Intracellular drug delivery agent

Abstract

In the present invention, an antibody or antigen-binding fragment thereof that specifically binds to an extracellular region of the CD179b protein expressed on a cell surface and has the ability to be internalized into cells expressing the CD179b protein on the cell surface can be used as an intracellular drug delivery agent for delivering a drug into cells expressing the CD179b protein on the cell surface.

Description

Intracellular drug delivery agents

[0001] The present invention relates to a drug delivery agent for cells expressing the CD179b protein on the cell surface, comprising an antibody or an antigen-binding fragment thereof as an active ingredient.

[0002] In recent years, various antibody drugs targeting antigen proteins on cells involved in disease have emerged as treatments for diseases. While these antibody drugs have attracted attention for their certain efficacy as targeted antigen-specific therapies, many of the targeted antigen proteins are also expressed on multiple normal cells. As a result of antibody administration, not only the cells involved in the disease but also the normal cells expressing the antigen are affected pharmacologically, and the resulting side effects are a problem. Therefore, it is expected that if antigens that are specifically expressed on the surface of cells involved in the disease can be identified and antibodies targeting these antigens can be used as pharmaceuticals, it will be possible to treat diseases with antibody drugs that have fewer side effects.

[0003] B cells are a type of lymphocyte (immune cell) that accounts for approximately 20-40% of lymphocytes in humans. B cells play a role in humoral immunity by producing antibodies (immunoglobulins). B cells differentiate through pro-B cells, pre-B cells, immature B cells, and mature B cells, ultimately differentiating into plasma cells and producing antibodies to eliminate foreign substances from the body. The CD179b protein is part of the surrogate light chain of immunoglobulins and is known to be expressed on the cell surface of B cells (pro-B cells and pre-B cells). The amino acid sequence of a part of the CD179b protein has a sequence very similar to that of the antibody light chain (Non-Patent Literature 1). The CD179b protein is known to be expressed in leukemia cells (pre-B cell leukemia) in which pre-B cells have become cancerous (Non-Patent Literature 2 and 3). Furthermore, the CD179b protein is also expressed in lymphoma cells that have become cancerous from pre-B cells (pre-B cell lymphoma), and it is known that it can be used as a diagnostic marker for pre-B cell lymphoma (Non-Patent Literature 4). In addition, there are cancers that specifically express the CD179b protein on the cell surface, and it is known that antibodies or antigen-binding fragments thereof that specifically bind to the extracellular domain of the CD179b protein can be used as pharmaceutical compositions for the treatment and / or prevention of such cancers (Patent Literature 1).

[0004] In recent years, research has been conducted to enhance the efficacy of antibody drugs for various diseases, and in particular, the development of antibody-drug conjugates (ADCs), which are formed by conjugating a drug with strong direct cell-killing ability with an antibody that targets those cells, is actively underway. In ADCs, the drug bound to the antibody circulates in the bloodstream and accumulates at the target disease site to exert its therapeutic effect. The release of the drug outside the disease site (detachment from the antibody) is not always desirable because it carries the risk of causing side effects. Therefore, it is desirable for the drug bound to the antibody to be taken up into cells. In other words, in ADCs, it is desirable that the antibody not only accumulates at the target disease site but also to be efficiently taken up into cells (antibody internalization ability). Since this ability is closely related to the therapeutic effect of ADCs, finding antibodies with high internalization ability against each antigen is a major challenge in the development of ADCs.

[0005] WO2010 / 005068

[0006] Science, Vol 316, Issue 5822 pp. 291-29 (2007) Adv. Immunol. , 63:1-41 (1996) Blood, 92:4317-4324 (1998) Modern Pathology, 17:423-429 (2004)

[0007] In developing ADCs that target cells expressing the CD179b protein on their cell surface, an antibody or its antigen-binding fragment with internalization activity into the cell is desirable. However, it was unknown whether an antibody or its antigen-binding fragment that specifically binds to the CD179b protein possesses such internalization activity into the cell.

[0008] The objective of the present invention is to identify an antibody or its antigen-binding fragment that targets cells expressing the CD179b protein on the cell surface and has internalization activity into such cells, and to utilize such antibody or its antigen-binding fragment as an intracellular drug delivery agent.

[0009] The present invention has the following features (1) to (24): (1) An intracellular drug delivery agent comprising an antibody or antigen-binding fragment thereof as an active ingredient, which specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells that express the CD179b protein on the cell surface. (2) The intracellular drug delivery agent according to (1), wherein the antibody or antigen-binding fragment thereof specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having 80% or more sequence identity with said amino acid sequence. (3) The intracellular drug delivery agent according to (1) or (2), wherein the antibody or antigen-binding fragment thereof specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having 80% or more sequence identity with said amino acid sequence. (4) The intracellular drug delivery agent according to any one of (1) to (3), wherein the cell is a B cell. (5) The intracellular drug delivery agent according to (4), wherein the B cell is a pro-B cell, pre-B cell, immature B cell, or mature B cell. (6) The intracellular drug delivery agent according to any one of (1) to (3), wherein the cells are cancer cells. (7) The intracellular drug delivery agent according to (6), wherein the cancer is leukemia or lymphoma. (8) The intracellular drug delivery agent according to any one of (1) to (7), wherein the antibody is a monoclonal antibody or a polyclonal antibody. (9) The intracellular drug delivery agent according to any one of (1) to (8), comprising a conjugate of the antibody or its antigen-binding fragment and a drug as an active ingredient. (10) The intracellular drug delivery agent according to (9), wherein the drug is a cytotoxic agent, an immunomodulator, or a radioisotope. (11) A pharmaceutical composition comprising an antibody or its antigen-binding fragment and a drug as active ingredients, which specifically bind to the extracellular domain of the CD179b protein expressed on the cell surface and have the ability to internalize the CD179b protein into cells expressing the CD179b protein on the cell surface. (12) The pharmaceutical composition according to (11), wherein the antibody or an antigen-binding fragment thereof and the drug conjugate are active ingredients.(13) A pharmaceutical composition for the treatment and / or prevention of a disease in which B cells exacerbate the condition, comprising an antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells that express the CD179b protein on the cell surface, and a drug as active ingredients. (14) The pharmaceutical composition according to (13), wherein the disease in which B cells exacerbate the condition is an autoimmune disease. (15) The pharmaceutical composition according to (14), wherein the autoimmune disease is systemic lupus erythematosus (SLE), SLE-associated pulmonary hypertension, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, systemic scleroderma, mixed connective tissue disease, ANCA-associated vasculitis, pemphigus, anti-NMDA receptor encephalitis, IgG4-related disease, cryoglobulinemia vasculitis, idiopathic thrombocytopenic purpura, primary biliary cholangitis, myasthenia gravis, polymyositis, dermatomyositis, IgA nephropathy, autoimmune hepatitis, chronic inflammatory demyelinating polyneuropathy, eosinophilic granulomatosis with polyangiitis, Hashimoto's disease, Graves' disease, antiphospholipid antibody syndrome, type 1 diabetes mellitus, Castleman disease, sarcoidosis, neuromyelitis optica, or autoimmune hemolytic anemia. (16) A pharmaceutical composition according to any one of (13) to (15), comprising a conjugate of the antibody or its antigen-binding fragment and the drug as an active ingredient. (17) A pharmaceutical composition for the treatment and / or prevention of cancer, comprising an antibody or its antigen-binding fragment that specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells that express the CD179b protein on the cell surface, and a drug as active ingredients. (18) The pharmaceutical composition according to (17), wherein the cancer is leukemia or lymphoma. (19) A pharmaceutical composition according to (17) or (18), comprising a conjugate of the antibody or its antigen-binding fragment and the drug as an active ingredient. (20) An intracellular drug delivery method comprising administering an antibody or an antigen-binding fragment thereof, and a drug, to a subject in vivo, which specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells that express the CD179b protein on the cell surface, and delivering the drug into cells expressing the CD179b protein on the cell surface within the subject's body.(21) The intracellular drug delivery method according to (20), wherein a conjugate of the antibody or its antigen-binding fragment and the drug is administered to a subject. (22) A method for treating and / or preventing a disease associated with cells expressing the CD179b protein on their cell surface, wherein an antibody or its antigen-binding fragment and a drug are administered to a subject, which specifically bind to the extracellular domain of the CD179b protein expressed on the cell surface and have the ability to be internalized into cells expressing the CD179b protein on their cell surface. (23) The method according to (22), wherein the disease is a disease in which B cells exacerbate the condition. (24) The method according to (22), wherein the disease is cancer.

[0010] The antibody or antigen-binding fragment used as the active ingredient of the present invention can be used as an antibody or fragment that delivers a drug into cells expressing the CD179b protein on the cell surface.

[0011] The present invention relates to the use of an antibody or its antigen-binding fragment (hereinafter collectively referred to as "the active ingredient antibody of the present invention") that specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to be internalized into the cell, as an intracellular drug delivery agent to cells that express CD179b on the cell surface.

[0012] One of the technical features of the antibody, which is the active ingredient of the present invention, is that it "specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface." This means that it specifically binds to the polypeptide, which is the extracellular domain of the CD179b protein expressed on the cell surface, through immunological reactivity.

[0013] The CD179b protein is expressed in various mammals, and information on its nucleotide and amino acid sequences can be obtained, for example, by accessing GenBank (NCBI, USA). CD179b is also known by other names such as λ5, IGLL1, and Vpreb2, but in this specification, "CD179b" will be used as a representative name. For example, the full-length amino acid sequence of the human CD179b protein is registered in GenBank as "NP_001356835".

[0014] It is known that in certain cells, a mature CD179b protein is expressed on the cell surface, in which the signal sequence has been removed from the full-length amino acid sequence of the CD179 protein. For example, in humans, it is known that the full-length mature human CD179b protein, consisting of the amino acid sequence of SEQ ID NO: 6, which has the signal sequence removed from the full-length amino acid sequence of the human CD179b protein, is expressed extracellularly. Therefore, when applying the present invention to humans, an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, or a polypeptide consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, is preferably used.

[0015] Furthermore, it is known that the C-terminal amino acid sequence of the CD179b protein is identical to that of the Immunoglobulin lambda light chain C region protein registered in GenBank as "CAA27229.1". For example, in humans, of the mature human CD179b protein consisting of the amino acid sequence represented by SEQ ID NO: 6, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8, which is the region from the N-terminus to 71 amino acid residues, is a specific extracellular region of the CD179b protein, and from the 72nd amino acid residue onward from the N-terminus, the amino acid sequence is identical to that of the Immunoglobulin lambda light chain C region protein registered in GenBank as "CAA27229.1". Therefore, when using the present invention to specifically introduce a drug into human cells expressing the CD179b protein on the cell surface, an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, is more preferably used as the antibody, which is the active ingredient of the present invention.

[0016] Furthermore, the base sequences of the DNA encoding the protein consisting of the amino acid sequences represented by SEQ ID NOs. 6 and 8 are the base sequences represented by SEQ ID NOs. 5 and 7, respectively.

[0017] Another technical feature of the antibody, which is the active ingredient of the present invention, is its ability to be internalized into cells that express the CD179b protein on their cell surface. The internalization ability of the antibody, which is the active ingredient of the present invention, can be clarified by evaluating the internalization into cells that express the CD179b protein on their cell surface in vitro, as will be described later.

[0018] Furthermore, the intracellular drug delivery agent of the present invention can be used to introduce a drug into cells that express the CD179b protein on their cell surface, utilizing an antibody having the aforementioned technical features. As an example of how to use it, the conjugate of the antibody and drug that constitute the active ingredient of the present invention can be brought into contact with cells that express the CD179b protein on their cell surface in vitro, or the conjugate can be administered in vivo to a subject (test animal) to introduce the drug into cells that express the CD179b protein on their cell surface. Whether the drug has been introduced into the cell can be confirmed by evaluating the effect of the drug on improving the disease state or inhibiting the proliferation of the cells in vitro or in vivo.

[0019] The following describes in detail the methods for carrying out the present invention.

[0020] <Preparation of Antibody Production Antigen> The protein or fragment thereof used as a sensitizing antigen to obtain the antibody, which is the active ingredient of the present invention, can be derived from mammals, specifically humans, dogs, cats, mice, cattle, horses, rats, chickens, etc., and the animal species from which it is derived is not limited. However, it is preferable to select a sensitizing antigen that has high sequence identity with the antigen expressed by the target cells. Generally, mammalian proteins are preferred, and human proteins are particularly preferred. For example, if the target CD179b protein is human CD179b protein, it is preferable to use human CD179b protein or its fragments, or cells that express human CD179b protein.

[0021] In this invention, the target protein is a sequence having 70-100%, preferably 80-100%, more preferably 90-100%, and even more preferably 95-100%, for example, 97-100%, 98-100%, 99-100%, or 99.5-100%, of the amino acid sequence of Sequence ID No. 6, which is the full-length amino acid sequence of mature human CD179b protein, as a reference. Here, "sequence identity" is calculated as the ratio of identical amino acids (or bases) to the total number of amino acids (or bases) when two sequences are aligned (sorted) to achieve the greatest similarity, with or without introducing gaps, and is expressed as a percentage (%).

[0022] A fragment of the CD179b protein has a length less than the full length of the protein, ranging from the amino acid length of the epitope (antigenic determinant), which is the smallest unit recognized by the antibody. An epitope refers to a polypeptide fragment that is antigenic or immunogenic in mammals, preferably humans, and its smallest unit consists of about 7 to 12 amino acids, for example, 8 to 11 amino acids, and includes polypeptides whose amino acid sequence has 80% or more, preferably 85% or more, more preferably 90% or more sequence identity with the amino acid sequence of the CD179b protein.

[0023] The CD179b protein and polypeptides containing its fragments, as described above, can be synthesized according to chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method) (Japanese Biochemical Society, ed., Biochemistry Experiment Course 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Tokyo Kagaku Dojin (Japan), 1981). They can also be synthesized by conventional methods using various commercially available peptide synthesizers. Furthermore, known genetic engineering methods (Sambrook et al., Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press; Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & By preparing DNA encoding the above polypeptide using Sons (or similar), incorporating the DNA into an expression vector and introducing it into a host cell, the target polypeptide can be obtained by causing the host cell to produce the polypeptide.

[0024] The DNA encoding the above polypeptide can be easily prepared by known genetic engineering techniques or by conventional methods using commercially available nucleic acid synthesizers. For example, DNA containing the base sequence of Sequence ID No. 1 can be prepared by performing PCR using human chromosomal DNA or a cDNA library as a template and a pair of primers designed to amplify the base sequence described in Sequence ID No. 1. The PCR reaction conditions can be set as appropriate, for example, by using a heat-stable DNA polymerase (e.g., Taq polymerase) and Mg ２＋Using the contained PCR buffer, one reaction cycle consists of 30 cycles of 30 seconds at 94°C (denaturation), 30 seconds to 1 minute at 55°C (annealing), and 1 minute at 72°C (extension), followed by a 7-minute reaction at 72°C. However, this is not an exhaustive condition. PCR methods and conditions are described, for example, in Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons (especially Chapter 15).

[0025] Furthermore, based on the nucleotide and amino acid sequence information represented by Sequence IDs 1 to 8 in the sequence listings herein, appropriate probes and primers can be prepared and used to screen a cDNA library, such as a human cDNA library, to isolate the desired DNA. The cDNA library is preferably prepared from cells, organs, or tissues expressing the CD179b protein. Examples of such cells or tissues include, but are not limited to, cells or tissues derived from normal or diseased sites such as bone marrow, peripheral blood mononuclear cells (PBMCs), cancer cells, cancerous tissue, and pathological tissue. The above-mentioned operations, such as the preparation of probes or primers, the construction of a cDNA library, the screening of the cDNA library, and the cloning of the target gene, are known to those skilled in the art and can be carried out in accordance with the methods described, for example, Sambrook et al., Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology (1989), Ausbel et al. (above), etc. From the DNA obtained in this way, DNA encoding the CD179b protein or its fragments can be obtained.

[0026] The host cells mentioned above can be any cells capable of expressing the polypeptide, and examples of prokaryotic cells include Escherichia coli, while examples of eukaryotic cells include mammalian cells such as monkey kidney cells COS1 and Chinese hamster ovary cells CHO, human fetal kidney cell line HEK293, mouse fetal skin cell line NIH3T3, yeast cells such as budding yeast and fission yeast, silkworm cells, and African clawed frog egg cells, but are not limited to these.

[0027] When prokaryotic cells are used as host cells, the expression vector preferably has an origin, promoter, ribosome binding site, multicloning site, terminator, drug resistance gene, nutrient complement gene, reporter gene, etc., that can be replicated in prokaryotic cells. Examples of expression vectors for E. coli include the pUC system, pBluescriptII, pET expression system, and pGEX expression system. By incorporating the DNA encoding the polypeptide into such an expression vector, transforming prokaryotic host cells with the vector, and then culturing the resulting transformants, the polypeptide encoded by the DNA can be expressed in the prokaryotic host cells. When constructing the expression vector, various tags and other proteins can be added to the polypeptide to design it, allowing it to be expressed as a fusion protein.

[0028] When eukaryotic cells are used as host cells, it is preferable to use an expression vector for eukaryotic cells that has a promoter, splicing region, poly(A) addition site, etc. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3.1, pSecTag(A, B, C), pYES2, etc. Similarly, by incorporating the DNA encoding the polypeptide into such an expression vector, transforming eukaryotic host cells with the vector, and then culturing the resulting transformants, the polypeptide encoded by the DNA can be expressed in the eukaryotic host cells. When constructing an expression vector, if the polypeptide is designed by adding various tags or other proteins, or if pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc. are used as the expression vector, the polypeptide can be expressed as a fusion protein to which various tags or other proteins such as His tags (e.g., (His)6 to (His)10), FLAG tags, myc tags, HA tags, GFP, etc. are added.

[0029] The expression vector can be introduced into host cells using well-known methods such as heat shock, electroporation, calcium phosphate, liposomes, DEAE dextran, microinjection, viral infection, lipofection, and binding to cell membrane-permeable peptides.

[0030] To isolate and purify a target polypeptide from host cells, a combination of known separation procedures can be performed. Examples of isolation and purification techniques include, but are not limited to, treatment with denaturing agents such as urea or surfactants, sonication, enzymatic digestion, salting out or solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration chromatography, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse-phase chromatography.

[0031] <Antibody Structure> The antibody, the active ingredient of the present invention, is typically a heteromultimeric glycoprotein containing at least two heavy chains and two light chains. IgG, a representative class of antibodies, is a heterotetrameric glycoprotein of approximately 150 kDa composed of two identical light (L) chains and two identical heavy (H) chains. Typically, the light chains are linked to the heavy chains by a single disulfide covalent bond, but the number of disulfide bonds between the heavy chains varies depending on the immunoglobulin isotype. In addition, both the heavy and light chains also have intrachain disulfide bonds. The heavy chain has a variable region (VH region) at its N-terminus and a constant region at its opposite C-terminus. Similarly, the light chain also has a variable region (VL region) at its N-terminus and a constant region at its opposite C-terminus. The domain configuration of each region is such that the variable region of the heavy chain, the variable region of the light chain, and the constant region of the light chain are all one, while the constant region of the heavy chain is multiple. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. The variable region of an antibody contains a region called the complementarity-determining region (CDR), which exhibits particularly high variability and functions as an antigen-binding site. The portion of the variable region that is relatively conserved relative to the CDR is called the framework region (FR). The variable regions of both the complete heavy chain and the light chain each contain four FRs linked by three CDRs. The three CDRs are called CDRH1, CDRH2, and CDRH3 in the heavy chain, in order from the N-terminus, and similarly CDRL1, CDRL2, and CDRL3 in the light chain. CDRH3 is the most important for the antibody's specificity in binding to the antigen. Furthermore, the CDRs of each chain are held together in close proximity by the FRs and, together with the CDR from the other chain, contribute to the formation of the antibody's antigen-binding site. The constant region does not directly contribute to antibody binding to antigens, but it contributes to various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC activity) and phagocytosis via binding to the Fcγ receptor, half-life / clearance rate via binding to the neonatal Fc receptor (FcRn), and complement-dependent cell-mediated cytotoxicity (CDC activity) via binding to the C1q component of the complement cascade.

[0032] Examples of antibodies include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, and single-chain antibodies. Antibodies also include, for example, antigen-binding fragments of the aforementioned antibodies (e.g., Fab or F(ab')). ２ This includes fragments such as monoclonal antibodies, polyclonal antibodies, or antigen-binding fragments thereof.

[0033] Furthermore, the antibody may be any class of immunoglobulin molecule, such as IgG, IgE, IgM, IgA, IgD, and IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.

[0034] The antibody, which is the active ingredient of the present invention, may be further modified by acetylation, formylation, amidation, phosphorylation, or PEGylation, in addition to glycosylation.

[0035] <Antibody Production> Examples of various antibody production methods are shown below.

[0036] Polyclonal antibodies usable in the present invention can be obtained, for example, as follows.

[0037] By immunizing mice, human antibody-producing mice, rabbits, and other small animals with naturally occurring CD179b protein, recombinant CD179b protein expressed in microorganisms such as E. coli as a fusion protein with GST, fragments thereof, or cells expressing CD179b on their cell surface (e.g., cancer cell line Nalm6), immune cells that produce anti-CD179b antibodies are induced, and serum containing the anti-CD179b antibodies produced by these immune cells is obtained. This serum is then prepared by purification using methods such as ammonium sulfate precipitation, protein A, protein G columns, DEAE ion exchange chromatography, or affinity columns coupled with CD179b protein or synthetic peptides. In the examples described later, mouse polyclonal antibodies against CD179b protein were produced, and the intracellular internalization activity of the mouse polyclonal anti-CD179b antibodies was confirmed.

[0038] Another example of an antibody usable in this invention is a monoclonal antibody. Methods for obtaining monoclonal antibodies include, for example, a method similar to obtaining polyclonal antibodies, in which a small animal is immunized with a sensitizing antigen to induce immune cells that produce anti-CD179b antibodies and a fusion cell (hybridoma) is established from the animal, and a method of isolating immune cells from the animal and cloning the antibody gene. In the method of establishing a hybridoma, the spleen is extracted from the animal, the cells are separated, and these cells are fused with myeloma cells. From the resulting fusion cell (hybridoma), a clone that produces an antibody with intracellular internalization activity is selected. The monoclonal antibody-producing hybridoma with intracellular internalization activity is isolated, the hybridoma is cultured, and the antibody is purified from the culture supernatant using a general affinity purification method.

[0039] A hybridoma that produces monoclonal antibodies can be created, for example, as follows:

[0040] First, animals are immunized with the sensitizing antigen according to known methods. Generally, this is done by injecting the sensitizing antigen intraperitoneally or subcutaneously into mammals. Specifically, the sensitizing antigen is diluted to an appropriate volume with PBS (Phosphate-Buffered Saline) or physiological saline, suspended, and then, if desired, mixed with an appropriate amount of a standard adjuvant, such as complete Freund's adjuvant. After emulsification, the immunized mammals are administered several times every 4 to 21 days. A suitable carrier can also be used during sensitizing antigen immunization.

[0041] After immunizing mammals in this manner and confirming an increase in the desired antibody levels in the serum, immune cells are collected from the mammals and subjected to cell fusion. Among the preferred immune cells are spleen cells in particular.

[0042] Mammalian myeloma cells are used as the other parent cells to be fused with the aforementioned immune cells. These myeloma cells are various known cell lines, such as P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), and P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler.G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. al., Nature (1978) 276, 269-270), F.O. Suitable examples include al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I.S.J. Exp.Med. (1978) 148, 313-323), R210 (Galfré, G. et al., Nature (1979) 277, 131-133), etc.

[0043] The cell fusion between the aforementioned immune cells and myeloma cells can be carried out in accordance with known methods, such as the method of Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46.

[0044] More specifically, the cell fusion is carried out, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ), and if desired, auxiliary agents such as dimethyl sulfoxide may be added to further enhance the fusion efficiency.

[0045] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, and other common culture media used for this type of cell culture can be used, and serum supplements such as fetal bovine serum (FBS) can also be used in combination.

[0046] Cell fusion is performed by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium, adding a PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C, usually at a concentration of 30 to 60% (w / v), and mixing to form the desired hybridoma. Subsequently, the cell fusion agent and other substances unfavorable to hybridoma growth are removed by repeatedly adding a suitable culture medium, centrifugating, and removing the supernatant.

[0047] The hybridomas obtained in this manner are selected by culturing them in a standard selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing in the HAT culture medium is continued for a sufficient amount of time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to be killed. Then, the standard limiting dilution method is performed to screen for hybridomas that produce the target antibody and to perform single cloning.

[0048] In a method for isolating immune cells from antigen-sensitized animals and cloning antibody genes, B cells are identified and unicellularized from peripheral blood or lymphoid tissue. The cDNA of the antibody gene is prepared by RT-PCR using antibody-specific primers and amplified by PCR. The obtained DNA is inserted into an expression vector, expressed in a recombinant expression system, and purified to obtain a monoclonal antibody (Zaibao Zhang, et al., Front Immunol 2017, 8:494). The target antibody is then cloned using the obtained monoclonal antibody.

[0049] In addition to obtaining the hybridomas described above by immunizing non-human animals with antigens, it is also possible to sensitize human lymphocytes, such as human lymphocytes infected with EB virus, in vitro with a protein, protein-expressing cells, or their lysates, and then fuse the sensitized lymphocytes with human-derived myeloma cells with permanent proliferative capacity, such as U266 (registration number TIB196), to obtain hybridomas that produce human antibodies with desired activity (e.g., cell proliferation inhibitory activity).

[0050] The hybridomas that produce monoclonal antibodies in this manner can be subcultured in a normal culture medium and can also be stored for long periods in liquid nitrogen.

[0051] In other words, it can be produced by using a desired antigen or cells expressing a desired antigen as a sensitizing antigen, immunizing them according to a standard immunization method, fusing the resulting immune cells with known parent cells using a standard cell fusion method, and then screening monoclonal antibody-producing cells (hybridoms) using a standard screening method.

[0052] Here, examples of human antibody-producing mice include KM mice (Kirin Pharma / Medarex) and Xeno mice (Amgen) (e.g., WO02 / 43478, WO02 / 092812, etc.). When such mice are immunized with the CD179b protein or a fragment thereof, fully human polyclonal antibodies can be obtained from the blood. Furthermore, spleen cells can be extracted from immunized mice and fully human monoclonal antibodies can be produced by fusing them with myeloma cells.

[0053] Furthermore, recombinant antibodies can be produced by cloning an antibody gene from a hybridoma, incorporating it into a suitable vector, introducing it into a host, and then using genetic recombination technology (see, for example, Carl, A.K. Borrebeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA of the variable region (V region) of the antibody is synthesized from the mRNA of the hybridoma using reverse transcriptase. Once the DNA encoding the V region of the target antibody is obtained, it is ligated with the DNA encoding the desired constant region (C region) of the antibody, and this is incorporated into an expression vector. Alternatively, the DNA encoding the V region of the antibody may be incorporated into an expression vector containing the DNA of the antibody C region. The expression vector is then designed to allow expression under the control of an expression regulatory region, such as an enhancer or promoter. Next, host cells can be transformed using this expression vector to express the antibody.

[0054] Monoclonal antibodies include human monoclonal antibodies and non-human animal monoclonal antibodies (e.g., mouse monoclonal antibodies, rat monoclonal antibodies, rabbit monoclonal antibodies, chicken monoclonal antibodies, etc.). Monoclonal antibodies can be produced by culturing hybridomas obtained by fusing splenocytes from non-human mammals (e.g., mice, human antibody-producing mice, rabbits, etc.) immunized with the CD179b protein or a fragment thereof with myeloma cells.

[0055] Chimeric antibodies are antibodies created by combining sequences from different animals. For example, an antibody consisting of the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody. Chimeric antibodies can be produced using known methods. For example, they can be obtained by ligating DNA encoding the V region of a non-human antibody with DNA encoding the C region of a human antibody, incorporating this into an expression vector, and introducing it into a host to induce production.

[0056] Polyclonal antibodies include antibodies obtained by immunizing human antibody-producing animals (e.g., mice and rabbits) with the CD179b protein or a fragment thereof. Polyclonal antibodies include antibodies produced from multiple types of antibody-producing cells, but antibodies produced by isolating one type of antibody-producing cell are called monoclonal antibodies.

[0057] Humanized antibodies are modified antibodies, also known as reshaped human antibodies. Humanized antibodies are constructed by substituting the CDR (Chronic Derived Residual Region) of an antibody derived from an immunized animal with the CDR of a human antibody. Common genetic engineering techniques for humanized antibodies are also known.

[0058] Specifically, a DNA sequence designed to link the CDR of a mouse or rabbit antibody with the framework region (FR) of a human antibody is synthesized by PCR from several oligonucleotides that have overlapping regions at their ends. The resulting DNA is then ligated to the DNA encoding the constant region of a human antibody, and subsequently incorporated into an expression vector, which is then introduced into a host to induce production (see EP239400 and WO96 / 02576). The FR of the human antibody linked via the CDR is selected from a CDR derived from an immunized animal that forms a good antigen-binding site. If necessary, amino acids in the framework region of the variable region of the antibody may be substituted so that the CDR of the reconstituted human antibody forms an appropriate antigen-binding site (Sato K. et al., Cancer Research 1993, 53:851-856). Alternatively, the framework region may be substituted with that derived from various human antibodies (see WO99 / 51743).

[0059] After producing chimeric antibodies or humanized antibodies, amino acids in the variable region (e.g., FR) or constant region may be substituted with other amino acids.

[0060] Amino acid substitutions are, for example, substitutions of less than 15, less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less amino acids, preferably 1 to 5 amino acids, more preferably 1 or 2 amino acids, and the substituted antibody should be functionally equivalent to the unsubstituted antibody. Conservative amino acid substitutions are preferred, which are substitutions between amino acids with similar properties such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties can be classified into, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acids (threonine, valine, isoleucine), and aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine).

[0061] Here, "functionally equivalent" refers to the technical characteristics of the antibody, which is the active ingredient of the present invention, namely its ability to specifically bind to the extracellular domain of the CD179b protein expressed on the cell surface, and its ability to internalize the CD179b protein into cells that express it on the cell surface. A method well known to those skilled in the art for preparing a polypeptide that is functionally equivalent to a given polypeptide is to introduce mutations into the polypeptide. For example, a person skilled in the art would know site-directed mutagenesis (Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271-275; Zoller, MJ., and Smith, M., (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W., and Fritz, HJ., (1987) Methods By appropriately introducing mutations into the antibody of the present invention using Enzymol. 154, 350-367, Kunkel, TA., (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766, etc., an antibody functionally equivalent to the antibody of the present invention can be prepared.

[0062] An antibody that recognizes the epitope of the CD179b protein recognized by the above-mentioned anti-CD179b antibody can be obtained by methods known to those skilled in the art. For example, it can be obtained by determining the epitope of the CD179b protein recognized by the anti-CD179b antibody by a conventional method (e.g., epitope mapping), and producing an antibody using a polypeptide having the amino acid sequence contained in the epitope as an immunogen, or by determining the epitope of an antibody produced by a conventional method and selecting an antibody with the same epitope as the anti-CD179b antibody. Here, "epitope" refers to an antigenic or immunogenic polypeptide fragment in mammals, preferably humans, and its minimum unit consists of about 7 to 12 amino acids, preferably 8 to 11 amino acids.

[0063] The antibody that is the active ingredient of the present invention is preferably an antibody that has a structure such that rejection reactions are almost or completely avoided in the target animal to which it is administered. Examples of such antibodies include, for example, when the target animal is human, human antibodies, humanized antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), single-chain antibodies, and bispecific antibodies. These antibodies are recombinant antibodies in which the variable regions of the heavy chain and light chain are derived from human antibodies, or the variable regions of the heavy chain and light chain consist of complementarity-determining regions (CDR1, CDR2, and CDR3) derived from non-human animal antibodies and a framework region derived from human antibodies, or the variable regions of the heavy chain and light chain are derived from non-human animal antibodies and the constant regions of the heavy chain and light chain are derived from human antibodies. The former two antibodies are preferred.

[0064] These recombinant antibodies can be produced as follows: DNA encoding monoclonal antibodies against the CD179b protein (e.g., human monoclonal antibody, mouse monoclonal antibody, rat monoclonal antibody, rabbit monoclonal antibody, chicken monoclonal antibody, etc.) is cloned from antibody-producing cells such as hybridomas. Using this as a template, DNA encoding the light chain variable region and heavy chain variable region of the antibody is prepared by RT-PCR or the like, and the sequences of the light chain and heavy chain variable regions, or the sequences of each CDR1, CDR2, and CDR3 are determined based on the Kabat EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991)).

[0065] Furthermore, DNA encoding each of these variable regions or DNA encoding each CDR is produced using genetic engineering technology (Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. Here, the above-mentioned human monoclonal antibody-producing hybridoma can be produced by immunizing human antibody-producing animals (e.g., mice or rabbits) with the CD179b protein, and then fusing splenocytes excised from the immunized animals with myeloma cells. Separately, if necessary, DNA encoding the variable and constant regions of the light or heavy chain derived from human antibodies is produced using genetic engineering technology or a DNA synthesizer.

[0066] In the case of humanized antibodies, DNA encoding the humanized antibody can be created by replacing the CDR coding sequence in the DNA encoding the variable region of the light or heavy chain derived from a human antibody with the corresponding CDR coding sequence of an antibody derived from a non-human animal (e.g., mouse, rabbit, rat, chicken, etc.), and then ligating the resulting DNA with the DNA encoding the constant region of the light or heavy chain derived from a human antibody.

[0067] In the case of chimeric antibodies, DNA encoding a chimeric antibody can be created by ligating the DNA encoding the variable region of the light chain or heavy chain of an antibody derived from an animal other than a human (e.g., mouse, rabbit, rat, chicken, etc.) with the DNA encoding the constant region of the light chain or heavy chain derived from a human antibody.

[0068] In the case of single-chain antibodies, the antibody is one in which the heavy chain variable region and the light chain variable region are linearly linked via a linker. DNA encoding the single-chain antibody can be produced by binding the DNA encoding the heavy chain variable region, the DNA encoding the linker, and the DNA encoding the light chain variable region. Here, both the heavy chain variable region and the light chain variable region are derived from human antibodies, or are derived from human antibodies in which only the CDR is replaced with the CDR of an antibody derived from an animal other than a human (e.g., mouse, rabbit, rat, chicken, etc.). The linker consists of 12 to 19 amino acids, for example, (G4S)3 of 15 amino acids (G.-B. Kim et al., Protein Engineering Design and Selection 2007, 20(9):425-432).

[0069] In the case of a bispecific antibody, this antibody is capable of specifically binding to two different epitopes. For example, DNA encoding a bispecific antibody can be produced by binding DNA encoding heavy chain variable region A, DNA encoding light chain variable region B, DNA encoding heavy chain variable region B, and DNA encoding light chain variable region A in this order (however, the DNA encoding light chain variable region B and the DNA encoding heavy chain variable region B are bound via DNA encoding a linker as described above). Here, both the heavy chain variable region and the light chain variable region are derived from human antibodies, or are derived from human antibodies in which only the CDR is replaced with the CDR of an antibody derived from an animal other than a human (e.g., mouse, rabbit, rat, chicken, etc.).

[0070] Recombinant antibodies can be produced by incorporating the recombinant DNA prepared as described above into one or more suitable vectors, introducing them into host cells (e.g., mammalian cells, yeast cells, insect cells, etc.), and (co)expressing them (P.J. Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997; WILEY, P. Shepherd and C. Dean., Monoclonal Antibodies., 2000; OXFORD UNIVERSITY PRESS; J.W. Goding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC). (PRESS). In addition, hybridomas capable of producing other human antibodies or non-human animal antibodies (e.g., mouse antibodies or rabbit antibodies) against the CD179b protein are constructed, and the monoclonal antibodies produced by the hybridomas are recovered. Immunological binding affinity to the CD179b protein and intracellular internalization activity are used as indicators to determine whether or not it is the target antibody. After identifying the target monoclonal antibody-producing hybridoma, as described above, DNA encoding the variable regions of the heavy and light chains of the target antibody is constructed from the hybridoma, sequenced, and this DNA is used to produce another antibody.

[0071] Furthermore, the antibody that is the active ingredient of the present invention may have one or several (preferably one or two) amino acid substitutions, deletions, or additions in the sequence of the framework region and / or the constant region of the antibody, insofar as it possesses the technical characteristics of the antibody described above. Here, several means two to five, preferably two or three.

[0072] Since the complementarity-determining regions (CDRs) encoded by these DNA sequences are the regions that determine the specificity of the antibody, the sequences encoding the other regions of the antibody (i.e., the constant region and framework region) may be sequences derived from other antibodies. Here, "other antibodies" include antibodies derived from organisms other than humans, but from the viewpoint of reducing side effects, human-derived antibodies are preferred. In other words, in the above DNA, it is preferable that the regions encoding the framework regions of the heavy chain and light chain, and the constant region, contain base sequences that encode the corresponding amino acid sequences derived from human antibodies.

[0073] The above DNA can be obtained, for example, by the method described above or the method described below. First, total RNA is prepared from a hybridoma related to the antibody of the present invention using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using random primers, etc. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides of sequences conserved in the variable regions of known mouse antibody heavy chain genes and light chain genes, respectively, as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The base sequence of the DNA can be determined by conventional methods, such as by incorporating it into a sequencing plasmid or phage.

[0074] <Antibody Characteristics> The antibody, which is the active ingredient of the present invention, is technically characterized by its specific binding to the extracellular domain of the CD179b protein expressed on the cell surface. Whether or not it specifically binds can be determined using binding assays such as ELISA, Western blotting, immunofluorescence, and flow cytometry, as described in the examples.

[0075] The antibody, which is the active ingredient of the present invention, is expected to exhibit stronger internalization ability the higher its binding affinity to the extracellular domain of the CD179b protein expressed on the cell surface. A high binding affinity is indicated by a binding constant (affinity constant) K. ａ (binding rate constant k ｏｎ / dissociation rate constant k ｏｆｆ Preferably, at least 10 ７ M －１, at least 10 ８ M －１ , at least 5 × 10 ８ M －１ , at least 10 ９ M －１ , at least 5 × 10 ９ M －１ , at least 10 １０ M －１ , at least 5 × 10 １０ M －１ , at least 10 １１ M －１ , at least 5 × 10 １１ M －１ , at least 10 １２ M －１ , or, at least 10 １３ M －１ is preferably.

[0076] The antibody which is the active ingredient of the present invention may be an antibody conjugate. Examples of the antibody conjugate include an antibody conjugated with various molecules such as polyethylene glycol (PEG). In the antibody conjugate of the present invention, the substance to be conjugated is not limited. Such an antibody conjugate can be obtained by chemically modifying the obtained antibody. These methods have already been established in this field.

[0077] <Intracellular drug delivery agent> The antibody which is the active ingredient of the present invention binds to the extracellular region of the CD179b protein expressed on the cell surface of cells related to a disease, and further has the ability to be internalized into the cells, and thus can be used for introducing a drug into cells for the purpose of treating the disease.

[0078] In the present invention, when a drug is introduced into cells expressing the CD179b protein on the cell surface, the antibody and the drug may be in a so-called composition state, but it is preferable that they take the form of a so-called conjugate, in which the antibody and the drug are bound together. Covalent bonding is preferred for the binding of the antibody and the drug, and although the antibody and the drug may be directly bound, binding via a linker is preferred. Binding of the antibody and the drug can be achieved via a linker having a group that is reactive with amino groups, carboxyl groups, hydroxyl groups, thiol groups, etc. (for example, imidyl succinate, formyl group, 2-pyridyldithio group, maleimidyl group, alkoxycarbonyl group, hydroxyl group, etc.).

[0079] A linker is a substance capable of binding an antibody to a drug. While linkers can be either cleaving or non-cleaving, cleaving linkers are preferably those specifically cleaved within cells, and more preferably those cleaved by intracellular peptidases or proteases. Furthermore, linkers may have the effect of improving the solubility and stability of the antibody-drug conjugate.

[0080] Details regarding linker types and joining methods can be found in publicly available information (for example, see Greg T. Hermanson, Bioconjugate Techniques, Third Edition, WO2004 / 010957 and WO2014 / 012479).

[0081] The following are examples of reactive groups used to form antibody-drug conjugates:

[0082] Reactive groups attached to the amino acid sequence of antibodies or glycoproteins modified with amino acids include primary amines (ε-amino acids), carboxyls, thiols (sulfhydryls), carbonyls (ketones or aldehydes), and hydroxyls, unless otherwise specially chemically modified. Primary amines are present at the N-terminus of polypeptides or in the side chains of lysine residues, are positively charged under physiological conditions, and are usually located on the outside of proteins, allowing them to be used for binding without altering the protein structure. Carboxyls are present at the C-terminus of polypeptides or in the side chains of aspartic acid and glutamic acid. Sulfhydryls are present in the side chains of cysteine ​​and form disulfide bonds that maintain the higher-order structure of proteins. Ketones or aldehydes are produced in glycoproteins by oxidizing glycosyl with sodium metaperiodate.

[0083] Examples of reactive groups attached to linkers and drugs include the following:

[0084] Reacting groups that can react with amines include N-hydroxysuccinimide (NHS) esters, imide esters, pentafluorophenyl esters, hydroxymethylphosphine, isothiocyanates, isocyanates, acyl azides, N-hydroxyl esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryls, carbodiimides, and carboxylic anhydrides.

[0085] Carbodiimides, diazoalkanes, diazoacetyl compounds, and carbonyldiimidazoles are reactive groups that can react with carboxyls and amines.

[0086] Reactive groups that can react with thiols include maleimide, haloacetamide, pyridyl disulfide, thiosulfone, vinyl sulfone, haloacetyl, aziridine, acryloyl, and aryl.

[0087] Hydrazides and alkoxyamines are reactive groups that can react with aldehydes. Epoxys, oxiranes, carbonyldiimidazoles, N,N'-disuccinimidyl carbonates, N-hydroxysuccinimidyl chloroformates, and isocyanates are reactive groups that can react with hydroxyls.

[0088] Isocyanates are reactive groups that can react with hydrochlor.

[0089] Diazirines, aryl azides, aryl compounds, benzophenols, and diazo compounds are examples of photoreactive reactive groups.

[0090] Examples of linkers having the above-mentioned reactive groups include the following:

[0091] As linkers having the same reactive group terminus, there are linkers with N-hydroxysucciimide esters as the reactive group (for example, Disuccinimide Glutarate (DSG), Disuccinimide Superate (DSS), Bis(sulfosuccinimide)Superate (BS3), Tris-(succinimide)Aminotriacetate (TSAT), PEGylated Bis(sulfosuccinimide)Superate (BS(PEG)). ５ BS (PEG) ９), Dithiobis (Succinimidyl Propionate) (DSP), 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), Sulfo-ethylene glycol bis(succinimidyl succinate (Sulfo-EGS), Dimethyl adipimidate・2HCl (DMA), Dimethyl pimelimidate・2HCl (DMP), Dimethyl suberimidate・2HCl (DMS), Dimethyl3,3'-dithiobispropionimidate・2H Cl (DTBP), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), Disuccinimidyl tartrate (DST), Bis[2-(Succinimidooxycarbonyloxy)ethyl]Sulfone (BSOCOES), and linkers with maleimide as the reactive group (e.g., Bismaleimidoethane (BMOE), 1,4-bismaleimidobutane (BMB), Bismaleimidohexane (BMH), Tris(2-maleimidothyl)amine (TMEA), 1,8-bismaleimido-(PEG)) ２ (BM(PEG) ２ ), 1,8-bismaleimido-(PEG) ３ (BM(PEG) ３ ), Dithiobismaleimidoethane (DTME) is used.

[0092] The main linkers with different reactive group ends include linkers with NHS esters and maleimides as reactive groups (e.g., AMAS, BMPS, GMBS, Sulfo-MBS, MBS, Sulfo-MBS, SMCC, Sulfo-SMCC, EMCS, Sulfo-EMCS, SMPB, Sulfo-SMPB, SMPH, LC-SMCC, Sulfo-KMUS, SM(PEG)). ２ SM (PEG) ４ SM (PEG)６ SM (PEG) ８ SM (PEG) １２ SM (PEG) ２４ ), linkers using NHS esters and pyridyldithiol as reactive groups (e.g., SPDP, LC-SPDP, Sulfo-LC-SPDP, SMPT, (PEG) ４ SPDP, PEG12-SPDP), linkers with NHS esters and haloacetyl as the reactive group (e.g., SIA, SBAP, SIAB, Sulfo-SIAB), linkers with NHS esters and aryl azides as the reactive group (e.g., ANB-NOS, Sulfo-SANPAH, ATFB), linkers with NHS esters and diazirine as the reactive group (e.g., SDA, Sulfo-SDA, LC-SDA, SDAD, Sulfo-SDAD), carbodiimi Linkers with a reactive group (e.g., DCC, EDC, EDAC, NHS, Sulfo-NHS), linkers with maleimide and hydrazide as reactive groups (e.g., BMPH, EMCH, MPBH, KMUH), linkers with pyridyldithiol and hydrazide as reactive groups (e.g., PDPH), linkers with isocyanate and maleimide as reactive groups (e.g., PMPI), and linkers with NHS ester and psoralen as reactive groups (e.g., SPB) are used.

[0093] Other linkers include polypeptide-containing linkers, such as Fmoc-Ala-Ala-Asn-PAB, Fmoc-Ala-Ala-Asn(Trt)-PAB, and Fmoc-PEG. ３ -Ala-Ala-Asn(Trt)-PAB, Fmoc-PEG ４ -Ala-Ala-Asn(Trt)-PAB, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Ala-Ala-Asn(Trt)-PAB-PNP, Fmoc-PEG ３ -Ala-Ala-Asn(Trt)-PAB-PNP, Azide-PEG ４ -Ala-Ala-Asn(Trt)-PAB-PNP, Mal-PEG ４-Ala-Ala-Asn(Trt)-PAB-PNP, Fmoc-Val-Cit-PAB-OH, Val-Cit-PAB-OH, Fmoc-Val-Cit-PAB-PNP, MC-Val-Cit-PAB, MC-Val-Cit-PAB-PNP, Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB-PNP, Fmoc-Gly3-Val-Cit-PAB, Fmoc-Gly3-Val-Cit-PAB-PNP, Ala-Ala-Asn-PAB TFA salt, etc. are used.

[0094] Also, Bis-PEG-acid, PEG Acid (e.g., Acid-PEG-TEMPO, Amino-PEG-acid, Amino-PEG-CH ２ CO ２ H, Aminoxy-PEG-acid, Azido-PEG-acid, Carboxy-PEG-sulfonic acid, Fmoc-N-amido-PEG-acid, Fmoc-N-amido-PEG-CH ２ CO ２ H, Fmoc-aminoxy-PEG-acid, Hydroxy-PEG-acid, Hydroxy-PEG-CH ２ CO ２ H, m-PEG-acid, m-PEG-(CH ２ ) ３ -acid, Methoxytrityl-N-PEG-acid, N-methyl-N-(t-Boc)-PEG-acid, Propargyl-PEG-acid, Propargyl-PEG-CH ２ CO ２ H, Propargyl-PEG-(CH ２ ) ３ -acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH ２ CO ２H, t-Boc-Aminoxy-PEG-acid, Acid-PEG-PFP ester, Miscellaneous PEG acid, ), PEG PFP ester (e.g., Acid-PEG-PFP ester, Bis-PEG-PFP ester), Bis-PEG-NHS, PEG Aldehyde (e.g., m-PEG-aldehyde, m-PEG-benzaldehyde, Ald-PEG-acid, Ald-PEG-amine, Ald-PEG-azide, Ald-PEG-NH-Boc, Ald-PEG-NHS ester, Ald-PEG-TFP ester, Ald-PEG-t-butyl ester), PEG Tosylate (e.g., Azido-PEG-Tos, Hydroxy-PEG-Tos, m-PEG-Tos, t-Boc-Aminoxy-PEG-Tos, Trifluoroethyl-PEG-Tos, Tos-PEG-acid, Tos-PEG-CH ２ CO ２ H, Tos-PEG-alkyne, Tos-PEG-t-butyl ester, Tos-PEG-CH ２ CO ２ tBu, Tos-PEG-Tos, S-acetyl-PEG6-Tos, N-Tos-N-(t-butoxycarbonyl)-aminoxy-PEG ４ -Tos, Ms-PEG-Ms, Ms-PEG-t-butyl ester, PEG-Ms, Propargyl-PEG-Ms), Boc-PEG (e.g., Amino-PEG-t-Boc-Hydrazide, Azido-PEG-t-Boc-Hydrazide, Boc-NH-PEG-NH-Boc, Bromoacetamido-PEG-Boc-amine, m-PEG-ONHBoc, Mal-Alkyl-t-Boc-amine, N-Boc-PEG-alcohol, N-Boc-PEG-bromide, N-methyl-N-(t-Boc)-PEG-acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH ２ CO ２H, t-Boc-N-Amido-PEG-amine, t-Boc-N-amido-PEG-azide, t-Boc-N-amido-PEG-NHS ester, t-Boc-N-amido-PEG-sulfonic acid), PEG NHS ester (e.g., Acid-PEG-NHS ester, Azido-PEG-NHS ester, Bis-PEG-NHS, Fmoc-PEG-NHS ester, m-PEG-NHS ester, m-PEG-NHS Carbonate, Mal-PEG-NHS ester, Propargyl-PEG-NHS ester, t-Boc-N-amido-PEG-NHS ester, t-Butoxycarbonyl-PEG-NHS ester), Fmoc-PEG (e.g., Fmoc-N-amido-PEG-acid, Fmoc-NH-PEG-CH ２ CO ２ H, Fmoc-PEG-NHS ester), Biotin PEG (e.g., Biotin PEG-acid, Biotin PEG-alcohol, Biotin PEG-alkyne, Biotin PEG-amine, Biotin PEG-azide, Biotin PEG-DBCO, Biotin PEG-hydrazide, Biotin-PEG-Mal, Biotin-PEG-NHS, Biotin-EDA-PEG-NHS, Biotin-PEG-oxyamine, Biotin-PEG-PFP, Biotin-EDA-PEG-PFP, Biotin-PEG-Tetrazine, Biotin-PEG-TFP, Azide-SS-biotin, Biotin-PEG ３ -SS-azide, DBCO-S-S-PEG ３ -Biotin, Dde Biotin-PEG4-Alkyne, Dde Biotin-PEG ４ -Azide, Dde Biotin-PEG ４ -DBCO, Diazo Biotin-PEG ３ -Alkyne, Diazo Biotin-PEG ３ -Azide, Diazo Biotin-PEG ３-DBCO、Diol Biotin-PEG ３ -Alkyne、Diol Biotin-PEG ３ -Azide、PC Biotin-PEG ３ -Alkyne、PC-Biotin-PEG ４ -PEG ４ -Alkyne、PC-Biotin-PEG ４ -PEG ４ -Alkyne、PC Biotin-PEG ３ -Azide、PC-Biotin-PEG4-PEG3-Azide、PC-Biotin-PEG ４ -NHS carbonate、PC DBCO-PEG ３ -Biotin、WSPC Biotin-PEG ３-DBCO, Fmoc-Lys (biotin-PEG)-OH, Fmoc-N-amido-(PEG-biotin)-acid, TAMRA-Azide-PEG-Biotin), PEG Phosphonate, Aminooxy PEG (e.g., Aminooxy-PEG-acid, Aminooxy-PEG-alcohol, Aminooxy-PEG-azide, Aminooxy-PEG-bromide, Aminooxy-PEG-methane, Aminooxy-PEG-Propargyl, Aminooxy-PEG-t-butyl ester, Aminooxy-PEG-Thiol, Bis-(Aminooxy)-PEG, t-Boc-Aminooxy-PEG-acid, t-Boc-Aminooxy-PEG-alcohol, t-Boc-Aminooxy-PEG-amine, t-Boc-Aminooxy-PEG-Azide, t-Boc-Aminooxy-PEG-Bromide, t-Boc-aminooxy-PEG-Methane, t-Boc-aminooxy-PEG-Propargyl, t-Boc-aminooxy-PEG-S-Ac, t-Boc-Aminooxy-PEG-Thiol, t-Boc-Aminooxy-PEG-Tos, Fmoc-aminooxy-PEG-acid, Trifluoroethyl-PEG-Aminooxy), Alkyne PEG (e.g., endo-BCN-PEG, exo-BCN-PEG, Propargyl-PEG-acid, Propargyl-PEG-CH ２ CO ２ H, Propargyl-PEG-(CH ２ ) ３ -acid, Propargyl-PEG-(CH ２ ) ３-methyl ester、Prropargyl-･Gmcrrylat e、Propargglll･･･alcohol、rrop argylmm･･mamine、.. G-methylamiine、| Propargylla. de、Propargyllm･･bromide、rro pargyl-･･omMaleimide、rropa gylmPEy-ｭs、Propargyllmm･om｢ｳester、PrropargylllEGsulffoiic I ester、Propargyl-･ym｣ｨ ２ 39 ２ tBu、Propargylll･･thiol、ｰcbd gummies carbonaate、 nooxy-EEorropargyl、 BismPropargyl-･1、mm PEGPropargyll) . ２ 39 ２ 2、2zido-E((3ｨ ２ ) ) ３ -methyl ester、｡zidomE･m｡crylatee｡ do-PEGalcoholl ２ ) ) ３ OH、｡zido-EGamine、｡zid o-PEmazide、｡zido-EMM I ylamine、｡zido-･mmethyll ester、｡zido-EE1mｮｳ esterr ２ 39 ２-NHS, Azido-PEG-oxazolidin-2-one, Azido-PEG-PFP ester, Azido-PEG-phosphonic acid, Azido-PEG-phosphonic acid ethyl ester, Azido-PEG-sulfonic acid, Azido-PEG-t-Boc-Hydrazide, Azido-PEG-t-butyl ester, Azido-PEG-CH ２ CO ２ -t-butyl ester, Azido-PEG-TFP ester, Azido-PEG-Tos, Aminooxy-PEG-azide, Bromo-PEG-azide, Bromoacetamido-PEG-azide, Carboxyrhodamine 110-PEG-Azide, Isothiocyanato-PEG-Azide, Isothiocyanato-PEG-Azide, m-PEG-azide, Propargyl-PEG-azide, TAMRA-PEG-Azide, t-Boc-N-Amino-PEG-Azide, t-Boc-Aminooxy-PEG-Azide, Thiol-PEG-Azide, Trifluoroethyl-PEG-Azide, Azido-PEG-amino acid, Azido-PEG ４ -4-nitrophenyl carbonate, S-Acetyl-PEG ３ -Azide, Azide, Trityl-PEG １０ -Azide), Alkyne PEG, DBCO-PEG, BCN-PEG, Propargyl-PEG, Bis-PEG-acid, Bis-PEG-NHS, Bis-PEG-PFP, Bis-Propargyl-PEG, Amine-PEG-Amine, Azido-PEG-azide, Bromo-PEG, Mal PEG are used.

[0095] Furthermore, Py-ds-Prp-Osu, Py-ds-dmBut-OSu, Py-ds-dmBut-OPFP, Py-ds-Prp-OPFP, MAL- HA-OSu, MAL-di-EG-OPFP, MAL-tri-EG-OPFP, MAL-tetra-EG-OPFP, N3-di-EG-OPFP, N3 -tri-EG-OPFP, N3-tetra-EG-OPFP, ALD-BZ-OSu, ALD-di-EG-OSu, ALD-tetra-EG-OSu, ALD-di-EG-OPFP, ALD-tetra-EG-OPFP, PHA-di-EG-OPFP, and PHA-tetra-EG-OPFP are used.

[0096] The conjugate can be obtained, for example, by subjecting it to gel filtration chromatography and separating the peak with higher molecular weight compared to the antibody before linker conjugation. To detect the mass of the conjugate while maintaining the intact bivalent antibody, the method described in WO2013 / 049410 can be used, for example.

[0097] The number of drugs bound to each antibody molecule in the conjugate can be quantified according to known methods such as mass spectrometry, ELISA, electrophoresis, and chromatography (HPLC).

[0098] The drugs introduced into cells by the intracellular drug delivery agent of the present invention include, but are not limited to, cytotoxic agents, immunomodulators, and radioisotopes. When the drug is a radioisotope, it is desirable that it is effective not only for the treatment of diseases but also for the diagnosis of diseases.

[0099] <Pharmaceutical Uses of Intracellular Drug Delivery Agents> When the intracellular drug delivery agent of the present invention is used for pharmaceutical purposes, the target cells of the intracellular drug delivery agent of the present invention are cells that express the CD179b protein on their cell surface, and it is preferable that the target cells are those associated with the disease to be treated, prevented (including prevention of recurrence), and / or diagnosed. That is, by using the intracellular drug delivery agent of the present invention in combination with a drug for the treatment, prevention (including prevention of recurrence), and / or diagnosis of a disease, it can be used as a pharmaceutical agent for treating, preventing (including prevention of recurrence), and / or diagnosing diseases associated with cells that express the CD179b protein on their cell surface.

[0100] Specific examples of cells that express the CD179b protein on their cell surface include B cells (pro-B cells, pre-B cells, immature B cells, or mature B cells), cancer cells which are B cells that have become cancerous, preferably leukemia cells or lymphoma cells.

[0101] Diseases associated with cells expressing the CD179b protein on the cell surface are preferably diseases in which B cells (pro-B cells, pre-B cells, immature B cells, or mature B cells) exacerbate the condition, cancer, more preferably autoimmune diseases in which B cells (pro-B cells, pre-B cells, immature B cells, or mature B cells) exacerbate the condition, leukemia, lymphoma, and these specific diseases include, for example, systemic lupus erythematosus (SLE), SLE-associated pulmonary hypertension, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, systemic sclerosis, mixed connective tissue disease, ANCA-associated vasculitis, pemphigus, anti-NMDA receptor encephalitis, and Ig G4-related diseases, cryoglobulinemia vasculitis, idiopathic thrombocytopenic purpura, primary biliary cholangitis, myasthenia gravis, polymyositis, dermatomyositis, IgA nephropathy, autoimmune hepatitis, chronic inflammatory demyelinating polyneuropathy, eosinophilic granulomatosis with polyangiitis, Hashimoto's disease, Graves' disease, antiphospholipid syndrome, type 1 diabetes, Castleman disease, sarcoidosis, neuromyelitis optica, autoimmune hemolytic anemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, aleukemic leukemia, leukocytic leukemia leukemia), basophilic leukemia, blastic leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonic cell leukemia, eosinophilic leukemia, gross leukemia, hemoblastic leukemia, hemoblastic leukemia leukemia), hemocytoblastic leukemia Leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphotropic leukemia, lymphoid leukemia, lymphosarcomacytic leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia Diseases, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli's leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, preleukemia and chronic myelomonocytic leukemia (CMML), non-Hodgkin lymphoma (Burkitt lymphoma (BL), small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL / CLL), mantle cell lymphoma (MCL)),Follicular lymphoma (FL), diffuse large B-cell lymphoma (DLCL), marginal zone lymphoma (MZL), pilaris cell leukemia (HCL), lymphoplasmacytic leukemia (LPL), extranodal marginal zone B-cell lymphoma of mucosal connective tissue (MALT), mediastinal large cell lymphoma, intravascular large cell lymphoma, primary exudative lymphoma, precursor B-cell lymphoblastic leukemia / lymphoma, precursor T-cell and NK-cell lymphoma (precursor T-cell lymphoblastic lymphoma, NK-blastic lymphoma), mature T and NK cell tumors (including peripheral T-cell lymphoma and leukemia (PTL)), adult T-cell leukemia / T-cell lymphoma This includes, but is not limited to, tumors and large granular lymphocytic leukemias, T-cell chronic lymphocytic leukemia / prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, extranodal T- / NK-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, unstructured large cell lymphoma (ALCL), angiocetric and angioimmunoblastic T-cell lymphoma, mycosis fungoides / Sézary syndrome, cutaneous T-cell lymphoma (CTCL), and Hodgkin lymphoma.

[0102] When administering the intracellular drug delivery agent and drug of the present invention in vivo to a subject (test animal) to introduce the drug into the subject's (test animal's) cells, the target test animal is preferably a mammal, including primates, pet animals, livestock, sports animals, and laboratory animals, with humans being particularly preferred.

[0103] When administering the intracellular drug delivery agent and drug of the present invention in vivo to a subject (test animal), it is possible to formulate them as a single formulation using methods known to those skilled in the art. For example, it can be used parenterally in the form of an injectable sterile solution or suspension with water or other pharmaceutically acceptable liquid. For example, it can be formulated by mixing it with a pharmacologically acceptable carrier or medium or additive, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., in a unit dose form generally accepted for pharmaceutical practice. The amount of active ingredient in these formulations should be such that an appropriate dose within the indicated range is obtained.

[0104] Sterile compositions for injection can be prepared using a vehicle such as distilled water for injection, following standard formulation procedures.

[0105] Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with a suitable solubilizer, such as alcohol, specifically ethanol, polyalcohol, such as propylene glycol and polyethylene glycol, and nonionic surfactant, such as polysorbate 80™ and HCO-60.

[0106] Examples of oily liquids include sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as solubilizers. It may also be combined with buffers such as phosphate buffer or sodium acetate buffer, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The prepared injection solution is usually filled into appropriate ampoules.

[0107] The above-mentioned formulations may be administered orally or parenterally, preferably parenterally, and specifically include injectable, nasal, pulmonary, and transdermal formulations. Examples of injectable formulations include intravenous, intramuscular, intraperitoneal, and subcutaneous injections, which can be administered systemically or locally. The antibodies of the present invention may also be directly administered to the disease site by injection, infusion, or implantation of sustained-release formulations.

[0108] Furthermore, the method of administering the preparation can be appropriately selected depending on the patient's age, weight, sex, symptoms, etc. The dosage of the preparation can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose, as the total amount of antibody and drug. Alternatively, the dosage can be selected in the range of 0.001 to 100000 mg / body per patient, but is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, sex, symptoms, etc., but a person skilled in the art can appropriately select them.

[0109] Furthermore, the intracellular drug delivery agent and the drug of the present invention may be prepared as separate formulations according to formulation methods known to those skilled in the art, and administered to the patient in combination. In this case, each formulation may be administered to the patient simultaneously or separately. When administered separately, the intracellular drug delivery agent may be administered first or second, and the administration interval, dosage, route of administration, and number of administrations may be appropriately selected by a specialist.

[0110] The present invention will be described more specifically below based on examples, but the scope of the present invention is not limited by these examples.

[0111] (Example 1) Production of mature human CD179b full-length protein (1) Cloning of cDNA encoding amino acids of human CD179b full-length protein The full-length cDNA encoding the amino acid sequence of human CD179b full-length protein was cloned using the following method based on the gene of Sequence ID No. 1. PCR was performed by adding 1 μl of cDNA derived from Nalm6 cells (human precursor B-cell acute lymphoblastic leukemia), which are known to express CD179b on the cell surface, to 0.4 μM each of two primers containing EcoRI and EcoRV restriction enzyme cleavage sequences (described in SEQ ID NOs. 9 and 10), 0.2 mM dNTPs, and 1.25 U of PrimeSTAR HS polymerase (Takara Shuzo) to a total volume of 50 μl. PCR was then performed using a Thermal Cycle (BIO RAD) by repeating a cycle of 10 seconds at 98°C, 15 seconds at 55°C, and 1 minute at 72°C 30 times. The two primers described above amplified the region encoding SEQ ID NO. 2 of the full-length human CD179b protein. After PCR, the amplified DNA was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 0.7 kbp was purified using the QIAquick Gel Extraction Kit (QIAGEN). The amplification product obtained from the above PCR reaction was ligated to pcDNA3.1 (ThermoFisher Scientific), into which a cDNA encoding the mouse IgG2a Fc protein was inserted, and this was used as an expression vector encoding the full-length human CD179b (including signal peptide) / mouse IgG2a Fc fusion protein (hereinafter referred to as hCD179b (including signal peptide)-mIgG2aFc) (hereinafter referred to as hCD179b (including signal peptide)-mIgG2aFc / pcDNA3.1). Furthermore, sequencing using a DNA sequencer confirmed that the cDNA sequence encodes the full-length hCD179b (including signal peptide)-mIgG2aFc.

[0112] The sequence represented by SEQ ID NO: 1 is the full-length nucleotide sequence of the human CD179b gene, the sequence represented by SEQ ID NO: 2 is the full-length amino acid sequence of the human CD179b protein, the sequence represented by SEQ ID NO: 11 is the nucleotide sequence encoding hCD179b full-length (including signal peptide)-mIgG2aFc, and the sequence represented by SEQ ID NO: 12 is the amino acid sequence of hCD179b full-length (mature)-mIgG2aFc expressed from the nucleotide sequence of SEQ ID NO: 11.

[0113] (2) Preparation of full-length (mature) hCD179-mIgG2aFc Full-length (mature) hCD179b-mIgG2aFc was prepared as an immunoantigen to produce antibodies against mature human CD179b full-length protein (SEQ ID NO: 6).

[0114] The expression vector hCD179b full length (including signal peptide)-mIgG2aFc / pcDNA3.1 was introduced into human embryonic kidney cell line HEK293 cells by lipofection, and hCD179b full length (mature)-mIgG2aFc was purified from the culture supernatant 7 days after introduction. The culture supernatant was applied to a Hi-Trap Protein A HP column (GE Healthcare Biosciences), washed with binding buffer (20 mM sodium phosphate (pH 7.0)), and eluted with elution buffer (0.1 M glycine-HCl (pH 2.7)). The eluate was immediately neutralized by eluting into a tube containing neutralization buffer (1 M Tris-HCl (pH 9.0)). Next, the buffer of the eluate obtained by the above method was replaced with physiological phosphate buffer (Nissui Pharmaceutical) using ultrafiltration NANOSEP 10K OMEGA (PALL), and then sterile filtration was performed using HT Tufflin Acrodisc 0.22 μm (PALL), and this was used in the following experiments.

[0115] (Example 2) Production of polyclonal antibodies against human CD179b full-length protein (1) Production of mouse polyclonal antibodies against human CD179b full-length protein To obtain antibodies that bind to mature human CD179b full-length protein (SEQ ID NO: 6), 0.1 mg of hCD179b full-length (mature)-mIgG2aFc produced above was used as an antigen and mixed with an equal volume of complete Freund's adjuvant (CFA) solution. This solution was administered subcutaneously to mice four times every two weeks. Blood was then collected to obtain antiserum containing polyclonal antibodies. This antiserum was further purified by passing it through a protein G support column (GE Healthcare Biosciences) and substituted with PBS to obtain an IgG-type polyclonal antibody against mature human CD179b full-length protein (SEQ ID NO: 6) (hereinafter referred to as "mouse polyclonal anti-human CD179 full-length sequence antibody").

[0116] (2) Immunological reactivity analysis of anti-human CD179b full-length antibody against CD179b protein expressed on the cell surface The mouse polyclonal anti-human CD179b full-length sequence antibody prepared above was analyzed to determine whether it showed specific reactivity to human CD179b expressed on the cell surface. Nalm6 cells (human precursor B-cell acute lymphoblastic leukemia cells) which are known to express human CD179b on the cell surface, and U937 cells (human acute myeloid leukemia cells) which do not express human CD179b protein on the cell surface were used in 10 samples each. ６Individual cells were centrifuged, and 0.1 μg (5 μl) of mouse polyclonal anti-human CD179b full-length sequence antibody was added. The cells were then suspended in PBS containing 95 μl of 3% fetal bovine serum and left to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the cells were suspended in PBS containing 0.5 μl of APC-labeled goat anti-mouse IgG antibody (BioLegend) and 99.5 μl of PBS containing 3% fetal bovine serum (FBS) and left to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the fluorescence intensity was measured using FACSVerse (Becton Dickinson). On the other hand, the same procedure as above was performed using purified IgG antibody (Fujifilm Wako Pure Chemical Industries) derived from normal mice that had not been immunized with a specific antigen instead of mouse polyclonal anti-human CD179b full-length sequence antibody, and this was used as a control. As a result, the mouse polyclonal anti-human CD179b full-length sequence antibody showed higher fluorescence intensity in Nalm6 cells compared to the control, and the fluorescence intensity in U937 cells was equivalent to that of the control. This confirmed that it is a polyclonal antibody that specifically reacts with the human CD179b protein expressed on the cell surface.

[0117] (Example 3) Production of mature human CD179b-specific sequence protein (1) Cloning of cDNA encoding human CD179b-specific sequence The full-length cDNA encoding human CD179b-specific sequence was cloned based on the gene of Sequence ID No. 1 by the following method. PCR was performed by adding 1 μl of cDNA derived from Nalm6 cells (human precursor B-cell acute lymphoblastic leukemia cells) known to express CD179b on the cell surface, 0.4 μM of two primers containing EcoRI and EcoRV restriction enzyme cleavage sequences (described in SEQ ID NOs. 9 and 13), 0.2 mM dNTPs, and 1.25 U of PrimeSTAR HS polymerase (Takara Shuzo) to a total volume of 50 μl. PCR was then performed using a Thermal Cycle (BIO RAD) by repeating a cycle of 10 seconds at 98°C, 15 seconds at 55°C, and 1 minute at 72°C 30 times. The two primers described above amplified the region encoding SEQ ID NO: 4, which is the region from the N-terminus to 108 amino acid residues containing the signal peptide, a CD179b-specific sequence, within the full-length human CD179b protein. After PCR, the amplified DNA was electrophoresed on a 1% agarose gel, and a DNA fragment of approximately 0.4 kbp was purified using the QIA Quick Gel Extraction Kit (QIAGEN). The amplification product obtained from the above PCR reaction was ligated to pcDNA3.1 (ThermoFisher Scientific), into which a cDNA encoding the mouse IgG2a Fc protein was inserted, and this was used as an expression vector encoding the human CD179b-specific sequence (including signal peptide) / mouse IgG2a Fc fusion protein (hereinafter referred to as hCD179b-specific sequence (including signal peptide)-mIgG2aFc) (hereinafter referred to as hCD179b-specific sequence (including signal peptide)-mIgG2aFc / pcDNA3.1). Furthermore, sequencing using a DNA sequencer confirmed that the cDNA sequence encodes the hCD179b-specific sequence (including the signal peptide)-mIgG2aFc.

[0118] The sequence represented by Sequence ID No. 3 is the nucleotide sequence of the region from the N-terminus to the 108th amino acid residue containing the signal peptide of the full-length human CD179b protein; the sequence represented by Sequence ID No. 4 is the amino acid sequence of the region from the N-terminus to the 108th amino acid residue containing the signal peptide of the full-length human CD179b protein; the sequence represented by Sequence ID No. 14 is the nucleotide sequence encoding the hCD179b-specific sequence (including signal peptide)-mIgG2aFc; and the sequence represented by Sequence ID No. 15 is the amino acid sequence of the hCD179b-specific sequence (mature)-mIgG2aFc expressed from the nucleotide sequence of Sequence ID No. 14.

[0119] (2) Preparation of hCD179b-specific sequence (mature)-mIgG2aFc A hCD179b-specific sequence (mature)-mIgG2aFc was prepared as an immunoantigen to produce antibodies against a mature human CD179b-specific sequence protein (SEQ ID NO: 8).

[0120] The expression vector hCD179b-specific sequence (including signal peptide)-mIgG2aFc / pcDNA3.1 was introduced into human embryonic kidney cell line HEK293 cells by lipofection. After 7 days of introduction, the hCD179b-specific sequence (mature)-mIgG2aFc was purified from the culture supernatant. The culture supernatant was applied to a Hi-Trap Protein A HP column (GE Healthcare Biosciences), washed with binding buffer (20 mM sodium phosphate (pH 7.0)), and eluted with elution buffer (0.1 M glycine-HCl (pH 2.7)). The eluate was immediately neutralized by eluting into a tube containing neutralization buffer (1 M Tris-HCl (pH 9.0)). Next, the buffer of the eluate obtained by the above method was replaced with physiological phosphate buffer (Nissui Pharmaceutical) using ultrafiltration NANOSEP 10K OMEGA (PALL), and then sterile filtration was performed using HT Tufflin Acrodisc 0.22 μm (PALL), and this was used in the following experiments.

[0121] (Example 4) Production of polyclonal antibodies against human CD179b-specific sequence proteins (1) Production of mouse polyclonal antibodies against human CD179b-specific sequence proteins To obtain antibodies that bind to mature human CD179b-specific sequence proteins (SEQ ID NO: 8), 0.1 mg of the hCD179b-specific sequence (mature)-mIgG2aFc produced above was used as an antigen and mixed with an equal volume of complete Freund's adjuvant (CFA) solution. This solution was administered subcutaneously to mice four times every two weeks. Blood was then collected to obtain antiserum containing polyclonal antibodies. This antiserum was further purified by passing it through a protein G support column (GE Healthcare Biosciences) and substituted with PBS to obtain IgG-type polyclonal antibodies against mature human CD179b-specific sequence proteins (SEQ ID NO: 8) (hereinafter referred to as "mouse polyclonal anti-human CD179-specific sequence antibody").

[0122] (2) Immunological reactivity analysis of anti-human CD179b-specific sequence antibodies against CD179b protein expressed on the cell surface The mouse polyclonal anti-human CD179b-specific sequence antibodies prepared above were analyzed to determine whether they showed specific reactivity against human CD179b expressed on the surface of cancer cells. Nalm6 cells (human precursor B-cell acute lymphoblastic leukemia cells) that are known to express human CD179b on the cell surface and U937 cells (human acute myeloid leukemia cells) which do not express human CD179b protein on the cell surface were used in 10 samples each. ６Individual cells were centrifuged, and 0.1 μg (5 μl) of mouse polyclonal anti-human CD179b-specific sequence antibody was added. The cells were then suspended in PBS containing 95 μl of 3% fetal bovine serum and left to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the cells were suspended in PBS containing 0.5 μl of APC-labeled goat anti-mouse IgG antibody (BioLegend) and 99.5 μl of PBS containing 3% fetal bovine serum (FBS) and left to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the fluorescence intensity was measured using FACSVerse (Becton Dickinson). On the other hand, the same procedure as above was performed using purified IgG antibody (Fujifilm Wako Pure Chemical Industries) derived from normal mice that had not been immunized with a specific antigen instead of mouse polyclonal anti-human CD179b-specific sequence antibody, and this was used as a control. As a result, the mouse polyclonal anti-human CD179b specific sequence antibody showed higher fluorescence intensity in Nalm6 cells compared to the control, and the fluorescence intensity in U937 cells was equivalent to that of the control. This confirmed that it is a polyclonal antibody that specifically reacts with the human CD179b protein expressed on the cell surface.

[0123] (Example 5) Evaluation of the intracellular internalization activity of anti-CD179b antibodies Next, we evaluated whether the mouse polyclonal anti-human CD179b full-length sequence antibody, mouse polyclonal anti-human CD179b specific sequence antibody, and commercially available mouse monoclonal anti-human CD179b antibody (BioLegend; Purified anti-human CD179b (Ig λ5) Antibody) prepared above exhibited intracellular internalization activity in cells expressing the CD179b protein on the cell surface. The evaluation was performed under conditions in which each antibody was added to cells at a concentration of 1 μg / ml. Nalm6 cells that express the CD179b protein on the cell surface and showed reactivity to the above antibodies were used. First, Nalm6 cells were dispensed into a flat-bottomed 96-well plate. Nalm6 cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS) to a concentration of 200,000 cells / well, and then added at a concentration of 90 μl / well. To the dispensed Nalm6 cells, 10 μl / well each of the following were added to form a complex: IgG antibody derived from a control normal mouse, mouse polyclonal anti-human CD179b full-length sequence antibody, mouse polyclonal anti-human CD179b specific sequence antibody, and commercially available mouse monoclonal anti-human CD179b antibody, all pre-incubated with Fabfluor-pH Red Antibody Dye (Sartorius, 4723) to a final concentration of 1 μg / ml, and then mixed with 5% CO2. ２ The samples were incubated at 37°C for 4 hours in the presence of FabFluor pH Red Antibody dye. The antibody-FabFluor pH Red Antibody dye complex exhibits red fluorescence at low pH, which is the intracellular environment; therefore, the amount of antibody internalized into the cell can be measured using fluorescence intensity as an indicator.

[0124] As a result, cells treated with mouse polyclonal anti-human CD179b-specific sequence antibody showed a 67% increase in fluorescence intensity compared to the control. Furthermore, cells treated with commercially available mouse monoclonal anti-human CD179b antibody showed a 238% increase in fluorescence intensity compared to the control. On the other hand, cells treated with mouse polyclonal anti-human CD179b full-length sequence antibody showed almost no increase in fluorescence intensity compared to the control. Therefore, it was revealed that both mouse polyclonal anti-human CD179b-specific sequence antibody and mouse monoclonal anti-human CD179b antibody can be internalized into cells expressing the CD179b protein on their cell surface.

[0125] The fluorescence intensity enhancement rate mentioned above is expressed as the rate of increase in the average fluorescence intensity (MFI value) in each cell, and was calculated using the following formula.

[0126] The percentage increase in average fluorescence intensity (fluorescence intensity enhancement rate) (%) = ((MFI value of cells reacted with anti-human CD179b antibody) - (control MFI value)) ÷ (control MFI value) × 100.

[0127] The intracellular drug delivery agent of the present invention is useful for the treatment and / or prevention of diseases in which cells expressing CD179b on their cell surface exacerbate the disease state.

[0128] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. An intracellular drug delivery agent comprising an antibody or its antigen-binding fragment as an active ingredient, which specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells expressing the CD179b protein on the cell surface.

2. The intracellular drug delivery agent according to claim 1, wherein the antibody or its antigen-binding fragment specifically binds to a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having 80% or more sequence identity with said amino acid sequence.

3. The intracellular drug delivery agent according to claim 1, wherein the antibody or its antigen-binding fragment specifically binds to a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having 80% or more sequence identity with said amino acid sequence.

4. The intracellular drug delivery agent according to claim 1, wherein the cell is a B cell.

5. The intracellular drug delivery agent according to claim 4, wherein the B cells are pro-B cells, pre-B cells, immature B cells, or mature B cells.

6. The intracellular drug delivery agent according to claim 1, wherein the cells are cancer cells.

7. The intracellular drug delivery agent according to claim 6, wherein the cancer is leukemia or lymphoma.

8. The intracellular drug delivery agent according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

9. An intracellular drug delivery agent according to any one of claims 1 to 8, comprising a conjugate of the antibody or its antigen-binding fragment and a drug as an active ingredient.

10. The intracellular drug delivery agent according to claim 9, wherein the drug is a cytotoxic agent, an immunomodulator, or a radioisotope.

11. A pharmaceutical composition comprising an antibody or its antigen-binding fragment and a drug as active ingredients, which specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells that express the CD179b protein on the cell surface.

12. The pharmaceutical composition according to claim 11, comprising a conjugate of the antibody or its antigen-binding fragment and the drug as an active ingredient.

13. A pharmaceutical composition for the treatment and / or prevention of diseases in which B cells exacerbate the condition, comprising an antibody or its antigen-binding fragment and a drug as active ingredients, which specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells that express the CD179b protein on the cell surface.

14. The pharmaceutical composition according to claim 13, wherein the disease in which the B cells exacerbate the condition is an autoimmune disease.

15. The pharmaceutical composition according to claim 14, wherein the autoimmune disease is systemic lupus erythematosus (SLE), SLE-associated pulmonary hypertension, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, systemic scleroderma, mixed connective tissue disease, ANCA-associated vasculitis, pemphigus, anti-NMDA receptor encephalitis, IgG4-related disease, cryoglobulinemia vasculitis, idiopathic thrombocytopenic purpura, primary biliary cholangitis, myasthenia gravis, polymyositis, dermatomyositis, IgA nephropathy, autoimmune hepatitis, chronic inflammatory demyelinating polyneuropathy, eosinophilic granulomatosis with polyangiitis, Hashimoto's disease, Graves' disease, antiphospholipid antibody syndrome, type 1 diabetes mellitus, Castleman disease, sarcoidosis, neuromyelitis optica, or autoimmune hemolytic anemia.

16. A pharmaceutical composition according to any one of claims 13 to 15, comprising a conjugate of the antibody or its antigen-binding fragment and the drug as an active ingredient.

17. A pharmaceutical composition for the treatment and / or prevention of cancer, comprising an antibody or its antigen-binding fragment and a drug as active ingredients, which specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells that express the CD179b protein on the cell surface.

18. The pharmaceutical composition according to claim 17, wherein the cancer is leukemia or lymphoma.

19. The pharmaceutical composition according to claim 17 or 18, comprising a conjugate of the antibody or its antigen-binding fragment and the drug as an active ingredient.

20. An intracellular drug delivery method comprising administering an antibody or its antigen-binding fragment and a drug in vivo to a subject, which specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells expressing the CD179b protein on the cell surface, thereby delivering the drug into cells expressing the CD179b protein on the cell surface within the subject's body.

21. The intracellular drug delivery method according to claim 20, comprising administering a conjugate of the antibody or its antigen-binding fragment and the drug to a subject.

22. A method for treating and / or preventing a disease associated with cells expressing the CD179b protein on their cell surface, comprising administering to a subject an antibody or its antigen-binding fragment and a drug that specifically binds to the extracellular domain of the CD179b protein expressed on the cell surface and has the ability to internalize the CD179b protein into cells expressing the CD179b protein on their cell surface.

23. The method according to claim 22, wherein the disease is a disease in which B cells exacerbate the pathological condition.

24. The method according to claim 22, wherein the disease is cancer.