Anti-integrin α8β1 antibody

Abstract

The present invention addresses the problem of providing a novel anti-integrin α8β1 antibody, and provides an anti-integrin α8β1 antibody including the following heavy chain and light chain: (i) a heavy chain which includes a heavy-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; and (ii) a light chain which includes a light-chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light-chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light-chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6.

Description

Anti-integrin α8β1 antibody

[0001] The present invention relates to an antibody that specifically binds to human integrin α8β1.

[0002] Fibrosis is generally known as a disease caused by the excessive deposition of matrix proteins such as collagen, which replaces normal tissue, hardening the tissue and causing a loss of normal function. Fibrosis occurs in the liver, lungs, kidneys, heart, skin, and other organs. For example, there is liver cirrhosis, in which extensive fibrosis occurs in liver tissue. There is also pulmonary fibrosis, in which lung tissue is damaged and hardened, resulting in a decline in lung function. Currently, there are very few drugs on the market as curative anti-fibrotic agents. A drug for nonalcoholic steatohepatitis was launched in 2024 as a treatment for liver fibrosis. However, these drugs are not sufficiently effective, and the possibility of serious drug interactions has been pointed out.

[0003] The integrin α8 chain forms a heterodimer with the β1 chain and is expressed on the cell membrane surface as integrin α8β1. Because the integrin α8 chain is highly expressed in fibrotic tissues, it is expected to be a target for suppressing various fibrotic diseases. However, there have been no reports of clinical development of drugs targeting integrin α8β1 that have confirmed their effectiveness.

[0004] Patent Document 1 discloses an integrin α8β1-binding antibody that inhibits the binding of integrin α8β1 to a ligand. Patent Document 2 discloses a novel and effective antifibrotic agent comprising an integrin α8β1-binding antibody. Non-Patent Documents 1 to 3 describe humanized antibodies. Non-Patent Document 4 discloses that integrin α8β1 enhances cell contractility and TGF-β activity in hepatic stellate cells.

[0005] International Publication No. WO 2011 / 049082 International Publication No. WO 2013 / 147076

[0006] Jones, PT., et al., Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321, 522-525, 1986Riechmann, L. et al., Reshaping human antibodies for therapy. Nature 332, 323-327, 1988Carter, P., et al., Humanization of an anti-p185 HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. 89, 4285-4289, 1992Nishimichi N et al., Induced hepatic stellate cell integrin, α8β1, enhances cellular contractility and TGFβ activity in liver fibrosis. J.Pathol. 253, 366-373, 2021

[0007] When using anti-integrin α8β1 antibodies as antibody drugs, immunogenicity in humans becomes an issue. Antibodies obtained from non-human mammalian species, such as mice, are generally humanized to prevent rejection by the human immune system (e.g., Non-Patent Document 1). The anti-integrin α8β1 antibodies disclosed in Patent Documents 1 and 2 are derived from chickens. Antibodies obtained from organisms other than mammals, such as mice, are more likely to elicit an immune response in the human immune system than antibodies obtained from mice. When these antibodies are humanized, it is difficult to maintain their function while minimizing their immunogenicity (e.g., Non-Patent Document 2). This is because antibodies derived from organisms other than mammals often have amino acid sequences and three-dimensional structures that differ from those of mammals, and it is technically difficult to adapt these unique sequences and structures to function in humans while minimizing immunogenicity. In particular, the present inventors have empirically observed that humanizing chicken antibodies not only reduces but completely eliminates antigen-binding ability, posing a significant challenge in the antibody humanization process. Furthermore, when antibodies are derived from organisms other than mammals, the production efficiency may decrease during the humanization process, making it difficult to obtain humanized antibodies with sufficient antibody production capacity.

[0008] Under these circumstances, it was considered necessary to further improve anti-integrin α8β1 antibodies in order to use them as effective antibody pharmaceuticals. In particular, antibodies with reduced immunogenicity, which is a problem, while maintaining antigen-binding ability are needed. The present invention relates to novel anti-integrin α8β1 antibodies that can solve the above-mentioned problems.

[0009] The present invention includes the following [1] to [8]. [1] An anti-integrin α8β1 antibody comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and (ii) a light chain variable region comprising a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. [2] The anti-integrin α8β1 antibody according to [1], wherein the antibody is humanized. [3] An anti-integrin α8β1 antibody comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises: (i-1) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (i-2) a serine at the position corresponding to position 28, an isoleucine at the position corresponding to position 29, a serine at the position corresponding to position 49, a phenylalanine at the position corresponding to position 67, a tyrosine at the position corresponding to position 73, a valine at the position corresponding to position 78, and a leucine at the position corresponding to position 82 according to the Kabat system; and the humanized light chain variable region comprises: (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; and (ii-2) The anti-integrin α8β1 antibody according to [2], which comprises a leucine at a position corresponding to position 4, a threonine at a position corresponding to position 46, a leucine at a position corresponding to position 47, a leucine at a position corresponding to position 66, a serine at a position corresponding to position 69, and a valine at a position corresponding to position 71 according to the Kabat system. [4] An anti-integrin α8β1 antibody comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises: (i-1) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3;(i-2) According to the Kabat system, a valine at a position corresponding to position 2, a leucine at a position corresponding to position 4, a glutamic acid at a position corresponding to position 6, a glycine at a position corresponding to position 9, a leucine at a position corresponding to position 20, a cysteine ​​at a position corresponding to position 22, an alanine at a position corresponding to position 24, a phenylalanine at a position corresponding to position 27, a serine at a position corresponding to position 28, an isoleucine at a position corresponding to position 29, a serine at a position corresponding to position 30, a tryptophan at a position corresponding to position 36, a valine at a position corresponding to position 37, an arginine at a position corresponding to position 38, a glutamine at a position corresponding to position 39, a leucine at a position corresponding to position 45, a tryptophan at a position corresponding to position 47, a valine at a position corresponding to position 48, and a serine at a position corresponding to position 49; a phenylalanine at a position corresponding to position 67, an isoleucine at a position corresponding to position 69, an arginine at a position corresponding to position 71, a tyrosine at a position corresponding to position 73, a valine at a position corresponding to position 78, a leucine at a position corresponding to position 80, a leucine at a position corresponding to position 82, a leucine at a position corresponding to position 82C, an aspartic acid at a position corresponding to position 86, a tyrosine at a position corresponding to position 90, a tyrosine at a position corresponding to position 91, a cysteine ​​at a position corresponding to position 92, an alanine at a position corresponding to position 93, a lysine at a position corresponding to position 94, a tryptophan at a position corresponding to position 103, a glycine at a position corresponding to position 104, a glycine at a position corresponding to position 106, a valine at a position corresponding to position 109, and a valine at a position corresponding to position 111; wherein the humanized light chain variable region comprises: (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:6;(ii-2) According to the Kabat system, leucine at a position corresponding to position 4, glutamine at a position corresponding to position 6, isoleucine at a position corresponding to position 21, cysteine ​​at a position corresponding to position 23, tryptophan at a position corresponding to position 35, tyrosine at a position corresponding to position 36, glutamine at a position corresponding to position 38, proline at a position corresponding to position 44, threonine at a position corresponding to position 46, leucine at a position corresponding to position 47, isoleucine at a position corresponding to position 48, tyrosine at a position corresponding to position 49, isoleucine at a position corresponding to position 58, phenylalanine at a position corresponding to position 62, glycine at a position corresponding to position 64, leucine at a position corresponding to position 66, and 67.

[0022] The anti-integrin α8β1 antibody according to [2], comprising a glycine at a position corresponding to position 8, a serine at a position corresponding to position 69, a valine at a position corresponding to position 71, a leucine at a position corresponding to position 73, an isoleucine at a position corresponding to position 75, a valine at a position corresponding to position 78, an aspartic acid at a position corresponding to position 82, a tyrosine at a position corresponding to position 86, a tyrosine at a position corresponding to position 87, a cysteine ​​at a position corresponding to position 88, a phenylalanine at a position corresponding to position 98, a glycine at a position corresponding to position 99, a glycine at a position corresponding to position 101, a threonine at a position corresponding to position 102, a leucine at a position corresponding to position 104, and a valine at a position corresponding to position 106.

[0023] The anti-integrin α8β1 antibody according to any one of [1] to [4], comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8.

[0024] The anti-integrin α8β1 antibody according to any one of [1] to [5], comprising the amino acid sequence set forth in SEQ ID NO: 3. [7] An anti-fibrotic agent comprising the anti-integrin α8β1 antibody according to any one of [1] to [5] or the antigen-binding fragment of the anti-integrin α8β1 antibody according to [6].[8] An anti-integrin α8β1 antibody according to any one of [1] to [5], which (a) binds to human integrin α8β1 with a dissociation rate constant (kd: the smaller the value, the less likely the antibody is to dissociate) that is the same as or smaller than that of a reference antibody, and (b) is less immunogenic in mammals than the reference antibody, wherein the reference antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 9 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 10.

[0010] The present invention provides a novel anti-integrin α8β1 antibody. The antibody provided by the present invention specifically binds to human integrin α8β1, has affinity for human integrin α8β1 equivalent to or higher than conventional antibodies, functions to inhibit human integrin α8β1, has low immunogenicity in mammals, and can be used to suppress tissue fibrosis.

[0011] Figure 1-1 shows the consensus sequence of the mutation introduction site of the phage antibody library. Figure 1-2 shows the results of competitive panning in Production Example 1 (1-3). Figure 1-3 shows the alignment of amino acid sequences. Figure 2 shows the results of Test Example 1. Figure 3 shows the results of Test Example 2. Figure 4 shows the results of Test Example 3. Figure 5 shows the test steps (A) and results (B) of Test Example 4. Figure 6 shows the test steps (A) and results (B) of Test Example 5. Figure 7 shows the test steps (A) and results (B) of Test Example 6-2. Figure 8 shows the test steps (A) and results (B) of Test Example 6-3. Figure 9-1 shows the results of Test Example 6-3. Figure 9-2 shows the results of Test Example 6-4. Figure 10 shows the results of Test Example 7. Figure 11 shows the results of Test Example 8. (A) Western blot results, (B) Measurement results of relative expression levels of α-SMA. Figure 12 shows the results of Test Example 8. Figure 13 shows the results of Test Example 9. Figure 14-1 shows the results of Test Example 10 (A). Figure 14-2 shows the results of Test Example 10 (B). Figure 14-3 shows the results of Test Example 10 (C). Figure 14-4 shows the results of Test Example 10 (D).

[0012] [Anti-integrin α8β1 antibody] The anti-integrin α8β1 antibody of the present invention (hereinafter sometimes simply referred to as "anti-integrin α8β1 antibody") comprises the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and (ii) a light chain variable region comprising a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0013] In one aspect, the anti-integrin α8β1 antibodies of the present invention are humanized and comprise the humanized heavy chain variable region and humanized light chain variable region described below. (i-1) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (i-2) a humanized heavy chain variable region comprising, according to the Kabat system, serine at the position corresponding to position 28, isoleucine at the position corresponding to position 29, serine at the position corresponding to position 49, phenylalanine at the position corresponding to position 67, tyrosine at the position corresponding to position 73, valine at the position corresponding to position 78, and leucine at the position corresponding to position 82; and (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; and (ii-2) a humanized light chain variable region comprising, according to the Kabat system, leucine at the position corresponding to position 4, threonine at the position corresponding to position 46, leucine at the position corresponding to position 47, leucine at the position corresponding to position 66, serine at the position corresponding to position 69, and valine at the position corresponding to position 71.

[0014] In another aspect, the humanized anti-integrin α8β1 antibody of the present invention is humanized and comprises the following humanized heavy chain variable region and humanized light chain variable region: (i-1) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; (i-2) According to the Kabat system, a valine at a position corresponding to position 2, a leucine at a position corresponding to position 4, a glutamic acid at a position corresponding to position 6, a glycine at a position corresponding to position 9, a leucine at a position corresponding to position 20, a cysteine ​​at a position corresponding to position 22, an alanine at a position corresponding to position 24, a phenylalanine at a position corresponding to position 27, a serine at a position corresponding to position 28, an isoleucine at a position corresponding to position 29, a serine at a position corresponding to position 30, a tryptophan at a position corresponding to position 36, a valine at a position corresponding to position 37, an arginine at a position corresponding to position 38, a glutamine at a position corresponding to position 39, a leucine at a position corresponding to position 45, a tryptophan at a position corresponding to position 47, a valine at a position corresponding to position 48, a serine at a position corresponding to position 49, and a glycine at a position corresponding to position 67. a phenylalanine at a position corresponding to position 69, an isoleucine at a position corresponding to position 71, an arginine at a position corresponding to position 73, a valine at a position corresponding to position 78, a leucine at a position corresponding to position 80, a leucine at a position corresponding to position 82, a leucine at a position corresponding to position 82C, an aspartic acid at a position corresponding to position 86, a tyrosine at a position corresponding to position 90, a tyrosine at a position corresponding to position 91, a cysteine ​​at a position corresponding to position 92, an alanine at a position corresponding to position 93, a lysine at a position corresponding to position 94, a tryptophan at a position corresponding to position 103, a glycine at a position corresponding to position 104, a glycine at a position corresponding to position 106, a valine at a position corresponding to position 109, and a valine at a position corresponding to position 111; and (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:6; and(ii-2) According to the Kabat system, leucine at a position corresponding to position 4, glutamine at a position corresponding to position 6, isoleucine at a position corresponding to position 21, cysteine ​​at a position corresponding to position 23, tryptophan at a position corresponding to position 35, tyrosine at a position corresponding to position 36, glutamine at a position corresponding to position 38, proline at a position corresponding to position 44, threonine at a position corresponding to position 46, leucine at a position corresponding to position 47, isoleucine at a position corresponding to position 48, tyrosine at a position corresponding to position 49, isoleucine at a position corresponding to position 58, phenylalanine at a position corresponding to position 62, glycine at a position corresponding to position 64, and leucine at a position corresponding to position 66 a humanized light chain variable region comprising: a cysteine ​​at a position corresponding to position 88; a phenylalanine at a position corresponding to position 99; a glycine at a position corresponding to position 101; a threonine at a position corresponding to position 102; a leucine at a position corresponding to position 104; and a valine at a position corresponding to position 106.

[0015] The anti-integrin α8β1 antibodies of the present invention specifically bind to human integrin α8β1 and have excellent properties, such as reduced immunogenicity in mammals, while having the same or higher affinity for human integrin α8β1 as conventional antibodies. The anti-integrin α8β1 antibodies of the present invention also bind to integrin α8β1, thereby inhibiting the function of integrin α8β1 and suppressing tissue fibrosis.

[0016] The term "immunogenicity," as used herein, refers to the ability of an antibody or antigen-binding fragment to elicit an immune response in a subject when administered to the subject with an antigen from a different species. This response is initiated when T cells from the subject mount an immune response against the administered antibody. The T cells then recruit B cells to produce heterologous antibodies. An "immune response" encompasses humoral and cellular immune responses.

[0017] <Integrin α8β1> The anti-integrin α8β1 antibody of the present invention specifically binds to human integrin α8β1. Integrin α8β1 is a heterodimer formed by the integrin α8 chain and the integrin β1 chain. Integrin α8β1 is known to be specific for ligands containing the RGD sequence, such as fibronectin, vitronectin, tenascin, and osteopontin. Integrin α8β1 is expressed in renal mesangial cells, vascular smooth muscle cells, fibroblasts, and the like. Furthermore, as shown in the Examples herein, integrin α8β1 has been confirmed to be expressed not only in renal fibroblasts but also in pulmonary fibroblasts. On the other hand, it has been reported that in normal liver, quiescent hepatic stellate cells do not express integrin α8β1, but that it is de novo expressed upon activation (Non-Patent Document 4). Hepatic stellate cells are myofibroblast precursor cells in the liver.

[0018] The anti-integrin α8β1 antibody of the present invention is an anti-integrin α8β1 antibody that inhibits the binding of integrin α8β1 to a ligand. The anti-integrin α8β1 antibody of the present invention specifically binds to R120 and its surrounding region in the amino acid sequence of the human integrin α8 chain shown in SEQ ID NO: 15. The amino acid sequence of the human integrin α8 chain shown in SEQ ID NO: 15 is the amino acid sequence of the integrin α8 chain when it contains a signal peptide. In the amino acid sequence of the human integrin α8 chain without the signal peptide, F39 is the amino acid residue at position 1, and the above-mentioned R120 is the amino acid residue at position 82.

[0019] The anti-integrin α8β1 antibodies of the present invention specifically bind to human integrin α8β1. However, because the amino acid sequence of R120 and its surrounding region of the human integrin α8 chain, to which the anti-integrin α8β1 antibodies of the present invention bind, is highly conserved among mammalian species, the anti-integrin α8β1 antibodies of the present invention also bind to integrin α8β1 derived from non-human mammals such as mice, rats, guinea pigs, rabbits, pigs, sheep, cows, cats, dogs, monkeys, and chimpanzees.

[0020] <Antibody> An antibody is an immunoglobulin protein and comprises a heavy chain and a light chain. The heavy chain and light chain each comprise a variable region and a constant region. That is, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region. Typically, an immunoglobulin protein has a known Y-shaped structure in which two heavy chains and two light chains are linked via disulfide bonds. When an antibody, particularly IgG, is digested with the protease papain, the two fragments comprising the N-terminal variable region are called the Fab region, and the single fragment at the C-terminal end is called the Fc region.

[0021] (Antigen-binding region) The variable region of an antibody is the antigen-binding region of the antibody and includes complementarity-determining regions (CDRs) and framework regions (FRs). The heavy chain variable region (VH region) consists of three complementarity-determining regions (CDRs): heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and four framework regions located at the most N-terminal side, between the CDRs, and at the most C-terminal side: heavy chain FR1 (HFR1), heavy chain FR2 (HFR2), heavy chain FR3 (HFR3), and heavy chain FR4 (HFR4). The light chain variable region (VL region) consists of three complementarity determining regions, namely, light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), and four framework regions, namely, light chain FR1 (HFR1), light chain FR2 (LFR2), light chain FR3 (LFR3), and light chain FR4 (LFR4), located at the most N-terminal side, between the CDRs, and at the most C-terminal side.

[0022] Complementarity-determining regions (CDRs), also known as hypervariable regions, are known to be involved in the binding and specificity of antibodies to antigens. Regions located between CDRs are called framework regions (FRs), and are known to support the three-dimensional structure of the antigen-binding site of antibodies. The three-dimensional structure of the antigen-binding site is important for binding to antigens and is involved in the binding and specificity to antigens. Complementarity-determining regions (CDRs) and framework regions (FRs) are numbered according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0023] According to the Kabat system, in the heavy chain variable region, heavy chain FR1 (HFR1) is composed of amino acid residues from position 1 to position 30, heavy chain FR2 (HFR2) is composed of amino acid residues from position 36 to position 49, heavy chain FR3 (HFR3) is composed of amino acid residues from position 66 to position 94, and heavy chain FR4 (HFR4) is composed of amino acid residues from position 103 to position 113. In the light chain variable region, light chain FR1 (LFR1) is composed of amino acid residues from position 1 to position 23, light chain FR2 (LFR2) is composed of amino acid residues from position 35 to position 49, light chain FR3 (LFR3) is composed of amino acid residues from position 57 to position 88, and light chain FR4 (LFR4) is composed of amino acid residues from position 98 to position 107.

[0024] (Amino acid sequences of complementarity determining regions (CDRs)) The anti-integrin α8β1 antibody of the present invention comprises: (i) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (ii) a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0025]

[0026] (Amino Acid Sequences of Heavy Chain Variable Region (VH Region) and Light Chain Variable Region (VL Region)) The anti-integrin α8β1 antibody of the present invention may have a heavy chain variable region comprising heavy chain CDR1 to 3 of the specific amino acid sequence described above, and a light chain variable region comprising light chain CDR1 to 3 of the specific amino acid sequence described above. Preferably, the heavy chain variable region comprises specific amino acids described below at specific positions, and the light chain variable region comprises specific amino acids described below at specific positions, and more preferably, the heavy chain variable region and light chain variable region comprise specific amino acid sequences described below.

[0027] One preferred embodiment of the anti-integrin α8β1 antibody of the present invention is a humanized antibody. In the present invention, a "humanized" antibody refers to an antibody in which the framework regions (FR) contained in the heavy chain variable region and the light chain variable region are sequences derived from human FRs. A sequence derived from human FRs is a natural human sequence or a modified sequence in which one or more amino acids have been substituted, deleted, and / or inserted in a natural human sequence. Preferably, the modified sequence is a natural human sequence in which one or more amino acids have been substituted with the corresponding amino acids in the chicken FR amino acid sequence so as to maintain high affinity for human integrin α8β1 without increasing immunogenicity in mammals (e.g., humans).

[0028] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13, 1619-1633, 2008, and also see, for example, Riechmann et al., Nature 332, 323-329, 1988; Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033, 1989; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36, 25-34, 2005 (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28, 489-498, 1991 (describing resurfacing); Dall'Acqua et al., Methods 36, 43-60, 2005 (describing FR shuffling); and Osbourn et al., Methods 36, 61-68, 2005 and Klimka et al., Br. J. Cancer, 83, 252-260, 2000 (describing a "guide selection" approach for FR shuffling).

[0029] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see Sims et al., J. Immunol. 151, 2296, 1993); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see Carter et al., Proc. Natl. Acad. Sci. USA 89, 4285, 1992 and Presta et al., J. Immunol. 151, 2623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13, 1619-1633, 2008); and framework regions derived from screening of FR libraries (see Baca et al., J. Biol. Chem. 272, 10678-10684, 1997 and Rosok et al., J. Biol. Chem. 271, 22611-22618, 1996).

[0030] The anti-integrin α8β1 antibody of the present invention more preferably comprises: (i-1) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and (i-2) a heavy chain CDR1 having a position corresponding to position 28, a heavy chain CDR2 having a position corresponding to position 29, a heavy chain CDR3 having a position corresponding to position 49, a heavy chain CDR4 having a position corresponding to position 67, a heavy chain CDR5 having a position corresponding to position 73, a heavy chain CDR6 having a position corresponding to position 78, a heavy chain CDR7 having a position corresponding to position 82, and a heavy chain CDR8 having a position corresponding to position 90, according to the Kabat system. and (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; and (ii-2) a humanized light chain variable region comprising, according to the Kabat system, leucine at a position corresponding to position 4, threonine at a position corresponding to position 46, leucine at a position corresponding to position 47, leucine at a position corresponding to position 66, serine at a position corresponding to position 69, and valine at a position corresponding to position 71.

[0031] The anti-integrin α8β1 antibody of the present invention is more preferably (i-1) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and (i-2) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and (i-3) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and (i-4) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and (i-5) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and (i-6) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, according to the Kabat system: valine at the position corresponding to position 2, leucine at the position corresponding to position 4, glutamic acid at the position corresponding to position 6, glycine at the position corresponding to position 9, leucine at the position corresponding to position 20, cystein lysine, arginine at a position corresponding to position 71, tyrosine at a position corresponding to position 73, valine at a position corresponding to position 78, leucine at a position corresponding to position 80, leucine at a position corresponding to position 82, leucine at a position corresponding to position 82C, aspartic acid at a position corresponding to position 86, tyrosine at a position corresponding to position 90, tyrosine at a position corresponding to position 91, cysteine ​​at a position corresponding to position 92, alanine at a position corresponding to position 93, lysine at a position corresponding to position 94, tryptophan at a position corresponding to position 103, and glutamic acid at a position corresponding to position 104. a humanized heavy chain variable region comprising: a lysine at a position corresponding to position 106; a glycine at a position corresponding to position 109; a valine at a position corresponding to position 111; (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; and (ii-2) a leucine at a position corresponding to position 4, a glutamine at a position corresponding to position 6, an isoleucine at a position corresponding to position 21, and a cysteine ​​at a position corresponding to position 23 according to the Kabat system;tryptophan at a position corresponding to position 35, tyrosine at a position corresponding to position 36, glutamine at a position corresponding to position 38, proline at a position corresponding to position 44, threonine at a position corresponding to position 46, leucine at a position corresponding to position 47, isoleucine at a position corresponding to position 48, tyrosine at a position corresponding to position 49, isoleucine at a position corresponding to position 58, phenylalanine at a position corresponding to position 62, glycine at a position corresponding to position 64, leucine at a position corresponding to position 66, glycine at a position corresponding to position 68, serine at a position corresponding to position 69, and a position corresponding to position 71 a valine at position 76, a leucine at a position corresponding to position 73, an isoleucine at a position corresponding to position 75, a valine at a position corresponding to position 78, an aspartic acid at a position corresponding to position 82, a tyrosine at a position corresponding to position 86, a tyrosine at a position corresponding to position 87, a cysteine ​​at a position corresponding to position 88, a phenylalanine at a position corresponding to position 98, a glycine at a position corresponding to position 99, a glycine at a position corresponding to position 101, a threonine at a position corresponding to position 102, a leucine at a position corresponding to position 104, and a valine at a position corresponding to position 106.

[0032] The anti-integrin α8β1 antibody of the present invention is particularly preferably an antibody comprising a humanized heavy chain variable region having the amino acid sequence shown in SEQ ID NO:7 and a humanized light chain variable region having the amino acid sequence shown in SEQ ID NO:8.

[0033]

[0034] The anti-integrin α8β1 antibodies of the present invention may be unmodified antibodies or modified antibodies that have been modified to include additional nonproteinaceous moieties known in the art and readily available. Examples of antibody modification moieties include, but are not limited to, water-soluble polymers. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), dextran or poly(N-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The number of polymers attached to an antibody is arbitrary, and when more than one polymer is attached, they may be the same molecule or different molecules. In general, the number and / or type of polymer used in the modification can be determined based on, but is not limited to, the particular property or function of the antibody to be improved, the conditions under which the modified antibody will be used, etc.

[0035] The modified portion of the antibody may also be a sugar chain, lipid, fluorescently labeled molecule, drug, etc. For example, a sugar chain may be attached to any amino acid residue in the Fc region or variable region of the antibody, thereby enabling enhancement of antibody-dependent cellular cytotoxicity (ADCC) and control of blood retention. Examples of sugar chains include fucose-deficient sugar chains, sialic acid-added sugar chains, and GalNAc-modified sugar chains. Furthermore, lipids such as liposome-constituting lipids may be attached to the amino group or thiol group of the antibody, which is expected to improve tissue affinity, stability, delivery efficiency, etc. The lipid may be attached via either a covalent or non-covalent bond. Furthermore, by attaching a fluorescently labeled molecule or a radioisotope, it can be used for diagnostic or tracer purposes. Examples of fluorescently labeled molecules include fluorescein, rhodamine, and the Alexa Fluor series (manufactured by Thermo Fischer Scientific). In addition, antibodies can be conjugated with cytotoxic substances, cytotoxic drugs, etc. to form antibody-drug conjugates (ADCs), which enable therapeutic applications that target specific cells.

[0036] The class of the anti-integrin α8β1 antibody of the present invention is not particularly limited, and may be IgG, IgM, IgA, IgD, or IgE, preferably IgG.

[0037] The anti-integrin α8β1 antibody of the present invention is preferably an anti-integrin α8β1 antibody that (a) binds to human integrin α8β1 with a dissociation rate constant (kd: the smaller the value, the less likely the antibody is to dissociate) that is the same as or smaller than that of a reference antibody, and (b) is less immunogenic in mammals than the reference antibody, wherein the reference antibody comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 9 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 10. Specifically, when compared to a reference antibody that is a chicken anti-integrin α8β1 antibody comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 9 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 10, the antibody has the characteristics of (a) binding to human integrin α8β1 with the same or a larger kd and (b) being less immunogenic in mammals than the reference antibody. The reference antibody is a chicken anti-integrin α8β1 antibody disclosed in Patent Document 2, and a plasmid containing a DNA sequence encoding the heavy chain of the reference antibody was domestically deposited with the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on October 16, 2009. Subsequently, the domestically deposited plasmid was transferred to an international deposit under the Budapest Treaty on October 12, 2010, under the accession number NITE BP-824.

[0038]

[0039] As used herein, the term "K" or "k" refers to the association rate constant of a particular antibody-antigen interaction, while the term "K" or "k" refers to the dissociation rate constant of a particular antibody-antigen interaction. As used herein, the term "K" refers to the dissociation constant, which is obtained from the ratio of k to k (i.e., k / k), and is expressed as a molar concentration (M). The K value of an antibody can be determined using methods well established in the art. Preferred methods for determining the k, k, and K of an antibody include methods using surface plasmon resonance, preferably using a biosensor system such as the BIACORE system described below, or a method combining biolayer interferometry with a dip and read assay, preferably using a biosensor system such as the BLITz system.

[0040] In one embodiment, to measure the binding activity of an antibody, for example, a ligand capture method using BIACORE™ T200 or BIACORE™ 4000 (GE Healthcare; Uppsala, Sweden), which relies on surface plasmon resonance analysis as the measurement principle, can be used. BIACORE™ Control Software can be used to operate the device. In one embodiment, an amine coupling kit (GE Healthcare; Uppsala, Sweden) is used according to the manufacturer's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare; Uppsala, Sweden). The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate for an appropriate injection time. Binding activity is measured using a buffer containing 0.05% polysorbate 20 (also known as Tween (trademark)-20) as the measurement buffer, at a flow rate of 10 to 30 μL / min, and at a measurement temperature of preferably 25°C or 37°C. In the case of measurements performed by capturing an antibody as a ligand on a ligand capture molecule, the antibody is injected and a target amount of antibody is captured, and then a serial dilution of antigen and / or Fc receptor (analyte) prepared using the measurement buffer is injected. In the case of measurements performed by capturing an antigen and / or Fc receptor as a ligand on a ligand capture molecule, the antigen and / or Fc receptor is injected and a target amount of antibody is captured, and then a serial dilution of antibody (analyte) prepared using the measurement buffer is injected.

[0041] In one embodiment, the measurement results are analyzed using BIACORE™ Evaluation Software. Kinetic parameters are calculated by simultaneously fitting the binding and dissociation sensorgrams using a 1:1 binding model to calculate the binding rate constant (k or k), the dissociation rate constant (k or k), and the equilibrium dissociation constant (K). When the binding activity is weak, particularly when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (K) may be calculated using a steady state model. As an additional parameter related to binding activity, the "amount of analyte bound per unit amount of ligand" may be calculated by dividing the amount of analyte bound at a specific concentration (resonance unit: RU) by the amount of captured ligand.

[0042] In one embodiment, the binding activity of an antibody is measured using a method that combines biolayer interferometry and a dip and read assay, for example, using the BLITz™ System (manufactured by Sartorius Japan Co., Ltd.), followed by analysis using BLITz Pro software.

[0043] The anti-integrin α8β1 antibodies of the present invention can be produced according to methods known in the art, for example, by recombinant methods similar to those described in U.S. Patent No. 4,816,567.

[0044] [Antigen-binding fragment of anti-integrin α8β1 antibody] The antigen-binding fragment of the present invention is an antigen-binding fragment of the above-mentioned anti-integrin α8β1 antibody, and comprises a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3. The antigen-binding fragment is not limited as long as it comprises a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3. Examples include Fv, Fab, F(ab')2, Fab', scFv, and dsFv.

[0045] [Anti-fibrotic Agent] The anti-fibrotic agent of the present invention contains the above-mentioned anti-integrin α8β1 antibody or an antigen-binding fragment of the above-mentioned anti-integrin α8β1 antibody as an active ingredient.

[0046] Antifibrotic agents can inhibit fibrosis. "Fibrosis" typically refers to a pathological condition caused by excessive deposition of matrix proteins such as collagen, replacing normal tissue, resulting in hardening of tissue and loss of normal function. In this specification, such a pathological condition is referred to as fibrosis.

[0047] Fibrosis occurs in, for example, the liver, lungs, kidneys, heart, skin, etc. Furthermore, for example, when a large amount of fibrosis occurs in liver tissue, it leads to liver cirrhosis. In addition to liver cirrhosis, malignant tumors may develop in various tissues as fibrosis progresses. In addition, in the lungs, there is pulmonary fibrosis, which causes a decrease in lung function.

[0048] Liver fibrosis includes, for example, cirrhosis, primary biliary cholangitis, nonalcoholic fatty liver disease (NAFLD) and alcoholic steatohepatitis (NASH), viral hepatitis, autoimmune liver disease, and genetic diseases. Pulmonary fibrosis includes, for example, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, interstitial pneumonia, occupational pulmonary fibrosis (e.g., aspergillosis), and drug-induced pulmonary fibrosis. Renal fibrosis is a cause of chronic kidney disease (CKD), and cardiac fibrosis is a cause of heart failure. Skin fibrosis includes scleroderma, keloids, and scars.

[0049] The anti-fibrotic agent can be used as a therapeutic drug for inhibiting fibrosis, a therapeutic drug for diseases that occur with the progression of fibrosis, a drug for inhibiting fibrosis for use in regenerative medicine, and the like.

[0050] The anti-fibrotic agent may be provided as a pharmaceutical composition by, for example, blending the antibody of the present invention with one or more pharmacologically acceptable carriers. Examples of pharmacologically acceptable carriers include aqueous buffers, pH adjusters such as acids and bases, stabilizers such as ascorbic acid and p-aminobenzoic acid, excipients such as D-mannitol, isotonicity agents, and preservatives. The dosage form is not particularly limited, but includes, for example, injections, capsules, tablets, and granules. The administration route may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The content of the active ingredient in the pharmaceutical composition is not particularly limited, and can be determined appropriately depending on the type of active ingredient, intended use, dosage form, and type of carrier. The content of the active ingredient in the pharmaceutical composition is, for example, 0.0001% by mass or more, preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and 100% by mass or less, preferably 90% by mass or less, and more preferably 50% by mass or less.

[0051] The dosage can be appropriately determined by those skilled in the art, as long as it is an amount that administers an effective amount of the antibody or antibody fragment, and is preferably 0.1 mg / kg or more, more preferably 1 mg / kg or more, even more preferably 10 mg / kg or more, and is preferably 5000 mg / kg or less, more preferably 500 mg / kg or less, even more preferably 100 mg / kg or less.

[0052] The administration method, dosage, and administration interval can be appropriately selected depending on the age, weight, symptoms, etc. of the subject.

[0053] The present invention further provides the following aspects related to anti-fibrotic agents: A method for treating fibrosis, comprising the step of administering an effective amount of the anti-integrin α8β1 antibody or an antigen-binding fragment of the anti-integrin α8β1 antibody to a subject in need thereof. Use of the anti-integrin α8β1 antibody or the antigen-binding fragment of the anti-integrin α8β1 antibody as an active ingredient in a therapeutic agent for fibrosis. Use of the anti-integrin α8β1 antibody or the antigen-binding fragment of the anti-integrin α8β1 antibody for the manufacture of a therapeutic agent for fibrosis.

[0054] The present invention further provides the following aspects: an expression vector comprising a polynucleotide encoding the amino acid sequence of the heavy chain of the anti-integrin α8β1 antibody and a polynucleotide encoding the amino acid sequence of the light chain of the anti-integrin α8β1 antibody; and a host cell comprising the expression vector.

[0055] The expression vector may be either a single expression vector containing a polynucleotide encoding the heavy chain amino acid sequence and a polynucleotide encoding the light chain amino acid sequence, or a combination of an expression vector containing a polynucleotide encoding the heavy chain amino acid sequence and an expression vector containing a polynucleotide encoding the light chain amino acid sequence.

[0056] Examples will be given below to specifically explain the present invention, but the present invention is not limited to these examples.

[0057] Production Example 1: Antibody Production (1-1) Preparation of Chicken Anti-Integrin α8β1 Antibody Mouse integrin α8 chain cDNA was introduced into the mammalian expression vector pEF6 V5-His A (Thermo Fisher Scientific; USA). The resulting expression vector was transfected into a chicken lymphoblastoid cell line by electroporation, and mouse integrin α8 chain-expressing cells were selected by adding a selection agent. Chickens were then hyperimmunized with the resulting mouse integrin α8 chain-expressing cell line. Antibody titers in chicken serum were measured by flow cytometry (FACS) analysis. FACS analysis was performed according to the standard protocol for FACSCalibur (BD Biosciences; USA). The amino acid sequence of the mouse integrin α8 chain used is shown in SEQ ID NO: 14.

[0058]

[0059] (1-2) Construction of an scFv phage antibody library and panning selection: Spleens were removed from chickens that had undergone immunization, and lymphocytes were isolated. RNA was extracted from the resulting lymphocytes, and cDNA was synthesized to construct an scFv phage antibody library. The phage antibody library was constructed according to the method described in Nakamura et al., J. Vet. Med. Sci. 2004 Jul, 66(7), 807-814.

[0060] As a pretreatment, the scFv phage antibody library was added to a cell line not expressing the mouse integrin α8 chain to allow absorption of nonspecific phages. The treated scFv phage antibody library was then reacted with a cell line expressing the mouse integrin α8 chain. After washing with organic solvent, phages that specifically bound to the cell line expressing the mouse integrin α8 chain were collected and infected with E. coli. After four rounds of panning, the reactivity of the enriched phage pool was confirmed by FACS analysis using a cell line expressing the mouse integrin α8 chain. Phages were cloned from the highly reactive phage pool, and positive clones were selected. Cell panning was performed according to the method described in Giordano et al., Nat Med. 2001 Nov, 7(11), 1249-1253.

[0061] Separately, a chicken lymphoblastoid cell line expressing the human integrin α8 chain was similarly prepared using the cDNA of the human integrin α8 chain instead of the mouse integrin α8 chain. The amino acid sequence of the human integrin α8 chain used is shown in SEQ ID NO: 15.

[0062]

[0063] Then, from the positive clones selected above, clones that also cross-react with the human integrin α8 chain-expressing cell line were selected by FACS analysis, and clones that cross-react with the human integrin α8 chain were selected.

[0064] Using the DNA strand encoding the scFv phage antibody obtained by the above method as a template, PCR amplification of the VH and VL regions was performed, followed by overlap PCR with the leader sequence and constant region of the chicken antibody, and cloning into the rIgY expression vector. The constructed H and L chain constructs were transfected into cultured mammalian cells, and the expressed antibody was purified. Recombination into the rIgY antibody was performed according to the method described by Shimamoto et al., Biologicals. 2005 Sep, 33(3), 169-174. Using the above procedure, the chicken anti-integrin α8β1 antibody YZ3, a monoclonal antibody, was produced.

[0065] In addition, a chicken-mouse chimeric YZ3 antibody was prepared, whose constant region is mouse IgG1 / κ. The chicken-mouse chimeric YZ3 antibody was prepared according to the method described in Tateishi et al., J. Vet. Med. Sci. 2008 Apr, 70(4), 397-400. The chicken-mouse chimeric YZ3 antibody was used in the administration test to mice described below.

[0066] The sequence of the heavy chain variable region of the prepared antibody YZ3 is SEQ ID NO: 9, and the sequence of the light chain variable region is SEQ ID NO: 10. Heavy chain CDR1, 2, and 3 of the heavy chain variable region are SYDMV (SEQ ID NO: 1), GIYSAGSGPQYAPAVKG (SEQ ID NO: 2), and AADSTYCASGSSCYAADSIDA (SEQ ID NO: 16), respectively, and light chain CDR1, 2, and 3 of the light chain variable region are SGGGSWYG (SEQ ID NO: 4), DNTNRPS (SEQ ID NO: 5), and GSADSTDAV (SEQ ID NO: 6), respectively.

[0067] (1-3) Preparation of humanized anti-integrin α8β1 antibodies To produce humanized antibodies, Expi cells were used. Antibody protein expression was induced by transient expression in Expi293 cells using an Expifectamine 293 transfection kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol.

[0068] First, the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 sequences of the chicken anti-integrin α8β1 antibody YZ3 obtained above were inserted into the framework sequence of a human heavy chain variable region, and the light chain CDR1, light chain CDR2, and light chain CDR3 sequences were inserted into the framework sequence of a human light chain variable region to produce the humanized anti-integrin α8β1 antibody hYZ3. Specifically, to humanize the chicken anti-integrin α8β1 antibody YZ3, the human germline V sequence most homologous to the YZ3 variable region was searched for in the human germline antibody variable region gene database VBASE2 (www.vbase2.org / vbase2.php). As a result, the VH sequence with the highest homology was V3-23*03 / J4*03, and the VL sequence with the highest homology was V3-25*03 / J7*01. The VH and VL framework sequences of YZ3 were replaced with the above-mentioned human framework sequences to produce humanized anti-integrin α8β1 antibody hYZ3. The amino acid sequences of the variable regions of the modified humanized anti-integrin α8β1 antibody hYZ3-1 are hYZ3VH1 and hYZ3VL shown in Figures 1-3. Specifically, the sequence of the heavy chain variable region of hYZ3 is SEQ ID NO: 11, and the sequence of the light chain variable region is SEQ ID NO: 12.

[0069] As described below, the resulting humanized hYZ3 antibody completely lost its ability to bind to α8. Therefore, a modified humanized anti-integrin α8β1 antibody, hYZ3-1, was produced by replacing five Vernier residues in the VH and four in the VL variable regions, which are involved in maintaining the three-dimensional structure of the antibody variable region in the frame, with chicken amino acids. Selection of the Vernier residues to be replaced was based on Nishibori et al., Mol. Immunol. 43, 634-642, 2006. The amino acid sequences of the variable regions of the modified humanized anti-integrin α8β1 antibody hYZ3-1 are hYZ3VH2 and hYZ3VL2 shown in Figures 1-3. Specifically, the sequence of the heavy chain variable region of hYZ3-1 is SEQ ID NO: 13, and the sequence of the light chain variable region is SEQ ID NO: 8. The amino acids substituted with chicken-derived amino acids in the humanized heavy chain variable region are serine at position 28, isoleucine at position 29, tyrosine at position 73, valine at position 78, and leucine at position 82 according to the Kabat system; and in the humanized light chain variable region are threonine at position 46, leucine at position 47, leucine at position 66, and serine at position 69 according to the Kabat system. In the humanized heavy chain variable region, the serine at position 49 and the phenylalanine at position 67 according to the Kabat system are human-derived amino acids, and in the humanized light chain variable region, the leucine at position 4 and the valine at position 71 according to the Kabat system are human-derived amino acids.

[0070] Furthermore, some amino acids in the CDR3 sequence of the heavy chain variable region of the improved humanized antibody hYZ3-1 obtained above were randomly altered, and antibody protein expression was induced by transient expression in Expi293 cells. Next, antibodies were obtained from the obtained antibodies by panning selection using a mouse α8-expressing cell line in which mouse α8 integrin was expressed in a human colon cancer cell line (SW480), and cross-reactive clones were further selected using a human α8-expressing cell line in which human α8 integrin was expressed in a human colon cancer cell line (SW480).

[0071] Specifically, clones were obtained as follows. In studies using the improved humanized antibody hYZ3-1, a mutant in which threonine at position 103 of the heavy chain amino acid sequence shown in SEQ ID NO: 13 was replaced with arginine was confirmed to be a binding-incompetent mutant in which the antibody's reactivity with α8 integrin was abolished. Based on this, a phage antibody library was constructed in which randomized mutations were introduced at eight positions near position 103. Figure 1-1 shows the amino acid sequence (SEQ ID NO: 18) near the corresponding hYZ3-1 heavy chain site, the nucleotide sequence encoding this sequence (SEQ ID NO: 17), and the consensus nucleotide sequence (SEQ ID NO: 19) into which mutations were introduced. The eight underlined amino acid positions are hotspot sites where mutations are known to occur frequently during the affinity maturation of antibodies. A total of four rounds of competitive panning were performed using the phage antibody library, with YZ3 reacted in advance with CHO cells overexpressing human α8 integrin. The results of FACS analysis of the phage population after panning are shown in Figure 1-2. Particularly after the second and fourth rounds of panning, it was confirmed that antibody clones that specifically bind to human α8 integrin were enriched in the library. Therefore, a total of 96 antibody clones were selected from the phage library after the second and fourth rounds of panning, and after each was expressed, their reactivity to human α8 integrin was evaluated by FACS. As a result, 17 clones exhibiting higher reactivity and 1 clone exhibiting lower reactivity compared to the original hYZ3-1 were obtained. Sequencing of the CDR3 sequences of the heavy chain variable region of these 18 clones confirmed that they consisted of 12 independent clones. The CDR3 amino acid sequences and reactivity to human α8 integrin of each selected clone are shown in Table 6-1. Reactivity to human α8 integrin is indicated by the specific activity relative to the hYZ3-1 antibody.

[0072]

[0073] Clone 2-70, which showed the highest reactivity, was selected as a representative clone to obtain the optimized humanized anti-integrin α8β1 antibody hYZ3-2. The amino acid sequences of the variable regions of the optimized humanized anti-integrin α8β1 antibody are hYZ3VH3 and hYZ3VL2 shown in Figures 1-3. Specifically, the sequence of the heavy chain variable region of hYZ3-2 is SEQ ID NO: 7, and the sequence of the light chain variable region is SEQ ID NO: 8.

[0074] The sequences of the antibodies used in this example are shown in Table 6-2.

[0075]

[0076] An alignment comparing each amino acid sequence is shown in Figures 1 to 3. In the figures, # indicates Vernier residues, which are amino acids presumed to be involved in maintaining the structure of the antibody variable region.

[0077] (1-4) Expression and Purification of Recombinant Antibody Chicken antibody YZ3 was produced using FreeStyle 293F cells. Antibody protein expression was transiently induced in FreeStyle 293F cells using a 293F transfection kit (trade name: FreeStyle 293 Expression System; Thermo Fisher Scientific, USA) according to the manufacturer's protocol. Heavy and light chain vectors were co-transfected at a 1:1 ratio. Three to four days after transfection, the recombinant antibody was purified from the cell culture supernatant.

[0078] Test Example 1: Binding Assay-1 The binding ability of each of the antibodies YZ3, hYZ3, and hYZ3-1 to the integrin α8 chain was measured by FACS analysis using the human colon cancer cell line SW480 (SW480 / hα8) overexpressing the human integrin α8 chain.

[0079] The results are shown in Figure 2. hYZ3 completely lost its binding ability to the human integrin α8 chain. This is thought to be due to the loss of antibody activity caused by the substitution with a heterologous animal-derived frame. On the other hand, modified hYZ3 (hYZ3-1), in which Vernier residues involved in maintaining the three-dimensional structure of the antibody variable region in the frame were re-substituted with chicken amino acids at five positions in VH and four positions in VL, restored binding ability to the human integrin α8 chain equivalent to that of YZ3.

[0080] Test Example 2: Binding Assay-2 The binding specificity of various integrin α8β1 antibodies to integrin α8β1 protein was evaluated. Each of the antibodies, YZ3, hYZ3-1, and hYZ3-2, was prepared at a concentration of 10 μg / mL and immobilized on a sensor chip. After immobilization, the binding and dissociation of each antibody with various concentrations of the fusion protein of the recombinant human α8β1 extracellular domain and human IgG-Fc domain was analyzed using the BLITz system and BLITz Pro software (both manufactured by Sartorius Japan K.K.), and the affinity was calculated.

[0081] The results are shown in Figure 3. hYZ3-1 had recovered to an affinity almost equivalent to that of the original antibody, YZ3. Furthermore, hYZ3-2 showed an affinity superior to that of YZ3. In particular, the kd value of hYZ3-2 was smaller than that of both YZ3 and hYZ3-1, meaning that it was less susceptible to dissociation.

[0082] Test Example 3: Comparison of immunogenicity in mice 20 μg of each of the YZ3, hYZ3-1, and hYZ3-2 antibodies was mixed with Gelb adjuvant and intraperitoneally administered to Balb / c mice. One week after administration, blood was collected from the tail vein and allowed to stand overnight at 4°C. The serum was then separated and collected by centrifugation at 1,000 × g for 25 minutes to obtain primary immunization serum. Two weeks after primary immunization, the antibodies were administered to mice under the same conditions, and one week after administration, the serum was separated and collected to obtain secondary immunization serum. The immunogenicity of each YZ3 variable region in mice was evaluated by ELISA. To evaluate the immunogenicity of the variable region, each YZ3 was prepared by recombinantly modifying the constant region with chicken IgY / Cλ, and this was immobilized overnight at 4°C on a 96-well plate at a concentration of 2.5 μg / mL. After blocking the wells with a blocking solution (25% Block Ace (KAC Corporation)), diluted serum (200, 500, 2,000, 5,000, and 20,000-fold dilutions) was added to the wells and allowed to react at room temperature for 2 hours. After washing the wells with a washing solution, HRPO-labeled anti-mouse IgG was diluted 10,000-fold and added to the wells, and allowed to react at room temperature in the dark for 30 minutes. After washing the wells with a washing solution, the HRPO substrate TMB was added to the wells and allowed to react at room temperature in the dark for 15 minutes to develop color. Finally, 2N sulfuric acid was added to the wells to stop the reaction, and the absorbance at 450 nm was measured using a plate reader.

[0083] The results are shown in Figure 4. hYZ3-1, in which some of the amino acids in the frame were re-substituted with chicken-derived amino acids, is a humanized antibody, but its immunogenicity was equivalent to that of YZ3, with no significant difference. In other words, hYZ3-1, in which some of the amino acids in the frame sequence were reverted to chicken-derived sequences, was equivalent to YZ3 in immunogenicity. However, compared with YZ3 and hYZ3-1, the immunogenicity of hYZ3-2 in mice was significantly reduced.

[0084] The above results indicate that a humanized antibody in which the variable region of the chicken antibody YZ3 was replaced with the most homologous human germline antibody variable region completely lost its binding ability to the human integrin α8 chain and its specificity. On the other hand, a humanized antibody in which some amino acids in the variable region of the human germline antibody were replaced with chicken amino acids completely restored its binding ability (specificity) to the human integrin α8 chain, but its immunogenicity in mammals (mice), which is important for a humanized antibody, was almost equivalent to that of a chicken antibody. However, hYZ3-2, a humanized antibody in which some amino acids in the heavy chain CDR3, which are thought to be important for the antibody's antigen-binding ability (specificity), have been modified, has an increased affinity for the human integrin α8 chain compared to the unmodified antibody YZ3-1 and the unhumanized YZ3. Its internal dissociation rate kd value, which determines affinity, is smaller than that of YZ3, meaning that it is less susceptible to dissociation. Furthermore, it has the excellent property of being significantly less immunogenic in mammals (mice) than YZ3 and hYZ3-1.

[0085] Test Example 4: Evaluation Using a Mouse Model of Renal Fibrosis The inhibitory effect of the antibody of the present invention on renal fibrosis in mice was evaluated using a mouse model of renal fibrosis induced by unilateral ureteral ligation (UUO). Eight- to nine-week-old C57BL / 6 female mice were subjected to abdominal section under deep anesthesia, and the ureter of the left kidney was carefully grasped with tweezers and ligated at two locations using nylon sutures (standard 5-0, manufactured by Natsume Seisakusho Co., Ltd.). After ligation, the abdomen was sutured, and the mice were warmed on a heat pad to awaken. One and four days after ligation, the hYZ3-2 antibody was administered subcutaneously at 10 mg / kg mouse body weight. A mouse control IgG1 (clone HKSP84) was administered in the same manner as a control antibody group. Seven days after ligation, the mice were dissected and the left kidney was removed. Total RNA was extracted from the excised kidney, and cDNA was synthesized. The expression levels of fibrosis marker genes Acta2, Tnc, and Trpc6, as well as the EMT (epithelial-mesenchymal transition) marker gene Snail were then measured by quantitative PCR. EMT has been reported to occur during the progression of renal fibrosis.

[0086] The test process is shown in Figure 5(A), and the results are shown in Figure 5(B). In the figure, * indicates the results of a significant difference test compared to the control antibody administration group, with Acta2 calculated as p = 0.0346, Tnc calculated as p = 0.0125, and Snail calculated as p = 0.008. Compared to the control antibody administration group, the expression of all marker genes in the hYZ3-2 antibody administration group showed a significant decrease or a tendency toward a decrease.

[0087] Test Example 4: Evaluation using a mouse model of liver fibrosis. The fibrosis-inhibitory effect of the antibody of the present invention on liver fibrosis in mice was evaluated using carbon tetrachloride (hereinafter referred to as CCl ４ The liver fibrosis model mice were evaluated using a mouse model created by administering CCl 4 diluted with olive oil to the peritoneal cavity of 8-9 week-old C57BL / 6 female mice. ４ to 1 mL CCl ４ hYZ3-2 or control IgG1 was administered intraperitoneally at 2 or 10 mg / kg of mouse body weight twice a week for a total of eight doses. ４ The drug was administered subcutaneously twice a week on the same day as the initial administration. ４ was administered in the morning, and hYZ3-2 or control IgG1 was administered in the afternoon. ４ There was an 8-hour interval between the first CC1 administration and the administration of hYZ3-2 or control IgG1. ４ Twenty-eight days after administration, the mice were dissected, and the livers were excised and sectioned. After staining with Sirius Red, the area of ​​the positive region was measured using ImageJ.

[0088] The test process is shown in Figure 6(A) and the results are shown in Figure 6(B). In the hYZ3-2 administration groups, both the 2 and 10 mg / kg groups, the Sirius Red-positive area, which indicates fibrotic sites, was significantly smaller than in the control antibody administration group.

[0089] Test Example 5: Evaluation using a mouse model of pulmonary fibrosis The inhibitory effect of the antibody of the present invention on pulmonary fibrosis in mice was evaluated using a mouse model of pulmonary fibrosis prepared by administration of bleomycin. Both prophylactic and therapeutic administration were evaluated.

[0090] 6-1) Preparation of antibody The hYZ3-2 antibody was prepared using an expression system of CHO-MK-derived cells. The antibody prepared using the expression system of CHO-MK-derived cells also has human IgG1 / κ in the constant region.

[0091] 6-2) Prophylactic Administration: Under deep anesthesia, the necks of 9-10 week-old C57BL / 6 female mice were incised, and bleomycin (Nippon Kayaku Co., Ltd.) diluted with saline was administered intratracheally at 1.5 U / kg body weight. After administration, the necks were sutured, and the mice were warmed on a heating pad to awaken. hYZ3-2 or human control IgG1 (clone 4F17) was administered subcutaneously at 10 mg / kg body weight twice a week starting the day after bleomycin administration. In the positive group, nintedanib ethanesulfonate (hereinafter also referred to as nintedanib; drug name: Ofev, obtained from Selleck), an existing drug for human pulmonary fibrosis, was orally administered at 50 mg / kg body weight four times in the first week and five times a week thereafter. Twenty-one days after the start of bleomycin administration, lungs were excised from the mice, sections were prepared, and collagen was stained by Masson's trichrome staining, and the area of ​​the positive region was measured using ImageJ.

[0092] The test process is shown in Figure 7(A), and the results are shown in Figure 7(B). In the figure, * indicates the result of a significant difference test relative to the human control IgG1 administration group, which was calculated as p = 0.0351. The hYZ3-2 administration group had a significantly smaller area of ​​fibrosis compared to the control antibody administration group. On the other hand, the nintedanib administration group showed no difference from the control antibody administration group.

[0093] 6-3) Therapeutic Administration: Bleomycin was administered intratracheally to mice in the same manner as in "6-2) Preventive Administration" above. Seven days after bleomycin administration, which corresponds to the onset of fibrosis, mice were divided into three groups: one group receiving subcutaneous administration of hYZ3-2 at 10 mg / kg body weight twice weekly, one group receiving subcutaneous administration of control IgG1 at 10 mg / kg body weight twice weekly, and one group receiving oral administration of nintedanib at 50 mg / kg body weight five times weekly. Twenty-one days after the start of bleomycin administration, lungs were excised from the mice, sectioned, and stained for collagen using Masson's trichrome staining. The area of ​​the stained region, which represented the site of fibrosis, was measured using ImageJ. Hydroxyproline levels, an indicator of collagen deposition, were also measured. Furthermore, the expression levels of the fibrosis marker genes Acta2, Col1a1, Col3a1, Tnc, Ctgf, and Postn were measured by quantitative PCR.

[0094] The test process is shown in Figure 8(A), the measurement results of the fibrosis area and hydroxyproline amount are shown in Figure 8(B), and the measurement results of the expression levels of fibrosis marker genes are shown in Figure 9-1. The fibrosis area and hydroxyproline amount were lower in the hYZ3-2 administration group than in the control antibody administration group and the nintedanib administration group. The fibrosis area showed a tendency to decrease. Meanwhile, there was no difference between the nintedanib administration group and the control antibody administration group. Compared to the control antibody administration group, the expression levels of fibrosis marker genes were significantly reduced in the hYZ3-2 administration group for Col1a1, Col3a1, Tnc, Ctgf, and Postn, and in the nintedanib administration group for Col1a1, Col3a1, Tnc, and Postn. The degree of reduction in expression levels was greater in the hYZ3-2 administration group than in the nintedanib administration group for all marker genes.

[0095] As described above, the test results using pulmonary fibrosis model mice showed that hYZ3-2 has a fibrosis-inhibiting effect equivalent to or greater than that of nintedanib.

[0096] 6-4) Therapeutic Low-Dose Administration Bleomycin was administered intratracheally to mice using the same method as in "6-3) Therapeutic Administration." On day 7 after administration, which corresponds to the time when fibrosis begins to occur, the mice were divided into four groups, and hYZ3-2 was administered subcutaneously to each group twice a week at 0, 3, 5, or 7.5 mg / kg of mouse body weight. Twenty-one days after the start of bleomycin administration, the mice were sacrificed, and the lungs were removed. The expression levels of fibrosis marker genes Acta2, Col1a1, Col3a1, Tnc, and Ctgf were measured by quantitative PCR.

[0097] The results are shown in Figure 9-2. Acta2 expression was significantly reduced in the 3 and 5 mg / kg groups, Col1a1, Tnc, and Ctgf expression in all groups, and Col3a1 expression in the 5 and 7.5 mg / kg groups compared to the non-administration group (0 mg / kg group). These results confirmed that hYZ3-2 exhibits anti-fibrosis effects even at doses lower than 10 mg / kg.

[0098] Test Example 7: Evaluation of the effect on TGF-β activation Rat lung fibroblasts were isolated from the lungs of 10-week-old male Wistar rats and cultured for 7 days. After that, the cultured cells were detached from the dish and plated at 2 × 10 cells per well. ４ Cells were seeded onto a 96-well plate, and 20 nM hYZ3-2, 20 nM control IgG1, 50 μM pirfenidone, or 75 nM nintedanib, an existing drug for human pulmonary fibrosis, was added and cultured for 2 hours. All addition amounts are final concentrations. As a control, DMSO alone was added and cultured for 2 hours. Then, 2 x 10 CCL64 reporter cells, which produce luciferase in response to TGF-β, were added per well. ４The cells were seeded with 100% TGF-β activators and co-cultured for 18 hours. CCL64 was kindly provided by Dr. Soichi Kojima of the Institute of Physical and Chemical Research. After culturing, the cells were lysed, Luciferase Assay Reagent (Promega) was added, and the luminescence intensity was measured as luciferase activity using a luminometer. The measurement conditions were a luminescence time of 1 second. The control antibody addition group was used as a control for the hYZ3-2 addition group, and the DMSO addition group was used as a control for the pirfenidone addition group and the nintedanib addition group, and the relative activation level was calculated relative to 100% TGF-β activation of the control.

[0099] The results are shown in Figure 10. In the figure, ** indicates the result of a significant difference test compared to the human control IgG1-added group, with a calculated p=0.0038. A significant decrease in luciferase activity was observed in the hYZ3-2-added group. On the other hand, no significant decrease in activity was observed in the pirfenidone-added group or the nintedanib-added group. The concentrations of pirfenidone and nintedanib added were set based on Ogura T et al., Safety and pharmacokinetics of nintedanib and pirfenidone in idiopathic pulmonary fibrosis. Eur. Respir. J., 2015, 45(5), 1382-1392.

[0100] Test Example 8: Evaluation of the effect on myofibroblast differentiation 8-1) Test of myofibroblast differentiation by TGF-β stimulation Rat lung fibroblasts isolated and cultured in the same manner as in Example 7 were detached and plated at 8 × 10 per well. ４Cells were seeded onto a 96-well plate and cultured for 24 hours. After culture, the medium was removed, the wells were washed twice with serum-free medium, and serum-free medium supplemented with 20 nM hYZ3-2, 20 nM control IgG1, 50 μM pirfenidone, or 75 nM nintedanib was added and incubated for 2 hours. All addition amounts are final concentrations. After incubation, recombinant human TGF-β1 protein was added to a final concentration of 5 ng / mL, and the cells were cultured for 72 hours with TGF-β stimulation to induce differentiation into myofibroblasts. After culture, proteins were extracted from the cells and subjected to Western blotting, and bands for the myofibroblast differentiation marker α-SMA and the control protein GAPDH were simultaneously detected. Band intensity relative to GAPDH expression level was measured using ImageJ, and the relative expression level of α-SMA to GAPDH expression level was calculated.

[0101] Figure 11(A) shows the results of Western blot, and Figure 11(B) shows the results of measuring the relative expression level of α-SMA. The expression level of α-SMA significantly increased with TGF-β stimulation, and this increase in expression level was significantly suppressed in the hYZ3-2-added group and the nintedanib-added group. No suppression of the increase in expression level was observed in the pirfenidone-added group. The concentrations of pirfenidone and nintedanib added were set in the same manner as in Test Example 7 above.

[0102] 8-2) Myofibroblast differentiation test by integrin α8β1 ligand signaling Fibroblasts were isolated from rat lung or kidney and cultured in the same manner as in Example 7. The medium for each cell was replaced with a serum-free medium, and after 24 hours of culture, the cells were detached and placed at 1.5 × 10 cells per well on a 6-well plate onto which nephronectin (hereinafter also referred to as NPNT), a ligand for integrin α8β1, had been immobilized. ５Cells were seeded on wells and cultured for 24 hours in the presence of 20 nM hYZ3-2 or 20 nM control IgG1. All concentrations are final concentrations. As a control, cells were seeded on wells immobilized with poly-L-lysine (PLL), which induces integrin-independent cell adhesion, and cultured in the same manner. After culture, total RNA was extracted from the cells, cDNA was synthesized, and the expression levels of the fibrosis marker genes Acta2 and Tnc were measured by quantitative PCR.

[0103] The results are shown in Figure 12. Expression levels of the fibrosis marker genes Acta2 and Tnc were significantly increased in both lung and renal fibroblasts when cultured on NPNT compared to culture on PLL. This increase in expression levels of fibrosis marker genes was almost completely inhibited by the addition of hYZ3-2. These results demonstrate that binding of integrin α8β1 to its ligand directly induces myofibroblast differentiation, and that the antibody of the present invention inhibits this induction.

[0104] Test Example 9: Antibody Accumulation in Fibrotic Tissue hYZ3-2 and control IgG1 were labeled with the fluorescent substance CF680 using VivoBrite Rapid Antibody Labeling Kits for Small Animal In Vivo Imaging CF680 (Biotium). The labeled antibodies were then administered to each fibrosis model mouse. Specifically, liver fibrosis mice prepared in the same manner as in Test Example 4 were treated with the fourth CCl ４ Two days after administration, 7 days after unilateral ureteral ligation for renal fibrosis mice prepared as in Test Example 5, and 16 days after bleomycin administration for pulmonary fibrosis mice prepared as in Test Example 6, each fluorescently labeled antibody was subcutaneously administered. Forty-eight hours after administration of the labeled antibody, the mice were dissected to remove the liver and kidneys, and fluorescent images of the kidneys and liver were obtained using NightWOL II LB983 (Berthold). 120 hours after administration of the labeled antibody, the mice were dissected to remove the lungs, and fluorescent images were obtained using an IVIS Imaging System (Revvity, obtained from Summit Pharmaceuticals International Corporation).

[0105] The results are shown in Figure 13. The arrows in the figure indicate strong fluorescent signals. "ND" indicates not determined. In mice administered with fluorescently labeled hYZ3-2, significantly strong fluorescent signals were observed in the fibrotic tissue in all fibrosis models, demonstrating the accumulation of hYZ3-2 specific to the fibrotic tissue.

[0106] Test Example 10: Identification of the transcription factor for the integrin α8 gene (Itga8 gene) Rat hepatic stellate cells (HSCs) are the primary cell source for myofibroblasts in the liver and play a central role in liver fibrosis. It is known that isolated HSCs become activated when cultured on plastic plates. This phenomenon is called "culture-induced activation" and is thought to mimic the activation of fibroblasts / stellate cells in tissue fibrosis (see, for example, Friedman, SL (2008). Physiological Reviews, 88, 125-172). It is known that α8 expression increases during this process, and the transcription factor for Itga8 was identified using this system as follows.

[0107] First, because culture activation does not occur on soft substrates such as collagen gel, we confirmed the involvement of the transcription factor YAP / TEAD, which is triggered by the hardness of the tissue microenvironment. The inhibitor verteporfin (VP) was added at a concentration of 2 μM on day 4 of culture, and the cells were cultured for three days. Changes in expression of Itga8 and Itga6 (control) were analyzed by q-PCR. α8 protein expression was analyzed by FACS after adding VP at a concentration of 2 μM to HSCs and culturing for three days from day 7 of culture. Next, ChIP sequencing data indicating binding to the Itga8 gene expression regulatory region, a more direct transcription factor effect, was searched on GEO (www.ncbi.nlm.nih.gov / gds / ) and confirmed with data from the ENCODE (www.encodeproject.org) project (see, for example, Croci, O. et al. (2017). Genes Dev., 31, 2017-2022). Finally, we examined whether TGF-β, known to be involved in integrin expression, induced α8 expression. Rat lung fibroblasts were stimulated with 5 ng / mL TGF-β for 3 days, and changes in α8 expression were analyzed by FACS.

[0108] The results are shown in Figures 14-1, 14-2, 14-3, and 14-4. Figure 14-1 ((A)) shows that Itga8 expression significantly increased after 3 days of culture, and that this increase was completely inhibited by VP. Meanwhile, Itga6 expression remained unchanged. Figure 14-2 ((B)) shows that, similar to gene expression, VP significantly reduced α8 protein expression, while α6 protein expression was unaffected. Figure 14-3 ((C)) shows ChIP-sequencing data demonstrating that YAP1 and TEAD1 bind to the Itga8 expression regulatory region. The expression regulatory region consists of a promoter region and an enhancer region. Binding of DNase, H3K4me1, H3K4me4, and H3K4ac27 antibodies also indicated that the chromatin in this region was open and activated. Finally, Figure 14-4 ((D) in the figure) shows that stimulation of rat lung fibroblasts with TGF-β did not change α8 expression. In the figure, α11 and α6 represent the positive and negative controls, respectively, and the numbers in the graph indicate the relative expression levels of each integrin compared to the negative control. These results indicate that integrin α8β1, which has anti-fibrotic activity, increases tissue stiffness as a result of its action, thereby inducing expression in a feedforward relationship. Therefore, inhibition of α8 function by the antibody of the present invention acts to block the feedforward loop, demonstrating its usefulness as a therapeutic agent for fibrosis.

Claims

1. An anti-integrin α8β1 antibody comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and (ii) a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:

6.

2. The anti-integrin α8β1 antibody of claim 1, wherein the antibody is humanized.

3. An anti-integrin α8β1 antibody comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises: (i-1) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (i-2) a Kabat sequence comprising serine at the position corresponding to position 28, isoleucine at the position corresponding to position 29, serine at the position corresponding to position 49, phenylalanine at the position corresponding to position 67, tyrosine at the position corresponding to position 73, valine at the position corresponding to position 78, and leucine at the position corresponding to position 82; and the humanized light chain variable region comprises: (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; and (ii-2) The anti-integrin α8β1 antibody of claim 2, comprising a leucine at a position corresponding to position 4, a threonine at a position corresponding to position 46, a leucine at a position corresponding to position 47, a leucine at a position corresponding to position 66, a serine at a position corresponding to position 69, and a valine at a position corresponding to position 71 according to the Kabat system.

4. An anti-integrin α8β1 antibody comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises: (i-1) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3; (i-2) According to the Kabat system, a valine at a position corresponding to position 2, a leucine at a position corresponding to position 4, a glutamic acid at a position corresponding to position 6, a glycine at a position corresponding to position 9, a leucine at a position corresponding to position 20, a cysteine ​​at a position corresponding to position 22, an alanine at a position corresponding to position 24, a phenylalanine at a position corresponding to position 27, a serine at a position corresponding to position 28, an isoleucine at a position corresponding to position 29, a serine at a position corresponding to position 30, a tryptophan at a position corresponding to position 36, a valine at a position corresponding to position 37, an arginine at a position corresponding to position 38, a glutamine at a position corresponding to position 39, a leucine at a position corresponding to position 45, a tryptophan at a position corresponding to position 47, a valine at a position corresponding to position 48, and a serine at a position corresponding to position 49; a phenylalanine at a position corresponding to position 67, an isoleucine at a position corresponding to position 69, an arginine at a position corresponding to position 71, a tyrosine at a position corresponding to position 73, a valine at a position corresponding to position 78, a leucine at a position corresponding to position 80, a leucine at a position corresponding to position 82, a leucine at a position corresponding to position 82C, an aspartic acid at a position corresponding to position 86, a tyrosine at a position corresponding to position 90, a tyrosine at a position corresponding to position 91, a cysteine ​​at a position corresponding to position 92, an alanine at a position corresponding to position 93, a lysine at a position corresponding to position 94, a tryptophan at a position corresponding to position 103, a glycine at a position corresponding to position 104, a glycine at a position corresponding to position 106, a valine at a position corresponding to position 109, and a valine at a position corresponding to position 111; wherein the humanized light chain variable region comprises: (ii-1) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:6;(ii-2) According to the Kabat system, a leucine at a position corresponding to position 4, a glutamine at a position corresponding to position 6, an isoleucine at a position corresponding to position 21, a cysteine ​​at a position corresponding to position 23, a tryptophan at a position corresponding to position 35, a tyrosine at a position corresponding to position 36, a glutamine at a position corresponding to position 38, a proline at a position corresponding to position 44, a threonine at a position corresponding to position 46, a leucine at a position corresponding to position 47, an isoleucine at a position corresponding to position 48, a tyrosine at a position corresponding to position 49, an isoleucine at a position corresponding to position 58, a phenylalanine at a position corresponding to position 62, a glycine at a position corresponding to position 64, a leucine at a position corresponding to position 66, and a cysteine ​​at a position corresponding to position 68 4, a glycine at a position corresponding to position 69, a serine at a position corresponding to position 71, a valine at a position corresponding to position 73, an isoleucine at a position corresponding to position 75, a valine at a position corresponding to position 78, an aspartic acid at a position corresponding to position 82, a tyrosine at a position corresponding to position 86, a tyrosine at a position corresponding to position 87, a cysteine ​​at a position corresponding to position 88, a phenylalanine at a position corresponding to position 98, a glycine at a position corresponding to position 99, a glycine at a position corresponding to position 101, a threonine at a position corresponding to position 102, a leucine at a position corresponding to position 104, and a valine at a position corresponding to position 106.

5. The anti-integrin α8β1 antibody of claim 1, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:7 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

8.

6. An antigen-binding fragment of the anti-integrin α8β1 antibody described in claim 1, comprising the amino acid sequence shown in SEQ ID NO:

3.

7. An anti-fibrotic agent comprising the anti-integrin α8β1 antibody according to any one of claims 1 to 5 or the antigen-binding fragment of the anti-integrin α8β1 antibody according to claim 6.

Images