Humanized anti-DNAM-1 antibody

Abstract

One aspect of the present invention is a humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof, having: a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 1, serving as HCDR1, an amino acid sequence represented by SEQ ID NO: 2, serving as HCDR2, and an amino acid sequence represented by SEQ ID NO: 3, serving as HCDR3; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 4, serving as LCDR1, an amino acid sequence represented by SEQ ID NO: 5, serving as LCDR2, and an amino acid sequence represented by SEQ ID NO: 6, serving as LCDR3.

Description

Technical Field

[0001] The present invention relates to a humanized anti-DNAM-1 antibody.

Background Art

[0002] DNAM-1, also known as CD226, is an adhesion molecule belonging to the immunoglobulin superfamily with a molecular weight of 65 kDa, and has been identified as an activating immunoreceptor expressed on hematopoietic cells such as CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and platelets. When DNAM-1 binds to its ligands CD155 or CD112, it mediates activation signals for cytotoxicity. DNAM-1 has been shown to be involved in various inflammatory diseases and cancer pathologies in human and mouse models. And anti-mouse DNAM-1 monoclonal antibodies have been reported to suppress the onset of experimental autoimmune encephalitis and acute graft-versus-host disease (GVHD) in mice, increase the regulatory T (Treg) cell population, and as a result, extend the survival period of mouse skin grafts. From these reports, anti-DNAM-1 monoclonal antibodies are considered to be useful for these diseases. The present inventors previously established a mouse anti-human DNAM-1 monoclonal antibody (Patent Document 1). Also, a humanized anti-DNAM-1 antibody has been reported (Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The mouse anti-human DNAM-1 monoclonal antibody described in Patent Document 1 activates regulatory T cells to suppress the immune response, and thus is said to be usable for the prevention or treatment of graft-versus-host disease, organ transplant rejection, autoimmune diseases, fibrotic diseases, inflammatory bowel disease, allergies, etc. However, what was established in Patent Document 1 was only a mouse antibody, and there remained room for improvement in terms of its therapeutic value in humans and the like. In addition, humanized antibodies generally have room for improvement in that their binding affinity to the target antigen is lower than that of their parent mouse antibody, and their immunogenicity tends to increase if the binding affinity to the antigen is maintained. Therefore, the present inventors aimed to obtain a novel mouse anti-human DNAM-1 monoclonal antibody and produce a humanized anti-DNAM-1 antibody with high therapeutic value in humans. An object of the present invention is to provide a humanized anti-DNAM-1 antibody that specifically binds to human DNAM-1.

Means for Solving the Problems

[0006] The present inventors intensively studied to solve the above problems. As a result, they found that the above problems can be solved by having the following configuration, and thus completed the present invention. The present invention relates to, for example, the following [1] to

[10] . [1] A heavy chain variable region comprising the following amino acid sequence; The amino acid sequence of SEQ ID NO: 1 as HCDR1, the amino acid sequence of SEQ ID NO: 2 as HCDR2, and the amino acid sequence of SEQ ID NO: 3 as HCDR3, and [2] A light chain variable region comprising the following amino acid sequence; The amino acid sequence of SEQ ID NO: 4 as LCDR1, the amino acid sequence of SEQ ID NO: 5 as LCDR2, and the amino acid sequence of SEQ ID NO: 6 as LCDR3, A humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof having the same. [3] The humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to [1], having a heavy chain variable region having an amino acid sequence 95% or more identical to SEQ ID NO: 10 and a light chain variable region having an amino acid sequence 95% or more identical to SEQ ID NO: 11. [4] A heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 11, or A heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 11, The humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to [1] or [2], having the same. [5] The humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to any one of [1] to [4], having a heavy chain having an amino acid sequence 95% or more identical to SEQ ID NO: 14 and a light chain having an amino acid sequence 95% or more identical to SEQ ID NO: 15. [6] A heavy chain having the amino acid sequence of SEQ ID NO: 14 and a light chain having the amino acid sequence of SEQ ID NO: 15, or A heavy chain having the amino acid sequence of SEQ ID NO: 13 and a light chain having the amino acid sequence of SEQ ID NO: 15 The humanized anti-DNAM-1 antibody according to any one of [1] to [5], having the same. [7] A nucleic acid encoding the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to any one of [1] to [6]. ​ [8] A vector containing the nucleic acid according to [7]. [9] A transformant containing the vector according to [8].

[10] An inhibitor for activating natural lymphocytes (ILC), comprising the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to any one of [1] to [6]. [Advantages of the Invention]

[0007] According to the present invention, a humanized anti-DNAM-1 antibody can be provided. In addition, an inhibitor for activating natural lymphocytes (ILC) can be provided. [Brief Description of the Drawings]

[0008]

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[0009] Next, the present invention will be specifically described. [Humanized Anti-DNAM-1 Antibody] The humanized anti-DNAM-1 antibody is used in the sense of a humanized antibody that specifically recognizes human DNAM-1. Specifically recognizing means binding to the human DNAM-1 protein but not binding to proteins other than the human DNAM-1 protein. The binding activity can be measured by known methods, such as immunoprecipitation, Western blotting, EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), flow cytometry, pull-down assay, and other methods.

[0010] A humanized antibody is an antibody in which the variable region consists, in principle, of complementarity-determining regions (CDRs) derived from antibodies other than humans and framework regions (FRs) derived from human antibodies, and the constant region consists of a constant region derived from a human antibody.

[0011] The humanized anti-DNAM-1 antibody, which is one aspect of the present invention, has a heavy chain variable region containing the following amino acid sequence; the amino acid sequence of SEQ ID NO: 1 as HCDR1, the amino acid sequence of SEQ ID NO: 2 as HCDR2, and the amino acid sequence of SEQ ID NO: 3 as HCDR3, and a light chain variable region containing the following amino acid sequence; having the amino acid sequence of SEQ ID NO: 4 as LCDR1, the amino acid sequence of SEQ ID NO: 5 as LCDR2, and the amino acid sequence of SEQ ID NO: 6 as LCDR3. The said humanized anti-DNAM-1 antibody is referred to as humanized anti-DNAM-1 antibody (A). HCDR is the CDR of the heavy chain, and LCDR is the CDR of the light chain.

[0012] The humanized anti-DNAM-1 antibody (A) may be an antibody that inhibits the binding of human DNAM-1 to its ligand or an antibody that does not inhibit the binding of human DNAM-1 to its ligand, but an antibody that inhibits the binding of human DNAM-1 to its ligand is preferred. Further, the humanized anti-DNAM-1 antibody (A) may be an antibody that inhibits or reduces the signal transduction of human DNAM-1 (neutralizing antibody), an antibody that does not change the signal transduction of human DNAM-1, or an antibody that promotes the signal transduction of human DNAM-1 (agonistic antibody), but an antibody that inhibits or reduces the signal transduction of human DNAM-1 (neutralizing antibody) is preferred.

[0013] The class and subclass of the humanized anti-DNAM-1 antibody (A) are not particularly limited as long as the effects of the present invention are exhibited, and may be any of IgG, IgM, IgA, IgD, and IgE, but IgG is preferred, and IgG1 is more preferred. The heavy chain of the humanized anti-DNAM-1 antibody (A) may be any of γ, μ, α, δ, and ε, but γ is preferred, and γ1 is more preferred. The light chain of the humanized anti-DNAM-1 antibody (A) may be either κ or λ, but κ is preferred.

[0014] The humanized anti-DNAM-1 antibody (A) may be used as it is, such as the antibody produced by the transformant or the culture supernatant of the transformant, or it may be used after purification. Purification can be carried out, for example, by subjecting it to affinity column chromatography such as saturated ammonium sulfate, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein A column, protein G column, etc.

[0015] The humanized anti-DNAM-1 antibody (A) may be a multispecific antibody, a recycling antibody, a sweeping antibody, a conjugate antibody, or the like. Further, the humanized anti-DNAM-1 antibody may be chemically or genetically conjugated with a functional molecule such as a non-peptidic polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular compound, a cytokine, a growth factor, albumin, an enzyme, or another antibody. These can be produced by known methods.

[0016] The method for producing the humanized anti-DNAM-1 antibody (A) is not particularly limited and can be produced by a known method. For example, the humanized anti-DNAM-1 antibody (A) can be obtained by transfecting a host cell with a vector containing a nucleic acid encoding the humanized anti-DNAM-1 antibody (A) to produce a transformant and causing the transformant to produce the humanized anti-DNAM-1 antibody (A).

[0017] The amino acid sequence of the humanized anti-DNAM-1 antibody (A) is not particularly limited as long as it specifically recognizes human DNAM-1 in regions other than HCDR1 to HCDR3 and regions other than LCDR1 to LCDR3.

[0018] The humanized anti-DNAM-1 antibody (A) may preferably have a reverse mutation of an amino acid residue in a region other than HCDR1 to HCDR3 of the heavy chain variable region and / or in a region other than LCDR1 to LCDR3 of the light chain variable region. The reverse mutation of an amino acid residue refers to the substitution of a single amino acid residue found in the human antibody framework with the corresponding amino acid residue found in the mouse antibody framework. By appropriate reverse mutations of amino acid residues, it is possible to achieve both low immunogenicity and high binding affinity to the antigen.

[0019] The reverse mutation of an amino acid residue in the heavy chain variable region is preferably 1 to 6, more preferably 1 to 3. The reverse mutation of an amino acid residue in the light chain variable region is preferably 1 to 5, more preferably 1 to 2. The humanized anti-DNAM-1 antibody (A) preferably has a reverse mutation of 1 to 3 amino acid residues in regions other than HCDR1 to HCDR3 of the heavy chain variable region and does not have a reverse mutation of amino acid residues in regions other than LCDR1 to LCDR3 of the light chain variable region.

[0020] The amino acid residues to be reversely mutated can be selected from amino acid residues that non-covalently directly bind to the antigen, amino acid residues adjacent to the CDR region, amino acid residues that interact with the CDR region, or amino acid residues involved in the VL-VH interface within the variable region. The reverse mutation of amino acid residues in the heavy chain variable region is preferably performed within a range of 5 amino acids, more preferably 2 amino acids, before and after HCDR1, HCDR2, or HCDR3.

[0021] The reverse mutation of amino acid residues in the heavy chain variable region is preferably performed at at least one amino acid residue selected from the 49th and 72nd amino acid residues of the heavy chain variable region. The reverse mutation of amino acid residues in the light chain variable region is preferably performed at the 67th amino acid residue of the light chain variable region. The humanized anti-DNAM-1 antibody (A) more preferably has reverse mutations at the 49th and 72nd amino acid residues of the heavy chain variable region and the 67th amino acid residue of the light chain variable region. In this specification, the Xth amino acid residue means the Xth amino acid residue counted from the N-terminus of the protein without including the signal peptide.

[0022] The reverse mutation of amino acid residues in the heavy chain variable region preferably has the 49th amino acid residue of the heavy chain variable region as M and the 72nd amino acid residue as R. The reverse mutation of amino acid residues in the light chain variable region preferably has the 67th amino acid residue of the light chain variable region as Y. The humanized anti-DNAM-1 antibody (A) more preferably has reverse mutations of I49M, V72R in the heavy chain variable region, and S67Y in the light chain variable region.

[0023] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain variable region having an amino acid sequence that is 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more identical to SEQ ID NO: 11.

[0024] The percentage of sequence identity is determined by the sequences of the antibodies maximally aligned according to the Kabat numbering rules. After alignment, when comparing the antibody region of interest (e.g., the light chain variable region) with the same region of the control antibody, the percentage of sequence identity between the antibody region of interest and the control antibody region is calculated by dividing the number of positions occupied by the same amino acid in both the antibody region of interest and the control antibody region by the total number of positions aligned in the two regions (gaps are not counted in the number), and multiplying by 100.

[0025] The humanized anti-DNAM-1 antibody (A) may be an antibody having a heavy chain variable region that is an amino acid sequence having 1 to 6 amino acid residue mutations in the region other than HCDR1 to HCDR3 of the amino acid sequence of SEQ ID NO: 10 and is at least 95% or more identical to SEQ ID NO: 10, and a light chain variable region that is an amino acid sequence having 1 to 5 amino acid residue mutations in the region other than LCDR1 to LCDR3 of the amino acid sequence of SEQ ID NO: 11 and is at least 95% or more identical to SEQ ID NO: 11. Amino acid residue mutation means substitution, insertion, or deletion of a single amino acid residue.

[0026] The humanized anti-DNAM-1 antibody (A) may be an antibody having a heavy chain variable region with an amino acid sequence having 1 to 3 amino acid residue mutations in regions other than HCDR1 to HCDR3 of the amino acid sequence of SEQ ID NO: 10 and being at least 97% identical to SEQ ID NO: 10, and a light chain variable region with an amino acid sequence having 1 to 3 amino acid residue mutations in regions other than LCDR1 to LCDR3 of the amino acid sequence of SEQ ID NO: 11 and being at least 97% identical to SEQ ID NO: 11.

[0027] The amino acid residue mutation is preferably a substitution, more preferably a conservative substitution among substitution, insertion, and deletion. "Conservative substitution" means substituting an amino acid residue with another chemically similar amino acid residue so as not to substantially modify the activity of the peptide. For example, when substituting one hydrophobic residue with another hydrophobic residue, or when substituting one polar residue with another polar residue having the same charge. Examples of such chemically similar amino acids for which such substitutions can be made include, as non-polar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of positively charged (basic) amino acids include arginine, histidine, lysine, etc. Also, examples of negatively charged (acidic) amino acids include aspartic acid, glutamic acid, etc.

[0028] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain variable region having an amino acid sequence 95% or more identical to SEQ ID NO: 10 and a light chain variable region having an amino acid sequence 95% or more identical to SEQ ID NO: 11, wherein the 49th and 72nd amino acid residues of the heavy chain variable region are M and R respectively, and the 67th amino acid residue of the light chain variable region is Y.

[0029] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 11, or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 11.

[0030] The humanized anti-DNAM-1 antibody (A) preferably has a mutation of an amino acid residue for abolishing the effector function of an IgG antibody. Examples of the mutation of an amino acid residue for abolishing the effector function of an IgG antibody include mutations in which a branched-chain amino acid such as V (valine), L (leucine), or I (isoleucine); a hydrophobic amino acid such as P (proline), M (methionine), or W (tryptophan); or an amino acid phosphorylated and involved in signal transduction such as Y (tyrosine), S (serine), or T (threonine) in the constant region of the heavy chain is substituted with A (alanine) or F (phenylalanine). Preferably, the mutation is a substitution of L with A or F, and more preferably, the mutation is a substitution of two consecutive Ls with AA, FF, AF, or FA. In the humanized anti-DNAM-1 antibody (A), the 238th and 239th amino acid residues of the heavy chain are preferably A or F, and more preferably both are A. Such a humanized anti-DNAM-1 antibody (A) abolishes the effector function of an IgG antibody and is less likely to aggregate platelets, and thus is useful for the treatment of human diseases.

[0031] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain having an amino acid sequence 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more identical to SEQ ID NO: 14 and a light chain having an amino acid sequence 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more identical to SEQ ID NO: 15.

[0032] The humanized anti-DNAM-1 antibody (A) preferably has an amino acid sequence having 1 to 22 amino acid residue mutations in regions other than HCDR1 to HCDR3 among the amino acid sequence of SEQ ID NO: 14, and is at least 95% identical to SEQ ID NO: 14. It has a heavy chain variable region having an amino acid sequence, and among the amino acid sequence of SEQ ID NO: 15, it has an amino acid sequence having 1 to 10 amino acid residue mutations in regions other than LCDR1 to LCDR3, and is at least 95% identical to SEQ ID NO: 15. It is an antibody having a light chain variable region having an amino acid sequence.

[0033] The humanized anti-DNAM-1 antibody (A) preferably has an amino acid sequence having 1 to 13 amino acid residue mutations in regions other than HCDR1 to HCDR3 among the amino acid sequence of SEQ ID NO: 14, and is at least 97% identical to SEQ ID NO: 14. It has a heavy chain variable region having an amino acid sequence, and among the amino acid sequence of SEQ ID NO: 15, it has an amino acid sequence having 1 to 6 amino acid residue mutations in regions other than LCDR1 to LCDR3, and is at least 97% identical to SEQ ID NO: 15. It is an antibody having a light chain variable region having an amino acid sequence.

[0034] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain having an amino acid sequence 95% or more identical to SEQ ID NO: 14 and a light chain having an amino acid sequence 95% or more identical to SEQ ID NO: 15, and the 49th and 72nd amino acid residues of the variable region of the heavy chain are M and R, respectively, and the 67th amino acid residue of the variable region of the light chain is Y.

[0035] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain having the amino acid sequence of SEQ ID NO: 14 and a light chain having the amino acid sequence of SEQ ID NO: 15, or a heavy chain having the amino acid sequence of SEQ ID NO: 13 and a light chain having the amino acid sequence of SEQ ID NO: 15.

[0036] [Antigen-binding fragment of humanized anti-DNAM-1 antibody] The antigen-binding fragment of a humanized anti-DNAM-1 antibody is a protein containing a part of the humanized anti-DNAM-1 antibody (A) and capable of binding to an antigen. Examples of the antigen-binding fragment include F(ab’)2, Fab’, Fab, disulfide bond-stabilized Fv (dsFv), single-chain antibody (scFv), diabody, and polymers thereof.

[0037] Fab is an antibody fragment having an antigen-binding activity with a molecular weight of about 50,000 among the fragments obtained by treating IgG with papain (a proteolytic enzyme). The Fab of the humanized anti-DNAM-1 antibody can be prepared by treating the humanized anti-DNAM-1 antibody with papain or inserting the DNA encoding the Fab of the antibody into an expression vector and introducing this vector into a prokaryote or eukaryote for expression.

[0038] F(ab')2 is an antibody fragment having an antigen-binding activity with a molecular weight of about 100,000 among the fragments obtained by treating IgG with pepsin (a proteolytic enzyme). The F(ab')2 of the humanized anti-DNAM-1 antibody can be prepared by treating the humanized anti-DNAM-1 antibody with pepsin or binding Fab' (described below) with a thioether bond or a disulfide bond.

[0039] Fab' is an antibody fragment having an antigen-binding activity with a molecular weight of about 50,000 obtained by cleaving the disulfide bond in the hinge region of F(ab')2. The Fab' of the humanized anti-DNAM-1 antibody can be prepared by treating the F(ab')2 of the humanized anti-DNAM-1 antibody with dithiothreitol or inserting the DNA encoding the Fab' of the antibody into an expression vector and introducing this vector into a prokaryote or eukaryote for expression.

[0040] An scFv is an antibody fragment that has antigen-binding activity with one VH and one VL linked using an appropriate peptide linker. The scFv of a humanized anti-DNAM-1 antibody can be prepared by obtaining cDNA encoding the VH and VL of the humanized anti-DNAM-1 antibody, constructing DNA encoding the scFv, inserting this DNA into an expression vector, and introducing this expression vector into prokaryotes or eukaryotes for expression.

[0041] A diabody is an antibody fragment in which scFvs dimerize and is an antibody fragment with bivalent antigen-binding activity. The diabody of a humanized anti-DNAM-1 antibody can be prepared by obtaining cDNA encoding the VH and VL of the humanized anti-DNAM-1 antibody, constructing DNA encoding the diabody, inserting this DNA into an expression vector, and introducing this expression vector into prokaryotes or eukaryotes for expression.

[0042] A dsFv is an antibody fragment in which a polypeptide with one amino acid residue in each of VH and VL replaced with a cysteine residue is bound via a disulfide bond between the cysteine residues. The dsFv of a humanized anti-DNAM-1 antibody can be prepared by obtaining cDNA encoding the VH and VL of the humanized anti-DNAM-1 antibody, constructing DNA encoding the dsFv, inserting this DNA into an expression vector, and introducing this expression vector into prokaryotes or eukaryotes for expression.

[0043] The antigen-binding fragment may be chemically or genetically conjugated with functional molecules such as non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular compounds, cytokines, growth factors, albumin, enzymes, and other antibodies. The antigen-binding fragment may be used alone or in combination of two or more. Also, it may be used in combination with a humanized anti-DNAM-1 antibody and its antigen-binding fragment.

[0044] [Nucleic acid encoding a humanized anti-DNAM-1 antibody or its antigen-binding fragment] One aspect of the present invention is a nucleic acid encoding a humanized anti-DNAM-1 antibody (A) or an antigen-binding fragment thereof. Examples of such nucleic acids include a nucleic acid encoding the heavy-chain variable region of the humanized anti-DNAM-1 antibody (A), a nucleic acid encoding the light-chain variable region of the humanized anti-DNAM-1 antibody (A), a nucleic acid encoding the heavy-chain variable region and a part of the constant region of the humanized anti-DNAM-1 antibody (A), a nucleic acid encoding the light-chain variable region and a part of the constant region of the humanized anti-DNAM-1 antibody (A), a nucleic acid encoding the full-length heavy chain of the humanized anti-DNAM-1 antibody (A), a nucleic acid encoding the full-length light chain of the humanized anti-DNAM-1 antibody (A), a nucleic acid encoding a scFv in which the heavy-chain variable region and the light-chain variable region of the humanized anti-DNAM-1 antibody (A) are linked with an appropriate linker, and the like. The nucleic acid can be produced using known genetic engineering techniques.

[0045] It is preferable to add a nucleic acid encoding a signal peptide to the nucleic acid encoding the humanized anti-DNAM-1 antibody (A) or an antigen-binding fragment thereof. By adding a signal peptide having an appropriate amino acid sequence, the expression level of the humanized anti-DNAM-1 antibody (A) or an antigen-binding fragment thereof in cells or the amount of the antibody or antigen-binding fragment secreted into the culture supernatant can be increased.

[0046] The amino acid sequence of the signal peptide is not particularly limited. In the heavy chain, it is preferably the amino acid sequence set forth in SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 31, and more preferably the amino acid sequence set forth in SEQ ID NO: 31. In the light chain, the amino acid sequence of the signal peptide is preferably the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 32, and more preferably the amino acid sequence set forth in SEQ ID NO: 32.

[0047] The nucleic acid encoding the full-length heavy chain with an added signal peptide is preferably a nucleic acid encoding the amino acid sequence described in SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 24, more preferably a nucleic acid encoding the amino acid sequence described in SEQ ID NO: 24, still more preferably a nucleic acid consisting of the nucleotide sequence described in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 26, and particularly preferably a nucleic acid consisting of the nucleotide sequence described in SEQ ID NO: 26. The nucleic acid encoding the full-length light chain with an added signal peptide is preferably a nucleic acid encoding the amino acid sequence described in SEQ ID NO: 18 or SEQ ID NO: 25, more preferably a nucleic acid encoding the amino acid sequence described in SEQ ID NO: 25, still more preferably a nucleic acid consisting of the nucleotide sequence described in SEQ ID NO: 21 or SEQ ID NO: 27, and particularly preferably a nucleic acid consisting of the nucleotide sequence described in SEQ ID NO: 27.

[0048] [Vector containing nucleic acid] One aspect of the present invention is a vector containing a nucleic acid encoding a humanized anti-DNAM-1 antibody (A) or an antigen-binding fragment thereof. In other words, it is a recombinant vector incorporating a nucleic acid encoding a humanized anti-DNAM-1 antibody (A) or an antigen-binding fragment thereof. The vector is not particularly limited and can include plasmid vectors and viral vectors. The vector may be a vector capable of expression in mammals, bacteria, insects, yeast, fungi, etc., but a vector capable of expression in eukaryotic cells is preferred, and a vector capable of expression in mammalian-derived cells is more preferred. Examples of vectors capable of expression in mammalian-derived cells include, for example, pUC series, pCAG, pEBMulti, pEGFP-C1, pEGFP-C1, pEF-BOS, pTRE-Myc, pMSCVpuro, pCEP4, and preferably pUC19.

[0049] The method for producing the vector is not particularly limited and can be produced by known gene recombination techniques. The vector may contain regulatory sequences capable of controlling replication and expression in the host and / or secretion from the host, in addition to the nucleic acid encoding the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment. Examples of regulatory sequences include promoter sequences such as the CMV promoter.

[0050] [Transformant containing the vector] One aspect of the present invention is a transformant containing a vector containing a nucleic acid encoding the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment. The humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment can be obtained from the transformant or its culture supernatant, etc.

[0051] The transformant can be obtained by introducing the above-described recombinant vector into a host. Examples of the host include cultured cells such as Escherichia coli, yeast, plant cells, insect cells, and animal cells; insect organisms such as silkworms; and plant bodies such as tobacco, etc., but animal cells are preferred. Examples of animal cells include mammalian cells such as NS0, Sp2 / 0, CHO, COS, HEK, fibroblasts, and myeloma cells, but CHO is preferred.

[0052] Introduction (transformation) of the recombinant vector into the host can be carried out using known methods. Examples of such methods include the competent cell method using calcium-treated cells and the electroporation method. In addition to plasmid vectors, methods of infecting and transforming the host with phage vectors, viral vectors, etc. may also be utilized.

[0053] [Inhibitor of natural lymphocyte (ILC) activation] One aspect of the present invention is an inhibitor of natural lymphocyte (ILC) activation, which contains the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment. One aspect of the present invention is the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment for use in inhibiting the activation of natural lymphocytes (ILC). One aspect of the present invention is a method for suppressing the activation of innate lymphoid cells (ILC), which comprises administering an effective amount of a humanized anti-DNAM-1 antibody (A) or an antigen-binding fragment thereof to a subject.

[0054] Innate lymphoid cells (ILC) are cells of the innate immune system derived from common lymphoid progenitors (CLP). Since ILC do not have either a T cell receptor or a B cell receptor, they are not activated antigen-specifically, but are known to be rapidly activated by antigen-independent stimuli and produce large amounts of cytokines. ILC can be classified into three subsets: ILC1, which differentiates in a T-bet-dependent manner and produces IFN-γ; ILC2, which differentiates in a GATA-3-dependent manner and produces IL-5, IL-9, and IL-13; and ILC3, which expresses RORγt and produces IL-22 or IL-17.

[0055] ILC are tissue-resident cells and interact not only with immune cells but also with non-immune cells to contribute to the maintenance of tissue homeostasis. Due to the property of ILC to localize in tissues and produce large amounts of cytokines in response to antigen-nonspecific stimuli, inappropriate activation of ILC is thought to lead to pathogenesis. However, the activation mechanism, such as through which molecules ILC are activated, is often not well understood.

[0056] ILC are thought to be related to lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis; inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis; infectious diseases such as viral infections, bacterial infections, parasitic infections, and protozoal infections; and acute and chronic liver injuries.

[0057] Activation of ILC can be indicated by an increase in the cell number of ILC, an increase in cytokines such as IFN-γ, IL-5, IL-9, IL-13, IL-22, IL-17, GM-CSF, TNF-α produced by ILC, and the like. The inhibitor of ILC activation may reduce any one of the above-mentioned indicators related to the activation of ILC. The inhibitor of ILC activation is preferably a cytokine production inhibitor, more preferably an IFN-γ production inhibitor or a TNF-α production inhibitor.

[0058] As described later in the examples, the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment can suppress the activation of natural lymphocytes, particularly cytokine production. Therefore, the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment is predicted to be effective in the prevention and treatment of diseases involving the activation of natural lymphocytes, such as lung diseases such as asthma, COPD (chronic obstructive pulmonary disease), and pulmonary fibrosis; inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis; infectious diseases such as viral infections, bacterial infections, parasitic infections, and protozoal infections, and acute and chronic liver damage.

[0059] Patent Document 1 describes that anti-DNAM-1 antibodies can be used for the prevention or treatment of graft-versus-host disease, organ transplant rejection, autoimmune diseases, fibrotic diseases, inflammatory bowel disease, allergies, etc., because they activate regulatory T cells and suppress the immune response. As described later in the Examples, the present inventors found that DNAM-1 is expressed in ILCs of the lung, small intestine, and PBMC. Furthermore, it was found that DNAM-1 is involved in the activation of ILCs, particularly cytokine production, and that a humanized anti-DNAM-1 antibody can suppress the activation of ILCs. Among the diseases for which anti-DNAM-1 antibodies have been considered to be usable for prevention or treatment, particularly in patients with lung diseases such as asthma, COPD, and pulmonary fibrosis; inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis; infectious diseases such as viral infections, bacterial infections, parasitic infections, and protozoal infections; and acute and chronic liver disorders, there are patients in whom ILCs are activated and patients in whom ILCs are not activated. Based on the new findings described in this specification, it has become possible to selectively administer the humanized anti-DNAM-1 antibody (A) to patients in whom ILCs are activated, so it is predicted that the treatment outcome will improve. In addition, it has become possible to determine the dosage, administration timing, and schedule of the humanized anti-DNAM-1 antibody (A) using the activation of ILCs as an index.

Examples

[0060] Next, examples of the present invention will be shown and described in more detail, but the present invention is not limited thereto. All experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of the Experimental Animal Resource Center, University of Tsukuba.

[0061] [Experimental Example 1] Preparation of a humanized anti-human DNAM-1 antibody [Preparation of an anti-human DNAM-1 monoclonal antibody (mAKB1)] The human DMAM-1 gene was introduced into the BW5147 cell line, which is a mouse lymphocyte cell line, to express the human DMAM-1 protein. Mice were immunized with these cells as antigens, spleen cells were collected from the mice, and hybridomas were obtained by fusing them with SP2 / 0 myeloma cells by a conventional method. Hybridomas were screened using the reactivity to human DNAM-1 protein as an index, and the selected clone was designated as clone mAKB1. The antibody produced by mAKB1 is referred to as anti-human DNAM-1 monoclonal antibody mAKB1 (simply referred to as mAKB1). The binding property with human DNAM-1 was analyzed using mAKB1 purified with a protein A Sepharose column from the culture supernatant of clone mAKB1.

[0062] [Analysis of binding property with human DNAM-1] Using the mouse lymphoblast BW5147 cell line (hereinafter sometimes referred to as "BW"), and BW5147 stably expressing human hDNAM-1 (hereinafter sometimes referred to as "hDNAM-1 / BW"), binding to human DNAM-1 was examined by flow cytometry. In PBS (FACS buffer) containing 0.5% BSA and 0.05% NaN 3 various concentrations of the test antibody were incubated with about 10 5 cells of hDNAM-1 / BW at 4°C for 1 hour. Thereafter, the cells were washed with ice-cold FACS Buffer and incubated with a PE-labeled goat anti-human IgG antibody (manufactured by SouthernBiotech) at 4°C for 30 minutes. After washing with FACS buffer, the stained cells were analyzed using a FACScan flow cytometer (manufactured by BD Biosciences).

[0063] The results of analyzing the binding property of mAKB1 with human DNAM-1 are shown in Figure 1. mAKB1 specifically binds to human DNAM-1, and the EC 50 was 6 μg / mL.

[0064] [Sequencing of mAKB1] From the clone mAKB1, the genes of the heavy chain variable region and the light chain variable region of the anti-human DNAM-1 monoclonal antibody mAKB1 were cloned by a conventional method, and the nucleotide sequences of the heavy chain variable region and the light chain variable region of mAKB1 were identified. The amino acid sequence was deduced from the nucleotide sequence, and the CDRs were determined by the Kabat method using abYsis.

[0065] [Design of VH and VL genes of humanized antibody hAKB1] Based on mAKB1, the amino acid sequences of VH (heavy chain variable region) and VL (light chain variable region) of the humanized antibody were designed as follows. First, a three-dimensional molecular model of the mAKB1 variable region was constructed. Using this molecular model, the framework amino acid residues important for the formation of the three-dimensional structure of the CDRs were identified.

[0066] A human VH sequence homologous to the framework of mAKB1 VH was searched in the GenBank database, and the VH sequence encoded by human FJ039783 cDNA (FJ039783 VH) was selected as the acceptor for humanization. The HCDR sequence of mAKB1 was grafted onto the corresponding position of FJ039783 VH. Since it was considered important for the formation of the CDR structure, the 30th, 49th, and 72nd amino acid residues of the heavy chain variable region were substituted from the amino acid residues of FJ039783 VH to the corresponding residues of mAKB1 VH. The VH of the obtained humanized antibody was designated as hAKB1-VH1. Furthermore, in order to reduce immunogenicity, hAKB1-VH2 was additionally designed in which the 30th amino acid residue was not reverted to the mouse amino acid residue. The amino acid sequences of mAKB1 VH (SEQ ID NO: 7), hAKB1-VH1 (SEQ ID NO: 9), hAKB1-VH2 (SEQ ID NO: 10), and FJ039783 VH are shown in Figure 2.

[0067] A human VL sequence homologous to the framework of mAKB1 VL was searched in the GenBank database, and the human Vκ region encoded by KU760971 cDNA (KU760971 VL) was selected as the acceptor for humanization. The LCDR sequence of mAKB1 was grafted onto the corresponding positions of KU760971 VL. Since it was considered important for the formation of the CDR structure, the 67th amino acid residue of the light chain variable region was substituted from the amino acid residue of KU760971 VL to the corresponding residue of mAKB1 VL. The obtained humanized antibody VL was designated as hAKB1-VL1. Furthermore, to reduce immunogenicity, hAKB1-VL2 was additionally designed in which the 67th amino acid residue was not reverted to the mouse amino acid residue. The amino acid sequences of VL of mAKB1 (SEQ ID NO: 8), hAKB1-VL1 (SEQ ID NO: 11), hAKB1-VL2 (SEQ ID NO: 12), and KU760971 VL are shown in Figure 3.

[0068] [Construction of VH and VL Genes of Humanized Antibody] Genes encoding hAKB1-VH1, hAKB1-VH2, hAKB1-VL1, and hAKB1-VL2 were synthesized including a signal peptide, a splice donor signal, and restriction enzyme sites at the 5' and 3' ends. The synthesized genes were incorporated into plasmids in the following combinations to prepare expression vectors.

[0069]

Table 1

[0070] Using polyethyleneimine, four expression vectors (phAKB1-A, phAKB1-B, phAKB1-C, phAKB1-D4) were each transfected into the human embryonic kidney cell line HEK293. HEK293 cells were cultured at 37°C in an incubator using DMEM medium containing 10% FBS (manufactured by HyClone) and 7.5% CO 2 and incubated.

[0071] The expression of the antibody in the culture supernatant of HEK293 cells transiently transfected was confirmed by ELISA. An ELISA plate was coated with 100 μl / well of goat anti-human IgG, Fcγ-specific polyclonal antibody (manufactured by Sigma-Aldrich) diluted 1 / 2000 with PBS at 4°C overnight, washed with Washing Buffer (PBS containing 0.05% Tween 20), and blocked with 300 μl / well of ELISA Buffer (PBS containing 2% skim milk and 0.05% Tween 20). After washing with Washing Buffer, 100 μl / well of the test antibody appropriately diluted with ELISA Buffer was applied to the ELISA plate. A humanized IgG1 / kappa antibody was used as a standard. The ELISA plate was incubated at room temperature for 1 hour, washed with Washing Buffer, and the bound antibody was detected using 100 μl / well of HRP-conjugated goat anti-human κ-chain polyclonal antibody (manufactured by Bethyl Laboratories) diluted 1 / 2000. After incubation at room temperature for 0.5 hour and washing with Washing Buffer, 100 μl / well of ABTS substrate (manufactured by Sigma-Aldrich) was added to initiate color development, and the reaction was stopped with 100 μl / well of 2% oxalic acid. The absorbance at 405 nm was read.

[0072] [Binding affinity of humanized antibody to antigen] The antibodies produced by cells transfected with phAKB1-A, phAKB1-B, phAKB1-C, or phAKB1-D were designated as hAKB1-A, hAKB1-B, hAKB1-C, and hAKB1-D, respectively. The binding affinity of these antibodies to human DNAM-1 was analyzed as described in the above-mentioned "Analysis of binding affinity to human DNAM-1". The results are shown in Figure 4. hAKB1-A and hAKB1-B had higher binding affinity to human DNAM-1 than hAKB1-C and hAKB1-D. For the antibodies containing hAKB1-VL1 (hAKB1-A, hAKB1-B) and the antibodies containing hAKB1-VL2 (hAKB1-C, hAKB1-D), there were differences in the binding affinity to the antigen. When the 67th amino acid residue of hAKB1-VL1 was changed from Tyr to Ser, the binding affinity decreased. No difference in the binding affinity to the antigen was observed between hAKB1-A and hAKB1-B.

[0073] [Establishment of cell lines stably producing antibodies] To obtain cell lines stably producing hAKB1-A or hAKB1-B, which have high binding affinity to human DNAM-1, the expression vectors phAKB1-A and phAKB1-B were introduced into the chromosomes of Chinese hamster ovary cell line CHO-K1 (obtained from ATCC) by the following method. CHO-K1 cells were cultured at 37°C in an incubator using SFM4CHO medium (manufactured by HyClone) with 7.5% CO 2 Transfection of CHO-K1 was performed by electroporation. Before transfection, each expression vector was linearized using FspI. Approximately 2.5×10 6 cells were transfected with 20 μg of the linearized plasmid, suspended in SFM4CHO medium, and after appropriately diluting the cells, they were plated in multiple 96-well plates. After 48 hours, 10 μg / ml of puromycin was added to isolate stable transfectants. Approximately 10 days after the start of selection, antibody production was measured by sandwich ELISA for the culture supernatants of the transfectants in the 96-well plates by the method described above. CHO-K1 stable transfectants producing hAKB1-A or hAKB1-B at a high level were selected and used for the next culture.

[0074] CHO-K1 stable transfectants were cultured in 450 ml of SFM4CHO at approximately 3×10 6Cultured in a roller bottle to a density of cells / ml, 50 ml of Cell Boost 4 (manufactured by HyClone) at 35 mg / ml was added, and further cultured until the cell viability reached 50% or less. After centrifugation and filtration, the culture supernatant was loaded onto a Protein A Sepharose column (HiTrap MabSelect SuRe, manufactured by GE Healthcare). After washing the column with PBS, the antibody was eluted with 0.1 M glycine-HCl buffer (pH 3.0) containing 0.1 M NaCl. After neutralization with 1 M Tris-HCl (pH 8.0), the buffer of the eluted antibody was exchanged to PBS by dialysis.

[0075] The purified hAKB1-A and hAKB1-B were characterized by SDS-PAGE according to standard procedures. As a result of analysis under reducing conditions, it was found that these antibodies were each composed of a heavy chain with a molecular weight of approximately 50 kDa and a light chain with a molecular weight of approximately 25 kDa. Also, the purity of each antibody was found to be 95% or more.

[0076] For the purified hAKB1-A and hAKB1-B, the EC with human DNAM-1 5 was determined by the method of "Analysis of binding to human DNAM-1" described above. The results are shown in Figure 5. The EC of hAKB1-A and hAKB1-B 50 was 5 μg / mL and 5 μg / mL, respectively, and the binding to human DNAM-1 was equal to or greater than that of mAKB1, which was the basis for humanization.

[0077] RNA was extracted from CHO-K1 / hAKB1-A and CHO-K1 / hAKB1-B, and PCR was performed to confirm the nucleotide sequences of the heavy and light chains of hAKB1-A and hAKB1-B produced by CHO-K1 / hAKB1-A and CHO-K1 / hAKB1-B. The nucleotide sequences of the coding regions of the heavy and light chains of the obtained hAKB1-A and hAKB1-B were completely identical to the corresponding nucleotide sequences of the phAKB1-A and phAKB1-B vectors. The nucleotide sequences of the coding regions of the heavy and light chains of hAKB1-A and hAKB1-B (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21) are shown in FIGS. 6 to 8, respectively. Also, the amino acid sequences were deduced from the nucleotide sequences, the variable regions were further determined, and the CDRs were determined by the Kabat method using abYsis. The results are shown in FIG. 9. The signal peptide is shown in white characters, the variable region is shown in bold, and the CDR is underlined.

[0078] The amino acid sequence of the heavy chain of hAKB1-A of SEQ ID NO: 16 (with signal peptide) is composed of the amino acid sequence of the signal peptide of the heavy chain of hAKB1-A (SEQ ID NO: 28) and the amino acid sequence of the heavy chain of hAKB1-A (without signal peptide: SEQ ID NO: 13). The amino acid sequence of the heavy chain of hAKB1-B of SEQ ID NO: 17 (with signal peptide) is composed of the amino acid sequence of the signal peptide of the hAKB1-B heavy chain (SEQ ID NO: 29) and the amino acid sequence of the heavy chain of hAKB1-B (without signal peptide: SEQ ID NO: 14). The amino acid sequences of the light chains of hAKB1-A and hAKB1-B of SEQ ID NO: 18 (with signal peptide) are composed of the amino acid sequences of the signal peptides of the hAKB1-A and hAKB1-B light chains (SEQ ID NO: 30) and the amino acid sequences of the light chains of hAKB1-A and hAKB1-B (without signal peptide: SEQ ID NO: 15).

[0079] The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of hAKB1-A and hAKB1-B were common and were set as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0080] [Establishment of hTKB1] In order to establish a cell line that stably and efficiently produces hAKB1-B and sufficiently secretes it extracellularly, the signal peptide sequences of the heavy and light chains of hAKB1-B were modified. Nucleic acid fragments consisting of sequences in which a nucleotide sequence encoding a modified signal peptide was tandemly incorporated into the expression vectors pWX068 and pWX069, respectively, upstream of the nucleotide sequences of the coding regions of the heavy and light chains of hAKB1-B. This was introduced into competent Escherichia coli TOP10 (manufactured by TIANGEN, CB104-2), and the expression vector incorporating the antibody gene was amplified and recovered. After linearizing this with FspI (manufactured by NEB, R0135L), it was introduced into the chromosome of CHO-K1. According to the method described above in "Establishment of a cell line that stably produces an antibody", a CHO-K1 stable transfectant into which the expression vector pWX069 that produces hAKB1-B at a high level and secretes it with high efficiency was introduced was selected. The obtained cell line was designated CHO-K1 / hTKB1, and the antibody produced by this cell line was designated hTKB1.

[0081] The nucleotide sequences of the coding regions of the heavy and light chains of hTKB1 (SEQ ID NO: 26, SEQ ID NO: 27) are shown in FIGS. 10 and 11, respectively. The signal peptide is shown in white characters, and the variable region is shown in bold. Also, the amino acid sequence was deduced from the nucleotide sequence, the variable region was further determined, and the CDRs were determined by the Kabat method using abYsis. The results are shown in FIG. 12. The signal peptide is shown in white characters, the variable region is shown in bold, and the CDRs are shown underlined.

[0082] The amino acid sequences of the heavy and light chains of hTKB1 were the same as those of the heavy and light chains of hAKB1-B, respectively, except for the signal peptide. That is, the amino acid sequence of SEQ ID NO: 22 and the amino acid sequence of SEQ ID NO: 14, and the amino acid sequence of SEQ ID NO: 23 and the amino acid sequence of SEQ ID NO: 15 are the same. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of hTKB1 were the same as those of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of hAKB1-A and hAKB1-B, respectively.

[0083] The amino acid sequence of the heavy chain of hTKB1 (with signal peptide) of SEQ ID NO: 24 is composed of the amino acid sequence of the signal peptide of the heavy chain of hTKB1 (SEQ ID NO: 31) and the amino acid sequence of the heavy chain of hTKB1 (without signal peptide: SEQ ID NO: 22). The amino acid sequence of the light chain of hTKB1 (with signal peptide) of SEQ ID NO: 25 is composed of the amino acid sequence of the signal peptide of the light chain of hTKB1 (SEQ ID NO: 32) and the amino acid sequence of the light chain of hTKB1 (without signal peptide: SEQ ID NO: 23).

[0084] [Analysis of DNAM-1 Expression in ILCs] (Method) The lungs of mice (C57BL / 6J, 8 - 10 weeks old, female) were perfused with PBS, then isolated, treated with collagenase IV (5 mg / ml; manufactured by Sigma) at 37°C for 1 hour, and then homogenized using a gentleMACS Dissociator (manufactured by Miltenyi Biotec). After removal, the mouse colon was incised and washed twice with phosphate-buffered saline (PBS) containing 0.5% BSA (manufactured by Wako) and 2 mM EDTA (manufactured by Junsei), and then incubated in RPMI1640 (manufactured by Invitrogen) containing 5% BSA, 5 mM EDTA, and 10 mM dithiothreitol (manufactured by Sigma Aldrich) for 30 minutes to remove the epithelial layer. The colon tissue with the epithelial layer removed was washed twice with PBS containing 0.5% BSA and 2 mM EDTA and with RPMI1640 containing 10 mM HEPES (manufactured by Sigma Aldrich), and then cut into pieces 3 - 5 mm in size. The cut colon pieces were digested using a lamina propria dissociation kit (manufactured by Miltenyi Biotec) and a gentleMACS Dissociator (manufactured by Miltenyi Biotec). The cell specimens obtained by digestion were filtered through a 70-μm nylon mesh to obtain a single-cell suspension.

[0085] Cells isolated from the lung and intestine were treated with anti-CD16 / 32 mAb (2.4G2; manufactured by Tonbo Biosciences) on ice for 10 minutes to avoid binding to FcγR, and then incubated for 10 minutes with a combination of monoclonal antibodies against CD45.2, CD11b, CD3ε, NK1.1, DX5, B220, CD44, CD90.2, Sca-1, RORγt and an anti-mouse DNAM-1 monoclonal antibody (clone TX42, manufactured by BioLegend), and analyzed by flow cytometry. A sample using rat IgG2a (manufactured by BioLegend) instead of the anti-mouse DNAM-1 monoclonal antibody was used as the Isotype control. Natural lymphocyte (ILC) subset 1 (ILC1) is CD45.2 + , CD11b - , CD3ε - , NK1.1 + , DX5 - Cells, and natural lymphocyte (ILC) subset 2 (ILC2) is CD45.2 + , CD11b - , B220 - , NK1.1 -, CD3ε - , CD44 + , CD90.2 + , Sca-1 + As cells that are CD45.2 + , CD11b - , B220 - , NK1.1 - , CD3ε - , CD44 + , CD90.2 + , RORγt + , Sca-1 - were identified as such cells.

[0086] The following antibodies were used. Anti-CD11b (M1 / 70) monoclonal antibody: BD Biosciences Anti-TCRβ monoclonal antibody (H57-597), anti-Thy1.2 monoclonal antibody (53-2.1): BD PharMingen CD45.2 monoclonal antibody (104), anti-CD4 monoclonal antibody (RM4-5), anti-CD8 monoclonal antibody (53-6.7), anti-CD127 monoclonal antibody (A7R34): BioLegend Anti-CD11c monoclonal antibody (N418): Tonbo biosciences Anti-CD3ε monoclonal antibody (17A2): BioLegend Anti-NK1.1 monoclonal antibody (S17016D): BioLegend Anti-DX5 monoclonal antibody (DX5): BioLegend Anti-B220 monoclonal antibody (RA3-6B2): BioLegend Anti-CD44 monoclonal antibody (3 / 23): BioLegend Anti-CD90.2 monoclonal antibody (30-H12): BioLegend Anti-Sca-1 monoclonal antibody (E13-161.7): BioLegend Anti-RORγt monoclonal antibody (RORg2): BioLegend

[0087] (Result) The results are shown in FIGS. 13A and 13B. The dotted line indicates the Isotype control, and the solid line indicates the case where an anti-mouse DNAM-1 monoclonal antibody was used. From FIGS. 13A and 13B, it was revealed that DNAM-1 is expressed in ILC1, ILC2, and ILC3 in the lung and intestine.

[0088] [Analysis of cytokine expression levels in ILCs] (Method) Wild-type (WT) mice (C57BL / 6J) and DNAM-1 gene-deficient mice (hereinafter also referred to as "DNAM-1KO") were each intratracheally administered a single dose of bleomycin hydrochloride (BLM, manufactured by Nippon Kayaku Co., Ltd.) dissolved in physiological saline at a dose of 6.5 mg / kg in a volume of 50 μL per mouse using an intratracheal nebulizer (manufactured by Natsume Seisakusho Co., Ltd.) to induce pulmonary fibrosis. ILCs (CD45.2 + , CD11b - , CD11c - , TCRβ - , CD127 + , Thy1.2 + ) present in the lungs before and after BLM administration were sorted using BD FACSAria III (manufactured by BD Biosciences) and homogenized in Isogen reagent (manufactured by Nippon Gene). Then, total RNA was isolated and first-strand DNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (manufactured by Thermo Fisher Scientific). Quantitative RT-PCR was performed using an ABI 7500 Fast real-time PCR system and ABI Power SYBR Green PCR Master Mix (both manufactured by Thermo Fisher Scientific). The relative amounts and copy numbers of the gene transcripts were normalized as values relative to the Actb transcript.

[0089] (Result) The results are shown in Fig. 14. As shown in Fig. 14, in ILCs of DNAM-1KO at 3 days, 5 days, and / or 14 days after BLM administration, the expression of mRNAs for IL17a, IL15, and IL13 was decreased compared with that in WT mice. From these results, it was revealed that DNAM-1 is involved in cytokine production in ILCs.

[0090] [Expression of DNAM-1 in human peripheral blood ILCs] (Method) Blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were collected using Lymphoprep TM (manufactured by STEMCELL Technologies) according to the manufacturer's instructions. PBMCs were stained with conventional lineage markers (antibodies against CD3, CD4, CD8, CD19, CD14, CD16, CD11b, CD11c, CD56, FcεRI), anti-CD127 antibody, CD45 antibody, and humanized anti-DNAM-1 antibody (hTKB1). The expression of DNAM-1 was analyzed by flow cytometry on cells gated as total ILCs (Lin - , CD45 + , CD127 + ). Cells using human IgG1 (manufactured by BioLegend) instead of the humanized anti-DNAM-1 antibody (hTKB1) were used as the Isotype control.

[0091] (Results) The results are shown in Fig. 15. The dotted line indicates the case using the Isotype control, and the solid line indicates the case using the humanized anti-DNAM-1 antibody (hTKB1). As shown in Fig. 15, it was revealed that DNAM-1 is expressed in ILCs of human PBMCs. In addition, it was confirmed that hTKB1 can detect DNAM-1 on ILCs.

[0092] [Inhibition of IFN-γ and TNF-α production from ILCs by anti-DNAM-1 humanized antibody] (Method) Blood was collected from healthy volunteers, and LymphoprepTM (manufactured by STEMCELL Technologies) was used to collect peripheral blood mononuclear cells (PBMCs) according to the manufacturer's instructions. Using a MACs LS column (manufactured by Miltenyi), CD4 + T cells, CD8 + T cells, and CD56 + NK cells were removed from the PBMCs. 2 - 3×10 6 of these T / NK cell - depleted PBMCs were cultured for 36 hours with 10 μg / mL of mouse IgG1, hAKB1 - A, hAKB1 - B, or hTKB1 in the presence of human IL - 2 (5 ng / mL), human IL - 12 (10 ng / mL), and human IL - 15 (50 ng / mL). Subsequently, the cells were stained with antibodies against lineage markers (Lin: CD3, CD4, CD8, CD19, CD14, CD16, CD11b, CD11c, FcεRI), anti - CD127 antibody, and anti - CD45 antibody. ILC (Lin - , CD127 + , CD45 + cells) were sorted using a FACS Aria III (manufactured by BD Biosciences). Total RNA was isolated from the sorted ILCs using an Isogen reagent according to the manufacturer's protocol (manufactured by Nippon Gene). For reverse transcription, a High - Capacity cDNA Reverse - Transcription Kit (manufactured by Applied Biosystems) was used. Quantitative PCR analysis of Ifng was performed using an ABI7500 sequence detector (manufactured by Applied Biosystems), Power SYBR Green PCR Master Mix (manufactured by Applied Biosystems), and the primers shown below. To normalize the data, the expression level of Gapdh was measured as an internal standard.

[0093] Gapdh forward; 5'-CTT CAC CAC CAT GGA GAA GGC-3' (SEQ ID NO: 33) Gapdh reverse; 5'-GGC ATG GAC TGT GGT CAT GAG-3' (SEQ ID NO: 34) Ifng forward; 5'-ACC AGA GCA TCC AAA AGA GTG T-3' (SEQ ID NO: 35) Ifng reverse; 5'-TTA GCT GCT GGC GAC AGT TC-3' (SEQ ID NO: 36) Tnfa forward; 5'-CAG CCT CTT CTC CTT CCT GAT-3' (SEQ ID NO: 37) Tnfa reverse; 5'-GCC AGA GGG CTG ATT AGA GA-3' (SEQ ID NO: 38)

[0094] (Results) The results are shown in Figure 16. The mouse monoclonal antibody mAKB1 had no effect on the expression of IFN-γ and TNF-α mRNA, but the humanized anti-DNAM-1 antibodies hAKB1-A, hAKB1-B, and hTKB1 suppressed the expression of IFN-γ and TNF-α mRNA (Figure 16).

[0095] SEQ ID NO: 1: Amino acid sequence of HCDR1 of hTKB1, hAKB1-A, and hAKB1-B SEQ ID NO: 2: Amino acid sequence of HCDR2 of hTKB1, hAKB1-A, and hAKB1-B SEQ ID NO: 3: Amino acid sequence of HCDR3 of hTKB1, hAKB1-A, and hAKB1-B SEQ ID NO: 4: Amino acid sequence of LCDR1 of hTKB1, hAKB1-A, and hAKB1-B SEQ ID NO: 5: Amino acid sequence of LCDR2 of hTKB1, hAKB1-A, and hAKB1-B SEQ ID NO: 6: Amino acid sequence of LCDR3 of hTKB1, hAKB1-A, and hAKB1-B SEQ ID NO: 7: Amino acid sequence of the heavy chain variable region of mAKB1 SEQ ID NO: 8: Amino acid sequence of the light chain variable region of mAKB1 SEQ ID NO: 9: Amino acid sequence of the variable region of VH1 of hAKB1 SEQ ID NO: 10: Amino acid sequence of the variable region of VH2 of hAKB1 Sequence number 11: Amino acid sequence of the variable region of VL1 of hAKB1 Sequence number 12: Amino acid sequence of the variable region of VL2 of hAKB1 Sequence number 13: Amino acid sequence of the heavy chain of hAKB1-A (without signal peptide) Sequence number 14: Amino acid sequence of the heavy chain of hAKB1-B (without signal peptide) Sequence number 15: Amino acid sequence of the light chains of hAKB1-A and hAKB1-B (without signal peptide) Sequence number 16: Amino acid sequence of the heavy chain of hAKB1-A (with signal peptide) Sequence number 17: Amino acid sequence of the heavy chain of hAKB1-B (with signal peptide) Sequence number 18: Amino acid sequence of the light chains of hAKB1-A and hAKB1-B (with signal peptide) Sequence number 19: Nucleotide sequence of the heavy chain coding region of hAKB1-A Sequence number 20: Nucleotide sequence of the heavy chain coding region of hAKB1-B Sequence number 21: Nucleotide sequence of the light chain coding regions of hAKB1-A and hAKB1-B Sequence number 22: Amino acid sequence of the heavy chain of hTKB1 (without signal peptide) Sequence number 23: Amino acid sequence of the light chain of hTKB1 (without signal peptide) Sequence number 24: Amino acid sequence of the heavy chain of hTKB1 (with signal peptide) Sequence number 25: Amino acid sequence of the light chain of hTKB1 (with signal peptide) Sequence number 26: Nucleotide sequence of the heavy chain coding region of hTKB1 Sequence number 27: Nucleotide sequence of the light chain coding region of hTKB1 Sequence number 28: Amino acid sequence of the signal peptide of the hAKB1-A heavy chain Sequence number 29: Amino acid sequence of the signal peptide of the hAKB1-B heavy chain Sequence number 30: Amino acid sequence of the signal peptides of the hAKB1-A and hAKB1-B light chains Sequence number 31: Amino acid sequence of the signal peptide of the hTKB1 heavy chain SEQ ID NO: 32: Amino acid sequence of the signal peptide of the light chain of hTKB1 SEQ ID NO: 33: Base sequence of the Gapdh forward primer SEQ ID NO: 34: Base sequence of the Gapdh reverse primer SEQ ID NO: 35: Base sequence of the Ifng forward primer SEQ ID NO: 36: Base sequence of the Ifng reverse primer SEQ ID NO: 37: Base sequence of the Tnfa forward primer SEQ ID NO: 38: Base sequence of the Tnfa reverse primer

Claims

1. A heavy chain variable region comprising the following amino acid sequence; The amino acid sequence of SEQ ID NO: 1 as HCDR1, the amino acid sequence of SEQ ID NO: 2 as HCDR2, and the amino acid sequence of SEQ ID NO: 3 as HCDR3, and A light chain variable region comprising the following amino acid sequence; The amino acid sequence of SEQ ID NO: 4 as LCDR1, the amino acid sequence of SEQ ID NO: 5 as LCDR2, and the amino acid sequence of SEQ ID NO: 6 as LCDR3, A humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof, having The heavy chain variable region has an amino acid sequence that is 95% or more identical to SEQ ID NO: 10, and The light chain variable region has an amino acid sequence that is 95% or more identical to SEQ ID NO: 11, The 49th and 72nd amino acid residues corresponding to the sequence of SEQ ID NO: 10 in the heavy chain variable region are M and R, respectively, and the 67th amino acid residue corresponding to the sequence of SEQ ID NO: 11 in the light chain variable region is Y, a humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof.

2. The heavy chain variable region has an amino acid sequence that is 99% or more identical to SEQ ID NO: 10, The light chain variable region has an amino acid sequence that is 99% or more identical to SEQ ID NO: 11, the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to claim 1.

3. The light chain variable region has the amino acid sequence of SEQ ID NO: 11, the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to claim 1 or 2.

4. The heavy chain variable region has the amino acid sequence of SEQ ID NO: 10, and The light chain variable region has the amino acid sequence of SEQ ID NO: 11, the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to claim 3.

5. The heavy chain variable region has the amino acid sequence of SEQ ID NO: 9, and The light chain variable region has the amino acid sequence of SEQ ID NO: 11, the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to claim 3.

6. The antibody comprises a heavy chain constant region of a class selected from the group consisting of IgG, IgM, IgA, IgD, and IgE, the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The heavy chain constant region is a human IgG1 constant region, the humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof according to claim 6.

8. The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to claim 7, wherein the human IgG1 constant region has one or more mutations that abolish effector function.

9. The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to claim 8, wherein the one or more mutations each contain amino acid A or F at amino acid positions 238 and 239 corresponding to the sequence of SEQ ID NO:

14.

10. The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to claim 8, wherein the one or more mutations each contain amino acid A at amino acid positions 238 and 239 corresponding to the sequence of SEQ ID NO:

14.

11. The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, having a heavy chain with an amino acid sequence that is at least 95% identical to SEQ ID NO: 14 and a light chain with an amino acid sequence that is at least 95% identical to SEQ ID NO:

15.

12. wherein the heavy chain has the amino acid sequence of SEQ ID NO: 14, and The humanized anti-DNAM-1 antibody according to claim 11, wherein the light chain has the amino acid sequence of SEQ ID NO:

15.

13. A nucleic acid encoding the humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. A vector containing the nucleic acid according to claim 13.

15. A host cell transfected with the vector according to claim 14.

16. A composition for use in a method of suppressing the activation of natural lymphocyte cells, comprising the humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

17. A composition for use in the treatment or prevention of inflammatory bowel disease, graft-versus-host disease, organ transplant rejection, autoimmune disease, fibrotic disease, ulcerative colitis, or allergy, comprising the humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

18. A composition for use in the treatment of inflammatory bowel disease, comprising the humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

19. The composition according to claim 18, wherein the inflammatory bowel disease is ulcerative colitis.

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