE cDNA Amplification Kit (Clontech Cat. No. 634924) under the following conditions. (1) 0.5 μg of total RNA, 1 μl of H-RT1 or L-RT1 (12 μM), and 3.75 μl of dH 2 O were mixed and reacted at 70° C. for 3 minutes and at 42° C. for 2 minutes. (2) To the reaction mixture, 1 μl of SMARTer II A Oligonucleotide (12 μM), 1 μl of DTT (20 mM), 1 μl of dNTP Mix (10 mM each), 2 μl of 5x First-Strand Buffer, 0.25 μl of RNase Inhibitor (40 U / μl), and 1 μl of SMARTScribe™ Reverse Transcriptase (100 U / μl) were added, and the mixture was incubated at 42°C for 90 minutes and then at 70°C for 10 minutes. (3) The reaction was stopped by adding 50 μl of Tricine-EDTA buffer, and the mixture was stored at -20°C.

[0085] 2. Mouse Antibody (IgG) Sequence-Specific RACE PCR Reaction 5' RACE PCR analysis was performed using the SMARTer™ RACE cDNA Amplification Kit. (1) Using the cDNA synthesized in 1 above (synthesized using a heavy chain-specific primer) as a template, a mouse antibody (IgG) heavy chain-specific primer was used as the reverse primer, and the UPM (Universal Primer Mix) included in the kit was used as the forward primer. Similarly, a RACE PCR reaction was performed using the cDNA (synthesized using a light chain-specific primer) as a template and a light chain-specific primer. PrimeSTAR (Takara Bio Inc.) was used as the PCR enzyme. The PCR reaction was performed according to the manufacturer's manual included with the kit. (2) The expected size of PCR product was confirmed by agarose gel electrophoresis. The PCR products were designated H-chain RT-PCR (SYN3460H) and L-chain RT-PCR (SYN3460L), excised from the gel, purified, and used for analysis.

[0086] 3. Cloning and Sequence Analysis (1) Each of the PCR products (SYN3460H and SYN3460L) purified as described above was ligated to the cloning plasmid pMD20-T (Takara Bio Inc.). (2) Transformation was performed using standard methods, and 48 clones were obtained for each PCR product. (3) The sequences of the inserts contained in the obtained clones were analyzed according to standard methods. Sequencing reactions were performed using the BigDye Terminators v3.1 Cycle Sequencing Kit (ABI) on an ABI3730 Sequencer (ABI) according to the manufacturer's instructions. (4) The sequence analysis results for 48 clones each for the heavy chain and light chain, as well as the sequences excluding the vector region and regions of low accuracy, were obtained.

[0087] 4. Sequencing Next, the following analysis was performed using the base sequences obtained in 3-(4) above. (1) Classification of Obtained Sequences and Obtaining Consensus Sequences The base sequences of the heavy and light chains were classified by homology. Homology comparison was performed using DNA Sequence Assembly software, SEQUENCHER™ (Gene Codes: Windows version). As a result, two contigs were obtained for the heavy chain and three for the light chain. A consensus sequence was obtained from the obtained contigs. (2) Candidate Sequences for the Target Gene Sequences that were considered to be candidates for the target gene were selected from the consensus sequences and sequences that did not form contigs. Here, all sequences that had a methionine residue without a stop codon upstream of the amino acid sequence of the antibody constant region gene were selected. (3) Estimation of Amino Acid Sequences Based on the number of sequences that formed contigs among the candidate sequences and the estimated gene lengths of the obtained sequences, it was determined that the major contigs for the heavy and light chains were likely to be the target sequences. Therefore, the amino acid sequences encoded by the consensus sequences of each major contig were determined to be the amino acid sequences of the heavy and light chains.

[0088] The amino acid sequences of the heavy and light chain variable regions of the ANAP antibody obtained by the above method are shown in Figure 10. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. According to the numbering system of Kabat et al. (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH.), in SEQ ID NO: 7, positions 1 to 19 correspond to a leader sequence, positions 20 and onwards correspond to a heavy chain variable region, positions 45 to 54 correspond to CDR1, positions 70 to 85 correspond to CDR2, and positions 118 to 127 correspond to CDR3 (see SEQ ID NOs: 1 to 3, respectively). Similarly, according to the numbering of Kabat et al., in SEQ ID NO: 8, positions 1 to 22 correspond to the leader sequence, positions 23 and onwards correspond to the light chain variable region, positions 46 to 55 correspond to CDR1, positions 71 to 77 correspond to CDR2, and positions 110 to 119 correspond to CDR3 (see SEQ ID NOs: 4 to 6, respectively). In Figure 10, CDRs 1 to 3 of the heavy and light chains are each underlined and written in bold.

[0089] The amino acid sequence of the ANAP antibody (mouse anti-human ITGA4 mAb) was modified to generate a mouse-human chimeric antibody (TNK1-G) and humanized mAb ANAP antibodies (TNK1-H-1 to 21). The chimeric antibody and 11 humanized antibodies bound to the NK / T lymphoma cell line NKL cells (Figure 11). The full amino acid sequences of the humanized ANAP antibodies TNK1-H-18, TNK1-H-20, and TNK1-H-21, which showed the strongest binding, are shown in Tables 1 and 2. TNK1-H-18 has the heavy and light chain constant regions of Example 1, TNK1-H-20 has the heavy and light chain constant regions of Example 2, and TNK1-H-21 has the heavy and light chain constant regions of Example 3. These three humanized antibodies exhibited 40% to 62% cytotoxic activity against NKL cells after 12 hours of incubation (Table 2).

[0090]

[0091]

[0092]

[0093] Sequence Listing SEQ ID NOs: 1 to 3 show the amino acid sequences of heavy chain CDR1 to 3 of the ANAP antibody, respectively. SEQ ID NOs: 4 to 6 show the amino acid sequences of light chain CDR1 to 3 of the ANAP antibody, respectively. SEQ ID NO: 7 shows the amino acid sequence of the heavy chain variable region of the ANAP antibody. SEQ ID NO: 8 shows the amino acid sequence of the light chain variable region of the ANAP antibody. SEQ ID NOs: 9 and 10 show the nucleotide sequences of primers specific to the heavy and light chains of a mouse antibody (IgG), respectively.

Claims

1. An antibody or antigen-binding fragment thereof that binds to integrin α4, wherein the antibody or antigen-binding fragment thereof: (a) has a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the amino acid sequence of the heavy chain variable region of SEQ ID NO: 7, and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3 in the amino acid sequence of the light chain variable region of SEQ ID NO: 8; (b) an antibody or antigen-binding fragment thereof having a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8; (c) an antibody or antigen-binding fragment thereof having a heavy chain variable region comprising heavy chain CDR1 to 3 of the heavy chain constant region of SEQ ID NO: 11 and a light chain variable region comprising light chain CDR1 to 3 of the light chain constant region of SEQ ID NO: 14; or (d) an antibody or antigen-binding fragment thereof having a heavy chain variable region comprising heavy chain CDR1 to 3 of the heavy chain constant region of SEQ ID NO: 12 and a light chain variable region comprising light chain CDR1 to 3 of the light chain constant region of SEQ ID NO: 15 (e) an antibody or antigen-binding fragment thereof having a heavy chain variable region having heavy chain CDR1 to CDR3 of the heavy chain constant region set forth in SEQ ID NO: 13 and a light chain variable region having light chain CDR1 to CDR3 of the light chain constant region set forth in SEQ ID NO: 16, or (f) an antibody or antigen-binding fragment thereof that competes with the antibody set forth in any of (b) and (c) to (e) above for binding to integrin α4 {with the proviso that the antibody is not the 9F10 antibody, nor an antibody having heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 of 9F10, nor an antibody containing mutations of 5 or fewer amino acids in the heavy chain and light chain CDR1 to CDR3.} 2. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in claim 1.

3. The pharmaceutical composition according to claim 2, wherein: (g) the antibody is an antibody having a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region having a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (h) the antibody is an antibody having a heavy chain constant region set forth in SEQ ID NO: 11 and a light chain constant region set forth in SEQ ID NO: 14; (i) an antibody having a heavy chain constant region set forth in SEQ ID NO: 12 and a light chain constant region set forth in SEQ ID NO: 15; (j) an antibody having a heavy chain constant region set forth in SEQ ID NO: 13 and a light chain constant region set forth in SEQ ID NO: 16; or (k) an antibody that competes with any of the antibodies set forth in (g) to (j) above for binding to integrin α4, but precipitates only α4 in the immunoprecipitation of α4β1.

4. A pharmaceutical composition comprising a conjugate of an antibody defined in any one of claims 1 to 3 and an anticancer agent.

5. A pharmaceutical composition according to any one of claims 2 to 4 for use in treating cancer.

6. The pharmaceutical composition according to claim 5, wherein the cancer is selected from the group consisting of hematopoietic tumors, malignant melanoma, and mesothelioma.

7. The pharmaceutical composition according to claim 6, wherein the hematopoietic tumor is leukemia, acute leukemia, chronic leukemia, monocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and / or lymphoma.

8. The pharmaceutical composition according to claim 7, wherein the hematopoietic tumor is Hodgkin's lymphoma, hairy cell leukemia, multiple myeloma, B-cell lymphoma and / or T-cell lymphoma.

9. The pharmaceutical composition according to claim 7, wherein the hematopoietic tumor is an NK / T cell tumor.

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