Antibody

Abstract

To provide an active ingredient of a pharmaceutical composition for treating myeloma, specifically binding to a myeloma cell or its precursor.SOLUTION: An antibody has an epitope in a region of the amino acid residue positions 20 to 109 of human integrin β7. The antibody does not recognize normal cells and is, therefore, useful as an active ingredient of a therapeutic agent for cancer (e.g., blood cancer). A chimeric antigen receptor T cell prepared by applying an antigen recognition site to a chimeric antigen receptor can be used as an active ingredient of the above pharmaceutical composition.SELECTED DRAWING: None

Description

[Technical Field]

[0001] New antibodies and their uses are disclosed. [Background technology]

[0002] Multiple myeloma, a representative example of a disease that causes neoplastic proliferation of plasma cells, is one of the most common cancers. Multiple myeloma accounts for approximately 1% of all hematologic malignancies and just over 10% of all hematologic malignancies. Plasma cells present in the nucleus become cancerous (resulting in abnormal plasma cells) and grow monoclonally. It is a disease that occurs.

[0003] In multiple myeloma, abnormal plasma cells (myeloma cells) spread to bone marrow throughout the body, causing bone marrow When abnormal plasma cells proliferate, various symptoms including bone destruction appear. Myeloma cells produce an abnormal immunoglobulin called M protein, which causes M in the blood. The increased protein concentration makes the blood thick.

[0004] M protein acts as an antibody, recognizing foreign substances such as pathogens that have entered the body. This affects many organs and leads to various Typical symptoms include bone pain and damage, hypercalcemia, kidney damage and failure, Anemia, etc.

[0005] Currently, proteasome inhibitors, thalidomide and its derivatives, are used to treat multiple myeloma. iMIDs such as lenalidomide, which is a vasopressin-resistant strain of cerebrospinal fluid (vasopressin), and combinations of melphalan and prednisone Chemotherapy and hematopoietic stem cell transplantation are the main treatments used.

[0006] However, myeloma cells almost always eventually develop resistance to these treatments. Therefore, with current treatment methods, the average survival time after onset is about 3 to 5 years, and bone The prognosis for myeloma patients is poor. Furthermore, these therapeutic drugs target tumor cells. As it does not act specifically on the target cells, it is also toxic to normal cells, resulting in However, there is a problem in that it is accompanied by serious side effects.

[0007] Attempts are being made to develop treatments for multiple myeloma using monoclonal antibodies. Antibodies such as anti-CS1 antibody and anti-CD38 antibody are considered promising (Non-Patent Documents 1 and 2). Patent Document 1 describes a drug for treating multiple myeloma, etc., which contains an anti-human CD48 monoclonal antibody as an active ingredient. A therapeutic agent for the treatment of

[0008] In vivo, integrins mainly form heterodimers of α and β chains, The α and β chains of such integrins function as receptors on the cell surface. The combinations are wide-ranging.

[0009] In addition, Non-Patent Documents 4 to 6 disclose a method for detecting a specific antigen containing an antigen recognition site having affinity for the specific antigen. Chimeric antigen receptor T cells (CAR-T cells) have been disclosed. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2010 / 117059 [Non-patent literature]

[0011] [Non-Patent Document 1] Journal of Clinical Oncology, 2012 Jun 1; 30(16): 1953-9. [Non-patent document 2] Journal of immunology, 2011 Feb 1; 186(3): 1840-8. [Non-patent document 3] J Biol Chem. 2012 May 4;287(19):15749-59. [Non-patent document 4] J Immunol. 2009 Nov 1;183(9):5563-74. [Non-Patent Document 5] N Engl J Med. 2014 Oct 16;371(16):1507-17. [Non-patent document 6] Nat Biotechnol. 2002 Jan;20(1):70-5. Summary of the Invention [Problem to be solved by the invention]

[0012] Although anti-CS1 antibody has relatively high specificity for myeloma cells, the antibody alone has a high anti-myeloma effect. However, its efficacy as a single agent has not been demonstrated in clinical trials. It was found that the antitumor effect of the anti-CS1 antibody is enhanced by this combination. On the other hand, CD38 is expressed on many normal blood cells, including CD34-positive hematopoietic progenitor cells. It is an antigen with low specificity as a therapeutic target for multiple myeloma because it is also expressed in cells. Under these circumstances, the treatment of diseases such as multiple myeloma that involve neoplastic proliferation of plasma cells is becoming increasingly difficult. One challenge is to provide more effective means. [Means for solving the problem]

[0013] The present inventors have conducted extensive research to solve these problems, and as a result, have discovered that myeloma cells and Screening was carried out using the specific binding to the precursor as an indicator, and the MMG49 antibody was obtained. It was then confirmed that the antibody binds to a specific region of human integrin β7. CAR-T cells engineered using the antigen-recognition site of a specific antibody are highly effective in treating myeloma. We also found that the epitope of the MMG49 antibody is located between positions 20 and 109 of human integrin β7. We also found that the region consists of the amino acid residues

[0014] The present invention has been completed based on these findings, and is an invention of the following broad aspects. Includes.

[0015] (I) Antibody The antibody (I) includes the antibodies shown in (I-1) to (I-25) below.

[0016] (I-1) An anti-human integrin β7 antibody, which is an antibody targeting amino acid residues 20 to 109 of human integrin β7. An antibody having an epitope in a region comprising the base. (1-1A) The epitope is located in the region consisting of amino acid residues 33-109 of human integrin β7. The antibody described in (I-1). (1-1B) The epitope is located in the region consisting of amino acid residues 20-90 of human integrin β7. The antibody described in (I-1). (1-1C) The epitope is located in the region consisting of amino acid residues 33-90 of human integrin β7. The antibody described in (I-1). (I-2) The presence of at least a portion of the region consisting of amino acid residues 379 to 721 of human integrin β7 The antibody according to (I-1), wherein the affinity for the epitope increases in the presence of a steroid hormone. (I-3) The presence of at least a portion of the region consisting of amino acid residues 417 to 721 of human integrin β7 The antibody according to (I-2), wherein the affinity for the epitope increases in the presence of a steroid hormone. (I-4) The presence of at least a portion of the region consisting of amino acid residues 564 to 721 of human integrin β7 The antibody according to (I-2), wherein the affinity for the epitope increases in the presence of a steroid hormone. (I-5) The presence of at least a portion of the region consisting of amino acid residues 379 to 563 of human integrin β7 The antibody according to (I-2), wherein the affinity for the epitope increases in the presence of a steroid hormone. (I-6) The presence of at least a portion of the region consisting of amino acid residues 417 to 563 of human integrin β7 The antibody according to (I-2), wherein the affinity for the epitope increases in the presence of a steroid hormone. (I-7) The presence of at least a portion of the region consisting of amino acid residues 379 to 416 of human integrin β7 The antibody according to (I-2), wherein the affinity for the epitope increases in the presence of a steroid hormone. (I-8) Activation of human integrin β7 increases affinity for the epitope. The antibody according to any one of I-1) to (I-7). (I-9) An anti-human integrin β7 antibody that inhibits human integrin β7 expressed on normal cells. This antibody has high affinity for human integrin β7, which is expressed on myeloma cells. (I-10) The antibody according to any one of (I-1) to (I-9), which has the same epitope as the MMG49 antibody. (I-11) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and / or Heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3 and / or a heavy chain variable region comprising a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:6; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and / or Light chain CDR3 having the amino acid sequence shown in SEQ ID NO:8 a light chain variable region comprising The antibody according to any one of (I-1) to (I-10), comprising: (I-12) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 4 and / or A light chain variable region having the amino acid sequence shown in SEQ ID NO:9 The antibody according to any one of (I-1) to (I-10), comprising: (I-13) Fv, scFv, diabody, triabody, tetrabody (I-1) The antibody according to any one of (I-1) to (I-12), which is a human IgG antibody, ... . (I-14) The antibody according to any one of (I-1) to (I-11), which comprises a constant region. (I-15) The antibody according to any one of (I-1) to (I-12) and (I-14), which is a chimeric antibody. (I-16) The antibody according to any one of (I-1) to (I-12) and (I-14), which is a humanized antibody. (I-17) The antibody according to any one of (I-1) to (I-12) and (I-14), which is a human antibody. (I-18) Immunoglobulin, Fab, F(ab')2, minibody, scFv-Fc, or any of these The antibody according to any one of (I-1) to (I-12) and (I-14) to (I-17), which is a combination. (I-19) IgA, IgD, IgE, IgG, or IgM, any of (I-1) to (I-12) and (I-14) to (I-18) The antibody described in item 1. (I-20) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 5 and / or the amino acid sequence set forth in SEQ ID NO: 10 any one of (I-1) to (I-12) and (I-14) to (I-19), comprising a light chain having the sequence antibody. (I-21) The antibody according to any one of (I-1) to (I-20), which has cytotoxic activity. (I-22) The antibody according to (I-21), wherein the cytotoxic activity is ADCC activity and / or CDC activity. (I-23) The antibody according to any one of (I-1) to (I-22), which is a multispecific antibody. (I-24) The antibody according to any one of (I-1) to (I-23), which is bound to a cytotoxin. (I-25) The antibody according to any one of (I-1) to (I-24), which is a monoclonal antibody.

[0017] (II) Polynucleotides The polynucleotide (II) includes the polynucleotide shown in (II-1) below. (II-1) A polynucleotide having a base sequence encoding the amino acid sequence of the antibody (I).

[0018] (III) Host cells The host cell (III) includes the host cells shown in (III-1) or (III-2) below. (III-1) A host cell harboring a polynucleotide (II). (III-2) The host cell according to (III-1), which is a eukaryotic cell.

[0019] (IV) Chimeric antigen receptor Chimeric antigen receptors (IV) include the chimeric antigen receptors shown in (IV-1) to (IV-5) below. (IV-1) A chimeric antigen receptor having the same epitope as the above antibody (I). (IV-2) A chimeric antigen receptor according to (IV-1), which comprises the antigen recognition site of the antibody (I). (IV-3) The antigen recognition site is a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and / or Heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3 and / or a heavy chain variable region comprising a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:6; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and / or Light chain CDR3 having the amino acid sequence shown in SEQ ID NO:8 a light chain variable region comprising The chimeric antigen receptor according to (IV-1) or (IV-2), comprising: (IV-4) The antigen recognition site is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 4 and / or A light chain variable region having the amino acid sequence shown in SEQ ID NO:9 The chimeric antigen receptor according to any one of (IV-1) to (IV-3), comprising: (IV-5) The chimera according to any one of (IV-1) to (IV-4), having the amino acid sequence shown in SEQ ID NO: 21. Antigen receptor.

[0020] (V) Polynucleotides The polynucleotide (V) is different from the polynucleotide (II) and is the following (V-1) or (V-2): ) is included. (V-1) A polynucleotide encoding the amino acid sequence of the chimeric antigen receptor (IV). (V-2) A polynucleotide according to (V-1), having the base sequence shown in SEQ ID NO: 22.

[0021] (VI) Cell The cell (VI) is different from the host cell (III) and is one of the following cells (VI-1) to (VI-4): Contains the cells. (VI-1) A cell harboring the above polynucleotide (V). (VI-2) The cell according to (VI-1), which is a eukaryotic cell. (VI-3) The cell according to (VI-1) or (VI-2), which is a T cell or an NK cell. (VI-4) Any of (VI-1) to (VI-3) which is a chimeric antigen receptor T cell or a chimeric antigen receptor NK cell 1. The cell according to claim 1.

[0022] (VII) Pharmaceutical Composition The pharmaceutical composition (VII) includes the pharmaceutical compositions shown in the following (VII-1) to (VII-5). (VII-1) A pharmaceutical composition comprising the above antibody (I) or the above cell (VI). (VII-2) The pharmaceutical composition according to (VII-1) above, wherein the cells are chimeric antigen receptor T cells (VI-4). (VII-3) The pharmaceutical composition according to (VII-1) or (VII-2), which is for treating cancer. (VII-4) The pharmaceutical composition according to (VII-3), wherein the cancer is a blood cancer. (VII-5) The pharmaceutical composition according to (VII-4), wherein the blood cancer is a disease that causes neoplastic proliferation of plasma cells.

[0023] (VIII) Methods for treating or preventing diseases The method for treating or preventing a disease (VIII) includes treating or preventing the diseases shown in the following (VIII-1) to (VIII-6). Prevention methods are included. (VIII-1) A method for treating a disease, comprising administering a therapeutically effective amount of the antibody (I) or the cell (VI) to a subject. Treatment or prevention methods. (VIII-2) The treatment or prevention method according to (VIII-1) above, wherein the cells are chimeric antigen receptor T cells (VI-4). method. (VIII-3) The disease is cancer, and the subject is a patient suffering from cancer or an animal likely to suffer from cancer. The method for treatment or prevention according to (VIII-1) or (VIII-2). (VIII-4) The method for treating or preventing cancer according to (VIII-3), wherein the cancer is a blood cancer. (VIII-5) The treatment or prevention method according to (VIII-4), wherein the blood cancer is a disease resulting in neoplastic proliferation of plasma cells. method. (VIII-6) A method for treating or preventing multiple myeloma that targets activated human integrin β7.

[0024] (IX) Use Use (IX) includes the uses shown in (IX-1) to (IX-5) below. (IX-1) Use of the above antibody (I) or the above cell (VI) for producing a pharmaceutical composition. (IX-2) The method for treating or preventing the disease according to (IX-1) above, wherein the cells are chimeric antigen receptor T cells (VI-4). Law. (IX-3) The use according to (IX-1) or (IX-2), which is for the treatment of cancer. (IX-4) The use according to (IX-3), wherein the cancer is a blood cancer. (IX-5) The use according to (IX-3), wherein the blood cancer is a disease resulting in neoplastic proliferation of plasma cells.

[0025] (X) Screening method The screening method (X) includes the following screening methods (X-1) to (X-5). (X-1) From the compound library, we have selected compounds that specifically bind to human integrin β7 and The method includes a step of selecting a candidate substance that binds to a region consisting of amino acid residues 20 to 109 of β7. A method for screening an active ingredient of a pharmaceutical composition for treating or preventing cancer. (X-2) The screening method according to (X-1) further comprises a step of selecting a substance having cytotoxic activity. How to do this. (X-3) The screening method according to (X-1) or (X-2), wherein the substance to be selected is a monoclonal antibody. method. (X-4) The screening method according to any one of (X-1) to (X-3), wherein the cancer is a blood cancer. (X-5) The screening method according to (X-4), wherein the blood cancer is a disease that causes neoplastic proliferation of plasma cells. Law.

[0026] (XI) Diagnostic methods The diagnostic method (XI) includes the diagnostic methods shown in (XI-1) to (XI-5) below. (XI-1) A method for diagnosing cancer, comprising the step of contacting a sample collected from a subject with the antibody (I). . (XI-2) A diagnostic method according to (XI-1), wherein the sample taken from the subject is blood or bone marrow fluid. (XI-3) When cells that bind to the above antibody (I) are detected, it is determined that the patient has or may have cancer. The diagnostic method according to (XI-1) or (XI-2), wherein the presence of the (XI-4) The diagnostic method according to (XI-3), wherein the cancer is a blood cancer. (XI-5) The cells are plasma cells, and the cancer is a disease resulting in neoplastic proliferation of plasma cells. (XI-4) The diagnostic method described in

[0027] (XII) Kit Kit (XII) includes the kits shown in (XII-1) to (XII-3) below. (XII-1) A cancer diagnostic kit comprising the above antibody (I). (XII-2) The diagnostic method according to (XII-1), wherein the cancer is a blood cancer. (XII-3) The kit according to (XII-2), wherein the cancer is a disease that causes neoplastic proliferation of plasma cells. [Effects of the Invention]

[0028] The antibody of the present invention does not recognize normal cells and is therefore useful as an active ingredient in pharmaceutical compositions. In particular, it is useful as an active ingredient in drugs for treating cancer (e.g., blood cancer).

[0029] The antibody of the present invention is produced by applying its antigen recognition site to a chimeric antigen receptor. The chimeric antigen receptor T cells can be used as an active ingredient of the pharmaceutical composition described above. This is useful because it allows [Brief explanation of the drawings]

[0030] [Figure 1] Results of FACS analysis of the binding of the MMG49 antibody to bone marrow cells derived from myeloma patients in Example 2. (Left) Diagram showing the method for identifying myeloma progenitor cells, myeloma plasma cells, and CD45+ leukocytes. (Right) Diagram showing the binding of the MMG49 antibody to each fraction. [Figure 2] FIG. 1 shows the results of FACS analysis of the binding of the MMG49 antibody to myeloma precursor cell fractions, myeloma plasma cell fractions, and CD45+ leukocytes of bone marrow cells (UPN1 to 5) derived from multiple myeloma patients in Example 2. [Figure 3] 1 shows the process of identifying the antigen protein recognized by the MMG49 antibody by expression cloning in Example 3. It also shows the process of enriching BaF3 cells that bind to the MMG49 antibody, which were initially less than 0.1%, by FACS sorting. [Figure 4] FIG. 1 shows the results of FACS analysis of ITGB7-deficient U266 cells prepared using the Crisp-cas9 system in Example 4, stained with MMG49 antibody or FIB27 antibody (commercially available anti-integrin β7 antibody). [Figure 5] In Example 4, immunoprecipitation was performed using MMG49 antibody or an isotype control antibody from a cell lysate derived from MM1s myeloma cells, followed by SDS-PAGE and Western blotting using a commercially available anti-integrin β7 antibody (Abcam). [Figure 6]FIG. 1 shows the results of FACS analysis of the binding of MMG49 antibody, FIB27 antibody, and FIB504 antibody to each cell fraction of peripheral blood cells from a healthy subject (showing, from left to right, B cells, T cells, monocytes, neutrophils, red blood cells, and platelets) in Example 5. [Figure 7] Figure 1 shows the results of FACS analysis of the binding of the MMG49 antibody to each cell fraction of bone marrow cells derived from a myeloma patient in Example 5. The left side shows the method for identifying each cell fraction, and the right side shows the binding of MMG49 to each fraction. A shows a comparison between hematopoietic stem cell and progenitor cell fractions and myeloma cells, and B shows a comparison between the B / T lymphocyte fraction and myeloma precursor cell and myeloma plasma cell fractions. [Figure 8] This figure shows the results of FACS analysis of the binding of MMG49 antibody and FIB27 antibody to various myeloma cell lines, peripheral blood-derived T cells, and B cells, respectively, in Example 6. Also shown are the results of FACS analysis confirming the expression of ITGA4 (binding of anti-integrin α4 antibody) and ITGAE (binding of anti-integrin αE antibody) in the above cells. [Figure 9] This figure shows the results of FACS analysis of the binding of MMG49 antibody and FIB27 antibody to U266 cells and ITGA4 (integrin α4)-deficient U266 cells in Example 6. The figure also shows the results of FACS analysis of ITGA4 expression (binding of anti-integrin α4 antibody) in the above cells. [Figure 10] In Example 7, integrin α4β7-expressing K562 cells and normal human peripheral blood-derived T cells were treated at 37°C for 20 minutes in the presence of Ca2+ / Mg2+ or Mn2+, reacted with MMG49 antibody or isotype antibody, and then stained with anti-mouse IgG antibody as a secondary antibody, and subjected to FACS analysis. [Figure 11] FIG. 10 shows the construction of a human / mouse chimeric integrin β7 protein in Example 8 and the presence or absence of binding of the MMG49 antibody to 293T cells transiently expressing the protein. [Figure 12]FIG. 10 shows the results of FACS analysis of the binding of the MMG49 antibody to 293T cells transiently expressing human / mouse chimeric integrin β7 protein in Example 8. [Figure 13] A diagram summarizing the results shown in Figure 12. In the graph in the figure, the vertical axis indicates the percentage of antibody-bound cells, and the horizontal axis indicates various human / mouse chimeric integrin β7 proteins. [Figure 14] FIG. 1 shows the results of staining MM1s cells and KMS12BM cells with a chimeric MMG49 antibody prepared by connecting the variable region of the MMG49 antibody to the constant region of a human IgG4 antibody. [Figure 15] Schematic showing the method for producing a CAR construct using the variable region of the MMG49 antibody. [Figure 16] FIG. 1 shows the results of staining T cells expressing a CAR construct using the variable region of the MMG49 antibody with a PE-anti-human F(ab')2 antibody. [Figure 17] Figure 11 shows the results of ELISA quantification of the amounts of IFN-γ and IL-2 produced by co-culture of MMG49 antibody-derived CAR-T cells or GFP-transfected T cells (control) with K562 cells that do not express integrin β7 or K562 cells that have been forced to express integrin α4β7, as described in Example 11. *: p<0.05. [Figure 18] FIG. 11 shows the results of ELISA quantification of the amount of IFN-γ produced by co-culture of MMG49 antibody-derived CAR-T cells or GFP-transfected T cells (control) with MMG49 antigen-expressing or non-expressing cells in Example 11. [Figure 19] FIG. 11 shows the results of ELISA quantification of the amount of IL2 produced by co-culture of MMG49 antibody-derived CAR-T cells or GFP-transfected T cells (control) with MMG49 antigen-expressing or non-expressing cells in Example 11. [Figure 20]This figure shows the results of measuring the degree of cytotoxicity of MMG49 antibody-derived CAR-T cells or GFP-transfected T cells (control) against K562 cells that do not express integrin β7 or K562 cells that have been forced to express integrin α4β7, using a 51Cr killing assay, in Example 11. The y-axis of the graph in the figure represents the cytotoxicity rate (%). [Figure 21] FIG. 11 shows the results of measuring the degree of cytotoxicity of MMG49 antibody-derived CAR-T cells or GFP-transfected T cells (control) against MMG49 antigen-expressing or non-expressing cells using a 51Cr killing assay in Example 11. [Figure 22] This figure shows the design and results of a therapeutic experiment conducted in Example 12 on the myeloma cell line MM1s engrafted in the bone marrow of NOG mice. One week after the transfer of MMG49 antibody-derived CAR-T cells or GFP-transfected T cells (control), bone marrow cells were collected and analyzed by FACS. MM1s cells can be identified as human CD138+ cells. MM1s cells in the bone marrow were almost completely eliminated in the group administered MMG49 antibody-derived CAR-T cells. [Figure 23] This figure shows the design and results of a therapeutic experiment on the myeloma cell line MM1s engrafted throughout the body of NOG mice in Example 12. The amount of myeloma cells before and after the transfer of MMG49 antibody-derived CAR-T cells or GFP-transfected T cells (control) was evaluated by measuring fluorescence intensity using IVIS imaging. MM1s cells in the bone marrow were almost completely eliminated in the group administered MMG49 antibody-derived CAR-T cells. [Figure 24] FIG. 1 shows a comparison of the amino acid sequences of human integrin β7 and mouse integrin β7. [Figure 25] FIG. 12 shows the construction of a human / mouse chimeric integrin β7 protein in Example 13 and the presence or absence of binding of the MMG49 antibody to 293T cells transiently expressing the protein. [Figure 26]1 shows the results of an experiment investigating the epitope of the MMG49 antibody in Example 14. MFI on the horizontal axis represents the binding strength to the MMG49 antibody, with higher values ​​indicating higher binding strength. DETAILED DESCRIPTION OF THE INVENTION

[0031] In this specification, "including" and "having" are so-called open language. However, these are concepts that include the closed-ended notion of "consisting only of" and In the form, it can be replaced with "consisting only of."

[0032] "Myeloma progenitor cells" are precursor cells that differentiate into myeloma plasma cells and have CD38. However, the absence of CD138, a marker specific to mature plasma cells, Therefore, myeloma progenitor cells are characterized by the expression of "CD38 ++ CD138 - cells" or "CD19 - CD3 8 ++ CD138 - Sometimes referred to as "cells."

[0033] "Myeloma plasma cells," commonly called myeloma cells, express an abnormal immunoglobulin called M Myeloma plasma cells express not only CD38 but also CD138. Therefore, myeloma plasma cells express "CD38 ++ CD138 + cells" or "CD19 - CD38 ++ CD138 + Sometimes referred to as "cells."

[0034] Myeloma precursor cells and myeloma plasma cells are neoplastic proliferations of plasma cells other than multiple myeloma. It also refers to tumor precursor cells and neoplastic plasma cells in diseases that cause proliferation.

[0035] "Hematopoietic progenitor cells" are cells that can differentiate into various blood cell lineage cells. Hematopoietic progenitor cells are characterized by the expression of CD34. + Sometimes referred to as "cells."

[0036] (I) Antibody The antibody (I) is preferably an anti-human integrin β7 antibody, and is It is an antibody having an epitope in the region consisting of amino acid residues 20 to 109 of

[0037] More preferably, the region consisting of amino acid residues 33 to 109 of human integrin β7 is Antibodies bearing a mitochondrial nucleotide sequence or a sequence consisting of amino acid residues 20-90 of human integrin β7 The most preferred antibody is an antibody having an epitope in the human integrin region. and an antibody having an epitope in the region consisting of amino acid residues 33 to 90 of phosphodiesterase β7. can be done.

[0038] Human integrin β7 is not particularly limited, and may be any of the following: It is a transmembrane protein that forms a heterodimer with integrin α. Specific examples of integrin α include integrin α4 and integrin α5. α E Examples include:

[0039] Specific amino acid sequences of human integrin β7 include the amino acid sequence shown in SEQ ID NO: 31 and the following: Also listed in the NCBI database, for example, ACCESSION:EAW96675; VERSION:EAW966 75.1,GI:119617081,ACCESSION:NM000889; VERSION:NM000889.2,GI:540344585,ACCESSIO N:XM005268851, VERSION:XM005268851.2,GI:767974096, ACCESSION:XM006719376, VERSI ON:XM006719376.2,GI:767974098, ACCESSION:XM005268852, VERSION:XM005268852.3,GI: 767974097 and the like.

[0040] The following description of human integrin β7 is based on the amino acid sequence shown in SEQ ID NO:31. However, the amino acid sequence of other human integrin β7 can be determined in silico as shown in SEQ ID NO: 31. The homology with the amino acid sequence of human integrin β7, which is described below, was confirmed. The region and / or site of the present invention does not differ from any region or site of the amino acid sequence of other human integrin β7. A person skilled in the art can easily determine whether or not a region corresponds to a site.

[0041] The region consisting of amino acid residues 1 to 19 of human integrin β7 is a signal peptide. It is a peptide fragment that does not exist when it functions as a membrane protein in vivo. When human integrin β7 functions as a membrane protein, the N-terminus is It is the 20th amino acid residue in the amino acid sequence.

[0042] The region consisting of amino acid residues 20 to 109 of human integrin β7 contains the PSI domain. Correlation between the PSI domain of human integrin β7 and that of mouse integrin β7 Although the sex ratio is known to be high at approximately 80% or more, the The region consisting of amino acid residues 20-109 that contains the PSI domain of mouse integrin β7 As shown in Figure 24, the amino acid residues at positions 23 and 26 of human integrin β7 are th, 28th, 30th, 32nd, 35th, 36th, 38th, 41st, 42nd, 48th, 93rd A total of 15 amino acid residues are different: the 94th, 102nd, and 109th amino acid residues. are.

[0043] Therefore, the epitope of antibody (I) consists of one or more of these 15 amino acid residues. It is preferable that the above relates to two or more, and more preferably three or more. The epitope of antibody (I) consists of amino acid residues 23 to 109 of human integrin β7. It is preferable that the region is located in the region consisting of amino acid residues 23 to 48 or 93 to 10. It is more preferable that it is present in the region consisting of the 9th amino acid residue.

[0044] In another more preferred embodiment, the epitope of antibody (I) consists of amino acid residues 23 to 48. the region consisting of amino acid residues 93-109, or the region consisting of amino acid residues 23-48 A three-dimensional region consisting of the region consisting of the 93rd to 109th amino acid residues and the region consisting of the 93rd to 109th amino acid residues. It may be a region.

[0045] The epitope of antibody (I) may be a linear epitope, but may also be a conformational epitope (non-linear epitope). A linear epitope is a sequence of consecutive amino acid residues that form an epitope. A conformational epitope is a heterologous epitope that is composed of non-contiguous amino acid residues. It is known to those skilled in the art that these epitopes are involved in the expression of ribonucleotides.

[0046] For example, the region consisting of the 23rd to 48th amino acid residues and the region consisting of the 93rd to 109th amino acid residues When the epitope is a three-dimensional region that combines regions consisting of bases, it is called a three-dimensional epitope. Examples of such a protein include those contained in the region consisting of amino acid residues 20 to 109. The above-mentioned steric epitope is also applicable when the epitope is a region consisting of non-contiguous amino acid residues contained in the region. It is included in the group.

[0047] Among the above epitopes, the 48th amino acid residue is the most potent epitope for antibody (I). It is preferred that the epitope is closely related to or included in the epitope of antibody (I).

[0048] For specific linear epitopes and conformational epitopes, see, for example, Patent Publication No. 2011-52 Publication No. 7572, Special Publication No. 2009-534401, "Dissecting antibodies with re gards to linear and conformational epitopes." Forsstrom B, Axnas BB, Rockberg J, Danielsson H, Bohlin A, Uhlen M.PLoS One. 2015 Mar 27;10(3):e0121673. doi: 10.1 371 / journal.pone.0121673. eCollection 2015. Those skilled in the art will understand It can be solved.

[0049] In other words, antibody (I) binds to the 20th to 109th amino acid residues of human integrin β7. It is an antibody that specifically binds to a region of the 23rd to 109th amino acid sequence. It is preferable that the nucleic acid specifically binds to the region of 23-48 and / or 93-109. is more preferable.

[0050] In addition, the epitope of antibody (I) is the amino acid residues 20 to 109 of integrin β7. The property of binding to a region consisting of the above is sometimes called affinity for the epitope. "Increased affinity to the epitope" means "increased specific binding ability to the epitope." " This is the same as "

[0051] The term "specific" can be distinguished from the term "selective."

[0052] In another embodiment of the antibody (I), the affinity of the antibody (I) for the above epitope is It is increased in the presence of at least a part of the region consisting of amino acid residues 379 to 721 of β7. It is preferable to set the following.

[0053] "At least a part of the region consisting of amino acid residues 379 to 721" means This means that it may be a region consisting of amino acid residues or a part of such a region. Specifically, "a part of the region" refers to, for example, the region between amino acids 417 and 721 of human integrin β7. At least a portion of the region consisting of acid residues, amino acids 564 to 721 of human integrin β7 At least a portion of the region consisting of amino acid residues 379 to 563 of human integrin β7 At least a portion of the region consisting of the base, amino acid residues 417 to 563 of human integrin β7 or at least a portion of the region consisting of amino acids 379 to 416 of human integrin β7 In particular, the present invention provides at least a portion of a region consisting of residues, i.e., in the presence of these regions: The affinity of antibody (I) for the above epitope can be increased.

[0054] The term "in the presence of" refers to the region consisting of amino acid residues 20 to 109 of human integrin β7. and at least a portion of the region consisting of amino acid residues 379 to 721 of human integrin β7. The region and the region can exist in the same molecule, and both regions exist in separate molecules. Preferably, both regions are present in the same molecule. It should be noted that the term "in the presence of" can also be read as "by."

[0055] The increase in affinity of the above-mentioned antibody (I) to the epitope is described in the following Examples. This can be easily confirmed by those skilled in the art using conventional immunological assays.

[0056] For example, various human / mouse chimeric integrin β7 proteins shown in Example 8 and comprising a region consisting of amino acid residues 1 to 109 of human integrin β7; a human / human integrin β7 region consisting of amino acid residues 722-798 of human integrin β7; We prepared cells expressing mouse chimeric integrin β7 protein (#4960) and The region consisting of amino acid residues 379-721 of human integrin β7 was cloned into mouse integrin β7. Human / mouse chimeric nucleotides substituted in the region consisting of amino acid residues 379-721 of integrin β7 The melanoma integrin β7 protein (#4961) was prepared. The degree of binding of antibody (I) was measured using the following method. By comparing cells expressing the latter (#4961) with cells expressing the former (#4960), Thus, an increase in the affinity of antibody (I) for the above epitope can be confirmed.

[0057] In another embodiment of the antibody (I), the affinity of the antibody (I) for the above epitope is It is preferable that the increase is caused by activating β7. Since integrin β7 has structural characteristics in the region containing the above epitope, the above-mentioned It is believed that the affinity to the epitope increases.

[0058] Methods for activating human integrin β7 are known. Expressing cells, such as plasma cells, NK cells, T cells, B cells, lymphoblasts, Burkitt lymphocytes PM is directed against either blood or immune cells, such as lymphoma-derived cells or dendritic cells. A, phorbol esters, manganese salts, etc. are used to induce the production of It can activate human integrin β7, which is expressed in the above specific cells. Not limited to this, cells expressing human integrin β7 were used to treat phorbol esters, manganese Human integrin β7 can also be activated by treatment with amine salts.

[0059] The affinity of antibody (I) for the above epitope activates human integrin β7. The increase in the amount of α-glucan can be determined by a conventional immunological assay as described in the Examples below. This can be easily confirmed by anyone.

[0060] For example, various human / mouse chimeric integrin β7 proteins shown in Example 8 The cells expressing #4960 or #4961, which contains the region consisting of amino acid residues 1 to 109, were The cells were then subjected to the integrin β7 activation method described in Example 7, and then subjected to immunoprecipitation. By measuring using an optical measuring means, the results before and after the activation treatment can be compared. It is possible to confirm the increase in affinity of antibody (I) to the above epitope on the cells.

[0061] In another embodiment of the antibody (I), the antibody (I) is a human integrin β7 antibody that binds to myeloma cells rather than human integrin β7 expressed on normal cells. It has been shown to have a higher affinity for human integrin β7 expressed on cells derived from The antibody can be an anti-human integrin β7 antibody.

[0062] The normal cells are not particularly limited as long as they are derived from a healthy individual, and for example, they may be derived from blood. Among such normal cells, normal plasma cells are preferred. It's nice.

[0063] Compared to the human integrin β7 expressed on normal cells, the expression of integrin β7 on myeloma cells is The method for confirming that the antibody has higher affinity for human integrin β7 is as follows: Those skilled in the art can easily carry out the assay using conventional immunological assay methods described in the Examples. This can be done.

[0064] "Conventional immunoassay" refers to a method of measuring using various antibodies regardless of the antigen. There is no particular limitation on the method, as long as it is suitable. For example, flow cytometry (FACS) and associated sorting, Western blotting, ELISA, immunoprecipitation, SPR, QCM, etc. Some examples include:

[0065] In another embodiment of the antibody (I), the antibody (I) has the same epitope as the MMG49 antibody disclosed in the Examples below. It is most preferable that the antibody has the same structure as the MMG49 antibody. For the method of producing the MMG49 antibody, see the Examples below.

[0066] Another embodiment of the antibody (I) is an antibody comprising a heavy chain variable region and / or a light chain variable region. That is, the antibody (I) may be composed of only the heavy chain variable region, or It may be a single chain variable region. Preferably, it contains a heavy chain variable region and a light chain variable region. It is an antibody.

[0067] The variable region is also called the antigen recognition site, and is an important part of an antibody's ability to recognize antigens. It is understood by those skilled in the art that such a variable region includes three hypervariable regions (complementarity determining regions (CDRs) These CDRs are most involved in the antigen recognition function of antibodies. It is also known to those skilled in the art that these regions are very important for the functioning of the immune system.

[0068] The heavy chain variable region included in another embodiment of the antibody (I) may comprise heavy chain CDR1, heavy chain CDR2, or heavy chain CDR3. That is, the heavy chain variable region includes one or more of heavy chain CDR1, heavy chain CDR2, or The heavy chain CDR3 may be contained alone, and preferably contains at least the heavy chain CDR3. More preferably, the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 are arranged in this order from the amino terminus (N-terminus). This is an embodiment including 3.

[0069] The light chain variable region may be the same as the heavy chain variable region, for example, light chain CDR1, light chain CDR2, or light chain CDR3, and preferably contains at least the light chain CDR3. It is preferable that the chain variable region contains, in order from the N-terminus, light chain CDR1, light chain CDR2, and light chain CDR3. .

[0070] The regions other than CDR1 to CDR3 in the heavy chain variable region and the light chain variable region are designated as FRs. More specifically, the region between the N-terminus and CDR1 is referred to as FR1, and the region between CDR1 and CDR2 is referred to as FR2. The region is called FR2, the region between CDR2 and CDR3 is called FR3, and the region between CDR3 and the carboxy terminus (C terminus) is called FR4. The region is called FR4, and is a designation given to the heavy chain variable region and the light chain variable region, respectively.

[0071] The amino acid sequences of the heavy chain CDR1 to 3 and the light chain CDR1 to 3 are not particularly limited. For example, heavy chain CDR1 to 3 or light chain CDR1 to 3 of MMG49 antibody, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2; a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:6; a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 7; a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 8; Examples include:

[0072] A preferred embodiment of the heavy chain variable region comprising the above-mentioned heavy chain CDRs 1 to 3 is, for example, the heavy chain variable region of the MMG49 antibody. An example of a variable region is a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 4. Furthermore, a preferred embodiment of the light chain variable region comprising the above-mentioned light chain CDRs 1 to 3 is, for example, the light chain variable region of the MMG49 antibody. An example of a light chain variable region is the light chain variable region having the amino acid sequence shown in SEQ ID NO: 9. Cut.

[0073] The amino acid sequences of the MMG49 antibodies shown in SEQ ID NOS: 1 to 4 and 6 to 9 are shown in Table 1 below. The amino acids of the heavy chain and variable regions are shown in SEQ ID NOs: 4 and 9 in the table. The underlined parts in the amino acid sequence indicate the parts located in CDR1, CDR2, and CDR3, in that order from the N-terminus. vinegar.

[0074] [Table 1]

[0075] The structure of the antibody (I) is not limited. Specific structures include Fv, scFv, diabody, dy), triabody, tetrabody, etc. It is also possible to use a structure in which these are combined as appropriate. These fragment antibodies include Fv. It can also be an artificially designed recombinant protein, and can be used to create biological components such as proteins. It can also be fused with a child.

[0076] Fv is also known as the smallest structural unit of an antibody, and is a non-covalent bond between the heavy chain variable region and the light chain variable region. The heavy chain variable region and the light chain variable region are assembled by intermolecular interactions. The structure is formed by disulfide bonds between the thiol groups of cysteine ​​residues present in the region. It is also possible to do so.

[0077] scFv has a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are connected by a linker. Also, the C-terminus and N-terminus connected by the linker are reversed. In addition, scFv may also be formed by association such as non-covalent intermolecular interactions, just like Fv. The structure can be formed by

[0078] Diabodies, triabodies, and tetrabodies are dimers of the above-mentioned scFv. They form trimers and tetramers, and like Fv, they form non-covalent intermolecular interfaces between the variable regions. This is the structure in which the molecules are assembled in the most structurally stable state due to interactions, etc.

[0079] Antibodies (I) having such various structures can be produced by conventional genetic engineering techniques using expression vectors. The vector is then transformed into a prokaryotic cell (such as Escherichia coli or Actinomyces cerevisiae) suitable for producing an antibody. developmental cells employing host cells such as eukaryotic cells (yeast cells, insect cells, mammalian cells, etc.) Those skilled in the art can easily produce the present invention by using a conventional cell-free expression system. The produced antibody can be purified to a high purity by appropriately subjecting it to a conventional purification process. It is also possible to obtain it.

[0080] In another embodiment of the antibody (I), a constant region may be contained. Those skilled in the art will recognize that a constant region includes CH1, CH2, and CH3, and a light chain constant region includes CL. It will be understood that the region containing CH2 and CH3 is sometimes also referred to as the Fc domain.

[0081] The origin of the specific constant region is not particularly limited. For example, it may be derived from human, mouse, or rat. Animal species that can withstand mass production, such as rabbits, monkeys, and chimpanzees , derived from animal species closely related to humans, or animal species that are unlikely to cause immunogenicity when administered to humans. Examples of constant regions include those that

[0082] Among the antibodies (I), the heavy chain variable region and / or the light chain variable region contain amino acids derived from mouse. When the antibody (I) has a sequence, it can be made into a protein by combining, for example, a human-derived constant region. The antibody may be a mAb.

[0083] In addition, in the above-mentioned chimeric antibody, heavy chain FR1 to FR4 and / or light chain FR1 to FR4 are derived from a human. By substituting the amino acid sequence, antibody (I) can be made into a humanized antibody.

[0084] Furthermore, the heavy chain CDR1 to 3 and / or the light chain CDR1 to 3 in the humanized antibody may be modified to have CDRs. The antibody (I) is produced by substituting amino acid sequences derived from humans only to the extent that the function of the antibody is not impaired. The term "human antibody" is also referred to as "fully humanized antibody." It may also be possible.

[0085] The structure of the antibody (I) in an embodiment comprising a constant region is a heavy chain having a heavy chain variable region and a heavy chain constant region. and a pair of light chains each having a light chain variable region and a light chain constant region, Not only immunoglobulins with a single chain structure, but also Fab, F(ab')2, and minibodies Furthermore, structures that combine these appropriately can be used. These combined structures are also called fragment antibodies. Such fragment antibodies are artificially designed constructs containing Fv. It can also be a recombinant protein, fused to a biomolecule such as a protein. It is also possible to do so.

[0086] Fab is a fragment of the heavy chain containing the heavy chain variable region and CH1 in the heavy chain constant region, and a fragment of the light chain variable region and and a light chain comprising a heavy chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region are non-covalently linked as described above. They associate through bonded intermolecular interactions or are linked by disulfide bonds. Furthermore, the cysteine ​​residues present in CH1 and CL are The thiol groups may be bonded together by disulfide bonds.

[0087] F(ab')2 is a peptide having a pair of the above-mentioned Fabs, in which the CH1s are linked by the cysteine ​​residues contained therein. It has a structure in which all groups are bonded together via disulfide bonds.

[0088] A minibody has a pair of antibody fragments containing the above scFv and CH3, and The structure is formed by the CH3s associating with each other through non-covalent intermolecular interactions.

[0089] The scFv-Fc has a pair of antibody fragments including the above scFv, CH2, and CH3, and is As with the ATPase, the CH3s associate with each other through non-covalent intermolecular interactions, and the It has a structure in which the thiol groups of the cysteine ​​residues contained therein are disulfide-bonded.

[0090] Antibodies (I) containing constant regions with such various structures and antibodies (I) not containing constant regions are also available. Similarly, an expression vector is constructed using conventional genetic engineering techniques, and the expression vector By using an expression system employing a host cell suitable for producing antibodies, those skilled in the art can The produced antibody can be easily purified by conventional purification steps. It is also possible to obtain a high

[0091] In the case of Fab, for example, IgG, an immunoglobulin, is purified by protease such as papain. Alternatively, F(ab')2 can be obtained by decomposing IgG using It can also be obtained by decomposing it using a protease such as pepsin.

[0092] Among the antibodies (I) containing the above constant region, the preferred structure is an immunoglobulin. The immunoglobulin subtypes are not particularly limited, and include, for example, IgA, IgD, IgE, and IgG. Among these, IgG is preferred, for example, mouse-derived In the case of IgG, IgG2 is preferred among the four subclasses.

[0093] Among the antibodies (I) comprising the above-mentioned constant region, an even more preferred embodiment of the antibody is the antibody represented by SEQ ID NO: 5. a heavy chain having the amino acid sequence shown in SEQ ID NO: 10 and / or a heavy chain having the amino acid sequence shown in SEQ ID NO: 11 The most preferred antibody is an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:5. and a light chain having the amino acid sequence shown in SEQ ID NO:10.

[0094] The above amino acid sequences may be subjected to mutations depending on the circumstances. Preferably, such mutations are not made to the heavy and light chain CDRs. When the antibody (I) contains a constant region, it is preferable that the heavy chain FR and the light chain FR are provided with the following. In addition to the above-described mutations for adjusting ADCC activity or CDC activity, further mutations have been made. It can be said that

[0095] The number of amino acid residues to be mutated is not particularly limited. The identity of the amino acid sequence before and after mutation is about 70%, preferably 75%. About 80%, more preferably about 85%, more preferably about 90% degree, more preferably about 95%, more preferably about 96%, more preferably about 97% More preferably, it is about 98%, and most preferably about 99%. The actual figures shall be obtained by rounding.

[0096] The term "identity" refers to the degree to which two or more comparable amino acid sequences have identical amino acids relative to each other. Therefore, the higher the identity of two amino acid sequences, the more similar their sequences are. It can be said that not only the identity of the columns but also their similarity is high.

[0097] Amino acid identity can be determined using commercially available or internet-available analytical tools (e.g., For example, this can be calculated using software such as FASTA, BLAST, PSI-BLAST, and SSEARCH. For example, the main initial conditions commonly used for BLAST searches are as follows: In Advanced BLAST 2.1, the program used was blastp, the Expect value was 10, and the Filter was all. Turn it OFF, use BLOSUM62 as the matrix, and use Gap existence cost, Per residue gap cost, and Lambda ratio are set to 11, 1, and 0.85 (default values), respectively, and other parameters are set to The amino acid sequence identity (%) is calculated by setting the default value. It can be put out.

[0098] The above-mentioned mutations introduced into the amino acid sequence include substitutions, deletions, insertions, etc. The introduction is not particularly limited as long as it can be achieved by employing a conventional method. For example, in the case of substitutions, conservative substitution techniques may be employed.

[0099] The term "conservative substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid having a similar side chain. This refers to the technique of being substituted with an acid residue.

[0100] For example, amino acid residues with basic side chains such as lysine, arginine, and histidine Conservative substitution techniques involve substitution between groups. amino acid residues with acidic side chains such as glycine, asparagine, glutamic acid, etc. amino acids with uncharged polar side chains such as threonine, serine, threonine, tyrosine, and cysteine Alanine, valine, leucine, isoleucine, proline, phenylalanine ... Amino acid residues with nonpolar side chains, such as thialanine, methionine, and tryptophan Amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine groups; aromatic groups such as tyrosine, phenylalanine, tryptophan, and histidine Substitution between amino acid residues having side chains is also a conservative substitution technique.

[0101] In another embodiment of the antibody (I), the antibody (I) may have cytotoxic activity. Cytotoxicity is the ability of an antibody to bind to a cell and cause some kind of damage to the cell. This refers to the activity of damaging the

[0102] Such cytotoxic activity includes, for example, ADCC activity, CDC activity, etc. DCC activity refers to Antibody-Dependent Cellular Cytotoxicity. It is an abbreviation for NK cell cytotoxicity, which expresses receptors specific to the constant region of antibodies. The antibody is then activated by recruiting cells with the activity to the vicinity of the antibody. It is an activity that induces damage to cells to which it binds.

[0103] The term "CDC activity" refers to complement-dependent cytotoxicity (CDC) activity. ) and the antibody recruits complement to its vicinity, and the action of the complement causes the antibody This refers to the activity of inducing damage to cells to which it is bound.

[0104] Here, both ADCC activity and CDC activity are as follows: Lazar GA et al., Proc Natl Acad Sci USA, 103: 400 5-10 (2006), Shields RL et al., J Biol Chem, 276: 6591-604 (2001)), Moore GL et al. al., J Immunol, 159:3613-21 (1997), An Z et al., MAbs, 1:572-9 (2009), etc. The activity can be adjusted by mutating the constant region, referring to the relevant references. do.

[0105] For example, if the constant region is human IgG1, it may be S239D, I332E, S239D / I332E, or S239D / I332E / A33 By introducing mutations such as 0L, S298A, K334A, S298A / K334A, and S298A / E333A / K334A, AD CC activity can be increased.

[0106] In addition, when the constant region is human IgG1, V234A / G237A, H268Q / V309L / A330S / P331S By introducing such mutations, ADCC activity can be reduced.

[0107] Regarding CDC activity, when the constant region is human IgG1, S267E, H268F, S324T, and S267E / H2 Mutations such as 68F, S267E / S324T, H268F / S324T, and S267E / H268F / S324T can improve the activity. It can be raised.

[0108] ADCC activity was measured by the method of Brunner KT et al. (Brunner, KT, et al., Immunology, 1968. 14: For example, myeloma cells can be cultured in RPMI 1640 supplemented with 10% FCS. Cultured in medium until the cell count reached 0.5 × 10 4 ~1.0×10 4 Add an appropriate amount of Na 2 51 CrO4 was added and the cells were incubated at 37°C for 1 hour. 51 Cr labeled and washed target cells The effector cells were bone marrow cells from SCID mice, which were cultured in 10% FBS, 10 ng / ml The cells were cultured for 6 days in RPMI1640 supplemented with mouse GM-CSF and 40 IU / ml human IL2. A 96-well plate can be used to measure the amount of test antibody or its isoform as a control. The antibody was added to a final concentration of 0.05-10 μg / mL, and the target cells (1.0 × 10 4 pieces) and and effector cells (5 × 10 5 After centrifugation, the supernatant was was released to 51 Cr is measured using a γ-counter. ADCC activity is calculated based on the following formula: It is possible to do this.

[0109] ADCC activity = {([from target cells 51 Cr release]-[spontaneous in the absence of antibodies 51 Cr release [Maximum with 1% Triton X-100] 51Cr release]-[autoimmune response in the absence of antibodies spontaneous 51 Cr release])}× 100

[0110] CDC activity was also measured by the method of Brunner KT et al. (Brunner, KT, et al., Immunology, 1999) 68. 14:181-96). For example, the target myeloma cells can be measured by The cells were cultured in RPMI1640 medium containing 0% FCS until the cell count reached 0.5 × 10 4 ~1.0×10 4 Prepared to be individual Add an appropriate amount of Na2 51 CrO4 was added and the cells were incubated at 37°C for 1 hour. 51 Label with Cr, The washed cells are used as target cells. The test antigen is suspended in RPMI1640 medium supplemented with fetal bovine serum. The antibody or control isotype antibody was added to 96 wells at a final concentration of 0.5 to 50 μg / mL. The reaction mixture is added to a plate, and then the target cells and complement are added and allowed to react for 1.5 hours. Centrifuged and released into the supernatant 51 Cr is measured using a γ-counter. CDC activity is measured using the following method. It can be calculated based on the formula.

[0111] CDC activity = {([from target cells 51 Cr release]-[spontaneous in the absence of antibodies 51 Cr release ]) / ([Maximum with 1% Triton X-100 51 Cr release]-[spontaneous release in the absence of antibodies target 51 Cr release])}× 100 The presence or absence of cytotoxic activity of an antibody is evaluated using, for example, the above-described method. The antibody can be obtained by selecting an antibody having the activity.

[0112] In another embodiment, the antibody (I) can be a multispecific antibody. Antigens other than the region consisting of amino acid residues 20 to 109 of tegrin β7 (hereinafter referred to as other antigens) It can be made to have binding ability with specificity to the target molecule (called a nucleotide).

[0113] The other antigen has a structural identity with the region consisting of amino acid residues 20-109 of human integrin β7. Preferably, the antigen is dissimilar to

[0114] Specific other antigens are not particularly limited. Examples include CD3, CD16, C1q, Adenovirus Knob Do Main, etc., and at least one of these can be used as another antigen in combination. Preferably, one of the antigens exemplified above can be used as the other antigen. That is, preferred multispecific antibodies are bispecific antibodies.

[0115] Such multispecific antibodies can be easily prepared by those skilled in the art by appropriately employing conventional techniques. For example, it can be produced from the 20th to 109th amino acid residues of human integrin β7. or a peptide fragment corresponding to the region of amino acids 20 to 109 of human integrin β7 Only the region consisting of residues is human-derived, and the rest is non-human-derived, such as mouse-derived. B cells obtained from animals immunized with cells expressing chimeric integrin β7 were used as a model. Hybridomas are prepared using antibody-producing cells such as cells, and then immunized with the other antigens described above. Hybridomas are produced separately using antibody-producing cells such as B cells obtained from the recipient animals. These hybridomas are then fused together to produce new hybridomas (two In the case of the production of polyspecific antibodies, this is also called a quadroma. Multispecific antibodies can be obtained by screening using the above antibodies.

[0116] In addition, for example, if it is a bispecific antibody, (1) The epitope is a region consisting of amino acid residues 20 to 109 of human integrin β7. An antibody having the above F(ab')2 structure is prepared: (2) On the other hand, antibodies with a F(ab')2 structure that specifically bind to other antigens are also produced in the same way: (3) The antibodies with the F(ab')2 structure obtained in (1) and (2) are each treated with a reducing agent such as DTT. After treatment with either of the treatments, the sample is further treated with Ellman's reagent: (4) The antibodies with F(ab')2 structure obtained in (3) after the treatment are mixed and reacted: Bispecific antibodies can also be produced by the procedures shown in (1) to (4).

[0117] (A) The epitope is a region consisting of amino acid residues 20 to 109 of human integrin β7. Produce antibodies that (B) On the other hand, antibodies that specifically bind to other antigens are also prepared in the same manner. (C) Amino acid sequences of the variable regions obtained in (A) and (B) and their corresponding sequences. The base sequence of the polynucleotide to be loaded is identified. (D) The polynucleotides having the respective base sequences identified in (C) are optionally identified. The expression vector is integrated together with a polynucleotide having the base sequence of the target site and a linker sequence. After the vector is produced, it is introduced into a host cell suitable for antibody production, such as a CHO cell. Bispecific antibodies can also be produced by the procedures shown in (A) to (D). .

[0118] Another embodiment of the antibody (I) is a cytotoxin (a substance having cytotoxic activity) conjugated antibody. Cytotoxins are substances that kill cells and suppress cell proliferation. There are no particular limitations on the substance, so long as it causes some kind of damage to cells, such as inhibiting cell proliferation.

[0119] Examples of such cytotoxins include cyclophosphamide hydrate and ifosfamide. thiazol-3, thiotepa, busulfan, melphalan, nimustine hydrochloride, ranimustine, dacaine Alkylating agents such as rupazine and temozolomide; methotrexate, pemetrexed sodium hydrate, fluorouracil, doxifluridine, capecitabine, tagafur, cytara iben, gemcitabine hydrochloride, fludarabine phosphate, nelarabine, cladribine, Antimetabolites such as calcium bofolinate; doxorubicin hydrochloride, daunorubicin hydrochloride Salt, pralubicin, epirubicin hydrochloride, idarubicin hydrochloride, aclarubicin hydrochloride, Amrubicin hydrochloride, mitoxantrone hydrochloride, mitomycin C, actinomycin D , bleomycin hydrochloride, pulperomacin hydrochloride, zinostatin stimalamer, calicheama Antibiotics such as isin, vincristine sulfate, vinblastine sulfate, vindesine sulfate antimicrobials such as benzodiazepines, benzodiazepines, and microtubule inhibitors such as paclitaxel; anastrozole, exemestane, and letrozole aromatase inhibitors such as cisplatin, carboplatin, and fadrozole hydrochloride hydrate; Platinum preparations such as platin, nedaplatin, and oxaliplatin; irinotecan hydrochloride hydrate, Topoisomerase inhibitors such as nogitecan hydrochloride, etoposide, and sobuzoxane, and prednisolone corticosteroids such as azolone and dexamethasone, thalidomide and its derivatives. lenalidomide, a protease inhibitor; bortezomib, a protease inhibitor; and radioactive isomers such as 90-Ittrium. The atomic elements can be mentioned.

[0120] Among these, calicheamicin, melphalan, and vincristine sulfate are preferable. Salt, doxorubicin hydrochloride, prednisolone, dexamethasone, thalidomide, lenalid amide, bortezomib, or more preferably, calichease, which has a proven track record of binding to antibodies. It is mycin.

[0121] These cytotoxins are all commercially available and one or two of the above are used. The above can be selected in appropriate combination.

[0122] The binding mode between the cytotoxin and the above-mentioned antibody is not particularly limited, and can be achieved by, for example, conventional genetic engineering. By appropriately employing conventional techniques or protein engineering techniques, one skilled in the art can convert the above-mentioned antibodies into cytokines. More specifically, the antibody (I) can be easily bound to the antibody (I) via a linker. The amino acid residue side chains of the amino acid groups, thiol groups, guanidyl groups, hydroxyl groups, carboxyl groups, etc. Examples of such methods include bonding to functional groups.

[0123] The antibody (I) may be a polyclonal antibody or a monoclonal antibody. or monoclonal antibodies.

[0124] The term "monoclonal" means obtained from a substantially homogeneous population. "Clonal antibody" means an antibody obtained from such a population. The individual antibodies in such a population may be subject to possible naturally occurring mutations that may be present in trace amounts. It is understood that apart from the differences, they are the same.

[0125] Furthermore, regarding the specific binding target (epitope) of the antibody, for example, in the case of antibody (I), It is present in the region consisting of amino acid residues 20 to 109 of human integrin β7. If the antibody is a polyclonal antibody, it is the amino acid residues 20 to 109 of human integrin β7. In contrast to monoclonal antibodies, which have multiple sites in a region consisting of a single base, It is advantageous in that it is a single site and therefore exhibits high specificity.

[0126] The modifier "monoclonal" refers to a substantially homogeneous population, as mentioned above. It should not be understood as a modifier specifying the manufacturing method. .

[0127] In addition to the above method, the antibody (I) can be obtained by the hybridoma method, the polynucleotide (II) shown below, (III) Recombinant DNA methods using host cells carrying the If employed, it can be easily manufactured by a person skilled in the art.

[0128] For example, a peptide corresponding to the region consisting of amino acid residues 20 to 109 of human integrin β7 The peptide is immunized into an animal suitable for antibody production, such as a mouse, rat, or rabbit, and then the B cells are After collecting the cells, they are subjected to the hybridoma method, and the function of the antibody (I) described above is used as an index. Examples of methods for producing antibody (I) include screening.

[0129] In addition, only the region consisting of amino acid residues 20 to 109 of integrin β7 was extracted from human. Chimeric integrants are derived from non-human sources, such as mice. We created cells that express β7 and used them to produce antibodies in mice, rats, rabbits, etc. The resulting animal (preferably a mouse) is immunized, and the B cells are then harvested to produce hybridomas. The antibody (I) is subjected to the method and screened using the function exhibited by the antibody (I) as an index. Examples of the method for producing I) include:

[0130] The function of antibody (I) is to bind to, for example, amino acids 20 to 109 of human integrin β7. The affinity for the region consisting of amino acid residues 380-721 of human integrin β7 The activation of human integrin β7 occurs under at least a portion of the region comprising the base of the β- Therefore, by using such a function, However, the antibody (I) can also be obtained by the method shown in the screening method (X) below.

[0131] Antibody (I) binds to the epitope region consisting of amino acid residues 20 to 109 of human integrin β7. Therefore, the antibody has the ADCC activity and the like against cells expressing integrin β7. Not only CDC activity, but also apoptosis-inducing activity, survival signal blocking activity, etc. Alternatively, two or more of them may be combined to exert cytotoxic activity against such cells. Therefore, the composition containing the antibody (I) is expected to have a pharmaceutical composition as described in detail below. It is useful as the compound (VII).

[0132] In particular, antibody (I) encodes a region consisting of amino acid residues 20 to 109 of human integrin β7. The affinity of antibody (I) to the epitope activates integrin β7. Activated integrin β7 is expressed in blood cells such as plasma cells. Therefore, antibody (I) is effective in preventing these cancers (e.g., blood cancers) and is therefore a promising candidate for pharmaceutical compositions. In particular, it is used to treat diseases that cause abnormalities in the above cells (e.g., myeloma, multiple myeloma, etc.). It can be effectively used as a pharmaceutical composition for the treatment of myeloma, myeloma, and other cancers.

[0133] (II) Polynucleotides The polynucleotide (II) is a polynucleotide having a base sequence encoding the amino acid sequence of the antibody (I). The term "polynucleotide" refers to, for example, ribonucleotides. , deoxyribonucleotides, or any of these nucleotides, etc., may be used as appropriate. It includes single-stranded or double-stranded forms that have been modified by the method.

[0134] The base sequence of polynucleotide (II) can be determined by a person skilled in the art, for example, in silico. The base sequence can be determined appropriately based on the amino acid sequence of the The type of codon used is not important. The codon frequency of the host in which the polynucleotide is used should be taken into consideration. Therefore, it is preferable to determine the base sequence.

[0135] The specific base sequence of the polynucleotide (II) is not particularly limited. Each SEQ ID NO: indicates an amino acid sequence identified as one of the amino acid sequences The correspondence of the sequence numbers showing the base sequences is shown in Table 2 below. The preferred base sequences are those shown in SEQ ID NOs: 11 to 20.

[0136] [Table 2]

[0137] The polynucleotide (II) may be incorporated into a vector. The vector is not particularly limited, and may be, for example, a cloning vector or an expression vector. It can be used in any way, and its purpose is not important.

[0138] In addition, expression vectors can be used for prokaryotic cells such as E. coli and actinomycetes. They can also be used as vectors for eukaryotic cells such as yeast cells, insect cells, and mammalian cells. Cut.

[0139] The 5'-terminal side of the polynucleotide (II) (the N-terminal side of the antibody (I)) may be appropriately signed. A base sequence encoding a null peptide can also be added.

[0140] The specific method of using the polynucleotide (II) is not particularly limited. For example, For example, the vector is introduced into cells (III) and used to express the antibody (I).

[0141] (III) Host cells The host cell (III) is a cell that retains the polynucleotide (II). means to maintain the state in which the polynucleotide (II) is present in the cell, and A state in which the cell does not spontaneously export the polynucleotide, whether actively or not. This means that

[0142] The mode in which the host cell (III) retains the polynucleotide (II) is not particularly limited. For example, the polynucleotide can be maintained in the cell in the form of a vector, or it can be expressed as a gene in the cell. The polynucleotide (II) can also be held in an integrated form in the genome. .

[0143] Specific cell types of the host cell (III) include eukaryotic cells such as yeast cells, insect cells, and mammalian cells. The cells may be prokaryotic cells such as Escherichia coli and actinomycetes, and are not particularly limited.

[0144] (IV) Chimeric antigen receptor Chimeric antigen receptors are artificial T cell receptor (TCR)-like proteins that act as T cell receptors. The antigen recognition site expressed on the vacuole membrane (corresponding to the extracellular domain) is converted to the desired antigen recognition site. This allows the T cells to more effectively exert their own functions, such as cytotoxicity. It is a protein designed to

[0145] The chimeric antigen receptor (IV) has the same epitope as the above-mentioned antibody (I), and More specifically, it is a protein containing the antigen recognition site of the above antibody (I). The epitope present in the antigen recognition site contained in the antigen receptor is described in detail in the above antibody (I). It can be similar to that.

[0146] More specifically, the antigen recognition site of the antibody (I) is The spacer sequence, transmembrane domain, costimulatory factors, and intracellular domain of the TCR are located in the It is a protein made up of

[0147] The antigen recognition site of the antibody (I) located in the chimeric antigen receptor (IV) is described in detail in the antibody (I). Specifically, the heavy chain variable region and / or the light chain variable region can be Among these, it is possible to obtain a scFv structure that has a heavy chain variable region and a light chain variable region. It is preferable that there is.

[0148] In such scFv, for example, there are suitably 10 amino acids between the heavy chain variable region and the light chain variable region. A spacer sequence consisting of about 25 amino acid residues can be provided. Such a spacer sequence is a chimeric antigen receptor (IV) sequence. The spacer sequence may be the same as or different from the spacer sequence arranged in the can.

[0149] The spacer sequence placed in the chimeric antigen receptor (IV) is not particularly limited. For example, It can be made up of about 10 to 25 amino acid residues. , about 15 to 18 pieces.

[0150] The transmembrane domain arranged in the chimeric antigen receptor (IV) is not particularly limited. The transmembrane domains derived from proteins such as CD28 and 4-1BB expressed in T cells are It can be adopted while allowing for appropriate introduction of mutations.

[0151] The costimulatory factor located in the chimeric antigen receptor (IV) is a costimulatory factor possessed by T cells, etc. For example, 4-1BB, OX40, CD28, etc. may be used after appropriate mutagenesis. It can be adopted while allowing for this to happen.

[0152] The intracellular domain of the TCR arranged in the chimeric antigen receptor (IV) is not particularly limited. For example, the intracellular domain derived from CD3, also known as the TCR ζ chain, is appropriately mutated. Regarding the introduction of mutations into CD3, ITAM (Immu Preferably, the tyrosine-based activation motif is included. I wish.

[0153] The chimeric antigen receptor (IV) preferably has the amino acid sequence shown in SEQ ID NO: 21. I wish.

[0154] The amino acid sequence specifying the chimeric antigen receptor is one that has been appropriately mutated. In addition, the above-mentioned transmembrane domain, costimulatory factor, and intracellular domain of TCR can be used. The number of mutations introduced into the main sequence can be similarly determined. It will not be done.

[0155] For example, if the amino acid sequence before and after mutation is about 70% identical, , preferably about 75%, more preferably about 80%, more preferably about 85%, Preferably, it is about 90%, more preferably about 95%, more preferably about 96%, and even more preferably Preferably, it is about 97%, more preferably about 98%, and most preferably about 99%. Such figures shall be obtained by rounding off.

[0156] The above-mentioned mutations introduced into the amino acid sequence include substitutions, deletions, insertions, etc. The insertion is not particularly limited as long as it can be achieved by adopting a conventional method. For example, if substitutions are required, conservative substitution techniques may be employed.

[0157] In addition, in order to produce such a chimeric antigen receptor, the methods described in Non-Patent Documents 4 to 6 and the like can be used. Those skilled in the art can easily produce such a compound by referring to the methods described above.

[0158] (V) Polynucleotides Polynucleotide (V) is different from the polynucleotide (II) and is a chimeric antigen receptor. A polynucleotide encoding the amino acid sequence of (IV).

[0159] The base sequence of polynucleotide (V) may be, for example, an intron, similar to that of polynucleotide (II). It can be appropriately determined in silico based on the amino acid sequence of the chimeric antigen receptor (IV). The type of codons used to determine the base sequence is not important. It is preferable to determine the base sequence taking into consideration the codon frequency of the target cell.

[0160] The specific base sequence is not particularly limited. For example, the amino acid sequence shown in SEQ ID NO: 21 The base sequence shown in SEQ ID NO: 22 is determined based on the amino acid sequence of the chimeric antigen receptor (IV) having Of course, polynucleotides based on such amino acid sequences can be used. The base sequence determined by the above sequence number is the same as the above sequence number, regardless of the type of codon used. It goes without saying that the present invention is not limited to the base sequence shown in No. 22.

[0161] The 5'-terminal side of the polynucleotide (V) (the N-terminal side of the chimeric antigen receptor (IV)) A base sequence encoding a signal peptide can be added as appropriate.

[0162] The specific method of using the polynucleotide (V) is not particularly limited. For example, the following cell (VI) ) and used to express the chimeric antigen receptor (IV). Cut.

[0163] (VI) Cell The cell (VI) is a cell that is different from the host cell (III) and that contains the polynucleotide (V). The term "maintaining" can be used in the same way as in the host cell (III) above. The type of cell can be the same as the host cell (III) above, but it can also be a cell that has cytotoxic activity. Examples include T cells, NK cells, and K cells, and among these, T cells are preferred. One type, killer T cells (also called cytotoxic T cells [CTLs]), is most preferred.

[0164] The polynucleotide (V) encoding the chimeric antigen receptor contained in the cell (VI) is expressed. By this, the antigen recognition site of the antibody (I) constituting the chimeric antigen receptor (IV) is exposed to the outside of the cell. The transmembrane domain constituting the chimeric antigen receptor (IV) is exposed to the side, the costimulatory factor mentioned above, if Preferably, the intracellular domain of the TCR is localized on the cell membrane or intracellularly.

[0165] These costimulatory factors or domains localized on the cell membrane or intracellularly can be used as antibodies (I) The antigen recognition site of this antibody binds to the region consisting of amino acid residues 20 to 109 of human integrin β7. When combined with human insulin, it activates a signal that induces cytotoxic activity within the cell. The affinity of antibody (I) for the region consisting of amino acid residues 20 to 109 of tegrin β7 was Therefore, antibody (I) activates activated integrin β7. It attacks or exerts cytotoxic activity against cells or tissues that express phospho-β7.

[0166] When the cells that perform this function are T cells, they are called chimeric antigen receptor T cells (VI- 4) Cells that have the potential to exert cytotoxic activity, such as NK cells, are also referred to as Similar to the antigen receptor T cells, the antigen recognition site is located at positions 20-109 of activated human integrin β7. and cytotoxic activity in the cell membrane or intracellular domain. By linking the activation of the signal that triggers it, similar to chimeric antigen receptor T cells These cells are called chimeric antigen receptor NK cells.

[0167] Thus, cells (VI) were cytosed against cells or tissues expressing activated integrin β7. Since the antibody (I) exhibits cell-damaging activity, the composition containing the cells (VI) as well as the antibody (I) is as follows: It can be said that it is useful as a pharmaceutical composition (IV) as described in detail. Since it is expressed in blood cells such as blood cells, it is useful for the preparation of pharmaceutical compositions for cancer (e.g., blood cancer). It is used as an active ingredient. In particular, it is used to treat diseases that cause abnormalities in the above cells (e.g., myeloma, It can be effectively used as a pharmaceutical composition for treating various diseases, such as multiple myeloma.

[0168] (VII) Pharmaceutical Composition The pharmaceutical composition (VII) contains the antibody (I) or the cell (VI). The cell (VI) is: Preferably, the cells are chimeric antigen receptor T cells (VI-4).

[0169] The content of the antibody (I) or the cells (VI) in the pharmaceutical composition (VII) is not particularly limited. For example, in the case of antibody (I), the amount is 0.001 to 10 parts by weight per 100 parts by weight of the pharmaceutical composition. In the case of cells (VI), the concentration can be 1 cell / mL to 10 4 cell / It can be about mL.

[0170] The method of administering the pharmaceutical composition (VII) is not particularly limited. Therefore, parenteral or non-enteral administration is preferred. Examples of administration include intramuscular administration and subcutaneous administration, and intravenous administration is preferred.

[0171] The dosage form of the pharmaceutical composition (VII) can be prepared by mixing it with a conventional pharmaceutically acceptable carrier depending on the above-mentioned administration method. In consideration of the above-mentioned preferred administration method, it is preferable to prepare the drug as an injection. preferable.

[0172] The target disease of the pharmaceutical composition (VII) is not particularly limited. Specific target diseases include, for example, Examples include cancer, preferably blood cancer, and more preferably a disease that causes neoplastic proliferation of plasma cells. The term "diseases resulting in neoplastic proliferation of plasma cells" refers to diseases resulting in neoplastic proliferation of abnormal plasma cells. It is a disease characterized by proliferation and an increase in abnormal proteins secreted by these cells. Such diseases include, for example, myeloma, multiple myeloma, plasma cell leukemia, plasmacytoma, Examples of the disease include H-chain disease and systemic AL-type amyloidosis. In this case, the target diseases of the pharmaceutical composition (VII) are malignant lymphoma, leukemia, and other hematological malignancies. It's okay to have it.

[0173] The subject to which the pharmaceutical composition (VII) is administered (the subject) is a patient suffering from the above-mentioned disease, or The term "possibly susceptible" refers to an animal that is susceptible to the disease, as described below. The animal may be, for example, a mammal. Preferably, the animal is a human.

[0174] The dosage of the pharmaceutical composition (VII) is determined based on the severity of the disease of the patient to be treated, the degree of the desired effect of the treatment, It depends on various factors such as weight, sex, age, and species, so it is not possible to make a general decision. For example, if the active ingredient is an antibody (I), the daily dose is usually 1 μg / kg (body weight) to 10 g / kg. kg (body weight). If the active ingredient is a cell (VI), it is usually 10 4 Thin cells / kg(weight)~10 9 It can be about cells / kg (body weight).

[0175] The administration schedule of the pharmaceutical composition (VII) as well as the dosage thereof will depend on the severity of the disease of the subject to which it is administered. It varies depending on various conditions such as the above, and cannot be determined in general. It is preferable that the dosage be administered once a day to once a month.

[0176] (VIII) Methods for treating or preventing diseases The method for treating or preventing a disease (VIII) comprises administering a therapeutically effective amount of the antibody (I) or the cell (VI). The present invention relates to a method for treating or preventing a disease, the method comprising administering to a subject the following: Chimeric antigen receptor T cells (VI-4) are preferred.

[0177] The subject may be the same as the pharmaceutical composition (VII) above. When the patient is a patient suffering from the disease, a therapeutically effective amount of the antibody (I) or the cell (VI) is administered. Therefore, the therapeutic effect is expected, and when the subject is an animal that may be susceptible to a disease, The prevention effect is expected. Prevention means that the conventional immunization method is used as shown in the following diagnostic method (XI). The values ​​measured by epidemiological methods do not reach the values ​​that are considered to be disease-causing. This means to make it so.

[0178] The disease can be the same as that of the above pharmaceutical composition (VII), and is exemplified by cancer, and is preferably New cancers include diseases that cause neoplastic proliferation of plasma cells (e.g., multiple myeloma). It is possible.

[0179] The therapeutically effective amount can be the same as the dose of the pharmaceutical composition (VII), and the therapeutically effective amount can be the same as the dose of the antibody (I) or Alternatively, the cells (VI) may be formulated in the same manner as the pharmaceutical composition (VII). The administration method and administration schedule of the antibody (I) or the cell (VI) may also be determined by the pharmaceutical composition. The composition can be as detailed in composition (VII).

[0180] The method for treating or preventing a disease (VIII) includes a method for treating or preventing a multiple myeloma that targets activated human integrin β7. The present invention also encompasses a method for treating or preventing myeloma. Alternatively, the application of the above-mentioned cells (VI) can be mentioned.

[0181] (IX) Use Use (IX) means use of the antibody (I) or the cell (IV) for producing a pharmaceutical composition. is.

[0182] The pharmaceutical composition may be the same as the pharmaceutical composition (VII) above. is preferably a chimeric antigen receptor T cell (VI-4).

[0183] The same applies to the target diseases of the pharmaceutical composition, for example, for the treatment of cancer, preferably blood cancer, More preferably, the present invention is directed to diseases that cause neoplastic proliferation of plasma cells (e.g., myeloma, multiple myeloma, etc.). ) are listed.

[0184] In addition, the content of the antibody (I) or cell (VI) as an active ingredient in the pharmaceutical composition, The dosage form, administration method, administration schedule, etc. of these are the same as those described in detail in the above pharmaceutical composition (VII). It can be the same as:

[0185] (X) Screening method Screening method (X) is for the treatment or prevention of diseases that cause neoplastic proliferation of plasma cells. A method for screening an active ingredient of a pharmaceutical composition, comprising: β7 integrin, and the amino acids 20 to 109 of human integrin β7 The method includes selecting candidate substances that bind to a region of residues.

[0186] The pharmaceutical composition may be the same as the pharmaceutical composition (VII) described above, and is effective in treating cancer, preferably blood cancer. and more preferably diseases that cause neoplastic proliferation of plasma cells (e.g., myeloma, multiple myeloma, etc.). The active ingredient of the pharmaceutical composition for treating or preventing the above-mentioned disease is, for example, the antibody (I). .

[0187] The compound library is not particularly limited, and an existing library can be used. Preferably, the antibody library is obtained from animals immunized with a desired antigen. The library is made up of hybridomas produced using antibody-producing cells such as B cells. It is preferable that:

[0188] Here, the desired antigen is not particularly limited, and may be, for example, the 20th to 109th amino acid sequence of human integrin β7. It is preferably a region consisting of amino acid residues. It is glynn β7.

[0189] The method for selecting candidate substances is not particularly limited. For example, Candidate substances that specifically bind to the 20th to 109th amino acid residues of human integrin β7 were selected. The binding of the compound to a peptide fragment corresponding to the region consisting of the group is determined by a conventional immunological assay. A means for checking and selecting the same can be adopted.

[0190] In addition, candidate substances that specifically bind to human integrin β7 are selected and further tested against the above antibody (I). Only the region consisting of amino acid residues 20 to 109 of integrin β7, which was previously described, was determined to be human-derived. The remaining parts are derived from non-human sources such as mice. Candidate substances that bind to cells expressing 7 are confirmed using conventional immunological assays, and A means for selecting this can be employed. A means for selecting this can be employed.

[0191] Furthermore, a candidate substance that specifically binds to human integrin β7 is selected, and the antibody (I) The region consisting of amino acid residues 20 to 109 of integrin β7 was extracted from human. The rest are chimeric integrins of non-human origin, such as mouse origin. Cells expressing β7 were treated with phorbol ester, manganese salt, etc. A method for identifying candidate substances whose binding level increases before and after treatment and selecting them will be adopted. It can also be done as follows.

[0192] Furthermore, a candidate substance that specifically binds to human integrin β7 is selected, and the antibody (I) The region consisting of amino acid residues 111 to 378 of human integrin β7, which was described in detail in The chimera expressing the β7-derived integrin was then transformed into a β7-derived chimera. A chimera was created in which the region consisting of amino acid residues was replaced with a mouse-derived one, and the degree of binding to the former was examined. It is also possible to employ means for identifying and selecting high potency candidate substances.

[0193] Furthermore, in the screening method (X), candidate substances are selected based on the cytotoxicity as an index. The method may also include a step of distinguishing between the target cells and the target cells for which specific cytotoxic activity is to be confirmed. There is no particular limitation to the above. For example, the PSI domain of the human integrin β7 is characterized as described above. Examples of such cells include blood cells that express activated human integrin β7.

[0194] When the candidate substance to be screened is an antibody, it is determined that the antibody has cytotoxic activity. a step of selecting candidate substances using the index, and a step of selecting antibodies having ADCC activity or CDC activity It may also be possible to use the following.

[0195] In this way, the candidate substance selected by the screening method (X) is an antibody. It is preferably a monoclonal antibody, more preferably a monoclonal antibody. The most preferred is the above antibody (I). do.

[0196] (XI) Diagnostic methods The diagnostic method (XI) is a method for diagnosing cancer, which comprises combining a sample collected from a subject with the antibody (I). The method includes contacting the

[0197] The subject may be the same as the subject described in detail in the method for treating or preventing a disease (VIII). can.

[0198] The sample taken from the subject may be blood or bone marrow fluid.

[0199] The specific diagnostic method is not particularly limited, but for example, cells that bind to the above antibody (I) can be detected. If the test is given, it may be judged that the patient has cancer or is at risk of developing cancer. .

[0200] The degree of binding can be readily determined by one skilled in the art by employing conventional immunological assays. Depending on the degree measured here, it can be determined whether the patient has cancer or not. It can be determined whether or not there is a possibility of

[0201] The specific cancer diagnosis is not particularly limited, but for example, blood cancer, more preferably, the above-mentioned antibody (I) When the cells that bind to the It can be used to diagnose whether a person has or may have a myeloma (myeloma, multiple myeloma, etc.) Cut.

[0202] (XII) Kit Kit (XII) is a cancer diagnostic kit containing the above-mentioned antibody (I).

[0203] The cancer is not particularly limited, and can be as described in detail in the above-mentioned pharmaceutical composition (VII). Preferably, the present invention is directed to blood cancer, more preferably to diseases that cause neoplastic proliferation of plasma cells (e.g., bone marrow cancer, myeloma, multiple myeloma, etc.

[0204] In addition, a manual may be attached to the kit (XII) as appropriate. The method detailed in the above diagnostic method (XI) can be described as the diagnostic criteria for cancer. Cut. [Example]

[0205] Examples are given below to explain the present invention in more detail. It goes without saying that the present invention is not limited to the examples.

[0206] Testing Method: Flow Cytometry and Sorting In the following examples, the flow cytometry (FACS) used for cell sorting was as follows: The procedure was as follows.

[0207] Bone marrow mononuclear cells were collected from the iliac bone of a myeloma patient who gave informed consent and were then added to ACK fluid (1 Erythrocytes were suspended in 50 mM NH4Cl and 10 mM KHCO3 and allowed to stand for 3 minutes at 4°C. The removed bone marrow mononuclear cells were washed with PBS containing 2% fetal bovine serum, and then non- To prevent specific antibody binding, the cells were incubated in PBS containing 10% human AB serum for 20 minutes at 4°C. Rocking was performed.

[0208] Then, fluorescently labeled antibodies (see below) were added to the mixture and incubated at 4°C for 30 minutes. After staining, the cells were washed with PBS and then resuspended in PBS containing 1 μg / ml of propidium iodide (PI). The cells were suspended in PBS and then subjected to FACS analysis. a Cell sorter (Becton Dickinson Immunocytometry Systems) was used.

[0209] The following monoclonal antibodies were appropriately selected and used for cell staining. APC-conjugated anti-human CD34 antibody (BD Pharmingen) ·PE-Cy7-conjugated anti-human CD34 antibody (manufactured by BD Pharmingen) ·APC / Cy7-conjugated anti-human CD19 antibody (manufactured by Biolegend) FITC-conjugated anti-human CD38 antibody (eBioscoience) ·APC-conjugated anti-human CD138 antibody (manufactured by Biolegend) ·PE / Cy7-conjugated anti-human CD3 antibody (manufactured by Biolegend) ·FITC-conjugated anti-human CD14 antibody (manufactured by BD Pharmingen) ·PE / Cy7-conjugated anti-human CD45 antibody (manufactured by Biolegend).

[0210] Example 1 Construction of a monoclonal antibody library that binds to myeloma cell lines but not to peripheral blood of healthy donors In antibody therapy for multiple myeloma, antibodies that bind to myeloma cells but not to normal blood cells are used. Therefore, it is important to use an antibody that does not bind to the IgG. First, we identified monoclonal antibodies that bind to various myeloma cell lines using the following method. More than 10,000 antibody clones were produced.

[0211] Six human myeloma cell lines (MM.1s, RPMI8226, INA6, U266, and OPM2) were used. Balb / c mice were immunized with the antigen (antigen) twice a week for 2-3 weeks in the footpads of the mice. Then, the popliteal lymph nodes were removed, a cell suspension was prepared, and the cells were then transferred to SP2 / 0 mouse myelin. Hybridomas were produced by cell fusion with a human ovarian cancer cell line. The procedure was carried out using a method using a PEG (PEG method). Hybridomas were selected by culturing the cells in thymidine medium (HAT medium) (> 10,000 clones).

[0212] Finally, the hybridoma culture supernatant was used to bind to the myeloma cell line used for immunization and to detect healthy The supernatant containing antibodies that do not bind to mononuclear cells derived from normal human peripheral blood was selected using FACS. Approximately 200 clones of antibodies specific to myeloma cells were obtained. The hybridomas were expanded and then cryopreserved.

[0213] Example 2 Identification of antibodies that specifically bind to myeloma cells in bone marrow of human multiple myeloma patients Approximately 200 candidate antibody clones obtained in Example 1 above were used to detect bone marrow-derived antibodies from myeloma patients. The marrow cells were stained and analyzed using FACS.

[0214] Each candidate antibody was added to bone marrow cells derived from multiple myeloma patients and incubated at 4°C for 30 minutes. After washing, PE-conjugated anti-mouse IgG antibody was added as a secondary antibody. After washing, the cells were incubated with APC-conjugated anti-human CD1 38 antibody, FITC-conjugated anti-human CD38, or PE / Cy7-conjugated anti-human As a negative control, staining was performed using CD45 instead of the candidate antibody. A sample containing ol was also prepared at the same time.

[0215] These were analyzed using FACS to identify CD45 - CD38 ++ CD138 + Myeloma plasma cells and C D45 - CD38 ++ CD138 - Binds to myeloma progenitor cells but not CD45 +Antibodies that do not bind to blood cells Selected.

[0216] As a result, the MMG49 antibody was identified as an antibody that met the above criteria (Figures 1 and 2). For each histogram in the figure, the Y axis represents the cell number, and the X axis represents the binding intensity of the MMG49 antibody. show

[0217] Example 3 Identification of the antigenic protein that MMG49 antibody binds to The antigen protein to which the MMG49 antibody binds was identified by expression cloning.

[0218] First, we identified superscript choice s from MM.1s cells, which are known to bind to the MMG49 antibody. A cDNA library was prepared using a system for cDNA synthesis (Invitrogen), and BstXI Using an adapter (Invitrogen), the pMXs retroviral vector (Institute of Medical Science, University of Tokyo) was inserted. The cDNA library thus constructed was inserted into a plasmid (provided by Professor Toshio Kitamura of the Institute of Genetics). The retrovirus obtained by transfecting at-E cells (provided by Dr. Toshio Kitamura) was transferred to BaF3 cells. to obtain BaF3 cells expressing the MM.1s-derived cDNA library.

[0219] Next, these cells were stained with MMG49 antibody and positive cells were sorted by FACS. After the third round of sorting, most of the cells were MMG4. The cells then became cells that bind to the antibody. After amplifying the fragment by PCR, the base sequence was identified by sequencing. The insert carried by the cells was identified as ITGB7.

[0220] Example 4 By generating ITGB7-deficient myeloma cells, we confirmed that the binding antigen of the MMG49 antibody is ITGB7-expressed protein. Confirmation with We generated ITGB7-deficient U266 myeloma cell lines using the Crisp-Cas9 system.

[0221] First, a double-stranded DNA sequence of an ITGB7-specific target sequence was inserted into the PX330 (addgene) vector. The vector was then transformed into linear hygro, a vector for drug selection. The cells were transfected with Nucleofector (registered trademark) together with a mycin-resistance gene expression vector (Clontech). The transfection was carried out into U266 cells using HIV-1 ... The expression of ITGB7 was examined by FIB27 antibody (anti-integrin β7 antibody) for clones that grew in the culture medium. ITGB7-deficient cells were identified by staining with a marker (Biolegend) and analyzing by FACS. did.

[0222] Next, the obtained ITGB7-deficient cells were stained with MMG49 antibody and analyzed by FACS. The MMG49 antibody binds to intact U266 cells, but not to ITGB7-deficient cells. This indicates that MMG49 is a cytosolic agent that expresses ITGB7 (integrin). This indicates that it binds only to β7).

[0223] Next, immunoprecipitation was performed using MMG49 antibody from the lysate of MM1s myeloma cells, followed by SDS-PAGE. Then, WB was performed using anti-integrin β7 antibody (Miltenyi). Integrin β7 was detected in the immunoprecipitates with antibody 9 (Fig. 5). 9 antibody binds to integrin β7.

[0224] Example 5 Measurement of the binding pattern of MMG49 antibody in each cell fraction of peripheral blood from healthy individuals and bone marrow from myeloma patients fixed Commercially available anti-integrin β7 antibody (FIB27 antibody; Biolegend) and MMG49 antibody The binding to various cell fractions in peripheral blood and bone marrow cells from healthy individuals was measured using the antibody.

[0225] After removing red blood cells from peripheral blood cells derived from healthy individuals using HES40, Fc receptor blocking A reagent (Miltenyi) was added to block nonspecific antibody binding, and then MMG49 The cells were incubated at 4°C for 30 minutes with either FIB27 antibody or FIB27 antibody, or mouse IgG2a as an isotype control. After incubation for 1 minute, the cells were washed and then incubated with PE-conjugated anti-mouse IgG as a secondary antibody. The antibody was added and the mixture was further incubated at 4°C for 30 minutes.

[0226] After washing, the cells were finally stained with APC / Cy7-conjugated anti-human CD19 antibody, FITC- conjugated anti-human CD14 antibody, PE / Cy7-conjugated anti-human CD3 antibody The stained cells were analyzed using FACS to determine the MMG49 activity in each fraction. The binding of the antibody and the FIB27 antibody was measured (Figure 6).

[0227] In addition, 1 μl of peripheral blood from a healthy individual was added to 100 μl of PBS (containing EDTA), and the mixture was similarly incubated with MMG49 antibody. The cells were stained with either the CD235 or FIB27 antibody, followed by Pacific blue-conjugated anti-human CD235. antibody (BD Pharmingen) or FITC-conjugated anti-human CD41 antibody (BD Pharmingen) By staining with erythromingen, CD235 + The presence of each antibody on red blood cells and platelets The FIB27 antibody was also examined by FACS analysis (Figure 6). The MMG49 antibody binds strongly to the above normal blood cells, whereas the MMG49 antibody binds very little to the above normal blood cells. It shows weakness.

[0228] Furthermore, there was no binding of the MMG49 antibody to normal cell fractions other than myeloma cells in the bone marrow. To clarify whether this is the case, bone marrow cells from myeloma patients were also stained with the MMG49 antibody. Finally, APC-conjugated anti-human CD34 antibody (BD Pharmingen) Alexa647-conjugated jugated human CD3 (manufactured by BD Pharmingen), Cy7APC-conjugated anti-CD19 human antibo dy (manufactured by BD Pharmingen), PE-Cy7-conjugated anti-CD38 human antibody (BD Pharmingen) BD Pharmingen) or FITC-conjugated anti-CD14 human antibody (BD Pharmingen) After staining, bone marrow cells from myeloma patients were analyzed using FACS. The binding of MMG49 antibody to each fraction was measured by the following method (Figure 7). binds strongly to myeloma cells, whereas it binds to all normal blood cells, including hematopoietic stem and progenitor cell fractions. This indicates that there is little binding to the sphere.

[0229] Example 6 Analysis of binding of MMG49 to various cell lines MMG49 antibody in various cell lines (MM1s cells, U266 cells, RPMI8226 cells, and JJN3 cells) The binding of the antibody and FIB27 antibody was analyzed using FACS. This is the same as in the case of peripheral blood.

[0230] Integrin β7 is a fusion protein of integrin α4 or integrin α E Forms a heterodimer with Since it is known that Alexa647-cojugated anti-human CD4 49d antibody (Biolegend) and APC-conjugated anti-human CD103 antibody (Biole The expression of these proteins was also analyzed simultaneously using FACS. Tegrin α E and CD49d refers to integrin α4. As in Example 5 above, Integrin α in peripheral blood from healthy individuals E and the expression level of integrin α4. (Figure 8).

[0231] As a result, ITGA4 was expressed in most myeloma cell lines, and ITGAE was expressed in all cell lines. The FIB27 antibody bound to all myeloma cell lines, whereas the MMG49 antibody did not. The expression level of the FIB27 antibody was not consistent with that of the FIB27 antibody. The binding of MMG49 antibody and FIB27 antibody to ITGA4-deficient U266 cells was examined by FACS. Both antibodies, MMG49 and FIB27, lost their binding to U266 cells due to ITGA4 deficiency. Both antibodies recognize β7 integrin, which is expressed as α4β7 integrin. Understood.

[0232] Example 7 Analysis of the relationship between integrin activation and MMG49 antibody binding Considering the unique binding mode of the MMG49 antibody described above, it is possible that the MMG49 antibody undergoes a structural change upon activation. It was speculated that it may recognize integrin β7.

[0233] Therefore, we used K562 cells that overexpressed α4β7 and a CD4 T cell enrichment kit (BD pharm Human normal peripheral blood CD4 T cells were enriched using a 5 mM EDTA / HBS solution and then washed with 1 mM Ca. 2+ / 1mM Mg 2+ / HBS (low activity buffer) or 2mM Mn 2+ / HBS (activation buffer) The cells were then incubated with MMG49 or FIB27 antibodies at room temperature for 30 minutes, and then washed. Then, PE-conjugated anti-mouse IgG antibody was added as a secondary antibody, and the mixture was left at room temperature for another 30 minutes. These were analyzed using FACS to detect integrin α4β7 The binding of the MMG49 and FIB27 antibodies to activated cells was measured.

[0234] As a result, Mn 2+ It was observed that the binding of the MMG49 antibody was enhanced in the presence of On the other hand, no similar changes were observed with the FIB27 antibody. This indicates that the MMG49 antibody This suggests that the antibody may be specific to activated integrin β7.

[0235] Example 8 Identification of the epitope essential for MMG49 antibody recognition To identify the epitope recognized by the MMG49 antibody, overlapping PCR was used. For the expression of eight types of human / mouse chimeric integrin β7 proteins, as shown in Figure 11, A vector was created.

[0236] Each expression vector was introduced into 293T cells by lipofection, and 48 hours later, MM cells were The presence or absence of binding of the G49 antibody was analyzed. The cells were suspended in PBS containing 1% fetal bovine serum, and the MMG49 antibody was After adding the antibody, the plate was left to stand at room temperature for 30 minutes. After washing, Alexa488-anti-mouse IgG antibody was added. After adding the eluate and leaving it at room temperature for 30 minutes, the cells were analyzed by FACS.

[0237] As a result, it was found that the region of the MMG49 antibody consisting of amino acid residues 110 to 721 is derived from mouse, And other than this (the region consisting of amino acid residues 20 to 109 and amino acid residues 722 to 798) Chimeric integrin β7 protein (#4960) in which the region consisting of amino acid residues is of human origin The binding was almost as strong as that of the full-length human chimeric integrin β7 protein (#4927). It was revealed that they bind to each other (Figures 11 to 13).

[0238] The region consisting of amino acid residues 722-798 is the transmembrane domain (TM) and the intracellular domain. The region consisting of amino acid residues 1 to 19 is a signal peptide. Considering that the PSI domain is a PSI domain, the region consisting of amino acid residues 20 to 109 is This indicates the presence of an epitope essential for the binding of the MMG49 antibody.

[0239] In addition, the region consisting of amino acid residues 110 to 378 is derived from mouse, and the region consisting of amino acid residues 20 to 109 is derived from mouse. The region consisting of the first amino acid residue and the region consisting of the 379th to 798th amino acid residues were derived from human The chimeric integrin β7 protein (#4961) developed The binding strength of the MMG49 antibody is slightly higher than that of the protein (#4960), and the full-length protein is human-derived. It was found that the binding level was exactly the same as that of the integrin β7 protein (#4927). This is what happened.

[0240] Furthermore, the amino acid residues 20 to 109, which were shown to contain the epitope of the above-mentioned MMG49 antibody, The region consisting of the nucleotide sequence and the signal peptide consisting of amino acid residues 1 to 19 The region consisting of amino acid residues 1 to 378, including the region, is of mouse origin, and the region consisting of amino acid residues 379 to 798 is of mouse origin. A chimeric integrin β7 protein (#4944) in which the amino acid residues of and the region consisting of amino acid residues 1 to 416 is of human origin, and amino acid residues 417 to 798 are of human origin. The chimeric integrin β7 protein (#4945), in which the amino acid residues are derived from humans, The region consisting of amino acid residues 1 to 563 is derived from mouse, and the region consisting of amino acid residues 564 to 798 is derived from mouse. a chimeric integrin β7 protein (#4946) in which the region of acid residues is of human origin; and The region consisting of amino acid residues 1 to 721 is derived from mouse, and the region consisting of amino acid residues 722 to 798 is derived from mouse. Compared to the chimeric integrin β7 protein (#4947) in which the region of residues is human-derived, It was also revealed that the binding ability of the MMG49 antibody was slightly increased.

[0241] In light of the above experimental results, the MMG49 antibody binds to the amino acid residues 20-109 of integrin β7. The specific binding strength, i.e., affinity, to the region consisting of 379-721 of human integrin β7 The region consisting of amino acid residues 379-721 of human integrin β7 It was also revealed that the activity increased in the presence of a region consisting of the first amino acid residue.

[0242] Example 9 Determination of the base sequence of the variable region of the MMG49 antibody molecule The MMG49 antibody subclass was confirmed using an Isotyping kit (Roche). Furthermore, the nucleotide sequence and amino acid sequence of the variable region of the MMG49 antibody were confirmed. The amino acid sequence was determined.

[0243] Sequencing was performed using the Smarter RACE cDNA amplification kit (Clontech). That is, cDNA prepared from mRNA derived from the hybridoma MMG49, which produces the MMG49 antibody. Using the above as a template, cDNA fragments of the H chain and κ chain variable region were amplified by PCR, and their base sequences were analyzed. The amino acid sequence, nucleotide sequence, and hypervariable region (CDR) of the decoded H chain variable region were 1 to 3) are shown in Tables 3 and 4 below.

[0244] The amino acid sequence, nucleotide sequence, and hypervariable region (CDR1-3) of the decoded L chain (κ chain) variable region were ) are also shown in Tables 3 and 4 below.

[0245] To confirm the specificity of the variable region sequence of the isolated MMG49 antibody, the variable region cDNA was cloned into human IgG 4 constant region and the constant region sequence of human IgL kappa chain to produce a chimeric antibody. Specifically, pFuse-CH-Ig-hG4 and pFuse were cloned using the In-Fusion cloning kit (Takara). Each variable region sequence was inserted into -CL-Ig-hk (Invivogen), and then the resulting vector was transfected with FreeStyle CHO-S cells. The chimeric antibody was then introduced into cells (Invitrogen) and the chimeric antibody secreted into the culture supernatant was collected. MM1s cells, which bind to MMG49 antibody, and KMS12BM cells, which do not bind to MMG49 antibody, were incubated with MMG49-hIgG4. After incubation in the buffer containing the antibody and washing, biotinylated anti-human IgG (Rockland) was added. ) was added as a secondary antibody, washed again, and streptavidin-PE (Biolegend) was added. The results showed that MMG49-hIgG4 was similar to the original MMG49 antibody. The staining pattern indicated that the obtained variable region sequence was correct (Fig. 1 4)

[0246] [Table 3]

[0247] [Table 4]

[0248] Example 10 Generation of chimeric antigen receptor T cells using the variable region of the MMG49 antibody Chimeric antigen receptor T cell (MMG49 antibody) using the variable region sequence of the MMG49 antibody molecule The production of these chimeric antigen receptor T cells (called somatic chimeric antigen receptor T cells) was carried out with reference to Non-Patent Documents 2 to 4. The following steps were taken:

[0249] (1) Cloning of CD28 and CD3z: RNA was extracted from Jurkat cells using Trizol (Invitrogen), and then purified with Superscript III. cDNA was prepared using a cDNA synthesis kit (Invitrogen). The cDNAs of CD28 and CD3z were amplified by PCR using the TA cloning kit (Invitrogen) The DNA was cloned using a DNA sequencing kit (Kanagawa University) and the base sequences were confirmed by sequencing.

[0250] (2) Combination of four fragments of VL / VH derived from MMG49 antibody and CD28 / CD3z: Using overlapping PCR, the VL and VH regions from the MMG49 antibody and the above The cloned CD28 and CD3z gene fragments were combined to prepare a chimeric cDNA. The procedure and the primers used are shown in Figure 15. The base sequences of the primers used are shown in Table 5 below. show.

[0251] [Table 5]

[0252] The ligated chimeric cDNA was cloned using a Zeroblunt PCR cloning kit (Invitrogen). After the DNA was cloned, sequencing was performed to confirm the base sequence. The amino acid sequence (SEQ ID NO: 21) and its base sequence (SEQ ID NO: 22) confirmed by the above procedure are shown in Table 1. The amino acid sequence shown in SEQ ID NO: 21 is the same as that shown in SEQ ID NO: 23 above. The amino acid sequence from the initiation codon (atg) immediately following it, without the sequence (gaattccacc) is converted into

[0253] (3) Insertion into expression vector: Next, the chimeric cDNA ligated in (2) was excised with two restriction enzymes, EcoRI / SalI, and It was inserted into the SCV-ires-GFP vector.

[0254] The MMG49 antibody-derived chimeric antigen receptor cDNA retroviral vector prepared above was used in gag / pol and VSV-G envelope expression vectors together with lipofectamine 2000 (Invitrogen). Retroviruses were produced by transfecting 293T cells with the vector. After a period of time, the supernatant was collected and used as a virus solution.

[0255] (4) Transduction into T cells: Next, the cDNA of the chimeric antigen receptor derived from the MMG49 antibody was introduced into human T cells as follows. Ta.

[0256] First, a 48-well plate was coated with anti-CD3 antibody (eBioscience). Human peripheral blood mononuclear cells were added and cultured for 72 hours. The culture medium was X-VIVO15 (Lonza) with 10% human Peripheral blood mononuclear cells were stimulated with AB type serum and IL-2 (175 IU / L). The virus solution prepared above was placed on a 48-well plate coated with ronectin (Takara). After adding the virus, the virus was adsorbed to Retronectin by centrifugation at 1700xg for 120 minutes. Peripheral blood mononuclear cells (including T cells) were added and gene transfer was performed. By continuing the culture, the MMG49 antibody-derived chimeric antigen receptor T cells were expanded and used for the following studies. T cells expressing a CAR construct using the variable region of the MMG49 antibody were incubated with PE-anti-human Staining using F(ab')2 antibody (Jackson Laboratory) confirmed that the construct was transfected. The expression of human F(ab')2 was detected in proportion to the expression of GFP (Fig. 16). It was confirmed that the CAR was expressed on the cell surface.

[0257] Example 11 Recognition and cytotoxicity of ITGB7-expressing tumor cells by MMG49 antibody-derived chimeric antigen receptor T cells analysis MMG49 antibody-derived chimeric antigen receptor T cells were prepared by the above method or GFP alone was introduced. The control T cells and K562 cells that do not express integrin β7 or integrin β7 were transfected with the control T cells. K562 cells overexpressing glyrin α4β7 were co-cultured and the amount of cytokines produced was quantified. Specifically, 1x10 T cells and 1x10 target cells were used. 5 into a 96-well plate After 24 hours, the supernatant was collected and the amount of IFN-γ produced was measured by ELISA. The results showed that the K562 cells expressing integrin α4β7 were Only in the co-culture of cells with MMG49 antibody-derived chimeric antigen receptor T cells, control (GFP-expressing Higher I than T cells obtained by similarly culturing peripheral blood mononuclear cells after stimulation with vectors FN-γ and IL2 production was observed (FIG. 17).

[0258] Next, we used MMG49 antibody-derived chimeric antigen receptor T cells or control T cells transfected with GFP alone. Myeloma cell lines (MM.1s cells, RPMI8226 cells, and JJN3 cells) that bind to human T cells and the MMG49 antibody cells) or cells to which the MMG49 antibody does not bind (KMS12BM, Molt4, and Raji cells). The amount of cytokines produced was also quantified. MM.1s, RPMI8226, and JJN3 cells and MMG49 antibody-derived chimeric antigen receptor T cells Only when co-cultured with control (GFP-expressing vector-transfected peripheral blood mononuclear cells after stimulation) The production of IFN-γ and IL-2 was higher than that of T cells obtained by similar culture of spheres (Figure 1 8 and 19). These results suggest that MMG49 antibody-derived chimeric antigen receptor T cells are It is activated by recognizing the antigen it recognizes (sometimes called the MMG49 antigen). This indicates that

[0259] moreover, 51 In a Cr cytotoxicity assay, MMG49 antibody-derived chimeric antigen receptor T cells inhibited myeloma cells We first investigated whether the target cells express integrin β7. K562 cells without or with integrin α4β7 overexpression were cultured in 10% FCS-supplemented medium. The cells were cultured in RPMI1640 medium supplemented with 0.5-1.0x10 4 It was prepared so that there were 1

[0260] Add an appropriate amount of Na2 51 CrO4 was added and the cells were incubated at 37°C for 2 hours. 51 Label with Cr, The washed cells were used as target cells. They were suspended in RPMI1640 medium supplemented with fetal bovine serum. The cells were mixed with MMG49 antibody-derived chimeric antigen receptor T cells and co-cultured for 4 hours.

[0261] It was then released into the supernatant 51 Cr was measured using a γ-counter. The cytotoxicity rate (%) was calculated as follows: was calculated based on equation (1).

[0262] (AB) / (CD)x100 (1) A: From the cells used in the experiment 51 Amount of Cr released B: Spontaneous in the absence of antibodies 51 Amount of Cr released C: Maximum with addition of 1% Triton X-100 51 Cr release amount D: Spontaneous in the absence of antibodies 51 Amount of Cr released.

[0263] As a result, in K562 cells forced to express integrin α4β7, which binds to the MMG49 antibody, MMG49 antibody-derived chimeric antigen receptor T cells express more GFP than T cells expressing only the control GFP. High cell damage was observed (Fig. 20).

[0264] Next, we used MMG49 antibody-derived chimeric antigen receptor T cells or control T cells transfected with GFP alone. Myeloma cell lines (MM1s cells, RPMI8226 cells, and JJN3 cells) that bind to human T cells and the MMG49 antibody ) or cells to which the MMG49 antibody does not bind (KMS12BM, Molt4, and Raji cells). As a result, the expression of integrin α4β7, which binds to the MMG49 antibody, was Only in K562 cells treated with MMG49 antibody were there significant differences in the expression of GFP-derived cytokines compared to control T cells expressing only GFP. High cytotoxicity by mela antigen receptor T cells was observed (Figure 21).

[0265] These results indicate that MMG49 antibody-derived chimeric antigen receptor T cells are capable of expressing the antigen recognized by MMG49 antibody. This indicates that it can specifically damage cells that express the antigen.

[0266] Example 12 Analysis of the in vivo ability of chimeric antigen receptor T cells derived from MMG49 antibody to eliminate bone marrow tumor cells MMG49 antibody-derived chimeric antigen receptor T cells were used to treat multiple myeloma in vivo. The therapeutic effect was investigated.

[0267] Myeloma cell line MM1s cells (4x10 5 pcs) Five days later, the mice were divided into two groups: one receiving MMG49 antibody-derived chimeric antigen receptor T cells and the other receiving control T cells. The mice were divided into groups receiving 5x10 6 Seven days later, Analysis revealed that all mice in the control T cell-administered group had significant bone marrow tumors. In the group administered MMG49 antibody-derived chimeric antigen receptor T cells, tumor cell proliferation was observed. These results suggest that the tumors disappeared almost completely after administration of MMG49 antibody-derived chimeric antigen receptor T cells. has the ability to eliminate tumors expressing the MMG49 antigen in vivo (Figure 22).

[0268] Furthermore, using a myeloma systemic dissemination model, we investigated the therapeutic effect on multiple myeloma in vivo. I looked it up.

[0269] Bone marrow of NOG mice irradiated with 2.4 Gy and intravenously transfected with luciferase gene tumor cell line MM1s cells (5x10 6 Five days after transplantation, the IVIS imaging system (Parkinson et al. The extent of tumor cell engraftment was measured using a chemiluminescence assay (Medtronic Elmer). The mice were divided into a group administered with somatic chimeric antigen receptor T cells and a group administered with control T cells, and 5 days after transplantation, and 7 days later, 3x10 6 Seven days after the second T cell injection, The tumor volume was measured again using the IS imaging system, and the tumor volume was In the MM49 group, significant myeloma cell proliferation was clearly observed in all mice, whereas in the MM49 group, significant myeloma cell proliferation was observed in all mice. In the group administered with somatic chimeric antigen receptor T cells, the tumors disappeared almost completely (Figure 23). These results suggest that administration of MMG49 antibody-derived chimeric antigen receptor T cells can also recognize the MMG49 antigen in vivo. These results demonstrate that the IL-16 receptor agonist has the ability to eliminate tumors that develop.

[0270] Example 13 The epitope of the MMG49 antibody was investigated in more detail based on the results of Example 8. Three types of human / mouse chimeric integrin β7 proteins were tested as shown in Figure 25. Expression vectors were prepared and transfected into 293T cells using the lipofection method. The cells were transfected into the cells, and 48 hours later, the presence or absence of binding of the MMG49 antibody was analyzed by FACS.

[0271] As a result, the MMG49 antibody targeted the integrin β7 protein at positions 1-32 and 91-798. The region consisting of amino acid residues is derived from mouse, and other than this (amino acid residues 33 to 90) The chimeric integrin β7 protein (the ch region in FIG. 25) is a sequence of human origin. 5.1), the full-length integrin β7 protein was almost the same as that of the human integrin β7 protein (#4927 in Figure 11). It was revealed that the binding was almost as strong as that of the hydroxybenzoates (Fig. 25).

[0272] Therefore, the epitope of the MMG49 antibody is located between the 33rd and 90th amino acids of the human integrin β7 protein. It was strongly suggested that the amino acid residues were involved.

[0273] Example 13 Human integrin β7, mouse integrin β7, and one or more of human integrin β7 Various mutants in which only two amino acids were mutated to the mouse-derived amino acid sequence (R35E / N36D, H38D, The vectors expressing the 293T cells were introduced into the cells by lipofection. After that, the experiment was carried out in the same manner as in Example 8. As a result, as shown in FIG. Only the variant showed significantly reduced binding to the MMG49 antibody compared to human integrin β7, and It was revealed that the values ​​were close to those of mouse integrin β7. The 48th amino acid residue of integrin β7 is strongly related to the epitope of the MMG49 antibody. It was revealed that this protein is included in the epitope of the MMG49 antibody.

[0274] The nucleotide sequences and amino acid sequences shown in this specification are shown below.

Claims

[Claim 1] The invention described in the specification.

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