B cell antibody receptor and its uses

Abstract

Provided are a B-cell antibody receptor (BAR), a BAR-T cell and others which are effective for the treatment of diseases associated with antibodies produced in the bodies of patients. The B-cell antibody receptor (BAR) according to the present invention comprises (a) an antibody binding domain, (b) a transmembrane domain, (c) an intracellular domain of a costimulating factor and (d) an intracellular domain of an activated receptor which are linked together.

Description

[Technical field]

[0001] The present invention relates to a novel artificial receptor (B-cell antibody receptor; BAR), immune cells, and regenerative medicine products (cellular medicines). [Background technology]

[0002] There are various proteins in the body of living organisms, and their functions maintain life activities. When a mutation occurs in a gene that codes for a protein, the protein can no longer be produced or its activity can be reduced, which can lead to the onset of disease.

[0003] Hemophilia is a hereditary disease caused by quantitative and qualitative abnormalities in blood clotting factors, and is classified into hemophilia A, hemophilia B, etc., depending on the type of blood clotting factor that causes the disease. Symptoms vary depending on the level of activity of the blood clotting factor, but severe cases can cause intra-articular and intramuscular bleeding, which can impair joint function and movement. Hemophilia is treated with replacement therapy, which replenishes missing blood clotting factors from outside the body.

[0004] Thrombotic thrombocytopenic purpura is a disease caused by quantitative and qualitative abnormalities in the function of ADAMTS13, a cleavage enzyme of von Willebrand factor (VWF). There are congenital and acquired cases, and this dysfunction induces excessive microthrombi, platelets are consumed and reduced in thrombus formation, and causes organ failure due to ischemic changes in various peripheral organs. In congenital cases, it has been reported that it is caused by a mutation in the gene that codes for the protein, and in acquired cases, it is caused by autoantibodies to this protein. As a treatment, for congenital cases, fresh frozen plasma 10ml / kg body weight is transfused every 2 weeks to replenish the ADAMTS13 enzyme and prevent the onset of the disease. For acquired cases, plasma exchange therapy is performed.

[0005] Lysosomal diseases are a group of diseases that currently include 60 types, and are caused by a genetic deficiency of acid hydrolase enzymes in lysosomes, intracellular organelles, resulting in the accumulation of large amounts of lipids or mucopolysaccharides in lysosomes, resulting in various symptoms such as liver and spleen enlargement, bone degeneration, and central nervous system disorders (Non-Patent Document 1).

[0006] Diseases classified as lysosomal diseases are distinguished by the type of enzyme that is deficient, and examples include Fabry disease, Gaucher disease, Pompe disease, and mucopolysaccharidoses.

[0007] Fabry disease, a lysosomal disease, is a disease that develops when glycoproteins GL-3 and Gb3 are not broken down due to a genetic deficiency or reduced activity of the enzyme α-galactosidase (α-GAL, hereafter also referred to simply as "GLA"), and the unbroken glycoproteins accumulate in tissues and organs. Symptoms include neurological symptoms (pain), corneal opacity, skin symptoms, cardiac dysfunction, renal dysfunction, etc. As a treatment, formulated α-GAL is administered intravenously to replenish the patient and break down the glycoproteins, thereby improving symptoms and inhibiting progression (Non-Patent Document 2).

[0008] Replacement therapy is used to treat diseases caused by gene mutations that prevent proteins from functioning properly, such as hemophilia and lysosomal diseases. However, in cases where the coagulation factor or responsible enzyme is missing or has a large defect, the coagulation factor or responsible enzyme supplemented from outside the body is regarded as a foreign substance by the patient's immune system, and there are quite a few cases where neutralizing antibodies against the coagulation factor or responsible enzyme supplemented from outside the body are produced. In cases where antibodies are produced in the patient's body, the enzyme cannot fully function, and the therapeutic effect is weakened. For example, in the treatment of Fabry disease, cases have been confirmed in which neutralizing antibodies against α-GAL are produced in the patient's body, and the antibodies bind to the administered α-GAL, thereby weakening the therapeutic effect (Non-Patent Documents 3 and 4). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Intractable Disease Information Center, Lysosomal storage disease (designated intractable disease 19): http: / / www.nanbyou.or.jp / entry / 4061 [Non-Patent Document 2] Sumitomo Dainippon Pharma website: https: / / healthcare.ds-pharma.jp / disease / fabry / index.html [Non-Patent Document 3] J Am Soc Nephrol 27:256-264,2016 [Non-Patent Document 4] Thromb Haematol 2005;94:760-769 Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, the production of antibodies against coagulation factors or responsible enzymes in the patient's body can lead to diseases or prevent treatments from being fully effective, so there is a demand for technology to control the function of these antibodies. [Means for solving the problem]

[0011] As a result of intensive research aimed at solving the above problems, the present inventors have found that the problem can be solved by eliminating the B cells that produce the neutralizing antibodies that cause the problem with immune cells that express a novel B cell antibody receptor. (1) A B cell antibody receptor that combines a) an antibody-binding domain, b) a transmembrane domain, c) the intracellular domain of a costimulatory factor, and d) the intracellular domain of an activating receptor. (2) The B cell antibody receptor described in (1), in which a linker is inserted into the binding site of a), b), c) or d). (3) The B cell antibody receptor described in (1) or (2), wherein the a) antibody binding domain is a protein or peptide containing an antibody recognition sequence that is specifically recognized by an antibody. (4) The B cell antibody receptor according to any one of (1) to (3), wherein the transmembrane domain b) is selected from the group consisting of CD28, CD3ζ, CD4, and CD8α. (5) The B cell antibody receptor according to any one of (1) to (4), wherein the intracellular domain of the costimulatory factor c) is selected from the group consisting of OX40, 4-1BB, CD27, CD278 and CD28. (6) The B cell antibody receptor according to any one of (1) to (5), wherein the activating receptor d) is selected from the group consisting of TCR and NKG2D. (7) A B cell antibody receptor described in any of (1) to (6), wherein the a) antibody binding domain comprises a full-length α-GAL protein, and the base sequence encoding the full-length α-GAL protein comprises the base sequence set forth in SEQ ID NO: 1, or a base sequence that is 95% or more identical to the base sequence set forth in SEQ ID NO: 1 and has the same function. (8) A B cell antibody receptor described in any of (1) to (6), wherein the a) antibody binding domain comprises the antibody recognition sequence region (D1 region) of α-GAL, and the base sequence encoding the antibody recognition sequence region (D1 region) of α-GAL comprises the base sequence set forth in SEQ ID NO: 2, or a base sequence that is 95% or more identical to the base sequence set forth in SEQ ID NO: 2 and has the same function. (9) A B cell antibody receptor described in any of (1) to (6), wherein the a) antibody binding domain comprises a spacer domain protein of ADAMTS13, and the base sequence encoding the spacer domain protein of ADAMTS13 comprises the base sequence set forth in SEQ ID NO: 3 or 37, or a base sequence that is 95% or more identical to the base sequence set forth in SEQ ID NO: 3 or 37 and has the same function. (10) A B cell antibody receptor described in any of (1) to (6), wherein the a) antibody binding domain comprises a spacer domain and a cysteine-rich domain of ADAMTS13, the base sequence encoding the spacer domain of ADAMTS13 comprises the base sequence set forth in SEQ ID NO: 3 or 37, or a base sequence that has the same function and is 95% or more identical to the base sequence set forth in SEQ ID NO: 3 or 37, and the base sequence encoding the cysteine-rich domain comprises the base sequence set forth in SEQ ID NO: 4, or a base sequence that has the same function and is 95% or more identical to the base sequence set forth in SEQ ID NO: 4. (11) The B cell antibody receptor described in any of (1) to (6), wherein the a) antibody-binding domain comprises a full-length protein or an antibody recognition sequence of blood coagulation factor VIII or blood coagulation factor IX. (12) A nucleic acid encoding the B cell antibody receptor according to any one of (1) to (8). (13) A vector comprising the nucleic acid according to (12). (14) An immune cell expressing a B cell antibody receptor gene transferred using the nucleic acid according to (12) or the vector according to (13). (15) The immune cell according to (14), wherein the immune cell is an αβ T cell, a CD8 T cell, a CD4 T cell, a γδ T cell, or a NK cell. (16) A regenerative medicine product (cell drug) containing the immune cell according to (14) or (15) as an active ingredient. (17) A regenerative medicine product (cellular drug) according to (16) for treating and / or preventing a lysosomal disease. (18) The regenerative medicine product (cellular pharmaceutical product) according to (17), wherein the lysosomal disease is Fabry disease. (19) A method for treating and / or preventing a lysosomal disease, comprising administering to a subject in need of treatment the immune cell according to (14) or (15), or the regenerative medicine product (cell drug) according to (17). (20) The method according to (19), wherein the lysosomal disease is Fabry disease. (21) A nucleic acid encoding a B cell antibody receptor according to (1) to (6), (9) or (10). (22) A vector comprising the nucleic acid according to (21). (23) An immune cell expressing a B cell antibody receptor, the B cell antibody receptor being genetically transferred using the nucleic acid according to (21) or the vector according to (22). (24) The immune cell according to (23), wherein the immune cell is an αβ T cell, a CD8 T cell, a CD4 T cell, a γδ T cell, or a NK cell. (25) A regenerative medicine product (cell drug) containing the immune cell according to (23) or (24) as an active ingredient. (26) A regenerative medicine product (cellular drug) according to (25) for treating and / or preventing thrombotic thrombocytopenic purpura. (27) A method for treating and / or preventing thrombotic thrombocytopenic purpura, comprising administering to a subject in need of treatment the immune cell according to (23) or (24), or the regenerative medicine product (cellular drug) according to (26). (28) A nucleic acid encoding the B cell antibody receptor according to (1) to (6) or (11). (29) A vector comprising the nucleic acid according to (28). (30) An immune cell expressing a B cell antibody receptor, the B cell antibody receptor being genetically transferred using the nucleic acid according to (28) or the vector according to (29). (31) The immune cell according to (30), wherein the immune cell is an αβ T cell, a CD8 T cell, a CD4 T cell, a γδ T cell, or a NK cell. (32) A regenerative medicine product (cell drug) containing the immune cell according to (30) or (31) as an active ingredient. (33) A regenerative medicine product (cellular drug) according to (32) for treating and / or preventing hemophilia. (34) A method for treating and / or preventing hemophilia, comprising administering to a subject in need of treatment the immune cell according to (30) or (31), or the regenerative medicine product (cellular drug) according to (33). Effect of the Invention

[0012] INDUSTRIAL APPLICABILITY The present invention provides an effective means for treating diseases caused by antibodies produced in the patient's body, such as lysosomal diseases. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows a conceptual diagram of therapy using BAR-T cells according to the present invention. [Diagram 2] Expression of BAR by BAR-T cells is shown. [Diagram 3] This shows the expression of BAR in BAR-T cells, which were human T cells transfected with BAR mRNA using the MaxCyte mRNA transfection protocol. [Figure 4] A method for producing a hybridoma that produces an anti-GLA monoclonal antibody is shown. [Diagram 5] 1 shows the generation of anti-GLA antibody scFv sequences. [Figure 6] An example of a plasmid vector into which the base sequence of the scFv of an anti-GLA antibody (SEQ ID NO: 9) has been inserted is shown. [Figure 7] An outline of the in vitro cytotoxic activity test is shown. [Figure 8] The results of in vitro cytotoxic activity (flow cytometry results) are shown. [Figure 9] a) The base sequence (SEQ ID NO: 1) encoding the amino acid sequence of the full-length protein of α-GAL, an example of an antibody-binding domain, and the lentiviral vector map of BAR into which this was inserted are shown. [Figure 10] a) The base sequence (sequence number 2) encoding the amino acid sequence of the antibody recognition sequence (D1 region) of α-GAL, which is an example of an antibody binding domain, and the lentiviral vector map of BAR into which this has been inserted are shown. [Figure 11] 1 shows the results of a GLA-mCherry binding assay targeting transfectants in which anti-GLA antibody scFv was forcibly expressed. [Figure 12] An example of a plasmid vector map for synthesizing mRNA of the full-length protein (FL)BAR of GLA is shown. [Figure 13]a) The base sequence (SEQ ID NO: 3) encoding the amino acid sequence of the spacer domain of ADAMTS13, which is an example of an antibody binding domain, and an example of a plasmid vector map of BAR into which this has been inserted are shown. [Figure 14] a) Shown is a base sequence (sequence number 3) encoding the amino acid sequence of the spacer domain of ADAMTS13, which is an example of an antibody binding domain, and a base sequence (sequence number 4) encoding the amino acid sequence of the cysteine-rich domain of ADAMTS13, as well as an example of a plasmid vector map of BAR into which these have been inserted. [Figure 15] a) The base sequence (SEQ ID NO: 1) encoding the amino acid sequence of the full-length protein of α-GAL, an example of an antibody-binding domain, and the retroviral vector map of BAR into which this was inserted are shown. [Figure 16] a) The base sequence (sequence number 2) encoding the amino acid sequence of the antibody recognition sequence (D1 region) of α-GAL, which is an example of an antibody binding domain, and the retroviral vector map of BAR into which this was inserted are shown. [Figure 17] An example of a plasmid vector map for synthesizing mRNA of the full-length protein (FL)BAR of GLA is shown. [Figure 18] a) The base sequence (SEQ ID NO: 3) encoding the amino acid sequence of the spacer domain of ADAMTS13, which is an example of an antibody binding domain, and an example of a plasmid vector map of BAR into which this has been inserted are shown. [Figure 19] a) Shown is a base sequence (sequence number 3) encoding the amino acid sequence of the spacer domain of ADAMTS13, which is an example of an antibody binding domain, and a base sequence (sequence number 4) encoding the amino acid sequence of the cysteine-rich domain of ADAMTS13, as well as an example of a plasmid vector map of BAR into which these have been inserted. [Figure 20] The base sequence (SEQ ID NO: 1) encoding the amino acid sequence of the full-length protein of α-GAL, which is an example of an antibody-binding domain, and the retroviral vector map of BAR into which this was inserted are shown. [Figure 21] The retroviral vector map into which the base sequence of the scFv of the anti-GLA antibody (SEQ ID NO: 36) has been inserted is shown. [Figure 22] The base sequence (SEQ ID NO: 37) encoding the amino acid sequence of ADAMTS13, which is an example of an antibody-binding domain, and the retroviral vector map of BAR into which this was inserted are shown. [Figure 23] The retroviral vector map into which the base sequence of the scFv of the anti-ADAMTS13 antibody (sequence number 41) has been inserted is shown. [Figure 24] 1 shows the results of a binding assay of hGLA-mCherry targeting Nalm6 expressing an anti-GLA antibody scFv. [Diagram 25] The results of confirming the expression of hGLA BAR using HA TAG are shown. [Figure 26] The results of confirming the expression of hGLA BAR using an anti-GLA antibody are shown. [Figure 27] These are the results of a CD69 detection test when BAR T cells (Jurkat-GLA-BAR2-EGFP: effector) and target cells (NALM6-scFv-GLA-mCherry: target) were mixed and cultured for 24 hours at ratios of 1:0, 2:1, and 1:1. [Figure 28] 1 shows the results of confirming the expression of ADAMTS13 BAR using HA TAG. [Figure 29] 1 shows the results of confirming the expression of ADAMTS13 BAR using HA TAG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention provides B cell antibody receptor-immune cells for treating diseases caused by the production in a patient's body of antibodies against therapeutic enzymes or coagulation factors supplemented from outside the body or endogenous proteins, and relates to immunotherapy using said cells. Here, "B cell antibody receptor-immune cells" (hereinafter also simply referred to as "BAR-immune cells") refer to immune cells that express a B cell antibody receptor (BAR), and when the immune cells are T cells, they are called "B cell antibody receptor-T cells" (hereinafter also simply referred to as "BAR-T cells").

[0015] [1] B cell antibody receptor (BAR) The BAR of the present invention is characterized by comprising a) an antibody-binding domain, b) a transmembrane domain, c) an intracellular domain of a costimulatory factor, and d) an intracellular domain of an activating receptor. a) It has high specificity and cytotoxicity against cells that overexpress the antibody to which the antibody-binding domain binds.

[0016] a) The antibody binding domain is not particularly limited as long as it is a substance to which an antibody causing a disease specifically binds. To damage or eliminate B cells that inhibit the therapeutic effect of enzyme replacement therapy for lysosomal diseases, etc., full-length proteins or peptides containing antibody recognition sequences, such as α-GAL for Fabry disease, glucocerebrosidase for Gaucher disease, α-glucosidase for Pompe disease, and α-L-iduronidase for mucopolysaccharidoses, are selected. The amino acid sequences or nucleotide sequences of these proteins are publicly available and can be obtained by keyword search from the database of the National Center for Biotechnology Information (NCBI). This antibody-binding domain binds to and interacts with disease-causing antibodies, thereby damaging and eliminating the B cells that produce those antibodies.

[0017] Furthermore, when damaging or eliminating B cells that produce antibodies that inhibit the therapeutic effect of administering blood coagulation factors, such as in hemophilia, the full-length proteins of blood coagulation factor VIII and blood coagulation factor IX, or peptides containing antibody recognition sequences, for example, are selected.

[0018] In the case of thrombotic thrombocytopenic purpura, examples include the full-length ADAMTS13 protein, or a peptide containing an antibody recognition sequence.

[0019] One embodiment of the antibody binding domain is a peptide containing the full-length protein (full length) of α-GAL or the antibody recognition sequence region (D1 region). The amino acid sequence of the full-length protein (full length) of α-GAL and the nucleotide sequence encoding it are shown in SEQ ID NO: 10 and SEQ ID NO: 1, and the amino acid sequence of the antibody recognition sequence region (D1 region) and the nucleotide sequence encoding it are shown in SEQ ID NO: 11 and SEQ ID NO: 2. The nucleotide sequence encoding the full-length protein (Full Length) or antibody recognition sequence region (D1 region) of α-GAL may be a substantially homologous nucleotide sequence having the same function. Here, the term "substantially homologous" encompasses two or more biological molecule sequences that are significantly similar to each other at the primary nucleotide sequence level. For example, in the context of two or more nucleic acid sequences, "substantially homologous" means at least about 75% identical, preferably at least about 80% identical, and more preferably at least about 85% identical or at least about 90% identical, and even more preferably at least about 95% identical, more preferably at least 97% identical, more preferably at least about 98% identical, and more preferably at least 99% identical.

[0020] One embodiment of the antibody binding domain is ADAMTS13. ADAMTS13 is a protein in which many domains are linked, and since it has been reported that most of the antibodies against ADAMTS13 bind to the spacer domain, it is considered preferable to use a spacer domain as the antibody binding domain. The amino acid sequence of the spacer domain of ADAMTS13 and the base sequence encoding it are shown in SEQ ID NOs: 12 and 3 (FIG. 13, FIG. 18). The base sequence encoding the spacer domain of ADAMTS13 may be a substantially homologous base sequence having the same function. It is also possible to use the spacer domain in combination with other parts. One embodiment of the other parts is a cysteine-rich domain, and the amino acid sequence of the cysteine-rich domain and the base sequence encoding it are shown in SEQ ID NOs: 13 and 4 (FIG. 14, FIG. 19).

[0021] b) The type of transmembrane domain is not limited as long as it does not inhibit the function of the B cell antibody receptor. For example, transmembrane domains such as CD28, CD3, CD4, and CD8α expressed in T cells can be used. The amino acid or nucleotide sequence information of these transmembrane domains is publicly available and can be obtained by keyword search from the NCBI database. These transmembrane domains may be appropriately mutated as long as it does not inhibit the function of the B cell antibody receptor. Here, the term "mutation" means any mutation including deletion, substitution, or addition of one or several to multiple amino acids. An oligopeptide linker or a polypeptide linker may be inserted into the binding site of a) the antibody binding domain, b) the cell transmembrane domain, c) the intracellular domain of the costimulatory factor, or d) the intracellular domain of the activating receptor, so as to link them together. Preferably, a linker consisting of 2 to 10 amino acids in length can be used. For example, a flexible linker having the amino acid sequence "GSGGG" can be used. As one embodiment of the transmembrane domain, the amino acid sequence of the transmembrane domain (TM) of CD3 and the nucleotide sequence encoding it are shown in SEQ ID NO: 15 and SEQ ID NO: 6, respectively. The BAR of the present invention may have a spacer sequence between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer sequence refers to any oligopeptide or polypeptide that functions to link the transmembrane domain and the extracellular domain and / or the transmembrane domain and the intracellular domain. The spacer sequence comprises up to 300 amino acids, preferably 10-100 amino acids, and most preferably 10-50 amino acids.

[0022] c) The type of intracellular domain of the costimulatory factor is not particularly limited, as long as it is an intracellular domain derived from a costimulatory factor possessed by immune cells or the like. For example, one or more intracellular domains selected from the group consisting of costimulatory factors such as OX40, 4-1BB, CD27, CD278, and CD28 can be appropriately selected and used. Information on the amino acid sequence or base sequence of the intracellular domain of these costimulatory factors is publicly available and can be obtained by keyword search from the NCBI database. The intracellular domain of these costimulatory factors may be appropriately mutated as long as it does not inhibit the function of the B cell antibody receptor. One embodiment of the intracellular domain of a costimulatory factor is 4-1BB, the amino acid sequence of which and the base sequence encoding it are shown in SEQ ID NO: 16 and SEQ ID NO: 7, respectively.

[0023] d) The type of intracellular domain of the activating receptor may be appropriately selected depending on the type of immune cell. For example, when T cells are used as immune cells, it is preferable to select CD3ζ (an intracellular domain derived from CD3, also called TCRζ chain), which is an intracellular domain of the T cell receptor (TCR). When NK cells are used, it is preferable to select an intracellular domain derived from NKG2D. Information on the amino acid or nucleotide sequence of these activating receptor domains is publicly available and can be obtained by keyword search from the NCBI database. Appropriate mutations may be introduced into these intracellular domains as long as they do not inhibit the function of the B cell antibody receptor. One embodiment of the intracellular domain of an activating receptor is CD3ζ, the amino acid sequence of which and the nucleotide sequence encoding it are shown in SEQ ID NO: 17 and SEQ ID NO: 8, respectively. The term "domain" as used herein refers to a region within a polypeptide. As used herein, "domain" refers to "antibody binding domain", "transmembrane domain", and "intracellular domain" depending on their location within the B cell antibody receptor molecule. A signal sequence can be added to support the transport of the intracellularly expressed BAR protein to the cell membrane, etc. Examples of signal sequences include Leader2 (SEQ ID NOs: 5 and 14) derived from a receptor gene called CSF2RA and Leader1 (SEQ ID NOs: 24 and 25) derived from CD8.

[0024] [2] Immune cells Examples of immune cells into which the B cell antibody receptor is introduced include αβT cells, CD8T cells, CD4T cells, γδT cells, and NK cells. First, peripheral blood mononuclear cells (PBMCs) are prepared as the raw material for αβT cells and the like. The method for collecting PBMCs is not particularly limited, but for example, PBMCs can be obtained by separating peripheral blood obtained by blood collection using density gradient centrifugation. The amount of blood collected at one time can be appropriately set depending on the treatment method, but for example, 1×1^7 to 3×10^7 PBMCs can be obtained by collecting 24 to 72 mL of blood. In addition, when it is necessary to secure a large amount of cells, mononuclear cell components can be collected using a blood component collection device.

[0025] When producing αβT cells, the collected PBMCs are suspended in a culture solution (medium), and interleukin 2 (IL-2) and anti-CD3 antibody are added to the obtained cell suspension, followed by culturing. The anti-CD3 antibody may be added to the medium or immobilized on the bottom surface of the culture vessel, but it is known that PBMCs can be cultured more effectively by seeding them in a culture vessel such as a flask on which the anti-CD3 antibody is immobilized (for example, Patent No. 3056230). IL-2 is preferably added to the medium at a concentration of 1000 to 2000 IU / mL.

[0026] Since the αβT cells prepared as described above contain CD8T cells and CD4T cells, when CD8T cells or CD4T cells are used as immune cells, they can be obtained from the prepared αβT cells by positive or negative selection using a cell separation method using magnetic beads or the like.

[0027] In the case of producing NK cells, a method of culturing PBMCs in the presence of an agonist of a molecule expressed in NK cells, such as an anti-CD16 antibody, or IL-2 is generally used (for example, Patent No. 4275680, etc.). The NK cells used in the present invention may be produced by any of these methods. In addition, CD16+ and / or CD56+ cells prepared by the method described in Patent Document WO2009 / 151183, etc. may be used as the NK cells.

[0028] When producing γδ T cells, the collected PBMCs are cultured in the presence of a phosphate antigen such as a bisphosphonate and IL-2, thereby selectively proliferating and activating the γδ T cells, thereby preparing a cell population containing γδ T cells at a high purity (for example, Patent Document WO2006 / 006720, etc.). The γδ T cells used in the present invention may be produced by any method.

[0029] The culture is carried out at a temperature of 34 to 38° C., preferably 37° C., and at a CO 2 concentration of 2 to 10%, preferably 5%, for a culture period of 1 to 20 days, and particularly preferably about 1 to 2 weeks.

[0030] The medium to be used is not particularly limited, and commercially available media used in cell culture, such as AIM-V medium (Invitrogen), RPMI-1640 medium (Invitrogen), Dulbecco's modified Eagle medium (Invitrogen), Iscove's medium (Invitrogen), KBM medium (Kohjin Bio), and ALys medium (Cell Science Institute), can be used. A human peripheral blood T cell medium (e.g., ALys505N; Cell Science Institute) to which IL-2 has been added in advance may also be used. In addition, 5 to 20% fetal bovine serum (FBS), fetal calf serum (FCS), human serum, human plasma (HBP), etc. can be added as necessary.

[0031] A gene encoding a B cell antibody receptor is introduced into the immune cells (αβT cells, CD8T cells, CD4T cells, γδT cells and / or NK cells) prepared as described above. The gene to be introduced can be selected from DNA or RNA. The introduction method can be appropriately selected from introduction using a viral vector, introduction using electroporation, introduction using lipofection, etc.

[0032] The immune cells thus prepared a) specifically bind to disease-causing antibody-producing B cells via the antibody through the antibody-binding portion, and are activated by activation signals and costimulatory signals, damaging and eliminating the B cells. As a result, the production of disease-causing antibodies is suppressed, and the disease is treated.

[0033] [3] Regenerative medicine products (cell medicines) and pharmaceutical compositions, or treatment and prevention methods using them According to the present invention, cells expressing a B cell antibody receptor (BAR) can be used to treat and / or prevent a disease, and typically, regenerative medicine products (cell medicines) and pharmaceutical compositions can be provided. Here, "treatment" includes alleviating (alleviating) symptoms characteristic of a target disease or associated symptoms, preventing or delaying the worsening of symptoms, and the like, and also includes amelioration of the disease. "Prevention" refers to preventing or delaying the onset / onset of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. In one aspect, the regenerative medicine product (cellular pharmaceutical product) of the present invention contains the cells of the present invention expressing BAR as an active ingredient, and may further contain appropriate excipients, etc. In another embodiment, the pharmaceutical composition of the present invention comprises an effective amount of the cells of the present invention as an active ingredient, and may further comprise a suitable pharma- ceutical acceptable excipient, etc. The excipient contained in the cell preparation and pharmaceutical composition includes various cell culture media, phosphate buffered saline, isotonic saline, etc. The term "regenerative medicine product" as used herein includes the following: 1) (i) The reconstruction, repair or formation of any structure or function of the human or animal body; or (ii) the treatment or prevention of disease in humans or animals; Among the products intended for use in the above, those that have been cultured or otherwise processed from human or animal cells; or 2) Products intended for use in the treatment of diseases in humans or animals that contain genes that are introduced into human or animal cells and expressed in the body. This refers to, but is not limited to, 1) Human cell-based products A. Human somatic cell processed products B. Human somatic stem cell processed products Human embryonic stem cell processed products D. Human induced pluripotent stem cell products 2)Animal cell-processed products A. Animal cell processed products B. Animal somatic stem cell processed products C. Animal embryonic stem cell processed products D. Animal induced pluripotent stem cell processed products 3) Gene Therapy Products A. Plasmid vector products B. Viral vector products Gene expression therapy products (excluding A and B) and regenerative medicine products that contain immune cells expressing the B cell antibody receptor of the present invention as active ingredients are classified as human somatic cell processed products.

[0034] [4] A nucleic acid encoding a B cell antibody receptor (BAR) of the present invention. According to the present invention, there is provided a nucleic acid encoding the amino acid sequence of the BAR described in [1] above. Unless otherwise specified, a "nucleic acid (or nucleotide sequence) encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate and encode the same amino acid sequence. Nucleic acids encoding BAR can be easily prepared from the cDNA sequence of the identified responsible enzyme by a conventional method. For example, among a) to d) constituting BAR, nucleic acids encoding a) can be obtained by, for example, extracting mRNA from somatic cells, preparing cDNA from the mRNA by reverse transcription, and using the cDNA as a template to amplify and obtain using PCR or the like. Nucleic acids encoding b), c), and d) can be obtained by, for example, extracting mRNA from immune cells, preparing cDNA from the mRNA by reverse transcription, and using the cDNA as a template to amplify and obtain using PCR or the like. The nucleic acid encoding BAR can be obtained by linking the nucleic acids encoding each domain obtained as described above. Linking of the nucleic acids encoding each domain can be performed using known methods such as cloning by homologous recombination and overlap extension method. The prepared BAR nucleic acid can be incorporated into a plasmid or viral vector for use, or mRNA can be synthesized and used.

[0035] [5] Method for producing immune cells expressing the B cell antibody receptor (BAR) of the present invention The method for producing immune cells expressing BAR of the present invention comprises the step of introducing the nucleic acid encoding BAR described in [4] above into immune cells. The step is carried out by a method of transforming immune cells ex vivo using a viral or non-viral vector containing a nucleic acid encoding BAR, or by electroporation. For example, they can be produced by transforming immune cells ex vivo using a viral vector or a plasmid vector containing a nucleic acid encoding a BAR according to the present invention, or the nucleic acid itself. The type and use of the vector are not particularly limited, and may be a plasmid vector or a virus vector (e.g., lentivirus, adenovirus, or retrovirus). The vector may be, for example, a cloning vector or an expression vector. Examples of the expression vector include vectors for prokaryotic cells such as Escherichia coli or actinomycetes, or vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells.

[0036] The equipment and method used for electroporation are not particularly limited, but MaxCyte GT (MaxCyte), Nepagene (Nepgene), Nucleofector (Lonza), Gene Pulser (Biorad), etc. can be used. Immune cells expressing B cell antibody receptors (BARs) a) recognize antibodies on B cells with their antibody-binding domain, then transmit the recognition signal to the inside of T cells and other cells via the ζ chain, and activate signals that induce cytotoxic activity within the cells via the transmembrane domain and costimulatory factors, thereby exerting cytotoxic activity against B cells that produce disease-causing antibodies.

[0037] [6] Immune cells expressing the B cell antibody receptor (BAR) of the present invention The immune cells expressing the BAR of the present invention are immune cells into which the nucleic acid encoding the BAR described in [4] above has been introduced and expressed by the production method described in [5] above.

[0038] The regenerative medicine product (cell drug) of the present invention is an injection (cell suspension) in which the immune cells of the present invention are suspended in a liquid (e.g., physiological saline) that can be used as a pharmaceutical, and can be used to treat and / or prevent diseases. This injection may be injected intravenously, intradermally, subcutaneously, etc., or may be directly injected into the affected area, or may be administered systemically as a drip infusion. It may contain a physiologically acceptable carrier or excipient.

[0039] The number of immune cells contained in the regenerative medicine product (cellular drug) of the present invention can be appropriately set depending on the administration method, type of disease, symptoms of the patient, age, sex, weight, severity of the disease, etc. Usually, it is set to 10^8 to 10^12 cells / person (preferably 10^9 cells / person).

[0040] The method for producing the regenerative medicine product (cell drug) of the present invention is not particularly limited. For example, the immune cells of the present invention can be produced as an infusion agent that can be administered intravenously by 1) recovering the immune cells of the present invention from a culture medium by centrifugation or the like; 2) washing the recovered immune cells with a washing solution (e.g., physiological saline, PBS, etc.); 3) recovering the washed immune cells from the washing solution by centrifugation or the like; and 4) suspending the recovered immune cells in a liquid that can be used as a pharmaceutical (e.g., physiological saline).

[0041] The target diseases for the regenerative medicine product (cell medicine) of the present invention include lysosomal diseases, including Fabry disease, Gaucher disease, Pompe disease, and mucopolysaccharidoses, as well as diseases for which replacement therapy is performed, such as hemophilia and thrombotic thrombocytopenic purpura.

[0042] The subject (subject) to which the regenerative medicine product (cellular drug) of the present invention is administered is, for example, an animal suffering from or likely to suffer from the above-mentioned disease. The animal is, for example, a mammal, preferably a human.

[0043] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0044] 1. Identification of the base and amino acid sequences of GLA The nucleotide sequence and amino acid sequence of GLA are publicly available, and the inventors obtained sequence information from the database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / protein / 4NXS_A). The nucleotide sequence of the D1 domain, which is the antibody recognition sequence region, was identified according to a non-patent document (J Mol Biol. 2004 Mar 19; 337(2): 319-35.). The nucleotide sequence encoding the full-length GLA protein is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the antibody recognition sequence region (D1 domain) of GLA is shown in SEQ ID NO: 2. MGC Human GLA Sequence-Verified cDNA (Clone ID: 3609235) (Dharmacon) in which the GLA sequence was inserted into a plasmid was purchased, and the plasmid was used as a template to isolate the full-length protein (Full Length) using the primer set shown in SEQ ID NO: 19 and SEQ ID NO: 20, and the nucleotide sequence of the antibody recognition sequence region (D1 region) using the primer set shown in SEQ ID NO: 19 and SEQ ID NO: 21.

[0045] 2. Identification of the base sequences of two types of BAR The amino acid sequences of the intracellular domain of the costimulatory factor (4-1BB), the intracellular domain of the activating factor (CD3ζ), the transmembrane domain (TM), and the spacer sequence that constitute BAR have already been demonstrated to be effective in CD19 CAR-T cells (Patent Document; WO2012079000A1, SEQ ID NO: 8). The base sequence combining the transmembrane domain, 4-1BB, and CD3ζ was synthesized by Integrated DNA Technologies' artificial gene synthesis service, and the base sequence encoding the transmembrane domain, 4-1BB, and CD3ζ was obtained using the primer set shown in SEQ ID NO: 22 and SEQ ID NO: 23 as a template. The nucleotide sequence of the full-length protein or antibody recognition sequence region (D1 region) described above, and the nucleotide sequence combining the transmembrane domain, 4-1BB, and CD3ζ were inserted into the XhoI-EcoRI site of the pMSCV plasmid by in-fusion cloning to clone the nucleotide sequence of BAR.

[0046] 3. Generation of GLA BAR-T cells The two types of BAR sequences identified in 2 above were subcloned into a general-purpose plasmid, and multiple restriction enzyme sites were introduced into the 5' side of the spacer of this sequence. The antigen sequence was recombined using these restriction enzyme sites. The base sequence of the full-length protein or D1 region of GLA, which is the antibody binding domain, was amplified by PCR, and after confirming that there were no mutations in the sequence by sequencing, it was recombined into the above-mentioned plasmid. Using the recombinant plasmids described above, the nucleotide sequence encoding the full-length GLA protein (Full Length: FL) BAR and the nucleotide sequence encoding the D1 region of the GLA BAR were inserted into a retroviral vector (Figures 15 and 16) or a lentiviral vector (Figures 9 and 10), respectively, to produce viral vectors. This vector was used to introduce genes into human T cells to generate FL BAR-T cells and D1 BAR-T cells. FACS was performed using an anti-GLA antibody. The results are shown in Figure 2. A rightward shift in the mean fluorescence intensity was observed, indicating that BAR was expressed in FL BAR-T cells and D1 BAR-T cells, resulting in increased fluorescence intensity. As another gene transfer method, mRNA transfection was performed. The base sequence constituting BAR was subcloned into a plasmid vector (pmRNAex, Figure 12, Figure 17) for in vitro transcription, and the BAR mRNA fragment was isolated using mMESSAGEmMACHINE T7 Ultra kit, Catalog#AM1345 (Thermo Fisher Science). BAR-encoding mRNA was introduced into human T cells using a MaxCyte electroporation device, and the protein expression level was confirmed by Western blotting. 14 hours after electroporation, the expression level of BAR was measured using β-actin as a housekeeping protein. From this result, it was confirmed that BAR was expressed by introducing GLA BAR mRNA into human T cells by electroporation. The BAR with the D1 region as the antibody binding domain showed extremely strong expression compared to the BAR with the FL as the antibody binding domain (Figure 3).

[0047] 4. Preparation of anti-GLA antibody overexpressing cells (anti-GLA scFv overexpressing M1 cells) We fused spleen cells from mice inoculated with human GLA protein with myeloma cells to obtain hybridomas, and positively selected the hybridoma population in HAT medium to obtain hybridomas that produce anti-GLA monoclonal antibodies (Figure 4). Next, antibody sequencing was performed on the anti-GLA monoclonal antibody-producing B cells to obtain the amino acid sequences of the heavy and light chain variable regions of the anti-GLA monoclonal antibody. Based on the obtained amino acid sequences, a linker was inserted between the sequences to create the scFv sequence of the anti-GLA antibody (Figure 5). The amino acid sequence of the scFv of the anti-GLA antibody and the base sequence encoding it are shown in SEQ ID NO: 18 and SEQ ID NO: 9, respectively. The base sequence of the scFv of the anti-GLA antibody identified above was subcloned and recombined into a plasmid vector (FIG. 6). The obtained plasmid vector was subcloned, recombined into a retroviral vector, and then infected into M1 cells, a mouse myeloid leukemia-derived cell line, to produce a transfectant that forcibly expressed the anti-GLA antibody scFv, which was used as a model cell for neutralizing antibody-producing B cells. The overexpression of anti-GLA antibody in the model cells was confirmed by a GLA-mCherry binding assay using these cells (Figure 11).

[0048] 5. Generation of CD19 CAR-T cells According to the Materials and Methods in the non-patent document (New England Journal of Medicine 2014;371(16):1507-1517), the base sequence encoding CD19 CAR-T in the patent document (WO2012079000A1, SEQ ID NO:8) was inserted into a retrovirus or lentivirus vector to prepare the retrovirus or lentivirus vector, and then gene transfer was performed into human T cells using RetroNectin to produce CD19 CAR-T cells.

[0049] 6.In Vitro Cytotoxicity Test (method) Figure 7 shows an outline of the cytotoxicity test for the two types of GLA BAR-T cells prepared in 2 and M1 cells in which the scFv of the anti-GLA antibody prepared in 3 was forcibly expressed. The subjects of the study were "FL BAR-T cells" that adopted the full-length protein of GLA as the antibody binding domain, and "D1 BAR-T cells" that adopted the D1 region of GLA. The immune cells were human primary T cells, and the T cell culture method and the protocol for the cytotoxicity test were the same as those used in the validation experiment of CD19 CAR-T cells (Non-Patent Document Frontiers in Immunology 8, 1956 (2018) or Non-Patent Document Science 353, 179-184 (2016)). The method was to obtain a mononuclear cell fraction from human peripheral blood by density centrifugation, transfer it to a plate attached with anti-CD3 / CD28 antibodies, and promote selective proliferation of T cells. The next day, T cells were stimulated to proliferate with IL-7:20ug / ml and IL-15:10ug / ml while suppressing differentiation into effector cells and maintaining them as memory (stem) cells. This cytokine has been reported to be the combination that most strongly maintains the cytotoxic activity of CAR-T cells and best retains the cytotoxic activity when administered in vivo. On day 2, genes were introduced into stimulated human T cells with recombinant retroviruses encoding anti-CD19 CAR or BAR, and on day 5, co-culture was started with M1 cells expressing scFv of anti-GLA antibody and M1 cells transfected with GFP as an infection control, and analysis was performed by FACS on days 0 and 4. Coculture of BAR-T cells with M1 cells transfected with GFP served as a transfection control, and coculture of CD19 CAR-T cells with M1 cells overexpressing the scFv of the anti-GLA antibody was used to exclude the possibility of nonspecific damage.

[0050] (result) The results are shown in Figure 8. Analysis by FACS was performed 4 days after the start of co-culture. The X-axis shows the co-cultured cells stained with APC anti-CD3 antibody and subjected to FACS. The viability of M1 cells was 72.3% in the co-culture of GFP-introduced M1 cells (control) with BAR-T cells (lower row GFP in Figure 8), and 66.9% in the co-culture of anti-GLA antibody scFv-overexpressed M1 cells with CD19 CAR-T cells (lower row CD19 in Figure 8), confirming the absence of non-specific reactions. On the other hand, the viability of M1 cells was 56.4% in the co-culture of anti-GLA antibody scFv-overexpressed M1 cells with FL BAR-T cells (lower row FL in Figure 8), and 50.8% in the co-culture of anti-GLA antibody scFv-overexpressed M1 cells with D1 BAR-T cells (lower row D1 in Figure 8), which successfully reduced the number of M1 cells significantly compared to the control. Furthermore, based on the results of this cytotoxicity test and the strength and stability of protein expression, it was determined that GLA BAR-T cells with the D1 region as their extracellular domain were more effective in specifically eliminating anti-GLA antibody-producing B cells.

[0051] 7. Preparation of target cells (anti-GLA scFv expressing cells) A retroviral vector (Figure 21) encoding the scFv of anti-GLA antibody was introduced into Nalm6 (RCB1933), a human leukemia precursor B cell line (purchased from Riken Cell Bank), to create a target model cell for BAR-T cells. After introducing the retroviral vector, sorting was performed using mCherry, and biotin-labeled anti-Fab2 antibody was reacted with the cell, followed by fluorescently labeled streptavidin. Analysis confirmed the expression of anti-GLA antibody by the target model cell (Figure 24).

[0052] 8. Preparation of GLA BAR-T Cells A retroviral vector (Figure 20) encoding the amino acid sequence of hGLA (SEQ ID NO: 10, DNA sequence SEQ ID NO: 1) was introduced into Jurkat, a human leukemia T cell line, to generate BAR-T cells. After gene introduction with the retroviral vector, the cells were sorted with GFP (Figure 25), reacted with anti-GLA antibody, and then reacted with a fluorescently labeled secondary antibody. Analysis confirmed the expression of BAR (Figure 26).

[0053] 9. Confirmation of BAR-T cell activation by mixed culture The BAR-T cells (Jurkat-GLA-BAR2-EGFP: Effector) prepared in 8 and the target cells (NALM6-scFv-GLA-mCherry: Target) prepared in 7 were mixed and cultured for 24 hours at a mixing ratio of 1:0, 2:1, and 1:1, and a CD69 detection test was performed. It is known that the expression of CD69 increases when Jurkat is activated, and if the BAR-T cells introduced into Jurkat bind to the scFv of the anti-GLA antibody of the target cells and an activation signal is received, the expression of CD69 is thought to increase depending on the mixing ratio. As a result of performing the mixed culture, the expression of CD69 increased in the presence of target cells compared to the control (E:T = 1:0) in which no target cells were present, and the expression of CD69 further increased when the target cells were increased (E:T = 2:1 → 1:1) (Figure 27). This confirmed that BAR-T cells are activated in an antigen-specific manner.

[0054] 10. Preparation of BAR-T cells (ADAMTS13) A retroviral vector encoding the amino acid sequence of ADAMTS13 (Figure 22) was introduced into Jurkat, a human leukemia T cell line, to generate BAR-T cells. After gene introduction with the retroviral vector, the cells were sorted by GFP and the expression of ADAMTS13 was confirmed by HA-TAG (Figures 28 and 29). [Industrial Applicability]

[0055] By using this invention, it is possible to specifically damage and eliminate B cells that produce specific antibodies, which is expected to improve the condition of patients with lysosomal diseases who, despite undergoing enzyme replacement therapy, are unable to obtain sufficient therapeutic effects due to the production of antibodies against the enzyme. This invention is also expected to be a therapeutic drug for diseases caused by antibodies. [Sequence List Free Text]

[0056] SEQ ID NO:1 <223> DNA encoding the full-length α-GAL protein SEQ ID NO:2 <223> DNA encoding the α-GAL D1 domain protein SEQ ID NO:3 <223> DNA encoding the spacer domain of ADAMTS13 SEQ ID NO:4 <223> DNA encoding the cysteine-rich domain of ADAMTS13 SEQ ID NO:5 <223> DNA encoding the signal sequence Leader2 SEQ ID NO:6 <223> DNA encoding the transmembrane domain SEQ ID NO:7 <223> DNA encoding 4-1BB SEQ ID NO:8 <223> DNA encoding CD3ζ SEQ ID NO:9 <223> DNA encoding the scFv of the anti-GLA antibody SEQ ID NO: 10 <223> α-GAL full length protein SEQ ID NO:11 <223> α-GAL D1 domain protein SEQ ID NO:12 <223> Spacer domain of ADAMTS13 SEQ ID NO:13 <223> Cysteine-rich domain of ADAMTS13 SEQ ID NO:14 <223> Signal sequence Leader2 protein SEQ ID NO:15 <223> Transmembrane domain proteins SEQ ID NO:16 <223> 4-1BB protein SEQ ID NO:17 <223> CD3ζ protein SEQ ID NO:18 <223> Anti-GLA antibody scFv protein SEQ ID NO:19 <223> α-GAL primer (before) SEQ ID NO: 20 <223> α-GAL primer (back: for full length) SEQ ID NO:21 <223> α-GAL primer (back: for D1 region) SEQ ID NO:22 <223> TM, 4-1BB, CD3ζ primer (first) SEQ ID NO:23 <223> TM, 4-1BB, CD3ζ primers (later) SEQ ID NO:24 <223> DNA encoding the signal sequence Leader1 SEQ ID NO:25 <223> Signal sequence Leader1 protein SEQ ID NO:26 <223> DNA encoding the human CD8 signal peptide SEQ ID NO:27 <223> Human CD8 signal peptide SEQ ID NO:28 <223> DNA encoding HA SEQ ID NO:29 <223> HA protein SEQ ID NO: 30 <223> DNA encoding the IgG4 antibody hinge region SEQ ID NO:31 <223> IgG4 antibody hinge region protein SEQ ID NO:32 <223> DNA encoding the light chain of human IgG anti-GLA antibody SEQ ID NO:33 <223> Human IgG anti-GLA antibody L chain protein SEQ ID NO:34 <223> DNA encoding mCherry SEQ ID NO:35 <223> mCherry protein SEQ ID NO:36 <223> DNA encoding the scFv of the anti-GLA antibody SEQ ID NO:37 <223> DNA encoding the spacer domain of ADAMTS13 SEQ ID NO:38 <223> Spacer domain of ADAMTS13 SEQ ID NO: 39 <223> DNA encoding the human IgG anti-ADAMTS13 antibody L chain signal peptide SEQ ID NO: 40 <223> Human IgG anti-ADAMTS13 antibody L chain signal peptide SEQ ID NO:41 <223> DNA encoding the scFv of the anti-ADAMTS13 antibody SEQ ID NO:42 <223> Anti-ADAMTS13 antibody scFv SEQ ID NO: 43 <223> DNA encoding the human CD8a transmembrane domain SEQ ID NO:44 <223> Human CD8a transmembrane domain

Claims

1. A B cell antibody receptor comprising: a) an antibody binding domain; b) a transmembrane domain; c) an intracellular domain of a costimulatory factor; and d) an intracellular domain of an activating receptor, a) the antibody-binding domain comprises a full-length α-GAL protein, and the nucleotide sequence encoding the full-length α-GAL protein comprises the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence having the same function and 95% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1; or a) the antibody-binding domain comprises an antibody recognition sequence region (D1 region) of α-GAL, and a base sequence encoding the antibody recognition sequence region (D1 region) of α-GAL comprises a base sequence set forth in SEQ ID NO: 2, or a base sequence having the same function and 95% or more identical to the base sequence set forth in SEQ ID NO: 2; B cell antibody receptor.

2. The B cell antibody receptor according to claim 1 , wherein a linker is inserted at the binding site of a), b), c) or d).

3. The B cell antibody receptor according to claim 1 or 2, wherein the a) antibody-binding domain is a protein or peptide comprising an antibody recognition sequence that is specifically recognized by an antibody.

4. The B cell antibody receptor according to any one of claims 1 to 3, wherein the b) transmembrane domain is selected from the group consisting of CD28, CD3ζ, CD4, and CD8α.

5. The B cell antibody receptor according to any one of claims 1 to 4, wherein the intracellular domain of the costimulatory factor c) is selected from the group consisting of OX40, 4-1BB, CD27, CD278 and CD28.

6. The B cell antibody receptor of any one of claims 1 to 5, wherein the activating receptor d) is selected from the group consisting of TCR or NKG2D.

7. A nucleic acid encoding the B cell antibody receptor according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid of claim 7.

9. An immune cell expressing a B cell antibody receptor gene transferred thereto using the nucleic acid of claim 7 or the vector of claim 8.

10. The immune cell according to claim 9 , wherein the immune cell is an αβ T cell, a CD8 T cell, a CD4 T cell, a γδ T cell or an NK cell.

11. A regenerative medicine product (cell drug) containing the immune cell according to claim 9 or 10 as an active ingredient.

12. The regenerative medicine product (cellular pharmaceutical product) according to claim 11 for treating and / or preventing a lysosomal disease.

13. The regenerative medicine product (cellular pharmaceutical product) according to claim 12, wherein the lysosomal disease is Fabry disease.

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