Humanized Anti-DNAM-1 antibody

Abstract

To provide a humanized anti-DNAM-1 antibody that specifically binds to human DNAM-1.SOLUTION: 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 including respective specific amino acid sequences, and a light chain variable region including respective specific amino acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] DNAM-1, also known as CD226, is an adhesion molecule of the immunoglobulin superfamily with a molecular weight of 65 kDa, and is expressed on CD4 + T cells, CD8 + It was identified as an activating immune receptor expressed on hematopoietic cells such as T cells, natural killer (NK) cells, and platelets. Upon binding to its ligands, CD155 or CD112, DNAM-1 mediates activation signals for cytotoxicity. DNAM-1 has been shown to be involved in the pathogenesis of various inflammatory diseases and cancers in humans and mouse models. Furthermore, anti-mouse DNAM-1 monoclonal antibodies have been reported to suppress the development of experimental autoimmune encephalitis and acute graft-versus-host disease (GVHD) in mice, increase regulatory T (Treg) cell populations, and consequently prolong the survival of skin grafts in mice. Based on these reports, anti-DNAM-1 monoclonal antibodies are considered to be useful in treating these diseases. The present inventors previously established a mouse anti-human DNAM-1 monoclonal antibody (Patent Document 1) and also reported a humanized anti-DNAM-1 antibody (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2017 / 183665 [Non-patent literature]

[0004] [Non-Patent Document 1] Yumi Yamashita-Kanemaru et.al., Suppression of Th1 and Th17 Proinflammatory Cytokines and Upregulation of FOXP3 Expression by a Humanized Anti-DNAM-1 Monoclonal Antibody, MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY, Volume 40, Number 2, 2021 Summary of the Invention [Problem to be solved by the invention]

[0005] The mouse anti-human DNAM-1 monoclonal antibody described in Patent Document 1 activates regulatory T cells and suppresses immune responses, and is therefore believed 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, the antibody established in Patent Document 1 was merely a mouse antibody, and there was still room for improvement in terms of its therapeutic value in humans, etc. Furthermore, humanized antibodies generally have reduced binding to target antigens compared to their parent mouse antibodies, and maintaining antigen binding tends to increase immunogenicity, leaving room for improvement. Therefore, the present inventors attempted to obtain a novel mouse anti-human DNAM-1 monoclonal antibody and to prepare a humanized anti-DNAM-1 antibody of high therapeutic value in humans. An objective of the present invention is to provide a humanized anti-DNAM-1 antibody that specifically binds to human DNAM-1. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by providing the following configuration, which has led to the completion of 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 a light chain variable region comprising the following amino acid sequence: LCDR1 is the amino acid sequence of SEQ ID NO: 4, LCDR2 is the amino acid sequence of SEQ ID NO: 5, and LCDR3 is the amino acid sequence of SEQ ID NO: 6; A humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof, having the following structure: [2] The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to [1], which has a heavy chain variable region having an amino acid sequence that is 95% or more identical to SEQ ID NO: 10 and a light chain variable region having an amino acid sequence that is 95% or more identical to SEQ ID NO: 11. [3] 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 antigen-binding fragment thereof according to [1] or [2], [4] The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of [1] to [3], wherein the amino acid residues at positions 238 and 239 of the heavy chain are A. [5] The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of [1] to [4], which has a heavy chain having an amino acid sequence that is 95% or more identical to SEQ ID NO: 14 and a light chain having an amino acid sequence that is 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 following structure: [7] A nucleic acid encoding the humanized anti-DNAM-1 antibody or 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 of innate lymphocyte (ILC) activation, comprising the humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of [1] to [6]. [Effects of the Invention]

[0007] According to the present invention, a humanized anti-DNAM-1 antibody and an inhibitor of innate lymphocyte (ILC) activation can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of analyzing the binding of mAKB1 to human DNAM-1. [Figure 2] Figure 2 shows the amino acid sequences of the heavy chain variable regions of mAKB1 and its humanized antibodies, with back-mutated amino acid residues in bold and CDRs underlined. [Figure 3] Figure 3 shows the amino acid sequences of the light chain variable regions of mAKB1 and its humanized antibodies, with back-mutated amino acid residues in bold and CDRs underlined. [Figure 4] FIG. 4 shows the results of analyzing the binding of hAKB1-A, hAKB1-B, hAKB1-C, and hAKB1-D to human DNAM-1. [Figure 5] FIG. 5 shows the results of analyzing the binding of hAKB1-A and hAKB1-B to human DNAM-1. [Figure 6] FIG. 6 shows the nucleotide sequence of the heavy chain coding region of hAKB1-A. [Figure 7] FIG. 7 shows the nucleotide sequence of the heavy chain coding region of hAKB1-B. [Figure 8] FIG. 8 shows the nucleotide sequences of the light chain coding regions of hAKB1-A and hAKB1-B. [Figure 9]FIG. 9 shows the amino acid sequences of the heavy chain of hAKB1-A, the heavy chain of hAKB1-B, and the light chains of hAKB1-A and hAKB1-B. [Figure 10] FIG. 10 shows the nucleotide sequence of the hTKB1 heavy chain coding region. [Figure 11] FIG. 11 shows the nucleotide sequence of the hTKB1 light chain coding region. [Figure 12] FIG. 12 shows the amino acid sequences of the heavy and light chains of hTKB1. [Figure 13A] FIG. 13A shows the results of examining DNAM-1 expression in pulmonary innate lymphoid cells (ILCs). [Figure 13B] FIG. 13B shows the results of examining DNAM-1 expression in innate lymphoid cells (ILCs) of the small intestine. [Figure 14] FIG. 14 shows the results of analyzing the expression levels of cytokines in ILCs using wild-type mice and DNAM-1 gene-deficient mice. [Figure 15] FIG. 15 shows the results of examining DNAM-1 expression in innate lymphoid cells (ILCs) of human peripheral blood mononuclear cells (PBMCs). [Figure 16] FIG. 16 shows the results of examining the effects of mAKB1, hAKB1-A, hAKB1-B, and hTKB1 on cytokine production in innate lymphoid cells (ILCs). DETAILED DESCRIPTION OF THE INVENTION

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

[0010] A humanized antibody is an antibody whose variable region is, in principle, composed of complementarity-determining regions (CDRs) derived from a non-human antibody and framework regions (FRs) derived from a human antibody, and whose constant region is composed of constant regions derived from a human antibody.

[0011] One embodiment of the humanized anti-DNAM-1 antibody of the present invention is a heavy chain variable region comprising the following amino acid sequences: HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3; and A light chain variable region comprising the following amino acid sequences: LCDR1 has the amino acid sequence of SEQ ID NO: 4, LCDR2 has the amino acid sequence of SEQ ID NO: 5, and LCDR3 has the amino acid sequence of SEQ ID NO: 6. The humanized anti-DNAM-1 antibody is referred to as humanized anti-DNAM-1 antibody (A). The HCDRs are the CDRs of the heavy chain and the LCDRs are the CDRs 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, although an antibody that inhibits the binding of human DNAM-1 to its ligand is preferred. Furthermore, the humanized anti-DNAM-1 antibody (A) may be an antibody that inhibits or reduces human DNAM-1 signal transduction (neutralizing antibody), an antibody that does not alter human DNAM-1 signal transduction, or an antibody that promotes human DNAM-1 signal transduction (agonistic antibody), although an antibody that inhibits or reduces human DNAM-1 signal transduction (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, with IgG being preferred, and IgG1 being more preferred. The heavy chain of the humanized anti-DNAM-1 antibody (A) may be any of gamma, mu, alpha, delta, and epsilon, with gamma being preferred, and gamma being more preferred. The light chain of the humanized anti-DNAM-1 antibody (A) may be either kappa or lambda, with kappa being preferred.

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

[0015] The humanized anti-DNAM-1 antibody (A) may be a multispecific antibody, a recycling antibody, a sweeping antibody, a conjugated antibody, or the like. Furthermore, the humanized anti-DNAM-1 antibody may be chemically or genetically conjugated with functional molecules such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors, albumin, enzymes, and other antibodies. These can be produced by known methods.

[0016] The method for producing the humanized anti-DNAM-1 antibody (A) is not particularly limited, and it can be produced by known methods. 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 then allowing 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 in the regions other than HCDR1 to HCDR3 and LCDR1 to LCDR3, as long as it specifically recognizes human DNAM-1.

[0018] The humanized anti-DNAM-1 antibody (A) may preferably have back mutations of amino acid residues in regions other than HCDR1 to HCDR3 of the heavy chain variable region and / or regions other than LCDR1 to LCDR3 of the light chain variable region. Backmutation of amino acid residues 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. Appropriate backmutation of amino acid residues can achieve both low immunogenicity and high antigen binding.

[0019] The number of backmutations of amino acid residues in the heavy chain variable region is preferably 1 to 6, more preferably 1 to 3. The number of backmutations of amino acid residues 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 backmutations of 1 to 3 amino acid residues in a region other than HCDR1 to HCDR3 of the heavy chain variable region, and has no backmutations of amino acid residues in a region other than LCDR1 to LCDR3 of the light chain variable region.

[0020] The amino acid residues to be backmutated can be selected from amino acid residues in the variable region that directly bind to an antigen via non-covalent bonding, 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. Backmutations of amino acid residues in the heavy chain variable region are preferably carried out within a range of 5 amino acids, more preferably 2 amino acids, before or after HCDR1, HCDR2, or HCDR3.

[0021] The backmutation of amino acid residues in the heavy chain variable region is preferably carried out at at least one amino acid residue selected from amino acid residues 49 and 72. The backmutation of amino acid residues in the light chain variable region is preferably carried out at amino acid residue 67. The humanized anti-DNAM-1 antibody (A) more preferably has back mutations at the 49th and 72nd amino acid residues in the heavy chain variable region and the 67th amino acid residue in 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, not including the signal peptide.

[0022] The backmutation of amino acid residues in the heavy chain variable region is preferably M at amino acid residue 49 and R at amino acid residue 72. The backmutation of amino acid residues in the light chain variable region is preferably Y at amino acid residue 67. The humanized anti-DNAM-1 antibody (A) more preferably has back mutations I49M and 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, or 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, or 99% or more identical to SEQ ID NO: 11.

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

[0025] The humanized anti-DNAM-1 antibody (A) may be an antibody having a heavy chain variable region having an amino acid sequence of SEQ ID NO: 10 with 1 to 6 amino acid residue mutations in a region other than HCDR1 to HCDR3, the amino acid sequence being at least 95% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence of SEQ ID NO: 11 with 1 to 5 amino acid residue mutations in a region other than LCDR1 to LCDR3, the amino acid sequence being at least 95% identical to SEQ ID NO: 11. "Amino acid residue mutation" refers to the 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 having an amino acid sequence of SEQ ID NO: 10 in which 1 to 3 amino acid residues have been mutated in a region other than HCDR1 to HCDR3, and which is at least 97% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence of SEQ ID NO: 11 in which 1 to 3 amino acid residues have been mutated in a region other than LCDR1 to LCDR3, and which is at least 97% identical to SEQ ID NO: 11.

[0027] Mutation of amino acid residues is preferably substitution, insertion, or deletion, with conservative substitution being more preferred. A "conservative substitution" refers to replacing an amino acid residue with another chemically similar amino acid residue without substantially altering the activity of the peptide. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Examples of chemically similar amino acids that can be substituted in this way include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0028] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain variable region having an amino acid sequence that is 95% or more identical to SEQ ID NO: 10 and a light chain variable region having an amino acid sequence that is 95% or more identical to SEQ ID NO: 11, in which 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 mutations in amino acid residues to eliminate the effector functions of IgG antibodies. Examples of amino acid residue mutations that eliminate the effector function of IgG antibodies include mutations in the heavy chain constant region that substitute branched-chain amino acids such as V (valine), L (leucine), and I (isoleucine); hydrophobic amino acids such as P (proline), M (methionine), and W (tryptophan); or amino acids that are phosphorylated and involved in signal transduction, such as Y (tyrosine), S (serine), and T (threonine), with A (alanine) or F (phenylalanine). Preferred are mutations that substitute L with A or F, and more preferred are mutations that substitute two consecutive Ls with AA, FF, AF, or FA. In the humanized anti-DNAM-1 antibody (A), the amino acid residues at positions 238 and 239 of the heavy chain are preferably A or F, and more preferably both are A. Such a humanized anti-DNAM-1 antibody (A) eliminates the effector function of IgG antibodies and is less likely to induce platelet aggregation, making it useful for treating human diseases.

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

[0032] The humanized anti-DNAM-1 antibody (A) is preferably an antibody having a heavy chain variable region having an amino acid sequence of SEQ ID NO: 14 in which 1 to 22 amino acid residues have been mutated in a region other than HCDR1 to HCDR3, and which is at least 95% identical to SEQ ID NO: 14, and a light chain variable region having an amino acid sequence of SEQ ID NO: 15 in which 1 to 10 amino acid residues have been mutated in a region other than LCDR1 to LCDR3, and which is at least 95% identical to SEQ ID NO: 15.

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

[0034] The humanized anti-DNAM-1 antibody (A) preferably has a heavy chain having an amino acid sequence that is 95% or more identical to SEQ ID NO: 14 and a light chain having an amino acid sequence that is 95% or more identical to SEQ ID NO: 15, in which the 49th and 72nd amino acid residues in the variable region of the heavy chain are M and R, respectively, and the 67th amino acid residue in 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] An antigen-binding fragment of a humanized anti-DNAM-1 antibody is a protein containing a portion of the humanized anti-DNAM-1 antibody (A) that can bind to an antigen. Examples of antigen-binding fragments include F(ab')2, Fab', Fab, disulfide-stabilized Fv (dsFv), single-chain antibody (scFv), diabody, and polymers thereof.

[0037] Fab is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by treating IgG with papain (a protease). The Fab of a humanized anti-DNAM-1 antibody can be produced by treating the humanized anti-DNAM-1 antibody with papain, or by inserting 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 with a molecular weight of approximately 100,000 that has antigen-binding activity and is obtained by treating IgG with pepsin (a protease). F(ab')2 of a humanized anti-DNAM-1 antibody can be produced by treating the humanized anti-DNAM-1 antibody with pepsin or by linking Fab' (described below) via a thioether bond or disulfide bond.

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

[0040] An scFv is an antibody fragment with antigen-binding activity in which one VH and one VL are linked using an appropriate peptide linker. The scFv of a humanized anti-DNAM-1 antibody can be produced 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 a prokaryote or eukaryote for expression.

[0041] A diabody is an antibody fragment formed by dimerization of scFv and has bivalent antigen-binding activity. Humanized anti-DNAM-1 antibody diabodies can be produced 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 a prokaryote or eukaryote for expression.

[0042] dsFv is an antibody fragment in which polypeptides in which one amino acid residue in each of VH and VL has been substituted with a cysteine ​​residue are linked via a disulfide bond between the cysteine ​​residues. dsFv of a humanized anti-DNAM-1 antibody can be produced 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 a prokaryote or eukaryote for expression.

[0043] The antigen-binding fragment may be chemically or genetically linked to a functional molecule such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor, albumin, an enzyme, or another antibody. One type of antigen-binding fragment may be used alone, or two or more types may be used in combination. Furthermore, a humanized anti-DNAM-1 antibody and its antigen-binding fragment may be used in combination.

[0044] [Nucleic acid encoding a humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof] 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 nucleic acids encoding the heavy chain variable region of humanized anti-DNAM-1 antibody (A), nucleic acids encoding the light chain variable region of humanized anti-DNAM-1 antibody (A), nucleic acids encoding a portion of the heavy chain variable region and constant region of humanized anti-DNAM-1 antibody (A), nucleic acids encoding a portion of the light chain variable region and constant region of humanized anti-DNAM-1 antibody (A), nucleic acids encoding the full-length heavy chain of humanized anti-DNAM-1 antibody (A), nucleic acids encoding the full-length light chain of humanized anti-DNAM-1 antibody (A), and nucleic acids encoding an scFv in which the heavy chain variable region and light chain variable region of humanized anti-DNAM-1 antibody (A) are linked by an appropriate linker. The nucleic acid can be produced using known genetic engineering techniques.

[0045] A nucleic acid encoding a signal peptide is preferably added to the nucleic acid encoding the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment. Addition of a signal peptide with an appropriate amino acid sequence can increase the expression level of the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment in cells or the amount of the antibody or antigen-binding fragment secreted into the culture supernatant.

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

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

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

[0049] There are no particular limitations on the method for producing the vector, and it can be produced by known gene recombination techniques. In addition to the nucleic acid encoding the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment, the vector may also contain regulatory control sequences capable of controlling replication and expression in a host and / or secretion from the host, such as a promoter sequence such as a CMV promoter.

[0050] [Transformants 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] A transformant can be obtained by introducing the above-mentioned recombinant vector into a host, such as cultured cells such as Escherichia coli, yeast, plant cells, insect cells, and animal cells; living insects such as silkworms; and plants such as tobacco, with animal cells being preferred. Animal cells include mammalian cells such as NS0, Sp2 / 0, CHO, COS, HEK, fibroblasts, and myeloma cells, with CHO being preferred.

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

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

[0054] Innate lymphoid cells (ILCs) are cells of the innate immune system derived from lymphoid progenitor cells (CLPs). Because ILCs lack T cell or B cell receptors, they are not activated by antigen-specific factors. However, they are rapidly activated by antigen-independent stimuli and produce large amounts of cytokines. ILCs can be classified into three subsets: ILC1, which differentiate in a T-bet-dependent manner and produce IFN-γ; ILC2, which differentiate in a GATA-3-dependent manner and produce IL-5, IL-9, and IL-13; and ILC3, which express RORγt and produce IL-22 or IL-17.

[0055] ILCs are tissue-resident cells that interact with not only immune cells but also non-immune cells to maintain tissue homeostasis. Because ILCs are localized in tissues and produce large amounts of cytokines in response to non-antigen-specific stimuli, their inappropriate activation is thought to lead to pathogenesis. However, the activation mechanisms of ILCs, such as the molecules through which they are activated, remain largely unknown.

[0056] ILCs are thought to be associated with a variety of conditions, including 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, bacterial, parasitic, and protozoan infections; and acute and chronic liver disorders.

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

[0058] As described below in the Examples, the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment can suppress the activation of innate lymphocytes, particularly cytokine production. Therefore, the humanized anti-DNAM-1 antibody (A) or its antigen-binding fragment is predicted to be effective in preventing and treating diseases involving the activation of innate lymphocytes, such as pulmonary 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 protozoan infections; and acute and chronic liver disorders.

[0059] Patent Document 1 describes that anti-DNAM-1 antibodies activate regulatory T cells and suppress immune responses, and thus can be used to prevent or treat graft-versus-host disease, organ transplant rejection, autoimmune diseases, fibrotic diseases, inflammatory bowel disease, allergies, and other conditions. As described in the Examples section below, the present inventors have discovered that DNAM-1 is expressed in ILCs in the lungs, small intestine, and PBMCs. Furthermore, they have found that DNAM-1 is involved in ILC activation, particularly cytokine production, and that humanized anti-DNAM-1 antibodies can suppress ILC activation. Among the diseases for which anti-DNAM-1 antibodies have been proposed for the prevention or treatment, some patients have activated ILCs, while others do not. These include pulmonary 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 protozoan infections; and acute and chronic liver disorders. The new findings described herein enable selective administration of humanized anti-DNAM-1 antibody (A) to patients with activated ILCs, which is expected to improve treatment outcomes. Furthermore, it has become possible to determine the dosage, timing, and schedule of administration of humanized anti-DNAM-1 antibody (A) using ILC activation as an indicator. [Example]

[0060] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. All experiments were performed in accordance with the guidelines of the Animal Ethics Committee of the Laboratory Animal Resource Center, University of Tsukuba.

[0061] [Experimental Example 1] Preparation of anti-human DNAM-1 humanized antibody [Production of anti-human DNAM-1 monoclonal antibody (mAKB1)] The human DMAM-1 gene was introduced into the mouse lymphocyte cell line BW5147, resulting in the expression of human DMAM-1 protein. Mice were immunized with these cells, and spleen cells were collected and fused with SP2 / 0 myeloma cells by standard methods to obtain hybridomas. Hybridomas were screened for reactivity to human DMAM-1 protein, and the selected clone was designated clone mAKB1. The antibody produced by mAKB1 is designated anti-human DMAM-1 monoclonal antibody mAKB1 (also simply referred to as mAKB1). The culture supernatant of clone mAKB1 was purified on a protein A Sepharose column, and the binding ability of mAKB1 to human DMAM-1 was analyzed.

[0062] [Analysis of binding to human DNAM-1] Binding to human DNAM-1 was examined by flow cytometry using the mouse lymphoblastoid cell line BW5147 (hereinafter referred to as "BW") and BW5147 stably expressing human hDNAM-1 (hereinafter referred to as "hDNAM-1 / BW"). Approximately 105 hDNAM-1 / BW cells were incubated with various concentrations of the test antibody in PBS containing 0.5% BSA and 0.05% NaN3 (FACS buffer) at 4°C for 1 hour. The cells were then washed with ice-cold FACS buffer and incubated with PE-conjugated goat anti-human IgG antibody (SouthernBiotech) for 30 minutes at 4°C. After washing with FACS buffer, the stained cells were analyzed using a FACScan flow cytometer (BD Biosciences).

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

[0064] [Sequencing of mAKB1] The heavy and light chain variable regions of the anti-human DNAM-1 monoclonal antibody mAKB1 were cloned from clone mAKB1 by standard methods, and the nucleotide sequences of the heavy and light chain variable regions of mAKB1 were identified. The amino acid sequences were deduced from the nucleotide sequences, and the CDRs were determined using abYsis according to the Kabat method.

[0065] [Design of VH and VL genes for humanized antibody hAKB1] Based on mAKB1, the amino acid sequences of the 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, framework amino acid residues important for forming the three-dimensional structure of the CDR were identified.

[0066] Human VH sequences homologous to the framework of mAKB1 VH were searched in the GenBank database, and the VH sequence encoded by human FJ039783 cDNA (FJ039783 VH) was selected as the acceptor for humanization. The mAKB1 HCDR sequence was grafted into the corresponding position of the FJ039783 VH. Because these residues were thought to be important for CDR structure formation, the 30th, 49th, and 72nd amino acid residues in the heavy chain variable region of the FJ039783 VH were substituted with the corresponding residues in the mAKB1 VH. The resulting humanized antibody VH was designated hAKB1-VH1. Furthermore, to reduce immunogenicity, an additional antibody, hAKB1-VH2, was designed without backmutating the 30th amino acid residue to the mouse amino acid residue. The amino acid sequences of the 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] Human VL sequences homologous to the framework of mAKB1 VL were 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 into the corresponding position of KU760971 VL. Because this residue was thought to be important for CDR structure formation, the 67th amino acid residue in the light chain variable region of KU760971 VL was substituted with the corresponding residue in mAKB1 VL. The resulting humanized antibody VL was designated hAKB1-VL1. Furthermore, to reduce immunogenicity, an additional antibody, hAKB1-VL2, was designed in which the 67th amino acid residue was not backmutated to the mouse amino acid residue. The amino acid sequences of mAKB1 VL (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 antibodies] 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 inserted into plasmids in the following combinations to create expression vectors.

[0069] [Table 1]

[0070] Four expression vectors (phAKB1-A, phAKB1-B, phAKB1-C, and phAKB1-D4) were transfected into human embryonic kidney cell line HEK293 using polyethyleneimine. HEK293 cells were cultured in DMEM medium containing 10% FBS (HyClone) at 37°C in a 7.5% CO2 incubator.

[0071] Antibody expression in the culture supernatant of transiently transfected HEK293 cells was confirmed by ELISA. ELISA plates were coated overnight at 4°C with 100 μl / well of a goat anti-human IgG, Fcγ-specific polyclonal antibody (Sigma-Aldrich) diluted 1 / 2000 in PBS, 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 in ELISA buffer, was applied to the ELISA plate. A humanized IgG1 / kappa antibody was used as a standard. The ELISA plate was incubated for 1 hour at room temperature and washed with washing buffer. Bound antibodies were detected with 100 μl / well of 1 / 2000 diluted HRP-conjugated goat anti-human kappa chain polyclonal antibody (Bethyl Laboratories). After 0.5 hour incubation at room temperature and washing with washing buffer, color development was initiated by adding 100 μl / well of ABTS substrate (Sigma-Aldrich) and stopped with 100 μl / well of 2% oxalic acid. Absorbance was read at 405 nm.

[0072] [Antigen binding of humanized antibodies] The antibodies produced by cells transfected with phAKB1-A, phAKB1-B, phAKB1-C, or phAKB1-D were designated hAKB1-A, hAKB1-B, hAKB1-C, and hAKB1-D, respectively. The binding of these antibodies to human DNAM-1 was analyzed as described above in "Analysis of binding 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. Antibodies containing hAKB1-VL1 (hAKB1-A, hAKB1-B) and those containing hAKB1-VL2 (hAKB1-C, hAKB1-D) showed differences in antigen binding, and the change of the 67th amino acid residue of hAKB1-VL1 from Tyr to Ser reduced the binding affinity. No difference in antigen binding was observed between hAKB1-A and hAKB1-B.

[0073] [Establishment of stable antibody-producing cell lines] 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 the Chinese hamster ovary cell line CHO-K1 (obtained from ATCC) using the following method. CHO-K1 cells were cultured in SFM4CHO medium (HyClone) at 37°C in a 7.5% CO2 incubator. Transfection into CHO-K1 cells was performed by electroporation. Prior to transfection, each expression vector was linearized with FspI. Approximately 2.5 x 10 6 20 μg of linearized plasmid was transfected into 100 cells, suspended in SFM4CHO medium, and appropriately diluted and plated in multiple 96-well plates. After 48 hours, 10 μg / ml puromycin was added to isolate stable transfectants. Approximately 10 days after the start of selection, antibody production was measured in the culture supernatant of the transfectants in the 96-well plates by sandwich ELISA as described above. CHO-K1 stable transfectants producing high levels of hAKB1-A or hAKB1-B were selected and subjected to further culture.

[0074] CHO-K1 stable transfectant was diluted to approximately 3 x 10 in 450 ml of SFM4CHO. 6The cells were cultured in roller bottles to a density of 1000 cells / ml, and 50 ml of 35 mg / ml Cell Boost 4 (HyClone) was added. The cells were further cultured until cell viability reached 50%. After centrifugation and filtration, the culture supernatant was loaded onto a Protein A Sepharose column (HiTrap MabSelect SuRe, 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 eluted antibody buffer was exchanged into PBS by dialysis.

[0075] Purified hAKB1-A and hAKB1-B were characterized by SDS-PAGE according to standard procedures. Analysis under reducing conditions revealed that each antibody consisted of a heavy chain with a molecular weight of approximately 50 kDa and a light chain with a molecular weight of approximately 25 kDa. The purity of each antibody was found to be greater than 95%.

[0076] The EC5 values ​​of purified hAKB1-A and hAKB1-B with human DNAM-1 were determined by the method described above in "Analysis of binding to human DNAM-1." The results are shown in FIG. hAKB1-A and hAKB1-B EC 50 were 5 μg / mL and 5 μg / mL, respectively, and their 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 base 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 heavy and light chain coding regions of hAKB1-A and hAKB1-B obtained were completely identical to the corresponding nucleotide sequences of the phAKB1-A and phAKB1-B vectors. The nucleotide sequences of the heavy and light chain coding regions of hAKB1-A and hAKB1-B (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21) are shown in Figures 6 to 8, respectively. Furthermore, the amino acid sequences were deduced from the nucleotide sequences, and the variable regions were determined. The CDRs were then determined by the Kabat method using abYsis. The results are shown in Figure 9. The signal peptide is shown in open text, the variable regions are shown in bold, and the CDRs are underlined.

[0078] The amino acid sequence of the heavy chain of hAKB1-A (with signal peptide) of SEQ ID NO: 16 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 (with signal peptide) of SEQ ID NO: 17 is composed of the amino acid sequence of the signal peptide of the heavy chain of hAKB1-B (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 sequence of the light chains of hAKB1-A and hAKB1-B (with signal peptide) of SEQ ID NO: 18 is composed of the amino acid sequence of the signal peptide of the hAKB1-A and hAKB1-B light chains (SEQ ID NO: 30) and the amino acid sequence 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 are common and are 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] To establish a cell line that can permanently and stably produce hAKB1-B efficiently and secrete it extracellularly, the signal peptide sequences of the heavy and light chains of hAKB1-B were modified. Nucleic acid fragments containing sequences encoding modified signal peptides were inserted upstream of the coding regions for the heavy and light chains of hAKB1-B into the expression vectors pWX068 and pWX069 in tandem. These fragments were then transformed into competent E. coli TOP10 (TIANGEN, CB104-2), and the expression vectors carrying the antibody genes were amplified and recovered. These fragments were linearized with FspI (NEB, R0135L) and then integrated into the chromosome of CHO-K1 cells. Following the method described above in "Establishment of a stable antibody-producing cell line," a CHO-K1 stable transfectant carrying the pWX069 expression vector was selected, which produced high levels of hAKB1-B and secreted it efficiently. The resulting 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 and SEQ ID NO: 27) are shown in Figures 10 and 11, respectively. The signal peptide is shown in white letters, and the variable region is shown in bold. The amino acid sequence was also deduced from the nucleotide sequence, and the variable region was determined. The CDRs were then determined using the Kabat method using abYsis. The results are shown in Figure 12. The signal peptide is shown in white, the variable region is shown in bold, and the CDRs are underlined.

[0082] The amino acid sequences of the heavy and light chains of hTKB1 were identical to those of hAKB1-B, except for the signal peptide. That is, the amino acid sequences of SEQ ID NO:22 and SEQ ID NO:14 were identical, and the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:15 were identical. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of hTKB1 were identical to the amino acid sequences 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 hTKB1 light chain (with signal peptide) of SEQ ID NO: 25 is composed of the amino acid sequence of the hTKB1 light chain signal peptide (SEQ ID NO: 32) and the amino acid sequence of the hTKB1 light chain (without signal peptide: SEQ ID NO: 23).

[0084] [Expression analysis of DNAM-1 in ILCs] (method) Lungs from mice (C57BL / 6J, 8-10 weeks old, female) were perfused with PBS, isolated, treated with collagenase IV (5 mg / ml; Sigma) at 37°C for 1 hour, and then homogenized using a gentleMACS Dissociator (Miltenyi Biotec). Mouse colons were removed, dissected, washed twice with phosphate-buffered saline (PBS) containing 0.5% BSA (Wako) and 2 mM EDTA (Junsei), and then incubated for 30 minutes in RPMI 1640 (Invitrogen) containing 5% BSA, 5 mM EDTA, and 10 mM dithiothreitol (Sigma-Aldrich) to remove the epithelial layer. The removed colon tissue was washed twice with PBS containing 0.5% BSA and 2 mM EDTA and RPMI 1640 containing 10 mM HEPES (Sigma-Aldrich), and then cut into 3-5 mm pieces. The cut colon pieces were digested using a lamina propria dissociation kit (Miltenyi Biotec) and a gentleMACS Dissociator (Miltenyi Biotec). The cell preparation obtained by digestion was 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; Tombo Biosciences) for 10 min on ice to avoid FcγR binding, then incubated with a combination of monoclonal antibodies against CD45.2, CD11b, CD3ε, NK1.1, DX5, B220, CD44, CD90.2, Sca-1, and RORγt, along with anti-mouse DNAM-1 monoclonal antibody (clone TX42; BioLegend), for 10 min, and analyzed by flow cytometry. Rat IgG2a (BioLegend) was used instead of anti-mouse DNAM-1 as an isotype control. Innate lymphoid cell (ILC) subset 1 (ILC1) expresses CD45.2 + , CD11b - , CD3ε - , NK1.1 + , DX5 - Innate lymphoid cell (ILC) subset 2 (ILC2) expresses CD45.2 + , CD11b - , B220 - , NK1.1 -, CD3ε - , CD44 + , CD90.2 + , Sca-1 + Innate lymphoid cell (ILC) subset 3 (ILC3) is CD45.2 + , CD11b - , B220 - , NK1.1 - , CD3ε - , CD44 + , CD90.2 + , RORγt + , Sca-1 - The cells were identified as:

[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 Figures 13A and 13B. The dotted line indicates the isotype control, and the solid line indicates the result when anti-mouse DNAM-1 monoclonal antibody was used. Figures 13A and 13B revealed that DNAM-1 was expressed in ILC1, ILC2, and ILC3 in the lung and small intestine.

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

[0089] (result) The results are shown in Figure 14. As shown in Figure 14, the expression of IL17a, IL15, and IL13 mRNA was reduced in ILCs from DNAM-1KO mice 3, 5, and / or 14 days after BLM administration compared to WT mice. These results demonstrated that DNAM-1 is involved in cytokine production in ILCs.

[0090] [DNAM-1 expression in human peripheral blood ILCs] (method) Blood was collected from healthy volunteers and treated with Lymphoprep. TM Peripheral blood mononuclear cells (PBMCs) were collected using a Stemcell Technologies kit according to the manufacturer's instructions. PBMCs were stained with conventional lineage markers (antibodies against CD3, CD4, CD8, CD19, CD14, CD16, CD11b, CD11c, CD56, and FcεRI), anti-CD127, CD45, and humanized anti-DNAM-1 (hTKB1) antibodies. DNAM-1 expression was measured in total ILCs (Lin et al., 2014). - , CD45 + , CD127 + ) were analyzed by flow cytometry. Human IgG1 (BioLegend) was used as an isotype control instead of the humanized anti-DNAM-1 antibody (hTKB1).

[0091] (result) The results are shown in Figure 15. The dotted line indicates the isotype control, and the solid line indicates the case where humanized anti-DNAM-1 antibody (hTKB1) was used. 15, it was revealed that DNAM-1 is expressed in ILCs of human PBMCs. It was also confirmed that hTKB1 can detect DNAM-1 on ILCs.

[0092] [Humanized anti-DNAM-1 antibody inhibits production of IFN-γ and TNF-α from ILCs] (method) Blood was collected from healthy volunteers and treated with Lymphoprep.TM Peripheral blood mononuclear cells (PBMCs) were collected using a PBS (STEMCELL Technologies) according to the manufacturer's instructions. CD4 + T cells, CD8 + T cells, and CD56 + NK cells were removed. 2-3 × 10 PBMCs were then cultured. 6 The cells 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). The cells were then stained with antibodies against lineage markers (Lin: CD3, CD4, CD8, CD19, CD14, CD16, CD11b, CD11c, FcεRI), anti-CD127, and anti-CD45. ILCs (Lin: - , CD127 + , CD45 + ILCs were sorted using a FACS Aria III (BD Biosciences). Total RNA was isolated from the sorted ILCs using Isogen reagent according to the manufacturer's protocol (Nippon Gene). A High-Capacity cDNA Reverse-Translation Kit (Applied Biosystems) was used for reverse transcription. Quantitative PCR analysis of Ifng was performed using an ABI7500 sequence detector (Applied Biosystems), Power SYBR Green PCR Master Mix (Applied Biosystems), and the primers listed below. To normalize the data, the expression level of Gapdh was measured as an internal control.

[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] (result) The results are shown in Figure 16. The mouse monoclonal antibody mAKB1 did not affect the expression of IFN-γ and TNF-α mRNA, whereas 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 sequences 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 SEQ ID NO: 11: Amino acid sequence of the variable region of VL1 of hAKB1 SEQ ID NO: 12: Amino acid sequence of the variable region of VL2 of hAKB1 SEQ ID NO: 13: Amino acid sequence of the heavy chain of hAKB1-A (without signal peptide) SEQ ID NO: 14: Amino acid sequence of the heavy chain of hAKB1-B (without signal peptide) SEQ ID NO: 15: Amino acid sequence of the light chain of hAKB1-A and hAKB1-B (without signal peptide) SEQ ID NO: 16: Amino acid sequence of the heavy chain of hAKB1-A (with signal peptide) SEQ ID NO: 17: Amino acid sequence of the heavy chain of hAKB1-B (with signal peptide) SEQ ID NO: 18: Amino acid sequence of the light chain of hAKB1-A and hAKB1-B (with signal peptide) SEQ ID NO: 19: Nucleotide sequence of the heavy chain coding region of hAKB1-A SEQ ID NO: 20: Nucleotide sequence of the heavy chain coding region of hAKB1-B SEQ ID NO: 21: Nucleotide sequence of the light chain coding region of hAKB1-A and hAKB1-B SEQ ID NO: 22: Amino acid sequence of the heavy chain of hTKB1 (without signal peptide) SEQ ID NO: 23: Amino acid sequence of the light chain of hTKB1 (without signal peptide) SEQ ID NO: 24: Amino acid sequence of the heavy chain of hTKB1 (with signal peptide) SEQ ID NO: 25: Amino acid sequence of the light chain of hTKB1 (with signal peptide) SEQ ID NO: 26: Nucleotide sequence of the heavy chain coding region of hTKB1 SEQ ID NO: 27: Nucleotide sequence of the light chain coding region of hTKB1 SEQ ID NO: 28: Amino acid sequence of the signal peptide of the hAKB1-A heavy chain SEQ ID NO: 29: Amino acid sequence of the signal peptide of the hAKB1-B heavy chain SEQ ID NO: 30: Amino acid sequence of the signal peptide of hAKB1-A and hAKB1-B light chains SEQ ID NO: 31: Amino acid sequence of the signal peptide of the heavy chain of hTKB1 SEQ ID NO: 32: Amino acid sequence of the signal peptide of the light chain of hTKB1 SEQ ID NO: 33: Nucleotide sequence of Gapdh forward primer SEQ ID NO: 34: Nucleotide sequence of Gapdh reverse primer SEQ ID NO: 35: Nucleotide sequence of Ifng forward primer SEQ ID NO: 36: Nucleotide sequence of Ifng reverse primer SEQ ID NO: 37: Nucleotide sequence of Tnfa forward primer SEQ ID NO: 38: Nucleotide sequence of 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: an amino acid sequence of SEQ ID NO: 4 as LCDR1, an amino acid sequence of SEQ ID NO: 5 as LCDR2, and an amino acid sequence of SEQ ID NO: 6 as LCDR3; A humanized anti-DNAM-1 antibody or an antigen-binding fragment thereof, comprising:

2. A humanized anti-DNAM-1 antibody or its antigen-binding fragment according to claim 1, having a heavy chain variable region having an amino acid sequence that is 95% or more identical to SEQ ID NO: 10 and a light chain variable region having an amino acid sequence that is 95% or more identical to SEQ ID NO:

11.

3. 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 antigen-binding fragment thereof according to claim 1 or 2, having the following structure:

4. The humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the 238th and 239th amino acid residues of the heavy chain are A.

5. A humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, having a heavy chain having an amino acid sequence that is 95% or more identical to SEQ ID NO: 14 and a light chain having an amino acid sequence that is 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 claims 1 to 5, having the following structure:

7. A nucleic acid encoding the humanized anti-DNAM-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

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

9. A transformant containing the vector according to claim 8.

10. An inhibitor of innate lymphocyte (ILC) activation, comprising the humanized anti-DNAM-1 antibody or its antigen-binding fragment according to any one of claims 1 to 6.

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