Antibody, nucleic acid, cell, and pharmaceutical

Abstract

To provide a novel antibody that specifically binds to human lysophosphatidic acid receptor 1 (human LPA1).SOLUTION: Provided is an antibody that specifically binds to the extracellular domain of human LPA1 but does not specifically bind to the extracellular domain of human LPA2 or the extracellular domain of human LPA3. The antibody preferably has activity to block LPA1-dependent cellular function. Also provided are: a nucleic acid encoding the antibody; a cell containing the nucleic acid; and a pharmaceutical containing the antibody as an active ingredient.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present disclosure relates to an antibody that specifically binds to the extracellular domain of human lysophosphatidic acid receptor 1 (human LPA1), a nucleic acid encoding the antibody, a cell containing the nucleic acid, and a pharmaceutical comprising the antibody as an active ingredient. [Background technology]

[0002] Lysophosphatidic acid (LPA) is a lysophospholipid in which one phosphate and one fatty acid are bound to a glycerol backbone. Initially, LPA was thought to be one of the intermediate products of lipid metabolism, but it is now recognized as a physiologically active lipid mediator that exhibits various physiological effects. For example, it is involved in cell functions such as cell proliferation, inhibition of apoptosis, cell migration, production of cytokines and chemokines, platelet aggregation, smooth muscle contraction, cell transformation, and neurite retraction (Non-Patent Documents 1 and 2).

[0003] LPA regulates intracellular signal transduction pathways by binding to G protein-coupled receptors on the cell surface, and exerts various physiological effects. Six subtypes of LPA receptors, LPA1, LPA2, LPA3, LPA4, LPA5, and LPA6, have been reported (Non-Patent Document 3). The three receptors, LPA1, LPA2, and LPA3, belong to the EDG (Endothelial Differentiation Gene) family, also called EDG2, EDG4, and EDG7, respectively, and have high structural homology with each other. LPA4 to LPA6 are non-EDG family members, and have low homology with the above-mentioned EDG family.

[0004] Research into the regulation of LPA receptor function using cells and animal models has examined its developmental biology and pathophysiological effects on organs such as the nervous system, cardiovascular system, reproductive system, lungs, liver, and kidneys, suggesting that LPA-dependent cell function disorders may be involved in diseases such as fibrosis, cancer, neuropsychiatric disorders, pain, cardiovascular disease, bone disorders, infertility, and obesity, including disorders of nervous and bone development (Non-Patent Documents 1, 2, 4, 5).

[0005] Tissue fibrosis is a disease in which excessive accumulation of extracellular matrix leads to tissue failure due to abnormal control of the tissue healing process. It has been reported that the LPA concentration in the alveolar lavage fluid of bleomycin-induced fibrosis model mice is elevated, and that fibrosis is suppressed in the same model by administration of LPA1 knockout mice or LPA1 antagonists (Non-Patent Documents 6, 7). It has also been shown that LPA1 antagonists BMS-986020 and BMS-986278 improve respiratory function and pulmonary fibrosis in patients with idiopathic pulmonary fibrosis (Non-Patent Documents 8, 9). Therefore, controlling abnormal LPA1-dependent cell function is useful for treating diseases caused by fibrosis (e.g., pulmonary fibrosis such as idiopathic pulmonary fibrosis, liver fibrosis such as non-alcoholic steatohepatitis, renal fibrosis such as diabetic nephropathy, skin fibrosis such as scleroderma, cardiovascular fibrosis, gastrointestinal fibrosis, etc.) (Non-Patent Documents 1, 2, 10, 11).

[0006] Other diseases for which it has been suggested that controlling LPA1-dependent cell functions is useful for treatment include, for example, neuropathic pain and inflammatory pain such as fibromyalgia, cancer pain, and diabetic neuropathy (Non-Patent Documents 12-14), inflammatory diseases and autoimmune diseases such as rheumatoid arthritis, sepsis, and Guillain-Barre syndrome (Non-Patent Documents 15-17), metabolic diseases including obesity and insulin-resistant diabetes (Non-Patent Document 18), cardiovascular disorders including atherosclerosis, cerebral infarction, and hypertensive nephropathy (Non-Patent Documents 19-21), nervous system disorders such as hydrocephalus, schizophrenia, depression, and dementia (Non-Patent Documents 22 and 23), cell proliferation diseases (tumor cell proliferation, tumor invasion and metastasis, and angiogenesis control) (Non-Patent Documents 6, 24, 25), urological diseases such as prostatic hypertrophy and urinary incontinence (Non-Patent Documents 26 and 27), and ophthalmological diseases such as ischemic retinopathy (Non-Patent Document 28).

[0007] LPA1, LPA2, and LPA3 are distributed differently in the body depending on the subtype, and each subtype contributes to different cellular functions and physiological actions (Non-Patent Documents 1 and 2). Therefore, as a useful active ingredient of a pharmaceutical with few side effects, a substance that specifically binds to human LPA1, does not specifically bind to human LPA2, and does not specifically bind to human LPA3 is desired. As shown by the LPA1 antagonist BMS-986020, there are cases where it binds to multiple LPA receptors and development has been discontinued due to side effects. It is still not easy to find a substance that specifically binds to human LPA1, does not specifically bind to human LPA2, and does not specifically bind to human LPA3.

[0008] Although several LPA1 antagonists made of low molecular weight compounds have been created so far, none have been put to practical use yet (Non-Patent Document 6). It would be useful to have an antibody that does not specifically bind to human LPA2 or LPA3, but specifically binds to LPA1 and controls LPA1-dependent cell functions, but there have been no reports on such an antibody. [Prior art documents] [Non-patent literature]

[0009] [Non-licensed document 1] Aikawa S, Hashimoto T, Kano K, Aoki J. "Lysophosphatidic acid as a lipid mediator with multiple biological actions." J Biochem. 2015; 157:81-89

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[0010] An object of the present disclosure is to provide a novel antibody that specifically binds to the extracellular domain of LPA1, and techniques relating thereto. [Means for solving the problem]

[0011] An antibody according to one embodiment of the present disclosure is an antibody that specifically binds to the extracellular domain of human LPA1, does not specifically bind to the extracellular domain of human LPA2, and does not specifically bind to the extracellular domain of human LPA3.

[0012] Preferably, the antibody has activity of blocking LPA1-dependent cellular functions.

[0013] Preferably, the antibody comprises a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, or 101; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, or 102; A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, or 103; A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, or 104; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, or 105; and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, or 106.

[0014] Preferably, the antibody is one of the following (A1) to (A11): (A1) 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, (A2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, (A3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, (A4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, (A5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43, (A6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53, (A7) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63, (A8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73, (A9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; (A10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93; (A11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103; Satisfy any of the following.

[0015] Preferably, the antibody is one of the following (B1) to (B11): (B1) 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, (B2) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16, (B3) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26, (B4) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, (B5) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46, (B6) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56; (B7) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66; (B8) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76; (B9) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86; (B10) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96; (B11) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106; Satisfy any of the following.

[0016] Preferably, the antibody is any of the following antibodies (AB1) to (AB11): (AB1) 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, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3, 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, (AB2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16, (AB3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26, (AB4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, (AB5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46, (AB6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56, (AB7) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66, (AB8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76, (AB9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86; (AB10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96, (AB11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106, Satisfy any of the following.

[0017] Preferably, the antibody is one of the following (C1) to (C11): (C1) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9, (C2) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19, (C3) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29, (C4) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 39, (C5) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 49, (C6) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 59; (C7) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 69; (C8) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 77, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 79; (C9) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89; (C10) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 99; (C11) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 107, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 109, Satisfy any of the following.

[0018] The antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human LPA1, does not specifically bind to the extracellular domain of human LPA2, and does not specifically bind to the extracellular domain of human LPA3, and has the following structures (C1') to (C11'): (C1') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 7, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 7; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 9, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 9. (C2') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 17; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 19, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 19. (C3') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:27, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:27; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:29, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:29. (C4') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 37; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 39. (C5') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 47, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 47; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 49, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 49. (C6') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 57, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 57; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:59, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:59. (C7') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 67, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 67; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:69, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:69. (C8') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 77, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 77; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 79, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 79. (C9') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 87, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 87; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 89, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 89. (C10') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 97, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 97; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 99, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 99. (C11') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 107, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 107; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 109, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 109. Satisfy any of the following.

[0019] Preferably, the antibody has activity of blocking LPA1-dependent cellular functions.

[0020] The second antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human LPA1, does not specifically bind to the extracellular domain of human LPA2, and does not specifically bind to the extracellular domain of human LPA3, and competitively inhibits the binding of the first antibody, which is the above antibody, to a receptor.

[0021] Preferably, the second antibody has an activity of blocking an LPA1-dependent cellular function.

[0022] Preferably, the antibody or second antibody is a humanized antibody or a chimeric antibody.

[0023] Preferably, the antibody or second antibody is a multispecific antibody.

[0024] Preferably, the antibody or second antibody is a modified antibody having another molecule attached thereto.

[0025] Preferably, the modified antibody is an antibody-drug conjugate.

[0026] A nucleic acid according to one aspect of the present disclosure encodes the antibody or second antibody described above.

[0027] A cell according to one embodiment of the present disclosure comprises the above-described nucleic acid.

[0028] A pharmaceutical according to one aspect of the present disclosure contains the above-mentioned antibody or second antibody as an active ingredient.

[0029] Preferably, the medicament is used for treating a disease, disorder, or condition in which impairment of LPA1-dependent cellular function is involved.

[0030] Preferably, the medicament is used for the treatment of tissue fibrosis, a cell proliferative disorder, pain, an inflammatory disorder, an autoimmune disorder, a metabolic disorder, a cardiovascular disorder, a urological disorder, or an ophthalmological disorder.

[0031] Preferably, the tissue fibrosis is liver fibrosis, kidney fibrosis, pulmonary fibrosis, skin fibrosis, cardiovascular fibrosis, or gastrointestinal fibrosis.

[0032] Preferably, the liver fibrosis is nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, ischemia-reperfusion, post-liver transplant injury, necrotizing hepatitis, hepatitis B, hepatitis C, primary biliary cirrhosis, or primary sclerosing cholangitis.

[0033] Preferably, the cirrhosis is alcohol-induced, drug-induced, or chemically-induced.

[0034] Preferably, the renal fibrosis is proliferative glomerulonephritis, sclerosing glomerulonephritis, nephrogenic fibrosing dermopathy, diabetic nephropathy, renal tubulointerstitial fibrosis, or focal segmental glomerulosclerosis.

[0035] Preferably, the pulmonary fibrosis is pulmonary interstitial fibrosis, drug-induced sarcoidosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar damage disease, pulmonary hypertension, or neonatal bronchopulmonary dysplasia.

[0036] Preferably, said skin fibrosis is scleroderma, keloid scarring, psoriasis, hypertrophic scarring, or pseudoscleroderma.

[0037] Preferably, said cardiovascular fibrosis is atherosclerosis, coronary restenosis, congestive cardiomyopathy, heart failure, heart transplantation, or myocardial fibrosis.

[0038] Preferably, said gastrointestinal fibrosis is collagenous colitis, villous atrophy, crypt hyperplasia, polyp formation, fibrosis of Crohn's disease, gastric ulcer healing, or post-abdominal adhesion scarring.

[0039] Preferably, said fibrosis comprises a condition resulting from a bone-related fibrotic disease, rheumatoid pannus formation.

[0040] Preferably, the cell proliferative disorder is the control of tumor cell proliferation, tumor invasion and metastasis, or angiogenesis.

[0041] Preferably, the cell proliferative disorder is a hematological cancer or a solid cancer.

[0042] Preferably, the solid cancer is breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, hepatocellular carcinoma, prostate cancer, urothelial carcinoma, renal cell carcinoma, or squamous cell carcinoma.

[0043] Preferably, the squamous cell carcinoma is oral squamous cell carcinoma, esophageal squamous cell carcinoma, or pharyngeal squamous cell carcinoma.

[0044] Preferably, the pain is fibromyalgia pain, cancer pain, or diabetic neuropathy pain.

[0045] Preferably, the inflammatory disease is rheumatoid arthritis, sepsis, chronic obstructive pulmonary disease, inflammatory bowel disease, transplant organ rejection, Guillain-Barre syndrome, or multiple sclerosis.

[0046] Preferably, the metabolic disease is obesity or insulin-resistant diabetes.

[0047] Preferably, the cardiovascular disorder is cerebral infarction, hypertensive nephropathy, or Raynaud's phenomenon.

[0048] Preferably, the nervous system disorder is hydrocephalus, schizophrenia, depression, or dementia.

[0049] Preferably, the urological disease is prostatic hyperplasia or urinary incontinence.

[0050] Preferably, the ophthalmic disease is ischemic retinopathy, diabetic retinopathy, or age-related macular degeneration. Effect of the Invention

[0051] According to the present disclosure, it is possible to provide a novel antibody that specifically binds to the extracellular domain of LPA1, and techniques related thereto. [Brief description of the drawings]

[0052] [Figure 1A] 1 is a histogram showing the results of flow cytometry performed in Example 4, showing the binding affinity of antibodies to human LPA1 to LPA3. [Figure 1B] 1 is a histogram showing the results of flow cytometry performed in Example 4, showing the binding affinity of antibodies to mouse LPA1 to LPA3. [Figure 2A] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-1-C) to CHO cells stably expressing human LPA1. [Figure 2B] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-1-G) to CHO cells stably expressing human LPA1. [Figure 2C] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-2-A) to CHO cells stably expressing human LPA1. [Figure 2D] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-2-D) to CHO cells stably expressing human LPA1. [Figure 2E] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-4-A) to CHO cells stably expressing human LPA1. [Figure 2F]1 is a graph showing the results of evaluating the binding ability of an antibody (210310-1-D) to CHO cells stably expressing human LPA1. [Figure 2G] 1 is a graph showing the results of evaluating the binding ability of an antibody (210420-4-E) to CHO cells stably expressing human LPA1. [Figure 2H] 1 is a graph showing the results of evaluating the binding ability of an antibody (210420-1-H) to CHO cells stably expressing human LPA1. [Figure 2I] 1 is a graph showing the results of evaluating the binding ability of an antibody (210420-3-E) to CHO cells stably expressing human LPA1. [Figure 2J] 1 is a graph showing the results of evaluating the binding ability of an antibody (211222-1-G) to CHO cells stably expressing human LPA1. [Figure 2K] 1 is a graph showing the results of evaluating the binding ability of an antibody (211222-1-A) to CHO cells stably expressing human LPA1. [Figure 3A] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-1-C) to CHO cells stably expressing mouse LPA1. [Figure 3B] Fig. 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-1-G) to CHO cells stably expressing mouse LPA1. [Figure 3C] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-2-A) to CHO cells stably expressing mouse LPA1. [Figure 3D] Fig. 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-2-D) to CHO cells stably expressing mouse LPA1. [Figure 3E] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-4-A) to CHO cells stably expressing mouse LPA1. [Figure 3F] Fig. 1 is a graph showing the results of evaluating the binding ability of an antibody (210310-1-D) to CHO cells stably expressing mouse LPA1. [Figure 3G] 1 is a graph showing the results of evaluating the binding ability of an antibody (210420-4-E) to CHO cells stably expressing mouse LPA1. [Figure 3H]1 is a graph showing the results of evaluating the binding ability of an antibody (210420-1-H) to CHO cells stably expressing mouse LPA1. [Figure 3I] 1 is a graph showing the results of evaluating the binding ability of an antibody (210420-3-E) to CHO cells stably expressing mouse LPA1. [Figure 3J] Fig. 1 is a graph showing the results of evaluating the binding ability of an antibody (211222-1-G) to CHO cells stably expressing mouse LPA1. [Figure 3K] 1 is a graph showing the results of evaluating the binding ability of an antibody (211222-1-A) to CHO cells stably expressing mouse LPA1. [Figure 4] 1 is a histogram showing the results of flow cytometry performed in Example 6, showing the binding affinity of the antibody to endogenous human LPA1. [Figure 5A] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-1-C) against human LPA1. [Figure 5B] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-1-G) against human LPA1. [Figure 5C] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-2-A) against human LPA1. [Figure 5D] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-2-D) against human LPA1. [Figure 5E] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-4-A) against human LPA1. [Figure 5F] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210310-1-D) against human LPA1. [Figure 5G] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210420-4-E) against human LPA1. [Figure 5H] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210420-1-H) against human LPA1. [Figure 5I]1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210420-3-E) against human LPA1. [Figure 5J] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (211222-1-G) against human LPA1. [Figure 5K] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (211222-1-A) against human LPA1. [Figure 6A] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-1-C) against mouse LPA1. [Figure 6B] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-1-G) against mouse LPA1. [Figure 6C] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-2-A) against mouse LPA1. [Figure 6D] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-2-D) against mouse LPA1. [Figure 6E] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-4-A) against mouse LPA1. [Figure 6F] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210310-1-D) against mouse LPA1. [Figure 6G] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210420-4-E) against mouse LPA1. [Figure 6H] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210420-1-H) against mouse LPA1. [Figure 6I] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210420-3-E) against mouse LPA1. [Figure 6J] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (211222-1-G) against mouse LPA1. [Figure 6K] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (211222-1-A) against mouse LPA1. [Figure 7A] 1 is a graph showing the dose dependency of the inhibitory activity of BMS-986278 against human LPA1. [Figure 7B] 1 is a graph showing the dose dependency of the inhibitory activity of BMS-986278 against mouse LPA1. [Figure 8] 1 is a graph showing the results of evaluating the effect of 210309-4-A on changes in intracellular cAMP concentration in the absence of an LPA1 ligand, using CHO cells stably expressing human LPA1. [Figure 9] 1 is a graph showing the dose-response curve of lysophosphatidic acid at each concentration of 210309-4-A. [Figure 10A] FIG. 1 is an explanatory diagram showing the alignment of the heavy chain variable region of a humanized anti-LPA1 antibody. [Figure 10B] FIG. 1 is an explanatory diagram showing the alignment of the light chain variable region of a humanized anti-LPA1 antibody. [Figure 11A] 1 is a graph showing the results of evaluating the binding ability of an antibody (h309-4-A-BP1) to CHO cells stably expressing human LPA1. [Figure 11B] 1 is a graph showing the results of evaluating the binding ability of an antibody (h309-4-A-SS1) to CHO cells stably expressing human LPA1. [Figure 11C] 1 is a graph showing the results of evaluating the binding ability of an antibody (h309-4-A-SG1) to CHO cells stably expressing human LPA1. [Figure 11D] 1 is a graph showing the results of evaluating the binding ability of an antibody (309-4-A-Chimera) to CHO cells stably expressing human LPA1. [Figure 11E] 1 is a graph showing the results of evaluating the binding ability of an antibody (210309-4-A) to CHO cells stably expressing human LPA1. [Figure 12A] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (h309-4-A-BP1) against human LPA1. [Figure 12B] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (h309-4-A-SS1) against human LPA1. [Figure 12C]1 is a graph showing the dose dependency of the inhibitory activity of an antibody (h309-4-A-SG1) against human LPA1. [Figure 12D] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (309-4-A-Chimera) against human LPA1. [Figure 12E] 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (210309-4-A) against human LPA1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] In the present disclosure, complementarity determining region is abbreviated as CDR. In the present disclosure, the heavy chain variable region may be abbreviated as VH, the heavy chain constant region as CH, the light chain variable region as VL, and the light chain constant region as CL. In the present disclosure, the term "antibody" may be replaced with "immunoglobulin". In the present disclosure, the term "nucleic acid" may be replaced with "DNA" or "gene".

[0054] <Human LPA1> Lysophosphatidic acid receptor 1 (LPA1) is a type of G protein-coupled receptor (GPCR), which penetrates the cell membrane seven times and exists with its N-terminus facing outside the cell and its C-terminus facing inside the cell. The gene (cDNA) encoding human LPA1 has already been isolated, and the amino acid sequence of human LPA1 is also known. The sequence information can be obtained from a gene database (for example, NCBI Reference Sequence: NP_001392). As an example, the amino acid sequence of human LPA1 is shown in SEQ ID NO: 111.

[0055] Each domain of human LPA1 is considered to correspond to the following portion in the amino acid sequence shown in SEQ ID NO: 111. The left side is the amino acid number, and the right side is each domain. Note that there may be some variation in the boundaries between each domain.

[0056] 1~ 50: N-terminal domain 76-83: Intracellular first loop domain 108-121: extracellular first loop domain 145-163: Intracellular second loop domain 185-204: extracellular second loop domain 226-255: Intracellular third loop domain 281-294: extracellular third loop domain 316-364: C-terminal domain

[0057] In addition to the one shown in SEQ ID NO: 111, various variants such as amino acid substitutions are known for human LPA1. The "human LPA1" in the present disclosure includes the above variants as long as they have an extracellular domain and function as LPA1.

[0058] The gene (cDNA) encoding human LPA2 has already been isolated, and the amino acid sequence of human LPA2 is also known. The sequence information can be obtained from a gene database (NP_004711, SEQ ID NO: 119). The gene (cDNA) encoding human LPA3 has already been isolated, and the amino acid sequence of human LPA3 is also known. The sequence information can be obtained from a gene database (NP_036284, SEQ ID NO: 121).

[0059] <Anti-LPA1 antibody> The antibody disclosed in the present application specifically binds to the extracellular domain of human LPA1, does not specifically bind to the extracellular domain of human LPA2, and does not specifically bind to the extracellular domain of human LPA3.

[0060] The antibody according to one embodiment comprises a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, or 101; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, or 102; A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, or 103; A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, or 104; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, or 105; and It has a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, or 106.

[0061] In one embodiment, heavy chain CDRs 1 to 3 of the antibody satisfy any one of the above (A1) to (A11).

[0062] In one embodiment, the light chain CDRs 1 to 3 of the antibody satisfy any one of the above (B1) to (B11).

[0063] In one embodiment, the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 of the antibody satisfy any one of the above (AB1) to (AB11).

[0064] In one embodiment, the heavy chain variable region and the light chain variable region of the antibody satisfy any one of the above (C1) to (C11).

[0065] The heavy chain variable region specified by (C1) (SEQ ID NO: 7) contains heavy chain CDRs 1 to 3 specified by (A1) or (AB1) (SEQ ID NO: 1 to 3). The light chain variable region specified by (C1) (SEQ ID NO: 9) contains light chain CDRs 1 to 3 specified by (B1) or (AB1) (SEQ ID NO: 4 to 6). An example of an antibody that satisfies (A1), (B1), (AB1), or (C1) is "210309-4-A" described in the Examples below.

[0066] The heavy chain variable region specified by (C2) (SEQ ID NO: 17) contains heavy chain CDRs 1 to 3 specified by (A2) or (AB2) (SEQ ID NO: 11 to 13). The light chain variable region specified by (C2) (SEQ ID NO: 19) contains light chain CDRs 1 to 3 specified by (B2) or (AB2) (SEQ ID NO: 14 to 16). An example of an antibody that satisfies (A2), (B2), (AB2), or (C2) is "210309-1-C" described in the Examples below.

[0067] The heavy chain variable region specified by (C3) (SEQ ID NO: 27) contains heavy chain CDRs 1 to 3 specified by (A3) or (AB3) (SEQ ID NO: 21 to 23). The light chain variable region specified by (C3) (SEQ ID NO: 29) contains light chain CDRs 1 to 3 specified by (B3) or (AB3) (SEQ ID NO: 24 to 26). An example of an antibody that satisfies (A3), (B3), (AB3), or (C3) is "210309-1-G" described in the Examples below.

[0068] The heavy chain variable region specified by (C4) (SEQ ID NO: 37) contains heavy chain CDRs 1 to 3 specified by (A4) or (AB4) (SEQ ID NO: 31 to 33). The light chain variable region specified by (C4) (SEQ ID NO: 39) contains light chain CDRs 1 to 3 specified by (B4) or (AB4) (SEQ ID NO: 34 to 36). An example of an antibody that satisfies (A4), (B4), (AB4), or (C4) is "210309-2-A" described in the Examples below.

[0069] The heavy chain variable region specified by (C5) (SEQ ID NO: 47) contains heavy chain CDRs 1 to 3 specified by (A5) or (AB5) (SEQ ID NO: 41 to 43). The light chain variable region specified by (C5) (SEQ ID NO: 49) contains light chain CDRs 1 to 3 specified by (B5) or (AB5) (SEQ ID NO: 44 to 46). An example of an antibody that satisfies (A5), (B5), (AB5), or (C5) is "210309-2-D" described in the Examples below.

[0070] The heavy chain variable region specified by (C6) (SEQ ID NO: 57) contains heavy chain CDRs 1 to 3 specified by (A6) or (AB6) (SEQ ID NO: 51 to 53). The light chain variable region specified by (C6) (SEQ ID NO: 59) contains light chain CDRs 1 to 3 specified by (B6) or (AB6) (SEQ ID NO: 54 to 56). An example of an antibody that satisfies (A6), (B6), (AB6), or (C6) is "210310-1-D" described in the Examples below.

[0071] The heavy chain variable region specified by (C7) (SEQ ID NO: 67) contains heavy chain CDRs 1 to 3 specified by (A7) or (AB7) (SEQ ID NO: 61 to 63). The light chain variable region specified by (C7) (SEQ ID NO: 69) contains light chain CDRs 1 to 3 specified by (B7) or (AB7) (SEQ ID NO: 64 to 66). An example of an antibody that satisfies (A7), (B7), (AB7), or (C7) is "210420-4-E" described in the Examples below.

[0072] The heavy chain variable region specified by (C8) (SEQ ID NO: 77) contains heavy chain CDRs 1 to 3 specified by (A8) or (AB8) (SEQ ID NO: 71 to 73). The light chain variable region specified by (C8) (SEQ ID NO: 79) contains light chain CDRs 1 to 3 specified by (B8) or (AB8) (SEQ ID NO: 74 to 76). An example of an antibody that satisfies (A8), (B8), (AB8), or (C8) is "210420-1-H" described in the Examples below.

[0073] The heavy chain variable region specified by (C9) (SEQ ID NO: 87) contains heavy chain CDRs 1 to 3 specified by (A9) or (AB9) (SEQ ID NO: 81 to 83). The light chain variable region specified by (C9) (SEQ ID NO: 89) contains light chain CDRs 1 to 3 specified by (B9) or (AB9) (SEQ ID NO: 84 to 86). An example of an antibody that satisfies (A9), (B9), (AB9), or (C9) is "210420-3-E" described in the Examples below.

[0074] The heavy chain variable region specified by (C10) (SEQ ID NO: 97) contains heavy chain CDRs 1 to 3 specified by (A10) or (AB10) (SEQ ID NO: 91 to 93). The light chain variable region specified by (C10) (SEQ ID NO: 99) contains light chain CDRs 1 to 3 specified by (B10) or (AB10) (SEQ ID NO: 94 to 96). An example of an antibody that satisfies (A10), (B10), (AB10), or (C10) is "211222-1-G" described in the Examples below.

[0075] The heavy chain variable region specified by (C11) (SEQ ID NO: 107) contains heavy chain CDRs 1 to 3 specified by (A11) or (AB11) (SEQ ID NO: 101 to 103). The light chain variable region specified by (C11) (SEQ ID NO: 109) contains light chain CDRs 1 to 3 specified by (B11) or (AB11) (SEQ ID NO: 104 to 106). An example of an antibody that satisfies (A11), (B11), (AB11), or (C11) is "211222-1-A" described in the Examples below.

[0076] The amino acid sequences of SEQ ID NO:1, SEQ ID NO:21, SEQ ID NO:31, and SEQ ID NO:41 are the same. The amino acid sequences of SEQ ID NO:2 and SEQ ID NO:32 are the same. The amino acid sequences of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, and SEQ ID NO:46 are the same. The amino acid sequences of SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:35, SEQ ID NO:45, SEQ ID NO:65, SEQ ID NO:75, SEQ ID NO:85, and SEQ ID NO:105 are the same. The amino acid sequences of SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:54, SEQ ID NO:94, and SEQ ID NO:104 are the same. The amino acid sequences of SEQ ID NO:51, SEQ ID NO:91, and SEQ ID NO:101 are the same. The amino acid sequences of SEQ ID NO:52 and SEQ ID NO:92 are the same. The amino acid sequences of SEQ ID NO:53, SEQ ID NO:93, and SEQ ID NO:103 are the same. The amino acid sequences of SEQ ID NO:55 and SEQ ID NO:95 are the same. The amino acid sequences of SEQ ID NO:56, SEQ ID NO:66, SEQ ID NO:76, SEQ ID NO:86, SEQ ID NO:96, and SEQ ID NO:106 are the same. The amino acid sequences of SEQ ID NO:61, SEQ ID NO:71, and SEQ ID NO:81 are the same. The amino acid sequences of SEQ ID NO:62, SEQ ID NO:72, and SEQ ID NO:82 are the same. The amino acid sequences of SEQ ID NO:63, SEQ ID NO:73, and SEQ ID NO:83 are the same. The amino acid sequences of SEQ ID NO:64, SEQ ID NO:74, and SEQ ID NO:84 are the same.

[0077] The antibody may be a functional fragment of an antibody. Here, the term "functional fragment of an antibody" refers to a partial fragment of an antibody (i.e., an immunoglobulin) that retains at least one action against an antigen. Examples of the partial fragment include F(ab')2, Fab, Fv, disulfide-linked Fv, single-chain antibody (scFv, VH-VL), VH, and polymers thereof, as well as fusions of these with the heavy chain CH3 region. In addition, examples include each CDR such as CDR1, CDR2, and CDR3, conjugates of these CDRs, and fusions of these CDRs or CDR conjugates with the heavy chain CH3 region. In other words, the antibody of the present disclosure also includes partial fragments of the antibody as described above. A partial fragment of an antibody may also be referred to as an "antibody fragment."

[0078] When the antibody is a functional fragment, it has the following effects, for example. That is, when applying the anti-LPA1 antibody of the present disclosure to a medicine as described below, if a full-length antibody such as an IgG type is used, in addition to inhibiting the cell function mediated by the target receptor, damage to the target tissue may occur, which may lead to side effects. In such a case, if a "functional fragment of an antibody" using only the variable region is adopted, it becomes easier to avoid the side effects described above.

[0079] The antibody may be a multispecific antibody. The multispecific antibody of this embodiment has at least a first specific binding property that specifically binds to the extracellular domain of human LPA1, does not specifically bind to the extracellular domain of human LPA2, and does not specifically bind to the extracellular domain of human LPA3, and a second specific binding property different from the first specific binding property. The second specific binding property may be for human LPA1 or for another target molecule. An example of a multispecific antibody is a diabody, which is a type of bispecific antibody.

[0080] The class (isotype) of the antibody is not particularly limited. For example, it may be any class such as IgG, IgM, IgA, IgD, IgE, etc. Furthermore, the subclass of the antibody is not particularly limited. For example, in the case of IgG, it may be any subclass such as IgG1, IgG2, IgG3, IgG4, etc.

[0081] In a preferred embodiment, the antibody has an activity of blocking LPA1-dependent cellular functions. LPA1-dependent cellular functions are functions induced by activation of proteins that conjugate with the intracellular portion of LPA1 and are responsible for intracellular signal transduction, such as trimer G protein and β-arrestin. Activation of LPA1-dependent cellular functions is induced by stimulation with an LPA1 ligand, or by high expression of LPA1 independent of an LPA1 ligand. Examples of cellular functions include fluctuations in intracellular cyclic adenosine monophosphate (cAMP), fluctuations in intracellular calcium ions, GTP binding to low molecular weight G protein Rho, cell proliferation, cell migration, production of cytokines and chemokines, and cell transformation.

[0082] The extracellular domain of LPA1 to which the antibody specifically binds may be any of the N-terminal domain, the extracellular loop 1 domain, the extracellular loop 2 domain, and the extracellular loop 3 domain. The antibody may bind to only one of these extracellular domains, or to two or more of them.

[0083] The present disclosure includes antibodies that are "functionally equivalent" to the above-mentioned anti-LPA1 antibodies. Functionally equivalent antibodies include antibodies that have the same epitope as the above-mentioned antibodies. For example, the epitopes of 11 types of anti-LPA1 antibodies specifically shown in the examples described below are analyzed by an epitope mapping method using partial peptides of LPA1, etc. Then, a synthetic peptide containing the identified epitope can be used as an antigen to obtain an anti-LPA1 antibody that binds to the same epitope as the above-mentioned 11 types of anti-LPA1 antibodies. Furthermore, the amino acid sequences of the heavy chain variable region and light chain variable region of the obtained anti-LPA1 antibody can be determined, and the amino acid sequences of the heavy chain CDR1-3 and the light chain CDR1-3 can be specified.

[0084] As an example of an antibody functionally equivalent to the above antibody, the present disclosure includes an antibody that specifically binds to the extracellular domain of human LPA1, does not specifically bind to the extracellular domain of human LPA2, and does not specifically bind to the extracellular domain of human LPA3, and satisfies any of the above (C1') to (C11'). The number of substituted, added, or deleted amino acids in (C1') to (C11') is preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3. The identity of the above amino acid sequence is preferably 92% or more, more preferably 95% or more, and particularly preferably 97% or more. In a preferred embodiment, the antibody has an activity of blocking LPA1-dependent cell function.

[0085] A method for examining whether the epitopes of two antibodies are the same or not includes a competitive experiment. For example, when the binding of the 11 types of anti-LPA1 antibodies or their functional fragments, which are the first antibodies, to a receptor is competitively inhibited by the second antibody to be tested, the second antibody can be said to bind to the same epitope as the first antibody. As an example of an antibody functionally equivalent to the above antibody, the present disclosure includes a second antibody that specifically binds to the extracellular domain of human LPA1, does not specifically bind to the extracellular domain of human LPA2, does not specifically bind to the extracellular domain of human LPA3, and competitively inhibits the binding of the above antibody (first antibody) to a receptor. In a preferred embodiment, the second antibody has the activity of blocking LPA1-dependent cellular functions.

[0086] <Humanized antibodies> The present disclosure includes the above antibodies that are humanized antibodies. A humanized antibody refers to an antibody in which the CDRs are derived from a non-human animal and the other regions (such as framework and constant regions) are derived from humans. Methods for constructing and producing humanized antibodies are described below.

[0087] <Chimeric antibodies> The present disclosure includes the above antibodies that are chimeric antibodies. A chimeric antibody refers to an antibody in which the heavy chain variable region (VH) and the light chain variable region (VL) are derived from an animal other than human, and other regions such as the heavy chain constant region (CH) and the light chain constant region (CL) are derived from human. Methods for constructing and producing chimeric antibodies are described below.

[0088] <Nucleic acid> The present disclosure includes nucleic acids (DNA) encoding the antibodies, including, for example, a first nucleic acid encoding a heavy chain variable region and / or a second nucleic acid encoding a light chain variable region.

[0089] The heavy chain variable region encoded by the first nucleic acid includes, for example, a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, or 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, or 102, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, or 103. The heavy chain variable region encoded by the first nucleic acid includes, for example, heavy chain CDR1 to 3 specified in any of (A1) to (A11) above. The heavy chain variable region encoded by the first nucleic acid is, for example, one specified in any of (C1) to (C11) above. The heavy chain variable region encoded by the first nucleic acid is, for example, any one of the above (C1') to (C11').

[0090] The light chain variable region encoded by the second nucleic acid includes, for example, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, or 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, or 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, or 106. The light chain variable region encoded by the second nucleic acid includes, for example, heavy chain CDR1 to 3 specified in any of (B1) to (B11) above. The light chain variable region encoded by the second nucleic acid includes, for example, any of (C1) to (C11) above. The light chain variable region encoded by the second nucleic acid is, for example, any one of those specified by (C1') to (C11') above.

[0091] The nucleic acid may be incorporated into a vector. The vector is appropriately selected depending on the type of host cell to be introduced. The vector includes a vector for gene therapy. In this case, the vector itself can be directly administered into the body.

[0092] <cell> The present disclosure encompasses cells containing the nucleic acid. For example, a cell containing a vector into which the nucleic acid has been incorporated is an example of the cell. The type of cell is not particularly limited as long as it is capable of expressing the nucleic acid, for example, the vector is functional. Examples include animal cells (COS cells, CHO cells, etc.), yeast, bacteria (Escherichia coli, etc.), plant cells, insect cells, etc.

[0093] <Method of producing antibodies> The antibody can be produced by recombinant gene techniques, that is, by constructing recombinant cells expressing the nucleic acid and obtaining the antibody from a culture of the cells.

[0094] <Construction and preparation of humanized antibodies> A method for constructing and producing a humanized antibody will be described. As an example, a method for constructing and producing a humanized antibody having heavy chain CDR1-3 and light chain CDR1-3 specified in (AB1) above will be described. First, DNA encoding the amino acid sequences shown in SEQ ID NOs: 1-6 is prepared as DNA encoding each CDR. The DNA can be prepared using a known method such as PCR. The DNA can also be prepared by chemical synthesis.

[0095] Next, these DNAs are used to prepare DNAs encoding variable regions in which heavy chain CDR1-3 are grafted into the framework region (FR) of VH in any human antibody. Similarly, DNAs encoding variable regions in which light chain CDR1-3 are grafted into the FR of VL in any human antibody are prepared. Each of the prepared DNAs is inserted into a vector having a sequence encoding CH or CL of a human antibody to construct a humanized antibody expression vector. The constructed expression vector is introduced into a host cell to obtain a recombinant cell that expresses the humanized antibody. The recombinant cell is then cultured, and the desired humanized antibody is obtained from the culture.

[0096] Humanized antibodies having heavy chain CDR1 to 3 and light chain CDR1 to 3 specified in the above (AB2) to (AB11) can also be constructed and produced in a similar manner.

[0097] <Construction and preparation of chimeric antibodies> A method for constructing and producing a chimeric antibody will be described. As an example, a method for constructing and producing a chimeric antibody having a heavy chain variable region (VH) and a light chain variable region (VL) as specified in (C1) above will be described. First, a DNA encoding the amino acid sequence shown in SEQ ID NO: 7 is prepared as a DNA encoding VH. In addition, a DNA encoding the amino acid sequence shown in SEQ ID NO: 9 is prepared as a DNA encoding VL. The DNA can be prepared using a known method such as PCR. The DNA can also be prepared by chemical synthesis.

[0098] The obtained DNA encoding VH or VL is inserted into a vector having a sequence encoding the CH or CL of a human antibody, respectively, to construct a chimeric antibody expression vector. Vectors having a sequence encoding the CH or CL of a human antibody are commercially available. The constructed expression vector is introduced into a host cell to obtain a recombinant cell expressing the chimeric antibody. The recombinant cell is then cultured, and the desired chimeric antibody is obtained from the culture.

[0099] Chimeric antibodies having the VH and VL specified in (C2) to (C11) above can also be constructed and produced in a similar manner.

[0100] <Method for producing polyspecific antibodies> A method for producing a multispecific antibody includes, for example, linking the anti-human LPA1 antibody or a fragment thereof of the present disclosure with another antibody or a fragment thereof. Another example includes co-expressing the anti-human LPA1 antibody or a fragment thereof of the present disclosure with another antibody or a fragment thereof in a host cell. Other linking methods include chemical coupling, gene fusion, non-covalent association, etc.

[0101] An example of a multispecific antibody (bispecific antibody) that has been put to practical use is B-LINCYTO (registered trademark), which binds to CD3 and CD19. Another example is Hemlibra (registered trademark), which binds to coagulation factor IXa and coagulation factor X. Techniques for producing multispecific antibodies are known in the art, and these known production techniques can also be applied to the anti-human LPA1 antibody of the present disclosure.

[0102] <Purification method> The method for purifying the antibody is not particularly limited, and known methods can be used. For example, the culture supernatant of the recombinant cell can be collected, and the antibody can be purified by combining known methods such as various types of chromatography, salting out, dialysis, and membrane separation. When the antibody isotype is IgG, it can be easily purified by affinity chromatography using protein A.

[0103] <Modified antibody> The antibody of the present disclosure may be a modified antibody to which another molecule is bound. Examples of the other molecule include peptides, proteins, low molecular weight compounds, radioisotopes, and light absorbers. Methods for binding to other molecules include chemical coupling, gene fusion, and non-covalent association. When the other molecule to be bound is an antibody, the modified antibody may be a multispecific antibody. In this respect, a multispecific antibody may be considered as a type of modified antibody.

[0104] In a preferred embodiment, the modified antibody is an antibody-drug conjugate (ADC). Drugs to be conjugated include antifibrotic agents, small molecule anticancer drugs (cytotoxins, chemotherapeutic drugs), proteins or polypeptides having biological activity such as cytokines, radioisotopes, light absorbers, etc. These drugs can be conjugated directly or indirectly via a linker or chelator to produce an antibody-drug conjugate.

[0105] Examples of antibody-drug conjugates that have been put to practical use include Enhertz (registered trademark), which is an antibody-drug conjugate conjugated with a low molecular weight anticancer drug, and which is an antibody-drug conjugate conjugated with a camptothecin derivative to trastuzumab. 90 Examples of the antibody-drug conjugates conjugated with a light absorber include Zevalin (registered trademark) labeled with IR Dye700DX. Examples of the antibody-drug conjugates conjugated with a light absorber include Acalux (registered trademark) in which cetuximab is bound with IR Dye700DX. The drugs contained in these antibody-drug conjugates can also be applied to the anti-human LPA1 antibody of the present disclosure. Furthermore, examples of suitable drugs used in antibody-drug conjugates are known in the art, and these known drugs can also be applied to the anti-human LPA1 antibody of the present disclosure.

[0106] <Medicine> The present disclosure includes a pharmaceutical agent containing the antibody as an active ingredient. The pharmaceutical agent may be a pharmaceutical composition containing the antibody and a pharma- ceutical acceptable carrier. Preferably, the pharmaceutical agent blocks LPA1-dependent cell function.

[0107] In a preferred embodiment, the medicament is used for treating a disease, disorder, or condition caused by the impairment of LPA1-dependent cell function. Examples of the disease, disorder, or condition caused by the impairment of LPA1-dependent cell function include fibrosis selected from the group consisting of liver fibrosis, renal fibrosis, pulmonary fibrosis, skin fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis, and other fibrotic diseases.

[0108] Examples of the liver fibrosis include fibrosis selected from the group consisting of nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver cirrhosis, ischemia-reperfusion, post-liver transplant injury, necrotizing hepatitis, hepatitis B, hepatitis C, primary biliary cirrhosis, and primary sclerosing cholangitis. Examples of the liver cirrhosis include at least one selected from the group consisting of alcohol-induced, drug-induced, and chemical-induced.

[0109] Examples of the renal fibrosis include fibrosis selected from the group consisting of proliferative glomerulonephritis, sclerosing glomerulonephritis, nephrogenic fibrosing dermopathy, diabetic nephropathy, renal tubulointerstitial fibrosis, and focal segmental glomerulosclerosis.

[0110] Examples of the pulmonary fibrosis include fibrosis selected from the group consisting of pulmonary interstitial fibrosis, drug-induced sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar damage disease, pulmonary hypertension, and neonatal bronchopulmonary dysplasia.

[0111] Examples of said skin fibrosis include fibrosis selected from the group consisting of scleroderma, keloid scarring, psoriasis, hypertrophic scarring, and pseudoscleroderma.

[0112] Examples of said cardiovascular fibrosis include fibrosis selected from the group consisting of atherosclerosis, coronary restenosis, congestive cardiomyopathy, heart failure, heart transplantation, and myocardial fibrosis.

[0113] Examples of said gastrointestinal fibrosis include fibrosis selected from the group consisting of collagenous colitis, villous atrophy, crypt hyperplasia, polyp formation, Crohn's disease fibrosis, gastric ulcer healing, and abdominal adhesion scarring.

[0114] The fibrosis includes conditions resulting from bone-related fibrotic diseases and may be rheumatoid pannus formation.

[0115] Other examples of diseases, disorders, or conditions in which impaired LPA1-dependent cell function is implicated include cell proliferative disorders such as blood cancers and solid cancers (control of tumor cell proliferation, tumor invasion and metastasis, and angiogenesis).

[0116] Examples of the blood cancer include blood cancers selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

[0117] Examples of the solid cancer include solid cancers selected from the group consisting of breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, hepatocellular carcinoma, prostate cancer, urothelial carcinoma, renal cell carcinoma, and various squamous cell carcinomas. Examples of the squamous cell carcinoma include squamous cell carcinomas selected from the group consisting of oral squamous cell carcinoma, esophageal squamous cell carcinoma, and pharyngeal squamous cell carcinoma.

[0118] Other examples of diseases, disorders or conditions in which impaired LPA1-dependent cellular function is implicated as a cause include pain including fibromyalgia, cancer pain and diabetic neuropathy; inflammatory and autoimmune diseases including rheumatoid arthritis, sepsis, chronic obstructive pulmonary disease, inflammatory bowel disease, transplant organ rejection, Guillain-Barré syndrome and multiple sclerosis; metabolic diseases including obesity and insulin-resistant diabetes; cardiovascular disorders including cerebral infarction, hypertensive nephropathy and Raynaud's phenomenon; nervous system disorders including hydrocephalus, schizophrenia, depression and dementia; urological diseases including prostatic hyperplasia and urinary incontinence; and eye diseases including ischemic retinopathy, diabetic retinopathy and age-related macular degeneration.

[0119] <Administration Method> The above-mentioned medicine can be administered orally or parenterally, systemically or locally. Examples of the administration form include injections, nasal administration, pulmonary administration, and transdermal administration. In the case of injections, the medicine can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. In addition, the administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the above-mentioned antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, in the range of 0.001 to 100,000 mg of antibody per patient. However, the dosage of the above-mentioned antibody is not limited to these ranges.

[0120] <Formulation> The above-mentioned medicines can be formulated according to conventional methods (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). The above-mentioned medicines can contain pharma- ceutically acceptable carriers and additives. Examples of the carriers and additives include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphate, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents, but are not limited to these, and other commonly used carriers and the like can be used as appropriate. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. In addition, other low molecular weight polypeptides, proteins such as serum albumin, gelatin, immunoglobulin, etc., amino acids such as glycine, glutamine, asparagine, arginine, lysine, etc. may be included.

[0121] When preparing an aqueous solution for injection, examples of the solution include physiological saline, isotonic solutions containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with an appropriate solubilizing agent, such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc. If necessary, the antibody can be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylic acid], etc.) or made into a colloid drug delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) (see, for example, "Remingto's Pharmaceutical Science 16th edition", Oslo Ed. 1980)).

[0122] Additionally, techniques for sustained release of drugs are known and may be applied to the above-mentioned pharmaceuticals (Langer et al., J. Biomed. Master. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:9(8-105 (1982); U.S. Pat. No. 3,773,919; European Patent Publication No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); European Patent Publication No. 133,988).

[0123] <Application to gene therapy> The above nucleic acid may be incorporated into a gene therapy vector or into mRNA that can be translated into a protein in vivo to produce a gene therapy drug. Methods for administering the gene therapy drug (recombinant vector) include direct administration using a naked plasmid, administration by packaging the vector in a liposome, administration by incorporating the vector into various virus vectors such as retrovirus vectors, adenovirus vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, and HVJ vectors (see Adolph, "Virus Genome Methods," CRC Press, Florid (1996)), administration by coating the vector on a bead carrier such as colloidal gold particles (e.g., International Publication WO 93 / 17706 pamphlet), and the like.

[0124] The gene therapy drug may be administered by any method as long as the antibody is expressed in vivo and can exert its action. Preferably, a sufficient amount is administered via a suitable parenteral route, such as intravenous, intraperitoneal, subcutaneous, intradermal, intraadipose tissue, intramammary tissue, inhalation, intramuscular, etc., by injection, infusion, gas-induced particle bombardment (by electron gun, etc.), mucosal route such as nasal drip, etc. Furthermore, the gene therapy drug may be administered ex vivo to cells using liposome transfection, particle bombardment (U.S. Pat. No. 4,945,050), or viral infection, and then reintroduced into the animal.

[0125] Furthermore, the present disclosure also includes a method and a therapeutic agent for treating a disease or disorder caused by abnormal enhancement of LPA1-dependent cell function in a mammal suffering from said disease. The term "treatment" as used herein means preventing or alleviating the progression and worsening of the pathological condition of a disease in a mammal that is at risk of contracting or is contracting the disease, and is used to mean a therapeutic procedure aimed at preventing or alleviating the progression and worsening of the symptoms of the disease. In addition, the term "disease" refers to diseases in general that develop due to abnormal enhancement of LPA1-dependent cell functions, and is not particularly limited, and includes, for example, hepatic fibrosis, renal fibrosis, pulmonary fibrosis, skin fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis, and other fibrotic diseases. In addition, the term includes blood cancer, solid cancer, pain, inflammatory disease, autoimmune disease, metabolic disease, cardiovascular disorder, nervous system disorder, urological disease, and ophthalmological disease. The "mammal" to be treated means any animal classified as a mammal, and is not particularly limited, and includes, for example, pet animals such as dogs, cats, and rabbits, as well as humans, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is human.

[0126] <Antibody immobilization carrier> The present disclosure includes a carrier on which the above-mentioned antibody is immobilized (antibody-immobilized carrier). In a preferred embodiment, the antibody-immobilized carrier is used to remove LPA1-expressing cells from a body fluid containing LPA1-expressing cells by contacting the carrier. An example of the body fluid is blood. The above-mentioned antibody immobilized on the carrier may be one type or two or more types.

[0127] Specific examples of the antibody-immobilized carrier of the present disclosure include those in which the antibody is immobilized on a water-insoluble carrier and filled in a container. Any material can be used as the water-insoluble carrier, but preferred materials in terms of moldability, sterilizability, and low cytotoxicity include synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, and polyurethane, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alginate, inorganic materials such as hydroxyapatite, glass, alumina, and titania, and metal materials such as stainless steel and titanium.

[0128] The shape of the carrier may be granular, cotton-like, woven fabric, nonwoven fabric, sponge-like porous body, flat plate, etc., but granular, cotton-like, woven fabric, nonwoven fabric, sponge-like porous body are preferred in terms of the large surface area per volume. For example, peripheral blood can be passed through a porous filter in which a water-insoluble carrier with an antibody immobilized thereon is packed in advance in a container, and LPA1 expressing cells involved in the disease can be efficiently removed.

[0129] A kit for removing LPA1-expressing cells can be prepared by combining the antibody-immobilized carrier of the present disclosure with other components, such as an anticoagulant and an extracorporeal circulation circuit.

[0130] <Other disclosures> The present disclosure includes a method for treating a disease, disorder, or condition involving impaired LPA1-dependent cellular function, comprising administering an effective amount of the antibody to a patient with the disease, disorder, or condition. The present disclosure includes the antibody for use in treating a disease, disorder, or condition involving impaired LPA1-dependent cellular function. The present disclosure includes the use of the antibody for producing a medicament for treating a disease, disorder, or condition involving impaired LPA1-dependent cellular function. The disease, disorder, or condition includes at least tissue fibrosis, cell proliferative disease, pain, inflammatory disease, autoimmune disease, metabolic disease, cardiovascular disorder, urological disease, or ophthalmological disease. EXAMPLES

[0131] [Example 1] Immunization of mice for producing anti-LPA1 antibodies (1) Construction of a genetic immunization vector expressing a fusion protein of LPA1 and GroEL subunits An artificial gene was synthesized by adding the GCTAGC sequence to the 5' end and the GTCGAC sequence to the 3' end based on human LPA1 (NP_001392, SEQ ID NO: 111) registered in the NCBI Gene Bank. After introducing this artificial gene into a cloning vector, the resulting vector was cleaved at the NheI and SalI sites to prepare a DNA fragment. pCI-hCCR7·GroEL (described in International Publication No. 2012 / 043533 and Japanese Patent No. 5315495) was digested with the restriction enzymes NheI and SalI, and the DNA fragment prepared above was inserted to construct the immunization vector pCI-hLPA1·GroEL. This vector expresses a fusion protein of human LPA1 and GroEL.

[0132] The same procedure was performed using mouse LPA1 (NP_034466, SEQ ID NO: 112) instead of human LPA1 to construct the immunization vector pCI-mLPA1·GroEL, which expresses a fusion protein between mouse LPA1 and GroEL.

[0133] (2) DNA immunity The vector pCI-hLPA1·GroEL or pCI-mLPA1·GroEL was dissolved in PBS to a concentration of 2 mg / mL to prepare an immunization composition. 25 μL of this immunization composition was applied to the thigh muscles of both legs of 8-week-old mice of various strains by in vivo electroporation (day 0). Thus, DNA immunization was performed with 50 μg of pCI-hLPA1·GroEL or pCI-mLPA1·GroEL in both legs, i.e., 100 μg per mouse per time. Thereafter, DNA immunization was performed in the same manner on days 14, 28, 42, and 56. Furthermore, blood was collected before immunization and on days 21, 35, and 63 after immunization, and serum was prepared.

[0134] (3) Preparation of cells stably expressing LPA1 An artificial gene was synthesized based on human LPA1 (NP_001392, SEQ ID NO: 111) registered in the NCBI Gene Bank, and introduced into a pCIneo vector (Promega) to construct pCIneo-hLPA1. pCIneo-hLPA1 was introduced into CHO-K1 cells using Lipofectamine 2000 (ThermoFisher Scientific). The resulting cells were cultured in a medium containing G418 (Promega), and cells stably expressing human LPA1 with G418 resistance were cloned (hereinafter referred to as "human LPA1 stably expressing CHO cells").

[0135] An artificial gene was synthesized based on mouse LPA1 (NP_034466, SEQ ID NO: 112) registered in the NCBI Gene Bank, and introduced into pEF5 / FRT / V5-DEST (ThermoFisher Scientific) to construct pEF-FRT-mLPA1. pEF-FRT-mLPA1 and pOG44 plasmid (ThermoFisher Scientific) were simultaneously introduced into Flp-In-CHO cells (ThermoFisher Scientific) using Lipofectamine 2000. The obtained cells were cultured in a medium containing hygromycin (ThermoFisher Scientific), and cells stably expressing mouse LPA1 with hygromycin resistance were cloned (hereinafter referred to as "mouse LPA1 stably expressing CHO cells").

[0136] (4) Flow cytometry to evaluate the binding of serum antibodies against human LPA1 and mouse LPA1 Human LPA1 stably expressed CHO cells, mouse LPA1 stably expressed CHO cells, and CHO-K1 cells or CHO-FlpIn cells (hereinafter referred to as negative control cells) were washed with FACS buffer (PBS containing 1% FBS), and Fc Block (Cytec Biosciences) was added in an amount of 1 / 500 of the cell suspension, and blocking was performed at 4°C for 30 minutes. After blocking, each cell was incubated with 50-fold diluted pre-immune and post-immune serum. Furthermore, each cell was washed with FACS buffer, and a phycoerythrin-labeled anti-mouse IgG antibody (Southern Biotech) was added as a secondary antibody, and the binding of each cell to the anti-human LPA1 antibody and anti-mouse LPA1 antibody in the serum was evaluated using a flow cytometer iQue (Sartorius). In the post-immunization serum, there were antibodies that bound to human LPA1 stably expressed CHO cells or mouse LPA1 stably expressed CHO cells, but did not bind to the negative control cells. Individuals with elevated specific antibody titers against LPA1 were subjected to boosting and sampling.

[0137] (5) Boosting with Transiently Expressing Cells and Sampling Vector pCI-hLPA1 or pCI-mLPA1 was transfected into HEK293FT cells (ThermoFisher Scientific) using Lipofectamine 2000 to transiently express human LPA1 or mouse LPA1. The cells were administered into the spleen and intraperitoneal cavity of individuals with elevated antibody titers, and 3 days later, the spleen, inguinal lymph nodes, and iliac lymph nodes were collected. Spleen cells and lymph node-derived cells were isolated from each collected tissue, suspended in CELLBANKER 1plus (Nihon Zenyaku Kogyo), and stored at -80°C until selection of specific antibody-producing lymphocytes.

[0138] [Example 2] Preparation of antibodies (1) Selection of specific antibody-producing lymphocytes using microwells Selection of specific antibody-producing lymphocytes using microwells was carried out according to the methods described in International Publication No. 2020 / 171020 and Japanese Patent No. 6881801. That is, human LPA1 stably expressing CHO cells or mouse LPA1 stably expressing CHO cells were suspended in F-12 medium (containing 10% FBS and penicillin / streptomycin) and 3.5 × 10 5 A cell suspension of 500 μL per cell was prepared. This cell suspension was filled into a microchamber (As One Co., Ltd.) for a cell picking system. The microchamber was centrifuged 5 times at 300 rpm for 1 minute, and prepared so that one or two human LPA1 stably expressed CHO cells or mouse LPA1 stably expressed CHO cells were stored in each microwell. After washing the microchamber with F-12 medium, 500 μL of F-12 medium was added. The cells were incubated at 37°C for 1 hour in a CO2 incubator, and the human LPA1 stably expressed CHO cells or mouse LPA1 stably expressed CHO cells were attached to the bottom of the microwell. A CytoRed solution (Dojindo Laboratories) adjusted to a concentration of 10 nM in F-12 medium was added, and the cells were further incubated at 37°C for 1 hour to stain the cells. After washing three times with F-12 medium to remove excess CytoRed, 1 mL of F-12 medium was filled into the microchamber.

[0139] Antibody-producing cells were enriched from the cells collected in Example 1(5) after DNA immunization using EasySep Mouse Biotin Positive Selection Kit (STEMCELL TECHNOLOGIES). 5The antibody-producing cells were suspended in 500 μL of F-12 medium and filled into the microchamber. The microchamber was centrifuged 5 times at 300 rpm for 1 minute to prepare the microwells so that 1 or 2 antibody-producing cells were stored in each well. After washing the microchamber with medium, an appropriate amount of medium was added and incubated at 37 ° C for 30 minutes to allow the antibody-producing cells to secrete the antibody. After washing the microwells and removing the supernatant, Alexa Fluor 488-labeled anti-mouse IgG antibody (Abcam) diluted 500-fold with RPMI1640 (containing 10% FBS) was added and incubated at 37 ° C for 30 minutes. After washing three times with RPMI1640 (containing 1% FBS without phenol red), 1 mL of RPMI1640 was added. The microchamber was set in a cell picking system (As One), and information on transmitted light images and two types of fluorescent images of all the microwells was obtained. Fluorescence detection of CytoRed was performed under conditions of an excitation wavelength of 543 nm and a fluorescence wavelength of 593 nm. Fluorescence detection of Alexa Fluor 488 was performed under conditions of an excitation wavelength of 482 nm and a fluorescence wavelength of 536 nm. From the microwells in which it was determined that antibodies binding to the surface of human LPA1 stably expressed CHO cells or mouse LPA1 stably expressed CHO cells were present based on the scanned images of transmitted light, CytoRed, and Alexa Fluor 488, antibody-producing cells were collected into a cell lysate using a capillary (As One Corporation) with a diameter of several μm to several tens of μm.

[0140] (2) Isolation of antibody genes from collected antibody-producing cells Antibody genes were obtained from antibody-producing cells by the MAGrahd method (Kurosawa N, Yoshioka M, Fujimoto R, Yamagishi F, Isobe M. "Rapidproduction of antigen-specific monoclonal antibodies from a variety of animals." BMC Biol. 2012; 10: 80). That is, 5 μL of the cell lysate collected in (1) was mixed with 5 μg of oligo dT magnet, and cell-derived mRNA was captured on the oligo dT magnet. Using a MAGrahd reactor tray and a neodymium magnet, cDNA synthesis was performed by reverse transcription after washing the oligo dT magnet with a washing solution. After further washing the magnet, a 5' terminal translational reaction was performed. Using the above synthesized cDNA, the genes of the antibody heavy chain variable region (VH region) and the antibody light chain variable region (VL region) were isolated and amplified by the 5' race PCR method. In order to increase the specificity of the amplified product, PCR was performed twice. In the first PCR, a first forward primer (SEQ ID NO: 113) that amplifies the VH region and the VL region in common, a VH first reverse primer (SEQ ID NO: 114) that specifically amplifies the VH region, and a VL first reverse primer (SEQ ID NO: 115) that specifically amplifies the VL region were mixed and used. In the second PCR, the amplification product of the first PCR was used as a template, and the second forward primer (SEQ ID NO: 116) and the VH second reverse primer (SEQ ID NO: 117) that specifically amplifies the VH region were used as primers for amplification of the VH region, and the second forward primer (SEQ ID NO: 116) and the VL second reverse primer (SEQ ID NO: 118) that specifically amplifies the VL region were used as primers for amplification of the VL region. When the sample after the second PCR was subjected to agarose gel electrophoresis, gene amplification products corresponding to the VH region at a position of about 750 bp and the VL region at a position of about 550 bp, respectively, were confirmed.

[0141] (3) Construction of antibody expression unit An antibody expression unit was constructed by the TS-jPCR method (Yoshioka M, Kurosawa N, Isobe M. "Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells." BMC Biotechnol. 2011; 11: 75). That is, the VH region gene amplified in (2), the antibody heavy chain constant region gene, and a gene containing a promoter region necessary for gene expression were fused by PCR to construct an antibody expression unit expressing a full-length antibody heavy chain. Similarly, the VL region gene amplified in (2), the antibody light chain constant region gene, and a gene containing a promoter region necessary for gene expression were fused by PCR to construct an antibody expression unit expressing a full-length antibody light chain.

[0142] (4) Introducing an antibody expression unit into mammalian cells Expi293F cells (ThermoFisher Scientific) were cultured at 7.5 × 10 in a 6-well cell culture plate. 6 The cells were seeded at 3 mL per well. The heavy and light chain antibody expression units constructed in (3) were co-transfected into Expi293F cells using Expifectamine 293 Reagent (ThermoFisher Scientific). The cell supernatant was collected 5 days after transfection and used to evaluate the binding of the produced antibodies.

[0143] (5) Evaluation of antibody binding using flow cytometry Human LPA1 stably expressing CHO cells or mouse LPA1 stably expressing CHO cells and negative control cells were washed with FACS buffer to obtain a cell concentration of 1 × 10 7The cells were suspended in FACS buffer at a concentration of 1 × 10 cells / mL. Fc Block (Cytec Biosciences) was added at a volume of 1 / 500 of the cell suspension, and blocking was performed at 4°C for 30 minutes. After blocking, 2 × 10 5 The cells were suspended at a ratio of cells / 50 μL. This cell suspension was mixed with the cell supernatant collected in (4) and incubated at 4°C for 1 hour. After incubation, the cells were washed twice with 100 μL of FACS buffer. 50 μL of a dilution of phycoerythrin-labeled anti-mouse IgG antibody (Southern Biotech) was added to each well as a secondary antibody, and incubated at 4°C for 1 hour. The cells were washed twice with 100 μL of FACS buffer, suspended in 80 μL of FACS buffer, and the binding of the antibody was evaluated by measuring the fluorescence intensity of the cell surface using a flow cytometer iQue (Sartorius). The antibody unit that bound to human LPA1 stably expressing CHO cells or mouse LPA1 stably expressing CHO cells and did not bind to the negative control cells was determined as the primary hit antibody.

[0144] [Example 3] Screening of antibodies by evaluating inhibition of intracellular cAMP signaling Purified antibodies were prepared from the culture supernatants corresponding to the primary hit antibodies in Example 2 by Protein A affinity purification according to standard methods and subjected to the following evaluations: Intracellular cyclic adenosine monophosphate (cAMP) concentration was measured using a LANCE Ultra cAMP Kit (PerkinElmer).

[0145] Human LPA1-stably expressing CHO cells and mouse LPA1-stably expressing CHO cells were cultured for 24 hours in FBS-free Ham's F-12 medium containing 0.5% BSA, 100 units / mL penicillin, and 100 μg / mL streptomycin. The cells were harvested from the cell culture dish with enzyme-free cell dissociation buffer (ThermoFisher Scientific) and washed with Hanks' Balanced Salt Solution (HBSS). The cells were cultured at 2 × 10 5 The cells were suspended at 1000 x 1000 / mL and 12.5 μL (2,500 cells) of cells were dispensed into an OptiPlate-96well (PerkinElmer). 6.25 μL of antibody diluted to 400 nM with stimulation buffer was added to each well (final concentration 100 nM) and left to stand at room temperature for 15 minutes. Next, 6.25 μL of a mixture of 12 μM forskolin (final concentration 3 μM) and 200 nM oleoyl-α-lysophosphatidic acid sodium salt (Merck) (final concentration 50 nM) was added and left to stand at room temperature for 30 minutes. Using the cAMP Detection Buffer provided with the kit, 12.5μL of Eu-cAMP tracer solution and ULight-anti-cAMP antibody solution, which were diluted according to the instruction manual, were added and left to stand at room temperature for 1 hour, after which TR-FRET was measured using a plate reader EnVision2104 (PerkinElmer). The inhibition rate (%) was calculated as the relative inhibition rate (%) of intracellular cAMP signaling, normalized by setting the actual measured value when forskolin and lysophosphatidic acid were added as 0% inhibition rate and the actual measured value when forskolin only was added as 100% inhibition rate.

[0146] Among the primary hits, 11 types of antibodies, namely 210309-1-C, 210309-1-G, 210309-2-A, 210309-2-D, 210309-4-A, 210310-1-D, 210420-4-E, 210420-1-H, 210420-3-E, 211222-1-G, and 211222-1-A, which inhibited lysophosphatidic acid-induced intracellular cAMP signaling in human LPA1-expressing cells and mouse LPA1-expressing cells, were selected as antibodies having the activity of blocking LPA1-dependent cellular functions.

[0147] Table 1 shows the types of antigens against which the 11 antibodies were obtained, as well as the inhibition rates of lysophosphatidic acid-induced intracellular cAMP signaling in human and mouse LPA1 expressing cells.

[0148] [Table 1]

[0149] [Example 4] Screening of antibodies by evaluating binding to lysophosphatidic acid receptors using flow cytometry

[0150] To evaluate the binding specificity of lysophosphatidic acid receptor subtypes to LPA1, LPA2, and LPA3, artificial genes were synthesized based on human LPA1 (SEQ ID NO: 111), mouse LPA1 (SEQ ID NO: 112), and human LPA2 (NP_004711, SEQ ID NO: 119), mouse LPA2 (NP_064412, SEQ ID NO: 120), human LPA3 (NP_036284, SEQ ID NO: 121), and mouse LPA3 (NP_075359, SEQ ID NO: 122) registered in the Gene Bank, with a FLAG tag sequence added to the 5' end of each gene. 10 μg of expression vectors containing each synthetic gene were introduced into HEK293FT cells using Lipofectamine 2000, and human LPA1 gene-introduced cells, mouse LPA1 gene-introduced cells, human LPA2 gene-introduced cells, mouse LPA2 gene-introduced cells, human LPA3 gene-introduced cells, and mouse LPA3 gene-introduced cells were prepared.

[0151] The next day, the cells were washed with PBS, detached from the cell culture dish using enzyme-free cell dissociation buffer (ThermoFisher Scientific), washed with PBS, and collected by centrifugation. Goat serum (ThermoFisher Scientific) was diluted 2-fold with FACS buffer (PBS containing 1% FBS) and diluted to 1 × 10 7 The cells were suspended at a concentration of 4 × 10 6 The cells were suspended in FACS buffer at a concentration of 1 × 10 / mL and 25 μL (1 × 10 5 The cells were then dispensed in aliquots of 1000 x 1000 cells.

[0152] 20μg / mL of 11 kinds of antibodies selected in Example 3 (210309-1-C, 210309-1-G, 210309-2-A, 210309-2-D, 210309-4-A, 210310-1-D, 210420-4-E, 210420-1-H, 210420-3-E, 211222-1-G, 211222-1-A), 20μg / mL of anti-FLAG mouse IgG antibody (Sigma-Aldrich) or mouse isotype control antibody (FUJIFILM Wako Pure Chemical Industries, Ltd.) were added at 25μL each and left to stand at 4℃ for 1 hour. Cells were collected by centrifugation and washed with 120μL of FACS buffer. This washing operation was performed twice. Secondary antibody, Goat Anti-Mouse IgG H&L (DyLight 650) (Abcam), diluted 500-fold with FACS buffer was dispensed in 25 μL portions into the cell pellet, suspended, and allowed to stand at 4°C for 1 hour. The cells were washed twice, suspended in 50 μL of FACS buffer, and the entire amount was transferred to a V-bottom 96-well plate (Greiner), and antibody binding was measured using a flow cytometer (Agilent Novocyte).

[0153] As a representative example, the histogram of the flow cytometer of 210309-4-A is shown in Figure 1. We investigated whether the histogram shifts to the right when transfected cells are used compared to when non-transfected cells are used. First, when anti-FLAG mouse IgG antibody was used as the primary antibody, a right shift was observed in all of the human LPA1 transfected cells, human LPA2 transfected cells, human LPA3 transfected cells, mouse LPA1 transfected cells, mouse LPA2 transfected cells, and mouse LPA3 transfected cells. On the other hand, when mouse isotype control antibody was used as the primary antibody or when only the secondary antibody was used, no right shift was observed in any of the transfected cells. This confirmed that all of the receptors were expressed on the cell surface.

[0154] On the other hand, when 210309-4-A was used as the primary antibody, a right shift in the histogram was observed only in the human LPA1 gene-transfected cells and the mouse LPA1 gene-transfected cells, indicating that 210309-4-A specifically binds to the extracellular domains of human LPA1 and mouse LPA, but does not bind to the extracellular domains of human LPA2, human LPA3, mouse LPA2, or mouse LPA3.

[0155] Results similar to those shown in FIG. 1 were obtained for antibodies other than 210309-4-A.

[0156] The binding specificity of 11 kinds of antibodies, including antibodies other than 210309-4-A, is shown in Table 2. Those in which a rightward shift in the histogram was observed are indicated as "+", and those in which no shift was observed are indicated as "-". All of the 11 kinds of antibodies were observed to specifically bind to the extracellular domains of human LPA1 and mouse LPA, but not to the extracellular domains of human LPA2, human LPA3, mouse LPA2, or mouse LPA3, and it was confirmed that these antibodies specifically bind to the extracellular domain of human LPA1, do not specifically bind to the extracellular domain of human LPA2, and do not specifically bind to the extracellular domain of human LPA3.

[0157] [Table 2]

[0158] [Example 5] Determination of CDR sequences of antibody heavy chain variable region and antibody light chain variable region The 11 types of antibody genes selected in Example 3, 210309-1-C, 210309-1-G, 210309-2-A, 210309-2-D, 210309-4-A, 210310-1-D, 210420-4-E, 210420-1-H, 210420-3-E, 211222-1-G, and 211222-1-A, were used and cloned into a pET vector by homologous sequence-selective recombination cloning (Kurosawa N, Yoshioka M, Isobe M. "Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells." BMC Biotechnol. 2011; 11: 39). Furthermore, in order to analyze the sequence of the cloned antibody heavy chain variable region (VH region), sequence analysis was performed using the VH second reverse primer (SEQ ID NO: 117) used in Example 2 to determine the sequence of the VH region. Similarly, in order to analyze the sequence of the cloned antibody light chain variable region (VL region), sequence analysis was performed using the VL second reverse primer (SEQ ID NO: 118) used in Example 2 to determine the sequence of the VL region. CDRs were determined by combining the Kabat numbering method and the IMGT numbering method, and the region comprehensively including the regions determined by both numbering methods was determined as the CDR.

[0159] The amino acid sequences (AA) of the heavy chain variable region, heavy chain CDR1 to 3, light chain variable region, and light chain CDR1 to 3 of each antibody, as well as the base sequences of the DNA encoding the heavy chain variable region and light chain variable region, respectively, are summarized in Tables 3-1 to 3-11.

[0160]

Table 3-1

[0161]

Table 3-2

[0162]

Table 3-3

[0163]

Table 3-4

[0164]

Table 3-5

[0165]

Table 3-6

[0166]

Table 3-7

[0167]

Table 3-8

[0168]

Table 3-9

[0169]

Table 3-10

[0170] [Table 3-11]

[0171] [Example 6] Evaluation of binding to LPA1 by flow cytometry Artificial genes were synthesized by connecting the heavy chain constant region and the κ chain constant region of mouse IgG1 to the VH and VL regions of 210309-1-C, 210309-1-G, 210309-2-A, 210309-2-D, 210309-4-A, 210310-1-D, 210420-4-E, 210420-1-H, 210420-3-E, 211222-1-G, and 211222-1-A determined in Example 5. The antibody genes were introduced into CHO cells, and purified antibodies were produced from the culture medium by affinity chromatography and subjected to the following tests. The purity of all purified antibodies was confirmed to be 90% or more when tested by SDS-PAGE.

[0172] Human LPA1- and mouse LPA1-stably expressed CHO cells were washed with PBS, detached from the cell culture dish using enzyme-free cell dissociation buffer (ThermoFisher Scientific), washed with PBS, and collected by centrifugation. Human LPA1- and mouse LPA1-stably expressed CHO cells were diluted 500-fold with FACS buffer to 1 × 10 7 The cells were suspended at a concentration of 4 × 10 6 The cells were suspended in FACS buffer to a concentration of 1 × 10 / mL and 25 μL (1 × 10 5The primary antibodies were 11 types of LPA1 antibodies produced and purified by the above method and mouse isotype control antibodies (FUJIFILM Wako Pure Chemical Corporation), which were serially diluted and added at 25 μL each, and then left to stand at 4 ° C for 1 hour. The cells were collected by centrifugation and washed with 120 μL of FACS buffer. This washing procedure was repeated twice. The secondary antibody, Goat Anti-Mouse IgG H&L (DyLight 650) (Abcam), which was diluted 500 times with FACS buffer, was dispensed at 25 μL each into the cell pellet, suspended, and then left to stand at 4 ° C for 1 hour. The cells were washed twice, suspended in 50 μL of FACS buffer, and antibody binding was measured using a flow cytometer (Agilent Novocyte).

[0173] A graph was created in which the antibody concentration is plotted on the horizontal axis and the median fluorescence intensity of the flow cytometer histogram is plotted on the vertical axis. All 11 types of antibodies were confirmed to bind to human LPA1 stably expressing CHO cells and mouse LPA1 stably expressing CHO cells in a dose-dependent manner. The results of binding of each antibody to human LPA1 stably expressing CHO cells are shown in Figures 2A to 2K, and the results of binding of each antibody to mouse LPA1 stably expressing CHO cells are shown in Figures 3A to 3K. The 50% binding concentration (EC50) was calculated and summarized in Table 4.

[0174] [Table 4]

[0175] Next, the binding of LPA1 antibodies to LPA1 endogenously expressed on the cell surface was evaluated. A human lung fibroblast cell line (IMR-90), in which human LPA1 gene expression has been confirmed, was used for verification. Using the same procedure as above, the binding of 10 μg / mL purified LPA1 antibodies and mouse isotype control antibodies was measured using a flow cytometer (Agilent Novocyte). As a result, it was confirmed that 11 types of antibodies specifically bound to IMR-90. A histogram comparing the 11 types of antibodies with the mouse isotype control antibody is shown in Figure 4.

[0176] [Example 7] Evaluation of inhibition of intracellular cAMP signaling The antibody produced in Example 6 was subjected to the following tests.

[0177] The change in intracellular cyclic adenosine monophosphate (cAMP) concentration was measured using the LANCE Ultra cAMP Kit (PerkinElmer) in accordance with the method described in Example 3. 5 12.5μL of human LPA1 stably expressed CHO cells and mouse LPA1 stably expressed CHO cells with a cell concentration of 100μL / mL were added with 6.25μL of serially diluted purified LPA1 antibody or LPA1 small molecule antagonist BMS-986278 (MedKoo) and left to stand at room temperature for 15 minutes. 6.25μL of a mixture of 12μM Forskolin (Merck) (final concentration 3μM) and 200nM oleoyl-L-α-lysophosphatidic acid sodium salt (final concentration 50nM) was added and left to stand at room temperature for 30 minutes, and the cAMP concentration was measured according to the method of Example 3. The inhibition rate (%) was calculated as the relative inhibition rate (%) of intracellular cAMP signaling by standardizing the actual measured value when Forskolin and oleoyl-L-α-lysophosphatidic acid were added as the inhibition rate of 0%, and the actual measured value when Forskolin alone was added as the inhibition rate of 100%. The horizontal axis represents the concentration of each antibody, and the vertical axis represents the inhibition rate (%). The dose-dependency of the inhibitory activity of each antibody against human LPA1 is shown in Figures 5A to 5K, and the dose-dependency of the inhibitory activity of each antibody against mouse LPA1 is shown in Figures 6A to 6K. As a result, it was confirmed that all 11 types of antibodies dose-dependently inhibited lysophosphatidic acid-induced intracellular cAMP signal transduction in human and mouse LPA1-expressing CHO cells, demonstrating that they have the activity of blocking LPA1-dependent cellular functions.

[0178] The median inhibitory concentration (IC) of intracellular cAMP signaling was determined in three independent experiments. 50) was calculated, and the average and standard deviation are shown in Table 5. The dose-dependence of BMS-986278 on the inhibition of intracellular cAMP signaling induced by lysophosphatidic acid in human and mouse LPA1-expressing CHO cells is shown in Figures 7A and 7B, respectively. The IC 50 The IC values ​​for the 11 antibodies were 83 nM and 232 nM, respectively. As a result, all of the 11 antibodies had IC values ​​of 83 nM and 232 nM for blocking human LPA1-dependent cell functions. 50 The results showed that the efficacy of IFN-γ ...α and β-aminobutyric acid (BMS-986278) was comparable to or superior to that of BMS-986278.

[0179] [Table 5]

[0180] [Example 8] Verification of the inverse agonist effect of LPA1 antibody on intracellular cAMP signaling Using human LPA1 stably expressing CHO cells, the effect of 210309-4-A on the change in intracellular cAMP concentration in the absence of LPA1 ligand was evaluated. 5 6.25 μL of 210309-4-A used in Example 6, which had been serially diluted, was added to 12.5 μL of human LPA1 stably expressing CHO cells at a cell concentration of 10 μM / mL, and the cells were left to stand at room temperature for 15 minutes. Furthermore, 6.25 μL of 12 μM Forskolin (final concentration 3 μM) was added, and the cells were left to stand at room temperature for 30 minutes, after which the cAMP concentration was measured. Figure 8 shows a graph plotting the antibody concentration on the horizontal axis and the cAMP concentration (nM) on the vertical axis. In Figure 8, the dotted line shows the cAMP concentration (2.9 nM) only with Forskolin stimulation. That is, 210309-4-A increased the cAMP level dose-dependently, thereby showing the characteristics of an inverse agonist. The same test was also performed on 10 other LPA1 antibodies, and as a result, they increased the cAMP level dose-dependently, showing the characteristics of an inverse agonist. It was shown that this antibody is an antibody that can block LPA1 receptor-dependent cell functions in an LPA1 ligand-independent manner.

[0181] [Example 9] Verification of the inhibitory mode of LPA1 antibody against intracellular cAMP signaling In addition, the effect of various concentrations of 210309-4-A on the dose-response curve of lysophosphatidic acid, an LPA1 ligand, was evaluated using human LPA1 stably expressing CHO cells. 5 6.25μL of 210309-4-A (final concentration 0, 5, 7.5, 10, 100nM) used in Example 6 was added to 12.5μL of human LPA1 stably expressed CHO cells with a cell concentration of 100μL / mL, and the mixture was left at room temperature for 15 minutes. Furthermore, 6.25μL of a mixture of 12μM Forskolin (final concentration 3μM) and oleoyl-L-α-lysophosphatidic acid sodium salt (final concentration 0.1-10000nM) was added, and the mixture was left at room temperature for 30 minutes, and then the cAMP concentration was measured. The dose-response curve of lysophosphatidic acid at each antibody concentration is shown in Figure 9. That is, as the antibody concentration increases, the dose-response curve shifts to the right, indicating the characteristics of competitive inhibition.

[0182] [Example 10] Preparation of humanized anti-LPA1 antibody Take the case of 210309-4-A as an example. To create a humanized antibody with grafted CDRs of 210309-4-A, a human framework was selected based on the homology between 210309-4-A and human germline VH and VK genes. Based on computer modeling, several antibodies, "h309-4-A-BP1", "h309-4-A-SS1", and "h309-4-A-SG1", were designed by adding mutations to the selected human germline VH and VL sequences so that the predicted antibody conformation of 210309-4-A can be supported. The alignment of the VH region of h309-4-A-BP1 (SEQ ID NO: 123), the VH region of h309-4-A-SS1 (SEQ ID NO: 125), the VH region of h309-4-A-SG1 (SEQ ID NO: 127), and the VH region of 210309-4-A (SEQ ID NO: 7) is shown in Figure 10A. The alignment of the VL region of h309-4-A-BP1 (SEQ ID NO: 124), the VL region of h309-4-A-SS1 (SEQ ID NO: 126), the VL region of h309-4-A-SG1 (SEQ ID NO: 128), and the VL region of 210309-4-A (SEQ ID NO: 9) is shown in Figure 10B.

[0183] An artificial gene was synthesized in which the heavy chain constant region of human IgG1 (including LALA mutation) (SEQ ID NO: 129) was connected to the VH region of h309-4-A-BP1, h309-4-A-SS1, and h309-4-A-SG1, and the κ chain constant region (SEQ ID NO: 130) was connected to the VL region. In addition, as a comparison, an artificial gene of a chimeric anti-LPA1 antibody "309-4-A-Chimera" was also synthesized in which the heavy chain constant region of human IgG1 (including LALA mutation) was connected to the VH region of 210309-4-A, and the κ chain constant region was connected to the VL region. The artificial gene was introduced into CHO cells, and purified antibodies were produced from the culture medium by affinity chromatography and subjected to the following tests. The purity of all purified antibodies was confirmed to be 90% or more when tested by SDS-PAGE.

[0184] Next, the binding activity to human LPA1 was measured using CHO cells stably expressing human LPA1 according to the method of Example 6. A graph was created by plotting the antibody concentration on the horizontal axis and the median value of the fluorescence intensity of the histogram of the flow cytometer on the vertical axis. The results of binding of each antibody to CHO cells stably expressing human LPA1 are shown in Figures 11A to 11E, and the calculated 50% binding concentration (EC50) is shown in Table 6. Furthermore, the inhibitory activity of intracellular cAMP signal transduction was measured according to the method of Example 7. A graph was created by plotting the concentration of each antibody on the horizontal axis and the inhibition rate (%) on the vertical axis. The dose dependency of the inhibitory activity of each antibody to human LPA1 is shown in Figures 12A to 12E, and the calculated 50% inhibitory concentration (IC 50 ) are shown in Table 7.

[0185] As shown by these results, the binding activity and inhibitory activity of 309-4-A-Chimera are equivalent to those of 210309-4-A, confirming that conversion to the constant region of the human antibody does not affect the binding activity and inhibitory activity. In addition, the binding activity and inhibitory activity of h309-4-A-BP1, h309-4-A-SS1, and h309-4-A-SG1, which were designed as humanized LPA1 antibodies, were equivalent to those of 309-4-A-Chimera, confirming that the binding activity and inhibitory activity were maintained after humanization in all designs.

[0186] [Table 6]

[0187] [Table 7]

[0188] The amino acid sequences of SEQ ID NOs: 123 to 130 are shown in Table 8.

[0189] [Table 8]

Claims

1. specifically binds to the extracellular domain of human LPA1; does not specifically bind to the extracellular domain of human LPA2; An antibody that does not specifically bind to the extracellular domain of human LPA3.

2. The antibody according to claim 1, which has activity of blocking LPA1-dependent cell function.

3. A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, or 101; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, or 102; A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, or 103; A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, or 104; A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, or 105; and having a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, or 106; The antibody according to claim 1 or 2.

4. The following (A1) to (A11): (A1) 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; (A2) having a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13; (A3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; (A4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; (A5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43; (A6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53; (A7) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63; (A8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73; (A9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; (A10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93; (A11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103; The antibody according to claim 1 or 2, which satisfies any one of the above.

5. The following (B1) to (B11): (B1) 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; (B2) having a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16; (B3) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26; (B4) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; (B5) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46; (B6) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56; (B7) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66; (B8) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76; (B9) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86; (B10) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96; (B11) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106; The antibody according to claim 1 or 2, which satisfies any one of the above.

6. The following (AB1) to (AB11): (AB1) 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, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3, 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, (AB2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16; (AB3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:22, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:23, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:26; (AB4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; (AB5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46, (AB6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:52, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:53, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:56; (AB7) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:62, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:63, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:66; (AB8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76; (AB9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86; (AB10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96; (AB11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106; The antibody according to claim 1 or 2, which satisfies any one of the above.

7. The following (C1) to (C11): (C1) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9; (C2) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19; (C3) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29; (C4) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 39; (C5) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 49; (C6) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 59; (C7) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 69; (C8) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 77, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 79; (C9) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89; (C10) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 99; (C11) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 107, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 109; The antibody according to claim 1 or 2, which satisfies any one of the above.

8. specifically binds to the extracellular domain of human LPA1; does not specifically bind to the extracellular domain of human LPA2; does not specifically bind to the extracellular domain of human LPA3; The following (C1') to (C11'): (C1') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 7, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 7; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:9, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:

9. (C2') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 17; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 19, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

19. (C3') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:27, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:27; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:29, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

29. (C4') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 37; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

39. (C5') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 47, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 47; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 49, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

49. (C6') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:57, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:57; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:59, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

59. (C7') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:67, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:67; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:69, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

69. (C8') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 77, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 77; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:79, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

79. (C9') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 87, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 87; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:89, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

89. (C10') a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:97, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:97; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO:99, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

99. (C11') A heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 107, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 107; and A light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 109, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:

109. An antibody that satisfies any one of the above criteria.

9. The antibody according to claim 8, having activity of blocking LPA1-dependent cell function.

10. specifically binds to the extracellular domain of human LPA1; does not specifically bind to the extracellular domain of human LPA2; does not specifically bind to the extracellular domain of human LPA3; A second antibody that competitively inhibits the binding of the first antibody, which is the antibody described in claim 7, to a receptor.

11. The second antibody described in claim 10, which has activity of blocking LPA1-dependent cell function.

12. 12. The antibody of claim 1, 2, 3, 4, 5, 6, 8, 9, 10, or 11, which is a humanized antibody or a chimeric antibody.

13. The antibody according to any one of claims 1 to 12, which is a multispecific antibody.

14. The antibody according to any one of claims 1 to 13, which is a modified antibody to which another molecule is bound.

15. The antibody of claim 14, wherein the modified antibody is an antibody-drug conjugate.

16. A nucleic acid encoding the antibody according to any one of claims 1 to 13.

17. A cell comprising the nucleic acid of claim 16.

18. A pharmaceutical comprising the antibody according to any one of claims 1 to 15 as an active ingredient.

19. The pharmaceutical composition according to claim 18, which is used to treat tissue fibrosis, cell proliferative disorders, pain, inflammatory disorders, autoimmune disorders, metabolic disorders, cardiovascular disorders, urological disorders, or ophthalmological disorders.

20. It is used to treat tissue fibrosis, The pharmaceutical composition of claim 19, wherein the tissue fibrosis is liver fibrosis, kidney fibrosis, pulmonary fibrosis, skin fibrosis, cardiovascular fibrosis, or gastrointestinal fibrosis.

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