Antibodies, nucleic acids, cells and drugs

Abstract

The present invention provides 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. It is preferable that the antibody has an activity of blocking an LPA1-dependent cell function. Also provided are a nucleic acid encoding the antibody, a cell comprising the nucleic acid, and a drug containing the antibody as an active ingredient.

Description

Technical Field

[0001] The present invention 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 drug containing the antibody as an active ingredient. Background Art

[0002] Lysophosphatidic acid (LPA) is a lysophospholipid in which a phosphate and a fatty acid are each bound to a glycerol backbone. Initially, LPA was considered to be one of the intermediate products of lipid metabolism, but now it is considered to be a bioactive 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 or chemokines, platelet aggregation, smooth muscle contraction, cell transformation, or retraction of neurites (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 exhibits various physiological effects. As LPA receptors, six subtypes, 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 and are also referred to as EDG2, EDG4, and EDG7, respectively, and have high structural homology to each other. LPA4 to LPA6 are non-EDG families and have low homology to the EDG family described above.

[0004] Based on studies of the functional regulation of LPA receptors using cells and animal models, the ontogeny and pathophysiological effects on visceral organs such as the nerves, cardiovascular system, reproductive system, lungs, liver, and kidneys have been studied, suggesting the possibility that disorders of LPA-dependent cell functions are related to the causes of diseases such as fibrosis including developmental disorders of nerves or bones, cancer, neuropsychiatric disorders, pain, cardiovascular diseases, bone disorders, infertility, and obesity (Non-Patent Documents 1, 2, 4, and 5).

[0005] Fibrosis of tissues is a disease in which incomplete tissues are caused by the excessive accumulation of extracellular matrix due to the abnormal control of the tissue healing process. An increase in the LPA concentration in the bronchoalveolar lavage fluid of bleomycin-induced fibrotic model mice has been reported, and fibrosis is inhibited by LPA1 knockout mice and administration of an LPA1 antagonist in this model (Non-Patent Documents 6 and 7). In addition, for patients with idiopathic pulmonary fibrosis, it has been shown that the LPA1 antagonists BMS-986020 or BMS-986278 improve respiratory function or pulmonary fibrosis (Non-Patent Documents 8 and 9). Therefore, for controlling abnormal LPA1-dependent cell functions, it is useful for the treatment of diseases caused by fibrosis (for example, 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, and 11).

[0006] As other diseases indicating the usefulness of controlling LPA1-dependent cell functions for treatment, for example, fibromyalgia, cancer pain, neuropathic pain such as diabetic neuropathy, inflammatory pain (Non-Patent Documents 12 to 14), inflammatory diseases and autoimmune diseases such as rheumatoid arthritis, sepsis, and Guillain-Barré syndrome (Non-Patent Documents 15 to 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 to 21), hydrocephalus, neurological disorders such as schizophrenia, depression, and dementia (Non-Patent Documents 22 and 23), cell proliferative diseases (control of tumor cell proliferation, tumor invasion and metastasis, and angiogenesis) (Non-Patent Documents 6, 24, and 25), urinary system diseases such as prostatic hypertrophy and urinary incontinence (Non-Patent Documents 26 and 27), and ophthalmic diseases such as ischemic retinopathy (Non-Patent Document 28).

[0007] LPA1, LPA2, and LPA3 have different distributions in the living body according to the subtype, and each subtype contributes to different cell functions and physiological effects (Non-Patent Documents 1 and 2). Therefore, as a substance useful as an active ingredient of a drug 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 binding to multiple LPA receptors has led to the discontinuation of development due to side effects. It is 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] To date, a variety of LPA1 antagonists composed of low-molecular compounds have been created, but they have not yet reached practical use (Non-Patent Document 6). It should be noted that if there are antibodies that specifically bind to LPA1 but do not specifically bind to human LPA2 and LPA3 to control LPA1-dependent cell functions, they would be useful, but no reports on such antibodies have been seen.

[0009] Prior Art Documents

[0010] Non-Patent Documents

[0011] Non-Patent Document 1: Aikawa S, Hashimoto T, Kano K, Aoki J. “Lysophospha tidicacid as a lipid mediator with multiple biological actions.” J Biochem. 2015; 157:81 - 89

[0012] Non-Patent Document 2: Yung YC, Stoddard NC, Chun J. “LPAreceptor signalin g: pharmacology, physiology, and pathophysiology.” J Lipid Res. 2014; 55:1192 - 1214

[0013] Non-Patent Document 3: Kihara Y, Maceyka M, Spiegel S, Chun J. “Lysophosph olipidreceptor nomenclature review: IUPHAR Review 8.” Br J Pharmacol. 2014; 171:3575 - 3594

[0014] Non-Patent Document 4: Kano K, Aoki J, Hla T. “Lysophospholipid Mediators in Healthand Disease.” Annu Rev Pathol. 2022; 17:459 - 483

[0015] Non-Patent Document 5: Meduri B, Pujar GV, Durai Ananda Kumar T, Akshatha HS, Sethu AK, Singh M, Kanagarla A, Mathew B. “Lysophosphatidic acid (LPA) receptor modulators: Structural features and recent development.” Eur J Med Chem. 2021;222:113574

[0016] Non-Patent Document 6: Tager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, Polosukhin V, Wain J, Karimi-Shah BA, Kim ND, Hart WK, Pardo A, Blackwell TS, Xu Y, Chun J, Luster AD. “The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak.” Nat Med. 2008;14:45 - 54

[0017] Non-Patent Document 7: Swaney JS, Chapman C, Correa LD, Stebbins KJ, Bundey RA, Prodanovich PC, Fagan P, Baccei CS, Santini AM, Hutchinson JH, Seiders TJ, Parr TA, Prasit P, Evans JF, Lorrain DS. “A novel, orally active LPA(1) receptor antagonist inhibits lung fibrosis in the mouse bleomycin model.” Br J Pharmacol. 2010;160:1699 - 1713

[0018] Non-Patent Document 8: Palmer SM, Snyder L, Todd JL, Soule B, Christian R, Anstrom K, Luo Y, Gagnon R, Rosen G. “Randomized, Double-Blind, Placebo-Controlled, Phase 2 Trial of BMS-986020, a Lysophosphatidic Acid Receptor Antagonist for the Treatment of Idiopathic Pulmonary Fibrosis.” Chest. 2018;154:1061-1069

[0019] Non-Patent Document 9: Cheng PTW, Kaltenbach RF 3rd, Zhang H, Shi J, Tao S, Li J, Kennedy LJ, Walker SJ, Shi Y, Wang Y, Dhanusu S, Reddigunta R, Kumaravel S, Jusuf S, Smith D, Krishnananthan S, Li J, Wang T, Heiry R, Sum CS, Kalinowski SS, Hung CP, Chu CH, Azzara AV, Ziegler M, Burns L, Zinker BA, Boehm S, Taylor J, Sapuppo J, Mosure K, Everlof G, Guarino V, Zhang L, Yang Y, Ruan Q, Xu C, Apedo A, Traeger SC, Cvijic ME, Lentz KA, Tirucherai G, Sivaraman L, Robl J, Ellsworth BA, Rosen G, Gordon DA, Soars MG, Gill M, Murphy BJ. “Discovery of an Oxycyclohexyl Acid Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist BMS-986278 for the Treatment of Pulmonary Fibrotic Diseases.” J Med Chem. 2021;64:15549-15581

[0020] Non-Patent Document 10: Kim D, Li HY, Lee JH, Oh YS, Jun HS. “Lysophosphatidic acid increases mesangial cell proliferation in models of diabetic nephropathy via Rac1 / MAPK / KLF5 signaling.” Exp Mol Med. 2019;51:1-10

[0021] Non-Patent Document 12: Ueda H. “LPA receptor signaling as a therapeutic target for radical treatment of neuropathic pain and fibromyalgia.” Pain Manag. 2020;10:43-53

[0022] Non-Patent Document 13: Ueda H, Neyama H, Matsushita Y. “Lysophosphatidic Acid Receptor 1- and 3-Mediated Hyperalgesia and Hypoalgesia in Diabetic Neuropathic Pain Models in Mice.” Cells. 2020;9:1906

[0023] Non-Patent Document 14: Srikanth M, Chew WS, Hind T, Lim SM, Hay NWJ, Lee JHM, Rivera R, Chun J, Ong WY, Herr DR. “Lysophosphatidic acid and its receptor LPA1 mediate carrageenan induced inflammatory pain in mice.” Eur J Pharmacol. 2018;841:49-56

[0024] Non-Patent Document 15: Miyabe Y, Miyabe C, Iwai Y, Takayasu A, Fukuda S, Yokoyama W, Nagai J, Jona M, Tokuhara Y, Ohkawa R, Albers HM, Ovaa H, Aoki J, Chun J, Yatomi Y, Ueda H, Miyasaka M, Miyasaka N, Nanki T. "Necessity of lysophosphatidic acid receptor 1 for development of arthritis." Arthritis Rheum. 2013; 65:2037-2047

[0025] Non-Patent Document 16: Zhao J, Wei J, Weathington N, Jacko AM, Huang H, Tsung A, Zhao Y. "Lysophosphatidic acid receptor 1 antagonist ki16425 blunts abdominal and systemic inflammation in a mouse model of peritoneal sepsis." Transl Res. 2015; 166:80-88

[0026] Non-Patent Document 17: Szepanowski F, Winkelhausen M, Steubing RD, Mausberg AK, Kleinschnitz C, Stettner M.J "LPA1 signaling drives Schwann cell dedifferentiation in experimental autoimmune neuritis." Neuroinflammation. 2021; 18:293

[0027] Non-Patent Document 18: D′Souza K, Paramel GV, Kienesberger PC. "Lysophosphatidic Acid Signaling in Obesity and Insulin Resistance." Nutrients. 2018; 10:399

[0028] Non - Patent Document 19: Zhou Y, Little PJ, Ta HT, Xu S, Kamato D. “Lysopho sphatidic acid and its receptors: pharmacology and therapeutic potential in atherosclerosis and vascular disease.” Pharmacol Ther. 2019;204:107404

[0029] Non - Patent Document 20: Gaire BP, Sapkota A, Choi JW. “BMS - 986020, a Specific LPA1 Antagonist, Provides Neuroprotection against Ischemic Stroke in Mice.” Antioxidants 2020;9:1097

[0030] Non - Patent Document 21: Naruse T, Otake H, Takahashi T. “Effects of a lysophosphatidic acid receptor 1 antagonist on hypertensive renal injury in Dahl - Iwai salt - sensitive rats.” J Pharmacol Sci. 2022;149:179 - 188

[0031] Non - Patent Document 22: Yung YC, Mutoh T, Lin ME, Noguchi K, Rivera RR, Choi JW, Kingsbury MA, Chun J. “Lysophosphatidic acid signaling may initiate fetal hydrocephalus.” Sci Transl Med. 2011;3:99ra87

[0032] Non-Patent Document 23: Moreno-Fernandez RD, Tabbai S, Castilla-Ortega E, Per ez-Martin M, Estivill-Torrus G, Rodriguez de Fonseca F, Santin LJ, PedrazaC. “Stress, Depression, Resilience and Ageing: A Role for the LPA-LPA1 Pathway.” CurrNeuropharmacol. 2018;16:271-283

[0033] Non-Patent Document 24: Boucharaba A, Serre CM, Guglielmi J, Bordet JC, Cle zardinP, Peyruchaud O. “The type 1 lysophosphatidic acid receptor is a tar get fortherapy in bone metastases.” Proc Natl Acad Sci U S A. 2006;103:9643-9648

[0034] Non-Patent Document 25: Zhao PF, Wu S, Li Y, Bao G, Pei JY, Wang YW, Ma Q, Sun HJ, Damirin A. “LPAreceptor1 antagonists as anticancer agents sup press human lungtumours.” Eur J Pharmacol. 2020;868:172886

[0035] Non-Patent Document 26: Sakamoto K, Noguchi Y, Ueshima K, Yamakuni H, Oh take A, SatoS, Ishizu K, Hosogai N, Kawaminami E, Takeda M, Masuda N. “Effect of ASP6432,aNovel Type 1 Lysophosphatidic Acid Receptor Antagonist, on Urethral Functionand Prostate Cell Proliferation.” J Pharmacol Exp Ther. 2018;366:390-396

[0036] Non-Patent Document 27: Sakamoto K, Noguchi Y, Ueshima K, Ohtake A, Sato S, Imazumi K, Takeda M, Masuda N. "Modulation of urinary frequency via type 1 lysophosphatidic acid receptors: Effect of the novel antagonist ASP 6432 in conscious rats." Eur J Pharmacol. 2019;853:11-17

[0037] Non-Patent Document 28: Yang C, Lafleur J, Mwaikambo BR, Zhu T, Gagnon C, Chemtob S, Di Polo A, Hardy P. "The role of lysophosphatidic acid receptor (LPA1) in the oxygen-induced retinal ganglion cell degeneration." Invest Ophthalmol Vis Sci. 2009;50:1290-1298 SUMMARY OF THE INVENTION

[0038] TECHNICAL PROBLEM TO BE SOLVED BY THE INVENTION

[0039] An object of the present invention is to provide a novel antibody that specifically binds to the extracellular domain of LPA1 and related technologies.

[0040] TECHNICAL MEANS FOR SOLVING THE PROBLEM

[0041] An antibody of one aspect of the present invention 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.

[0042] The antibody preferably has an activity of blocking LPA1-dependent cellular functions.

[0043] The antibody preferably has: 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;

[0044] 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;

[0045] The 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;

[0046] The 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;

[0047] The 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

[0048] The 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.

[0049] Preferably, the antibody satisfies any one of the following (A1) to (A11):

[0050] (A1) having: the heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO.1, the heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO.2, and the heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO.3;

[0051] (A2) having: the heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO.11, the heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO.12, and the heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO.13;

[0052] (A3) having: the heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO.21, the heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO.22, and the heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO.23;

[0053] (A4) having: the heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO.31, the heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO.32, and the heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO.33;

[0054] (A5) having: the heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO.41, the heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO.42, and the heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO.43;

[0055] (A6) has: 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;

[0056] (A7) has: 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;

[0057] (A8) has: 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;

[0058] (A9) has: 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;

[0059] (A10) has: 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;

[0060] (A11) has: 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.

[0061] The antibody preferably satisfies any one of the following (B1) to (B11):

[0062] (B1) has: 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;

[0063] (B2) has: 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;

[0064] (B3) has: 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;

[0065] (B4) has: 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;

[0066] (B5) has: 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;

[0067] (B6) has: 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;

[0068] (B7) has: 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;

[0069] (B8) has: 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;

[0070] (B9) has: 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;

[0071] (B10) has: 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;

[0072] (B11) has: 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.

[0073] The antibody preferably satisfies any one of the following (AB1) to (AB11):

[0074] (AB1) has: 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;

[0075] (AB2) has: 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;

[0076] (AB3) has: 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;

[0077] (AB4) has: 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;

[0078] (AB5) has: 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;

[0079] (AB6) has: 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;

[0080] (AB7) has: 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;

[0081] (AB8) has: 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;

[0082] (AB9) has: 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;

[0083] (AB10) has: 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;

[0084] (AB11) has: 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.

[0085] The antibody preferably satisfies any one of the following (C1) to (C11):

[0086] (C1) has: 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;

[0087] (C2) having: 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;

[0088] (C3) having: 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;

[0089] (C4) having: 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;

[0090] (C5) having: 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;

[0091] (C6) having: 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;

[0092] (C7) having: 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;

[0093] (C8) having: 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;

[0094] (C9) having: 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;

[0095] (C10) having: 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;

[0096] (C11) having: 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.

[0097] An antibody of one embodiment of the present invention 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 the antibody satisfies any one of the following (C1') to (C11'):

[0098] (C1’) has: 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 with the amino acid sequence represented by SEQ ID NO.7; and

[0099] 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,

[0100] (C2’) has: 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 with the amino acid sequence represented by SEQ ID NO.17; and

[0101] 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 with the amino acid sequence represented by SEQ ID NO.19,

[0102] (C3’) has: 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 with the amino acid sequence represented by SEQ ID NO.27; and

[0103] 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 with the amino acid sequence represented by SEQ ID NO.29,

[0104] (C4’) has: 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 with the amino acid sequence represented by SEQ ID NO.37; and

[0105] A light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 39,

[0106] (C5’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 47; and

[0107] A light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 49,

[0108] (C6’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 57; and

[0109] A light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 59,

[0110] (C7’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 67; and

[0111] A light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 69,

[0112] (C8’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 77; and

[0113] A light chain variable region having 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 having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 79,

[0114] (C9’) having: a heavy chain variable region having 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 having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 87; and

[0115] a light chain variable region having 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 having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 89,

[0116] (C10’) having: a heavy chain variable region having 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 having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 97; and

[0117] a light chain variable region having 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 having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 99,

[0118] (C11’) having: a heavy chain variable region having 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 having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 107; and

[0119] a light chain variable region having 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 having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 109.

[0120] The antibody preferably has the activity of blocking LPA1-dependent cellular functions.

[0121] A second antibody according to one embodiment of the present invention 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 first antibody to the receptor. The first antibody is the antibody described above.

[0122] Preferably, the second antibody has the activity of blocking LPA1-dependent cellular functions.

[0123] Preferably, the antibody or the second antibody is a humanized antibody or a chimeric antibody.

[0124] Preferably, the antibody or the second antibody is a multispecific antibody.

[0125] Preferably, the antibody or the second antibody is a modified antibody conjugated with other molecules.

[0126] Preferably, the modified antibody is an antibody-drug conjugate.

[0127] A nucleic acid according to one embodiment of the present invention encodes the antibody or the second antibody.

[0128] A cell according to one embodiment of the present invention contains the nucleic acid.

[0129] A drug according to one embodiment of the present invention contains the antibody or the second antibody as an active ingredient.

[0130] Preferably, the drug is used for the treatment of diseases, disorders or conditions related to dysfunctions of LPA1-dependent cellular functions.

[0131] Preferably, the drug is used for the treatment of tissue fibrosis, cell proliferative diseases, pain, inflammatory diseases, autoimmune diseases, metabolic diseases, cardiovascular disorders, urinary system diseases or ophthalmic diseases.

[0132] Preferably, the tissue fibrosis is liver fibrosis, renal fibrosis, pulmonary fibrosis, skin fibrosis, cardiovascular fibrosis or gastrointestinal fibrosis.

[0133] Preferably, the liver fibrosis is non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, ischemia-reperfusion, post-liver transplantation injury, necrotic hepatitis, hepatitis B, hepatitis C, primary biliary cirrhosis or primary sclerosing cholangitis.

[0134] Preferably, the cirrhosis is cirrhosis caused by alcohol-based induction, drug-based induction or chemical induction.

[0135] Preferably, the renal fibrosis is proliferative glomerulonephritis, sclerosing glomerulonephritis, nephrogenic fibrosing dermopathy, diabetic nephropathy, tubulointerstitial fibrosis or focal segmental glomerulosclerosis.

[0136] The pulmonary fibrosis is preferably interstitial pulmonary fibrosis, drug-induced sarcoidosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar damage disease, pulmonary hypertension, or neonatal bronchopulmonary dysplasia.

[0137] The skin fibrosis is preferably scleroderma, keloid scarring, psoriasis, hypertrophic scar, or pseudoscleroderma.

[0138] The cardiovascular fibrosis is preferably atherosclerosis, coronary restenosis, congestive cardiomyopathy, heart failure, heart transplantation, or myocardial fibrosis.

[0139] The gastrointestinal fibrosis is preferably collagenous colitis, villous atrophy, crypt hyperplasia, polyp formation, Crohn's disease fibrosis, gastric ulcer healing, or scar after abdominal adhesion surgery.

[0140] The fibrosis preferably has a state resulting from fibrotic diseases related to bone, which is rheumatoid pannus formation.

[0141] The cell proliferative disease is preferably tumor cell proliferation, tumor invasion and metastasis, or regulation of angiogenesis.

[0142] The cell proliferative disease is preferably blood cancer or solid cancer.

[0143] The solid cancer is preferably 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.

[0144] The squamous cell carcinoma is preferably oral squamous cell carcinoma, esophageal squamous cell carcinoma, or pharyngeal squamous cell carcinoma.

[0145] The pain is preferably pain caused by fibromyalgia, cancer pain, or pain caused by diabetic neuropathy.

[0146] The inflammatory disease is preferably rheumatoid arthritis, sepsis, chronic obstructive pulmonary disease, inflammatory bowel disease, transplant organ rejection, Guillain-Barré syndrome, or multiple sclerosis.

[0147] The metabolic disease is preferably obesity or insulin-resistant diabetes.

[0148] The cardiovascular disorder is preferably cerebral infarction, hypertensive nephropathy, or Raynaud's phenomenon.

[0149] The neurological disorder is preferably hydrocephalus, schizophrenia, depression, or dementia.

[0150] The urinary system disease is preferably prostate hypertrophy or urinary incontinence.

[0151] The ophthalmic disease is preferably ischemic retinopathy, diabetic retinopathy or age-related macular degeneration.

[0152] Effects of the Invention

[0153] According to the present invention, novel antibodies specifically binding to the extracellular domain of LPA1 and related technologies can be provided. Brief Description of the Drawings

[0154] Figure 1A is a histogram showing the results of flow cytometry performed in Example 4, indicating the binding of the antibody to human LPA1-LPA3.

[0155] Figure 1B is a histogram showing the results of flow cytometry performed in Example 4, indicating the binding of the antibody to mouse LPA1-LPA3.

[0156] Figure 2A is a graph showing the evaluation results of the binding of the antibody (210309-1-C) to human LPA1-stably expressing CHO cells.

[0157] Figure 2B is a graph showing the evaluation results of the binding of the antibody (210309-1-G) to human LPA1-stably expressing CHO cells.

[0158] Figure 2C is a graph showing the evaluation results of the binding of the antibody (210309-2-A) to human LPA1-stably expressing CHO cells.

[0159] Figure 2D is a graph showing the evaluation results of the binding of the antibody (210309-2-D) to human LPA1-stably expressing CHO cells.

[0160] Figure 2E is a graph showing the evaluation results of the binding of the antibody (210309-4-A) to human LPA1-stably expressing CHO cells.

[0161] Figure 2F is a graph showing the evaluation results of the binding of the antibody (210310-1-D) to human LPA1-stably expressing CHO cells.

[0162] Figure 2G is a graph showing the evaluation results of the binding of the antibody (210420-4-E) to human LPA1-stably expressing CHO cells.

[0163] Figure 2H ​​​​​​​​​​is a graph showing the results of the binding evaluation of antibody (210420-1-H) to CHO cells stably expressing human LPA1.

[0164] Figure 2I is a graph showing the results of the binding evaluation of antibody (210420-3-E) to CHO cells stably expressing human LPA1.

[0165] Figure 2J is a graph showing the results of the binding evaluation of antibody (211222-1-G) to CHO cells stably expressing human LPA1.

[0166] Figure 2K is a graph showing the results of the binding evaluation of antibody (211222-1-A) to CHO cells stably expressing human LPA1.

[0167] Figure 3A is a graph showing the results of the binding evaluation of antibody (210309-1-C) to CHO cells stably expressing mouse LPA1.

[0168] Figure 3B is a graph showing the results of the binding evaluation of antibody (210309-1-G) to CHO cells stably expressing mouse LPA1.

[0169] Figure 3C is a graph showing the results of the binding evaluation of antibody (210309-2-A) to CHO cells stably expressing mouse LPA1.

[0170] Figure 3D is a graph showing the results of the binding evaluation of antibody (210309-2-D) to CHO cells stably expressing mouse LPA1.

[0171] Figure 3E is a graph showing the results of the binding evaluation of antibody (210309-4-A) to CHO cells stably expressing mouse LPA1.

[0172] Figure 3F is a graph showing the results of the binding evaluation of antibody (210310-1-D) to CHO cells stably expressing mouse LPA1.

[0173] Figure 3G is a graph showing the results of the binding evaluation of antibody (210420-4-E) to CHO cells stably expressing mouse LPA1.

[0174] Figure 3H is a graph showing the results of the binding evaluation of antibody (210420-1-H) to CHO cells stably expressing mouse LPA1.

[0175] ​​​​​​​​​​​Figure 3I is a graph showing the results of evaluating the binding of antibody (210420-3-E) to CHO cells stably expressing mouse LPA1.

[0176] Figure 3J is a graph showing the results of evaluating the binding of antibody (211222-1-G) to CHO cells stably expressing mouse LPA1.

[0177] Figure 3K is a graph showing the results of evaluating the binding of antibody (211222-1-A) to CHO cells stably expressing mouse LPA1.

[0178] Figure 4 is a histogram showing the results of the flow cytometry performed in Example 6, demonstrating the binding of the antibody to endogenous human LPA1.

[0179] Figure 5A is a graph showing the dose-dependence of the inhibitory activity of antibody (210309-1-C) against human LPA1.

[0180] Figure 5B is a graph showing the dose-dependence of the inhibitory activity of antibody (210309-1-G) against human LPA1.

[0181] Figure 5C is a graph showing the dose-dependence of the inhibitory activity of antibody (210309-2-A) against human LPA1.

[0182] Figure 5D is a graph showing the dose-dependence of the inhibitory activity of antibody (210309-2-D) against human LPA1.

[0183] Figure 5E is a graph showing the dose-dependence of the inhibitory activity of antibody (210309-4-A) against human LPA1.

[0184] Figure 5F is a graph showing the dose-dependence of the inhibitory activity of antibody (210310-1-D) against human LPA1.

[0185] Figure 5G is a graph showing the dose-dependence of the inhibitory activity of antibody (210420-4-E) against human LPA1.

[0186] Figure 5H is a graph showing the dose-dependence of the inhibitory activity of antibody (210420-1-H) against human LPA1.

[0187] Figure 5I ​​​​​​​​​​​​​is a graph showing the dose-dependence of the inhibitory activity of the antibody (210420-3-E) against human LPA1.

[0188] Figure 5J is a graph showing the dose-dependence of the inhibitory activity of the antibody (211222-1-G) against human LPA1.

[0189] Figure 5K is a graph showing the dose-dependence of the inhibitory activity of the antibody (211222-1-A) against human LPA1.

[0190] Figure 6A is a graph showing the dose-dependence of the inhibitory activity of the antibody (210309-1-C) against mouse LPA1.

[0191] Figure 6B is a graph showing the dose-dependence of the inhibitory activity of the antibody (210309-1-G) against mouse LPA1.

[0192] Figure 6C is a graph showing the dose-dependence of the inhibitory activity of the antibody (210309-2-A) against mouse LPA1.

[0193] Figure 6D is a graph showing the dose-dependence of the inhibitory activity of the antibody (210309-2-D) against mouse LPA1.

[0194] Figure 6E is a graph showing the dose-dependence of the inhibitory activity of the antibody (210309-4-A) against mouse LPA1.

[0195] Figure 6F is a graph showing the dose-dependence of the inhibitory activity of the antibody (210310-1-D) against mouse LPA1.

[0196] Figure 6G is a graph showing the dose-dependence of the inhibitory activity of the antibody (210420-4-E) against mouse LPA1.

[0197] Figure 6H is a graph showing the dose-dependence of the inhibitory activity of the antibody (210420-1-H) against mouse LPA1.

[0198] Figure 6I is a graph showing the dose-dependence of the inhibitory activity of the antibody (210420-3-E) against mouse LPA1.

[0199] Figure 6J is a graph showing the dose-dependence of the inhibitory activity of the antibody (211222-1-G) against mouse LPA1. ​​​​​​​​​​​​

[0200] Figure 6K is a graph showing the dose-dependence of the inhibitory activity of the antibody (211222-1-A) against mouse LPA1.

[0201] Figure 7A is a graph showing the dose-dependence of the inhibitory activity of BMS-986278 against human LPA1.

[0202] Figure 7B is a graph showing the dose-dependence of the inhibitory activity of BMS-986278 against mouse LPA1.

[0203] Figure 8 is a graph showing the results of evaluating the effect of 210309-4-A on the change in intracellular cAMP concentration in the absence of an LPA1 ligand using human LPA1 stably expressing CHO cells.

[0204] Figure 9 is a graph showing the dose-response curve of lysophosphatidic acid at each concentration of 210309-4-A.

[0205] Figure 10A is an explanatory diagram showing the alignment of the heavy chain variable region of a humanized anti-LPA1 antibody.

[0206] Figure 10B is an explanatory diagram showing the alignment of the light chain variable region of a humanized anti-LPA1 antibody.

[0207] Figure 11A is a graph showing the results of evaluating the binding of the antibody (h309-4-A-BP1) to human LPA1 stably expressing CHO cells.

[0208] Figure 11B is a graph showing the results of evaluating the binding of the antibody (h309-4-A-SS1) to human LPA1 stably expressing CHO cells.

[0209] Figure 11C is a graph showing the results of evaluating the binding of the antibody (h309-4-A-SG1) to human LPA1 stably expressing CHO cells.

[0210] Figure 11D is a graph showing the results of evaluating the binding of the antibody (309-4-A-Chimera) to human LPA1 stably expressing CHO cells.

[0211] Figure 11E is a graph showing the results of evaluating the binding of the antibody (210309-4-A) to human LPA1 stably expressing CHO cells.

[0212] Figure 12A ​​​​​​​​​​​​​is a graph showing the dose-dependence of the inhibitory activity of the antibody (h309-4-A-BP1) against human LPA1.

[0213] Figure 12B is a graph showing the dose-dependence of the inhibitory activity of the antibody (h309-4-A-SS1) against human LPA1.

[0214] Figure 12C is a graph showing the dose-dependence of the inhibitory activity of the antibody (h309-4-A-SG1) against human LPA1.

[0215] Figure 12D is a graph showing the dose-dependence of the inhibitory activity of the antibody (309-4-A-Chimera) against human LPA1.

[0216] Figure 12E is a graph showing the dose-dependence of the inhibitory activity of the antibody (210309-4-A) against human LPA1. Detailed implementation mode

[0217] In the present invention, the complementarity determining region is abbreviated as CDR. In the present invention, the heavy chain variable region is sometimes abbreviated as VH, the heavy chain constant region is abbreviated as CH, the light chain variable region is abbreviated as VL, and the light chain constant region is abbreviated as CL. In the present invention, the term "antibody" can be replaced by "immunoglobulin". In the present invention, the term "nucleic acid" can be replaced by "DNA" or "gene".

[0218] <Human LPA1>

[0219] Lysophosphatidic acid receptor 1 (LPA1) is a type of G protein-coupled receptor (GPCR), which traverses the cell membrane 7 times, with its N-terminus facing the outside of the cell and its C-terminus facing the inside of the cell. The gene (cDNA) encoding human LPA1 has been isolated, and the amino acid sequence of human LPA1 is also known. This 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.

[0220] It is considered that each domain of human LPA1 corresponds to the following parts 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. It should be noted that there may be some deviations at the boundaries between each domain.

[0221] ​​​​1 - 50: N - terminal domain

[0222] 76 - 83: Intracellular loop 1 domain

[0223] 108 - 121: Extracellular loop 1 domain

[0224] 145 - 163: Intracellular loop 2 domain

[0225] 185 - 204: Extracellular loop 2 domain

[0226] 226 - 255: Intracellular loop 3 domain

[0227] 281 - 294: Extracellular loop 3 domain

[0228] 316 - 364: C - terminal domain

[0229] In human LPA1, various variants such as amino acid substituents are also known in addition to those shown in SEQ ID NO.111. In the "human LPA1" of the present invention, as long as it has an extracellular domain and has the function of LP A1, the variants are included.

[0230] The gene (cDNA) encoding human LPA2 has 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 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)

[0231] <Anti - LPA1 antibody>

[0232] 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.

[0233] An antibody of one mode has:

[0234] 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;

[0235] 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;

[0236] The 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;

[0237] The 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;

[0238] The 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

[0239] The 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.

[0240] The heavy chain CDR1-3 of the antibody in one mode satisfies any one of the above (A1)-(A11).

[0241] The light chain CDR1-3 of the antibody in one mode satisfies any one of the above (B1)-(B11).

[0242] The heavy chain CDR1-3 and the light chain CDR1-3 of the antibody in one mode satisfy any one of the above (AB1)-(AB11).

[0243] The heavy chain variable region and the light chain variable region of the antibody in one mode satisfy any one of the above (C1)-(C11).

[0244] (C1) The determined heavy chain variable region (SEQ ID NO.7) contains the heavy chain CDR1-3 (SEQ ID NO.1-3) determined by (A1) or (AB1). (C1) The determined light chain variable region (SEQ ID NO.9) contains the light chain CDR1-3 (SEQ ID NO.4-6) determined by (B1) or (AB1). As an example of the antibody satisfying (A1), (B1), (AB1) or (C1), "210309-4-A" described in the following examples can be cited.

[0245] The heavy chain variable region (SEQ ID NO.17) determined by (C2) contains the heavy chain CDR1-3 (SEQ ID NOs.11-13) determined by (A2) or (AB2). The light chain variable region (SEQ ID NO.19) determined by (C2) contains the light chain CDR1-3 (SEQ ID NOs.14-16) determined by (B2) or (AB2). As an example of an antibody satisfying (A2), (B2), (AB2) or (C2), there can be mentioned "210309-1-C" described in the examples below.

[0246] The heavy chain variable region (SEQ ID NO.27) determined by (C3) contains the heavy chain CDR1-3 (SEQ ID NOs.21-23) determined by (A3) or (AB3). The light chain variable region (SEQ ID NO.29) determined by (C3) contains the light chain CDR1-3 (SEQ ID NOs.24-26) determined by (B3) or (AB3). As an example of an antibody satisfying (A3), (B3), (AB3) or (C3), there can be mentioned "210309-1-G" described in the examples below.

[0247] The heavy chain variable region (SEQ ID NO.37) determined by (C4) contains the heavy chain CDR1-3 (SEQ ID NOs.31-33) determined by (A4) or (AB4). The light chain variable region (SEQ ID NO.39) determined by (C4) contains the light chain CDR1-3 (SEQ ID NOs.34-36) determined by (B4) or (AB4). As an example of an antibody satisfying (A4), (B4), (AB4) or (C4), there can be mentioned "210309-2-A" described in the examples below.

[0248] The heavy chain variable region (SEQ ID NO.47) determined by (C5) contains the heavy chain CDR1-3 (SEQ ID NOs.41-43) determined by (A5) or (AB5). The light chain variable region (SEQ ID NO.49) determined by (C5) contains the light chain CDR1-3 (SEQ ID NOs.44-46) determined by (B5) or (AB5). As an example of an antibody satisfying (A5), (B5), (AB5) or (C5), there can be mentioned "210309-2-D" described in the examples below.

[0249] (C6) The heavy chain variable region (SEQ ID NO.57) determined contains the heavy chain CDR1-3 (SEQ ID NO.51-53) determined by (A6) or (AB6). The light chain variable region (SEQ ID NO.59) determined by (C6) contains the light chain CDR1-3 (SEQ ID NO.54-56) determined by (B6) or (AB6). As an example of an antibody satisfying (A6), (B6), (AB6), or (C6), there can be cited "210310-1-D" described in the following Examples.

[0250] (C7) The heavy chain variable region (SEQ ID NO.67) determined contains the heavy chain CDR1-3 (SEQ ID NO.61-63) determined by (A7) or (AB7). The light chain variable region (SEQ ID NO.69) determined by (C7) contains the light chain CDR1-3 (SEQ ID NO.64-66) determined by (B7) or (AB7). As an example of an antibody satisfying (A7), (B7), (AB7), or (C7), there can be cited "210420-4-E" described in the following Examples.

[0251] (C8) The heavy chain variable region (SEQ ID NO.77) determined contains the heavy chain CDR1-3 (SEQ ID NO.71-73) determined by (A8) or (AB8). The light chain variable region (SEQ ID NO.79) determined by (C8) contains the light chain CDR1-3 (SEQ ID NO.74-76) determined by (B8) or (AB8). As an example of an antibody satisfying (A8), (B8), (AB8), or (C8), there can be cited "210420-1-H" described in the following Examples.

[0252] (C9) The heavy chain variable region (SEQ ID NO.87) determined contains the heavy chain CDR1-3 (SEQ ID NO.81-83) determined by (A9) or (AB9). The light chain variable region (SEQ ID NO.89) determined by (C9) contains the light chain CDR1-3 (SEQ ID NO.84-86) determined by (B9) or (AB9). As an example of an antibody satisfying (A9), (B9), (AB9), or (C9), there can be cited "210420-3-E" described in the following Examples.

[0253] (C10) The determined heavy chain variable region (SEQ ID NO. 97) contains the heavy chain CDR1-3 (SEQ ID NOs. 91-93) determined by (A10) or (AB10). The determined light chain variable region (SEQ ID NO. 99) contains the light chain CDR1-3 (SEQ ID NOs. 94-96) determined by (B10) or (AB10). As an example of an antibody satisfying (A10), (B10), (AB10) or (C10), there can be mentioned "211222-1-G" described in the examples below.

[0254] (C11) The determined heavy chain variable region (SEQ ID NO. 107) contains the heavy chain CDR1-3 (SEQ ID NOs. 101-103) determined by (A11) or (AB11). The determined light chain variable region (SEQ ID NO. 109) contains the light chain CDR1-3 (SEQ ID NOs. 104-106) determined by (B11) or (AB11). As an example of an antibody satisfying (A11), (B11), (AB11) or (C11), there can be mentioned "211222-1-A" described in the examples below.

[0255] 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.

[0256] The antibody may be a functional fragment of an antibody. Here, a "functional fragment of an antibody" refers to a partial fragment of an antibody (i.e., an immunoglobulin) that retains at least one function against an antigen. Examples of such partial fragments include F(ab’)2, Fab, Fv, disulfide-bonded Fv, single-chain antibodies (scFv, VH-VL), VH, and their polymers, as well as fusions thereof with the heavy-chain CH3 region. In addition, examples include each CDR such as CDR1, CDR2, CDR3, and linkers of these CDRs, as well as fusions of these CDRs or CDR linkers with the heavy-chain CH3 region. That is, the antibodies of the present invention also include partial fragments of antibodies as described above. Sometimes, partial fragments of antibodies are also referred to as "antibody fragments."

[0257] When the antibody is a functional fragment, for example, it has the following effects. That is, when the anti-LPA1 antibody of the present invention is applied to a drug as described below, when a full-length antibody such as IgG type is used, in addition to inhibiting cell function through the target receptor, it sometimes causes damage in the target tissue, resulting in side effects. In such a case, when using a "functional fragment of an antibody" that only uses the variable region, it is easy to avoid side effects as described above.

[0258] The antibody may be a multispecific antibody. For the multispecific antibody of this mode, it has at least a first specific binding property of specifically binding to the extracellular domain of human LPA1 and not specifically binding to the extracellular domain of human LPA2 or 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 a specific binding property to human LPA1 or a specific binding property to other target molecules. Examples of multispecific antibodies include diabody, which is a type of bispecific antibody.

[0259] 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. In addition, the subtype of the antibody is not particularly limited. For example, if it is IgG, it may be any subtype such as IgG1, IgG2, IgG3, IgG4, etc.

[0260] In a preferred embodiment, the antibody has the activity of blocking LPA1-dependent cellular functions. LPA1-dependent cellular functions are functions induced by the activation of proteins that undertake intracellular signal transduction through coupling with the intracellular portion of LPA1 via trimeric G proteins or β-arrestin. Activation of LPA1-dependent cellular functions is induced by stimulation with an LPA1 ligand or by overexpression independent of the LPA1 ligand. Examples of cellular functions include changes in intracellular cyclic adenosine monophosphate (cAMP), changes in intracellular calcium ions, GTP binding to the low molecular weight G protein Rho, cell proliferation, cell migration, production of cytokines or chemokines, and cell transformation.

[0261] The extracellular domain of LPA1 to which the antibody specifically binds can be any one of the N-terminal domain, extracellular loop 1 domain, extracellular loop 2 domain, and extracellular loop 3 domain. In addition, the antibody can bind to only any one of these extracellular domains or can bind to two or more of them.

[0262] The present invention includes antibodies "functionally equivalent" to the anti-LPA1 antibody. Functionally equivalent antibodies include antibodies having the same epitope as the antibody. For example, the epitopes of 11 anti-LPA1 antibodies specifically shown in the following examples were analyzed by epitope mapping using a partial peptide of LPA1 or the like. And a synthetic peptide containing the determined epitope can be used as an antigen to obtain an anti-LPA1 antibody that binds to the same epitope as the 11 anti-LPA1 antibodies. In addition, the amino acid sequences of the heavy chain variable region and the light chain variable region of the obtained anti-LPA1 antibody can be determined, and the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 can be determined.

[0263] As an example of an antibody functionally equivalent to the antibody, the present invention includes an 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 satisfies any one of (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 amino acid sequences is preferably 92% or more, more preferably 95% or more, and particularly preferably 97% or more. In a preferred embodiment, the antibody has the activity of blocking LPA1-dependent cellular functions.

[0264] As a method for studying whether the epitopes of two antibodies are the same, a method based on a competition experiment can be cited. For example, in the case where the binding of the 11 anti-LPA1 antibodies or their functional fragments as the first antibody to the receptor is competitively inhibited by the second antibody as the test subject, it can be said that the second antibody binds to the same epitope as the first antibody. Further, as an example of an antibody functionally equivalent to the antibody, the present invention 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 antibody (the first antibody) to the receptor. In a preferred embodiment, the second antibody has the activity of blocking LPA1-dependent cellular functions.

[0265] <Humanized antibody>

[0266] The present invention includes the antibody as a humanized antibody. A humanized antibody refers to an antibody in which the CDRs are derived from non-human animals and other regions (framework regions, constant regions, etc.) are derived from humans. Methods for constructing and producing humanized antibodies will be described later.

[0267] <Chimeric antibody>

[0268] The present invention includes the antibody as a chimeric antibody. 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 non-human animals and other regions such as the heavy chain constant region (CH) and the light chain constant region (CL) are derived from humans. Methods for constructing and producing chimeric antibodies will be described later.

[0269] <Nucleic acid>

[0270] The present invention includes a nucleic acid (DNA) encoding the antibody. With respect to the nucleic acid, for example, it includes: a first nucleic acid encoding the heavy chain variable region and / or a second nucleic acid encoding the light chain variable region.

[0271] For the first nucleic acid-encoded heavy chain variable region, for example, it includes: heavy chain CDR1, which contains the amino acid sequence represented by SEQ ID NO.1, 11, 21, 31, 41, 51, 61, 71, 81, 91 or 101; heavy chain CDR2, which contains the amino acid sequence represented by SEQ ID NO.2, 12, 22, 32, 42, 52, 62, 72, 82, 92 or 102; and heavy chain CDR3, which contains the amino acid sequence represented by SEQ ID NO.3, 13, 23, 33, 43, 53, 63, 73, 83, 93 or 103. The first nucleic acid-encoded heavy chain variable region, for example, includes heavy chain CDR1-3 determined by any one of the above (A1)-(A11). The first nucleic acid-encoded heavy chain variable region is determined by any one of the above (C1)-(C11), for example. The first nucleic acid-encoded heavy chain variable region is determined by any one of the above (C1')-(C11'), for example.

[0272] For the second nucleic acid-encoded light chain variable region, for example, it includes: light chain CDR1, which contains the amino acid sequence represented by SEQ ID NO.4, 14, 24, 34, 44, 54, 64, 74, 84, 94 or 104; light chain CDR2, which contains the amino acid sequence represented by SEQ ID NO.5, 15, 25, 35, 45, 55, 65, 75, 85, 95 or 105; and light chain CDR3, which contains the amino acid sequence represented by SEQ ID NO.6, 16, 26, 36, 46, 56, 66, 76, 86, 96 or 106. The second nucleic acid-encoded light chain variable region, for example, includes heavy chain CDR1-3 determined by any one of the above (B1)-(B11). The second nucleic acid-encoded light chain variable region is determined by any one of the above (C1)-(C11), for example. The second nucleic acid-encoded light chain variable region is determined by any one of the above (C1')-(C11'), for example.

[0273] The nucleic acid can be integrated into a vector. The vector is appropriately selected according to the type of host cell into which it is introduced, etc. The vector includes a vector for gene therapy. In this case, the vector itself can be directly administered into a living body.

[0274] <Cell>

[0275] The present invention includes cells containing the nucleic acid. For example, a cell containing a vector integrated with the nucleic acid is an example of such a cell. As the type of cell, as long as it is a cell capable of expressing the nucleic acid, for example, a cell in which the vector functions, there is no particular limitation. As examples, animal cells (COS cells, CHO cells, etc.), yeast, bacteria (E. coli, etc.), plant cells, insect cells, etc. can be mentioned.

[0276] <Method for manufacturing an antibody>

[0277] The antibody can be produced by a genetic recombination method. That is, a recombinant cell expressing the nucleic acid can be constructed, and the antibody can be obtained from the culture of the cell.

[0278] <Construction and Preparation of Humanized Antibody>

[0279] The method for constructing and producing a humanized antibody will be described later. As an example, the method for constructing and producing a humanized antibody having the heavy chain CDR1-3 and light chain CDR1-3 determined by the above (AB1) will be described. First, as the DNA encoding each CDR, the DNA encoding the amino acid sequences represented by SEQ ID NOs. 1 to 6 is prepared. The preparation of the DNA can be carried out by a known method such as PCR. The DNA can also be prepared by chemical synthesis.

[0280] Next, using these DNAs, the DNA encoding the variable region in which the heavy chain CDR1-3 is transplanted into the framework region (FR) of VH in an arbitrary human antibody is prepared. Similarly, the DNA encoding the variable region in which the light chain CDR1-3 is transplanted into the FR of VL in an arbitrary human antibody is prepared. Each prepared DNA is inserted into a vector having a sequence encoding CH or CL of a human antibody to construct a humanized antibody expression vector. By introducing the constructed expression vector into a host cell, a recombinant cell expressing the humanized antibody is obtained. Then, the recombinant cell is cultured, and the desired humanized antibody is obtained from the culture.

[0281] For the humanized antibody having the heavy chain CDR1-3 and light chain CDR1-3 determined by the above (AB2)-(AB11), it can be constructed and produced by the same method.

[0282] <Construction and Preparation of Chimeric Antibody>

[0283] The method for constructing and producing a chimeric antibody will be described later. As an example, the method for constructing and producing a chimeric antibody having the heavy chain variable region (VH) and light chain variable region (VL) determined in the above (C1) will be described. First, as the DNA encoding VH, the DNA encoding the amino acid sequence represented by SEQ ID NO. 7 is prepared. In addition, as the DNA encoding VL, the DNA encoding the amino acid sequence represented by SEQ ID NO. 9 is prepared. The preparation of the DNA can be carried out by a known method such as PCR. The DNA can also be prepared by chemical synthesis.

[0284] The DNA encoding the obtained VH or VL is respectively inserted into a vector having a sequence encoding CH or CL of a human antibody to construct a chimeric antibody expression vector. It should be noted that the vector having a sequence encoding CH or CL of a human antibody can be obtained from the market. By introducing the constructed expression vector into a host cell, a recombinant cell expressing the chimeric antibody is obtained. Then, the recombinant cell is cultured, and the desired chimeric antibody is obtained from the culture.

[0285] For the chimeric antibody having VH and VL determined by the above (C2) to (C11), it can be constructed and produced by the same method.

[0286] <Method for Preparing Multispecific Antibody>

[0287] As a method for manufacturing a multispecific antibody, for example, it can be exemplified that the anti-human LPA1 antibody or its fragment of the present invention is linked to another antibody or its fragment. As other examples, it can be exemplified that the anti-human LPA1 antibody or its fragment of the present invention and another antibody or its fragment are co-expressed in a host cell. As other linking methods, chemical coupling, gene fusion, non-covalent association, etc. can be cited.

[0288] It should be noted that as an example of a practical multispecific antibody (bispecific antibody), BLINCYTO (registered trademark) that binds CD3 and CD19 can be cited. As other examples, HEMLIBRA (registered trademark) that binds coagulation factor IXa and coagulation factor X can be cited. The manufacturing techniques of multispecific antibodies are well-known in the technical field, and these well-known manufacturing techniques can be applied to the anti-human LPA1 antibody of the present invention.

[0289] <Purification Method>

[0290] As the purification method of the above antibody, there is no particular limitation, and a well-known method can be adopted. For example, the culture supernatant of the above recombinant cell can be recovered, and various well-known methods such as chromatography, salting out, dialysis, membrane separation, etc. can be combined to purify the antibody. When the isotype of the antibody is IgG, it can be simply purified by affinity chromatography using protein A.

[0291] <Modified Antibody>

[0292] The antibody of the present invention can be a modified antibody conjugated with other molecules. As examples of other molecules, peptides, proteins, low molecular weight compounds, radioisotopes, light absorbers, etc. can be cited. As methods for binding to other molecules, chemical coupling, gene fusion, non-covalent association, etc. can be cited. It should be noted that when the other molecule bound is an antibody, the modified antibody can become a multispecific antibody. In this regard, multispecific antibodies can be understood as a type of modified antibody.

[0293] In a preferred embodiment, the modified antibody is an antibody-drug conjugate (ADC). As the drug to be conjugated, examples include anti-fibrotic agents; small molecule anti-cancer agents (cytotoxins, chemotherapeutic drugs); biologically active proteins or polypeptides such as cytokines; radioisotopes; light absorbers, etc. These drugs can be conjugated directly or indirectly via a linker or chelator to prepare an antibody-drug conjugate.

[0294] It should be noted that, as an example of a practical antibody-drug conjugate, as an antibody-drug conjugate conjugated with a small molecule anti-cancer agent, ENHERTU (registered trademark), in which camptothecin derivative is bound to trastuzumab, can be cited. As an antibody-drug conjugate conjugated with a radioisotope, Zevalin (registered trademark), in which yttrium 90 ( 90 Y) is labeled on an anti-CD20 monoclonal antibody, can be cited. As an antibody-drug conjugate conjugated with a light absorber, Akalux (registered trademark), in which IR Dye700DX is bound to cetuximab, can be cited. The drugs contained in these antibody-drug conjugates are also applicable to the anti-human LPA1 antibody of the present invention. In addition, examples of suitable drugs used in antibody-drug conjugates are well-known in the art, and these well-known drugs can be applied to the anti-human LPA1 antibody of the present invention.

[0295] <Drug>

[0296] The present invention includes a drug containing the antibody as an active ingredient. The drug may be a pharmaceutical composition containing the antibody and a pharmaceutically acceptable carrier. The drug preferably blocks LPA1-dependent cellular functions.

[0297] In a preferred embodiment, the drug is preferably used for the treatment of diseases, disorders or conditions related to the causative agents of LPA1-dependent cellular dysfunction. As examples of diseases, disorders or conditions related to LPA1-dependent cellular dysfunction, fibrosis selected from liver fibrosis, kidney fibrosis, lung fibrosis, skin fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis and other fibrotic diseases can be cited.

[0298] As an example of the liver fibrosis, fibrosis selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver cirrhosis, ischemia-reperfusion, post-liver transplantation injury, necrotic hepatitis, hepatitis B, hepatitis C, primary biliary cirrhosis and primary sclerosing cholangitis can be cited. As an example of the liver cirrhosis, it can be caused by at least one selected from alcohol-based induction, drug-based induction or chemical induction.

[0299] As an example of the renal fibrosis, the fibrosis selected from proliferative glomerulonephritis, sclerosing glomerulonephritis, nephrogenic fibrosing dermopathy, diabetic nephropathy, tubulointerstitial fibrosis or focal segmental glomerulosclerosis can be cited.

[0300] As an example of the pulmonary fibrosis, the fibrosis selected from pulmonary interstitial fibrosis, drug-induced sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar damage disease, pulmonary arterial hypertension and neonatal bronchopulmonary dysplasia can be cited.

[0301] As an example of the skin fibrosis, the fibrosis selected from scleroderma, keloid scarring, psoriasis, hypertrophic scar and pseudoscleroderma can be cited.

[0302] As an example of the cardiovascular fibrosis, the fibrosis selected from atherosclerosis, coronary restenosis, congestive cardiomyopathy, heart failure, heart transplantation and myocardial fibrosis can be cited.

[0303] As an example of the gastrointestinal fibrosis, the fibrosis in collagenous colitis, villous atrophy, crypt hyperplasia, polyp formation, Crohn's disease fibrosis, gastric ulcer healing and scar after abdominal adhesion surgery can be cited.

[0304] The fibrosis may have a state produced by a fibrotic disease related to bone, which is rheumatoid pannus formation.

[0305] As other examples of diseases, disorders or conditions related to the impairment of LPA1-dependent cell functions, cell proliferative diseases such as blood cancer and solid cancer (regulation of tumor cell proliferation, tumor invasion and metastasis, angiogenesis) can be cited.

[0306] As an example of the blood cancer, the blood cancer selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma can be cited.

[0307] As an example of the solid cancer, the solid cancer selected from 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, various squamous cell carcinomas can be cited. As an example of the squamous cell carcinoma, the squamous cell carcinoma selected from oral squamous cell carcinoma, esophageal squamous cell carcinoma or pharyngeal squamous cell carcinoma can be cited.

[0308] As other examples of diseases, disorders or conditions associated with dysfunctions of LPA1-dependent cellular functions, the following can be cited: pain including fibromyalgia, cancer pain, diabetic neuropathy; inflammatory diseases and autoimmune diseases including rheumatoid arthritis, sepsis, chronic obstructive pulmonary disease, inflammatory bowel disease, transplanted organ rejection, Guillain-Barré syndrome, multiple sclerosis; metabolic diseases including obesity, insulin-resistant diabetes; cardiovascular disorders including cerebral infarction, hypertensive nephropathy, Raynaud's phenomenon; neurological disorders including hydrocephalus, schizophrenia, depression, dementia; urinary system diseases including prostatic hypertrophy, urinary incontinence; ophthalmic diseases including ischemic retinopathy, diabetic retinopathy, age-related macular degeneration.

[0309] <Method of administration>

[0310] The drug can be administered orally or parenterally, systemically or locally. As the mode of administration, injection dosage forms, nasal administration dosage forms, pulmonary administration dosage forms, transdermal administration dosage forms, etc. can be cited. In the case of injection dosage forms, for example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. In addition, the method of administration can be appropriately selected according to the age and symptoms of the patient. As the dosage of the antibody, for example, it can be selected within the range of 0.0001 mg to 1000 mg per 1 kg body weight each time. Or, for example, the dosage can be selected within the range of 0.001 to 100000 mg / body of the antibody per patient. However, the dosage of the antibody is not limited to these ranges.

[0311] <Formulation>

[0312] The drug can be formulated by conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A). The drug may contain pharmaceutically acceptable carriers and additives. Examples of the carrier or the additive include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorants, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity promoters, flavor correctives, etc., but are not limited to these, and other commonly used carriers, etc. may be appropriately used. Specifically, examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. In addition, other low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; and amino acids such as glycine, glutamine, asparagine, arginine, and lysine may be included.

[0313] In the case of preparing an aqueous injection solution, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used in combination with appropriate solubilizing aids, such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. In addition, if necessary, the antibody can be encapsulated in microcapsules (such as microcapsules of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.), or made into a colloidal delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) (refer to "Remingto's Pharmaceutical Science 16th edition", Oslo Ed. 1980, etc.).

[0314] In addition, techniques for sustained release of pharmaceuticals are known and can be applied to the above-mentioned drugs (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech. 12: 98-105 (1982); U.S. Patent No. 3,773,919; European Patent Application Publication No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); European Patent Application Publication No. 133,988).

[0315] <Applications of Gene Therapy>

[0316] It is also possible to consider integrating the nucleic acid into a vector for gene therapy or mRNA that can be translated into a protein in a living body as a gene therapy drug. As a method of administering the gene therapy drug (recombinant vector), in addition to direct administration based on a naked plasmid, methods of administering it packaged in liposomes, etc., methods of administering it integrated into various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, HVJ vectors (refer to Adolph 《Viral Genome Methods》, CRC Press, Florida (1996)), methods of administering it coated on bead vectors such as colloidal gold particles (pamphlet of International Publication No. 93 / 17706, etc.) can be cited.

[0317] As long as the gene therapy drug can express the antibody in a living body and exert its function, it can be administered by any method. It is preferably administered in a sufficient amount through an appropriate parenteral route, such as intravenous, intraperitoneal, subcutaneous, intradermal, within adipose tissue, within mammary tissue, inhalation, intramuscular, etc., through methods such as injection, infusion, gas-induced particle bombardment method (using an electron gun, etc.), mucosal routes such as nasal sprays. In addition, the gene therapy drug can also be administered to cells by lipofection, particle bombardment method (U.S. Patent No. 4,945,050) or viral infection in ex vivo, and the cells are then re-introduced into an animal for administration.

[0318] In addition, the present invention also includes a treatment method and a therapeutic drug related to a disease in a mammal suffering from or likely to suffer from a disease caused by abnormal hyperfunction of LPA1-dependent cell functions.

[0319] Here, "treatment" means, in a mammal that may have a disease or has a disease, preventing or alleviating the development and deterioration of the pathological condition of the disease, and thus is used in the sense of a therapeutic treatment aimed at preventing or alleviating the development and deterioration of each symptom, etc., of the disease.

[0320] In addition, "disease" refers to all diseases caused by abnormal hyperfunction of LPA1-dependent cell functions, without particular limitation. For example, it is a concept including liver fibrosis, kidney fibrosis, lung fibrosis, skin fibrosis, cardiovascular fibrosis, digestive tract fibrosis, and other fibrosis diseases. In addition, it is a concept including blood cancer, solid cancer, pain, inflammatory diseases, autoimmune diseases, metabolic diseases, cardiovascular disorders, nervous system disorders, urinary system diseases, and ophthalmic diseases. The "mammal" to be treated refers to any animal classified as a mammal, without particular limitation. For example, it is a human, pet animals such as dogs, cats, rabbits, livestock animals such as cows, pigs, sheep, horses, etc. A particularly preferred "mammal" is a human.

[0321] <Antibody-immobilized carrier>

[0322] The present invention includes a carrier immobilized with the antibody (antibody-immobilized carrier). In a preferred embodiment, the antibody-immobilized carrier is used to bring a body fluid containing LPA1-expressing cells into contact and remove LPA1-expressing cells from the body fluid. As an example of the body fluid, blood can be mentioned. The antibody immobilized on the carrier can be only one kind or two or more kinds.

[0323] As a specific form of the antibody-immobilized carrier of the present invention, for example, an antibody-immobilized carrier in which the antibody is immobilized on a water-insoluble carrier and filled in a container can be mentioned. Here, as the water-insoluble carrier, any material can be used. From the viewpoints of moldability, sterilizability, and low cytotoxicity, synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resins, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, alginate, inorganic materials such as hydroxyapatite, glass, alumina, titanium dioxide, and metal materials such as stainless steel and titanium are preferably selected.

[0324] As the shape of the carrier, granular, cotton-like, woven fabric, non-woven fabric, sponge-like porous body, flat plate-like, etc. can be mentioned. From the aspect of a large surface area per unit volume, granular, cotton-like, woven fabric, non-woven fabric, sponge-like porous body are preferred. For example, peripheral blood can be efficiently passed through a porous filter in a container pre-filled with a water-insoluble carrier immobilized with an antibody to remove LPA1-expressing cells related to the disease.

[0325] The antibody-immobilized carrier of the present invention can be combined with other components to prepare a kit for removing LPA1-expressing cells. As the other components, anticoagulants, extracorporeal circulation circuits, etc. can be mentioned.

[0326] <Other disclosures>

[0327] The present invention includes a method for treating the disease, disorder or condition, which comprises administering an effective amount of the antibody to a patient with a disease, disorder or condition associated with a disorder of LPA1-dependent cellular function. The present invention includes the antibody for treating a disease, disorder or condition associated with a disorder of LPA1-dependent cellular function. The present invention includes the use of the antibody for manufacturing a medicament used in the treatment of a disease, disorder or condition associated with a disorder of LPA1-dependent cellular function. The disease, disorder or condition at least includes tissue fibrosis, cell proliferative disease, pain, inflammatory disease, autoimmune disease, metabolic disease, cardiovascular disorder, urinary system disease or ophthalmic disease.

[0328] Example

[0329] [Example 1] Immunization of Mice for Preparing Anti-LPA1 Antibody

[0330] (1) Construction of Gene Immunization Vector Expressing Fusion Protein of LPA1 and GroEL Subunit

[0331] Based on human LPA1 (NP_001392, SEQ ID NO.111) registered in the NCBI gene bank, an artificial gene with a GCTAGC sequence added at the 5'-end and a GTCGAC sequence added at the 3'-end was synthesized. After introducing this artificial gene into a cloning vector, the obtained vector was cut 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 restriction enzymes NheI and SalI, and the prepared DNA fragment was inserted to construct an immunization vector pCI-hLPA1·GroEL. This vector expresses a fusion protein of human LPA1 and GroEL.

[0332] The same operation was performed using mouse LPA1 (NP_034466, SEQ ID NO.112) instead of human LPA1 to construct an immunization vector pCI-mLPA1·GroEL. This vector expresses a fusion protein of mouse LPA1 and GroEL.

[0333] (2) DNA Immunization

[0334] The vector pCI-hLPA1·GroEL or pCI-mLPA1·GroEL was dissolved in PBS to a concentration of 2 mg / mL to prepare an immunizing composition. By in vivo electroporation, 25 μL of this immunizing composition was immunized into the thigh muscles of both legs of 8-week-old mice of various systems (day 0). Thus, 50 μg of pCI-hLPA1·GroEL or pCI-mLPA1·GroEL was immunized into each leg, that is, 100 μg of DNA was immunized per mouse per time. Thereafter, DNA immunization was also performed on days 14, 28, 42, and 56 in the same manner. In addition, blood was collected before immunization and on days 21, 35, and 63 after immunization to prepare serum.

[0335] (3) Preparation of LPA1 stably expressing cells

[0336] An artificial gene was synthesized based on the human LPA1 (NP_001392, SEQ ID NO.111) registered in the NCBI gene bank, introduced into the pCIneo vector (PROMEGA), and pCIneo-hLPA1 was constructed. pCIneo-hLPA1 was introduced into CHO-K1 cells using Lipofectamine 2000 (ThermoFisher Scientific). The obtained 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").

[0337] An artificial gene was synthesized based on the mouse LPA1 (NP_034466, SEQ ID NO.112) registered in the NCBI gene bank, introduced into pEF5 / FRT / V5-DEST (ThermoFisher Scientific), and pEF-FRT-mLPA1 was constructed. Using Lipofectamine 2000, pEF-FRT-mLPA1 and the pOG44 plasmid (ThermoFisher Scientific) were simultaneously introduced into Flp-In-CHO cells (ThermoFisher Scientific). 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").

[0338] (4) Evaluation of the binding of antibodies in serum to human LPA1 and mouse LPA1 by flow cytometry

[0339] Human LPA1 stable expression CHO cells, mouse LPA1 stable expression 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 1 / 500 volume of Fc Block (Cytek Biosciences) was added to the cell suspension and blocked at 4°C for 30 minutes. After blocking, the pre-immune and post-immune sera diluted 50-fold were incubated with each cell. In addition, each cell was washed with FACS buffer, and after adding phycoerythrin-labeled anti-mouse IgG antibody (Southern Biotech) as a secondary antibody, the binding of each cell to anti-human LPA1 antibody and anti-mouse LPA1 antibody in the serum was evaluated using a flow cytometer iQue (Sartorius). In the post-immune serum, there were antibodies that bound to human LPA1 stable expression CHO cells or mouse LPA1 stable expression CHO cells but not to negative control cells. Individuals with an increase in the titer of specific antibodies against LPA1 were subjected to booster immunization and sampling.

[0340] (5) Booster immunization and sampling using transient expression cells

[0341] The 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 intraperitoneum of individuals with an increase in antibody titer, and the spleen, inguinal lymph nodes, and iliac lymph nodes were collected 3 days later. Splenocytes and lymph node-derived cells were isolated from each sampled tissue, suspended in CELLBANKER 1plus (Nippon Zenyaku Kogyo Co., Ltd., Japan), and stored at -80°C until screening for specific antibody-producing lymphocytes.

[0342] [Example 2] Preparation of antibodies

[0343] (1) Screening for specific antibody-producing lymphocytes using a microplate

[0344] The screening for specific antibody-producing lymphocytes using a microplate was carried out according to the methods described in International Publication No. 2020 / 171020 and Japanese Patent No. 6881801. That is, human LPA1 stable expression CHO cells or mouse LPA1 stable expression CHO cells were suspended in F-12 medium (containing 10% FBS, Penicillin / Streptomycin) to prepare 3.5×10 5Cells / 500 μL of cell suspension. The cell suspension was filled into a micro-chamber (AS ONE Corporation) for a cell picking system (Cell Picking System). The micro-chamber was centrifuged at 300 rpm for 1 minute, 5 times, so that the human LPA1-stably expressing CHO cells or mouse LPA1-stably expressing CHO cells were prepared in such a way that 1 or 2 cells were contained in each micropore. After washing the micro-chamber with F-12 medium, 500 μL of F-12 medium was added. The micro-chamber was incubated at 37 °C in a CO2 incubator for 1 hour to allow the human LPA1-stably expressing CHO cells or mouse LPA1-stably expressing CHO cells to adhere to the bottom surface of the micropores. 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 3 times with F-12 medium to remove the remaining CytoRed, 1 mL of F-12 medium was filled into the micro-chamber.

[0345] Among the cells recovered from Example 1(5) after DNA immunization, antibody-producing cells were concentrated using an EasySep Mouse Biotin Positive Selection Kit (STEMCELL TECHNOLOGIES). 1.1×10 5Antibody-producing cells were suspended in 500 μL of F-12 medium and filled into the microwells. The microwells were centrifuged at 300 rpm for 1 minute, 5 times, so that 1 or 2 antibody-producing cells were placed in each micropore. After washing the microwells with the medium, an appropriate amount of medium was added, and the cells were incubated at 37 °C for 30 minutes to secrete antibodies from the antibody-producing cells. After washing the microplate to remove the supernatant, Alexa Fluor 488-labeled anti-mouse IgG antibody (ABCAM) diluted 500-fold with RPMI1640 (containing 10% FBS) was added, and the cells were incubated at 37 °C for 30 minutes. After washing 3 times with RPMI1640 (without phenol red, containing 1% FBS), 1 mL of RPMI1640 was added. The microwells were set in a cell picking system (AS ONE), and information on transmitted light images and two fluorescence images of the entire microplate was obtained. Fluorescence detection of CytoRed was performed under the conditions of an excitation wavelength of 543 nm and a fluorescence wavelength of 593 nm. Fluorescence detection of Alexa Fluor 488 was performed under the conditions of an excitation wavelength of 482 nm and a fluorescence wavelength of 536 nm. Based on the scanned images of transmitted light, CytoRed, and Alexa Fluor 488, antibody-producing cells were recovered into the cell lysate from the micropores where antibodies that could be judged to bind to the surface of human LPA1-stably expressing CHO cells or mouse LPA1-stably expressing CHO cells were present, using a capillary (AS ONE) with a diameter of several μm to several tens of μm.

[0346] (2) Isolate the antibody gene from the recovered antibody-producing cells

[0347] The antibody gene is obtained from antibody-producing cells by the MAGrahd method (Kurosawa N, Yoshioka M, Fujimoto R, Yamagishi F, Isobe M. “Rapid production of antigen-specific monoclonal antibodies from a variety of animals.” BMC Biol. 2012; 10:80). That is, 5 μL of the cell lysate recovered in (1) is mixed with 5 μg of oligo dT magnetic beads to capture the cell-derived mRNA onto the oligo dT magnetic beads. After washing the oligo dT magnetic beads with a washing solution using a MAGrahd reactor tray and neodymium magnetic beads, cDNA synthesis based on reverse transcription reaction is carried out. After further washing the magnetic beads, a 5'-end translation reaction is implemented. Using the synthesized cDNA, the genes of the antibody heavy chain variable region (VH region) and the antibody light chain variable region (VL region) are isolated and amplified by the 5' race PCR method. It should be noted that in order to improve the specificity of the amplification product, PCR is carried out twice. In the first PCR, the following primers are used in combination: the first forward primer (SEQ ID NO. 113) that co-amplifies the VH region and the VL region, the VH first reverse primer (SEQ ID NO. 114) that specifically amplifies the VH region, and the VL first reverse primer (SEQ ID NO. 115) that specifically amplifies the VL region. In the second PCR, using the amplification product of the first time as a template, for the amplification of the VH region, the second forward primer (SEQ ID NO. 116) and the VH second reverse primer (SEQ ID NO. 117) that specifically amplifies the VH region are used as primers respectively, and for the amplification of the VL region, the second forward primer (SEQ ID NO. 116) and the VL second reverse primer (SEQ ID NO. 118) that specifically amplifies the VL region are used as primers respectively. When agarose gel electrophoresis is performed on the sample after the second PCR, a gene amplification product corresponding to the VH region can be confirmed at about 750 bp, and a gene amplification product corresponding to the VL region can be confirmed at about 550 bp.

[0348] (3) Construction of the antibody expression unit

[0349] 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 gene of the VH region amplified in (2), the gene of the antibody heavy chain constant region, and the gene containing the promoter region required for gene expression were fused using PCR to construct an antibody expression unit expressing the full-length antibody heavy chain. Similarly, the gene of the VL region amplified in (2), the gene of the antibody light chain constant region, and the gene containing the promoter region required for gene expression were fused using PCR to construct an antibody expression unit expressing the full-length antibody light chain.

[0350] (4) Introduce the antibody expression unit into mammalian cells

[0351] In a 6-well plate for cell culture, Expi293F cells (Thermo Fisher Scientific) were inoculated at a density of 7.5×10 6 cells / 3 mL / well. Using Expifectamine 293 Reagent (Thermo Fisher Scientific), the two antibody expression units of the heavy chain and the light chain constructed in (3) were co-introduced into Expi293F cells. On the 5th day after the introduction, the cell supernatant was collected for evaluating the binding property of the antibody to be produced.

[0352] (5) Evaluation of the binding property of the antibody using flow cytometry

[0353] Human LPA1 stable expression CHO cells or mouse LPA1 stable expression CHO cells and negative control cells were washed with FACS buffer to suspend them at a cell concentration of 1×10 7 cells / mL with FACS buffer. 1 / 500 volume of Fc Block (Cytek Biosciences) was added and blocked at 4 °C for 30 minutes. After blocking, to make it 2×10 5Suspend the cells at a density of cells / 50 μL. Mix the cell suspension with the cell supernatant recovered in (4) and incubate at 4°C for 1 hour. After incubation, wash the cells twice with 100 μL of FACS buffer. Add 50 μL of a dilution of phycoerythrin-labeled anti-mouse IgG antibody (Southern Biotech) as the secondary antibody to each well and incubate at 4°C for 1 hour. After washing the cells twice with 100 μL of FACS buffer, suspend them in 80 μL of FACS buffer and measure the fluorescence intensity on the cell surface using a flow cytometer iQue (Sartorius) to evaluate the antibody binding. Antibody units that bind to human LPA1 stably expressing CHO cells or mouse LPA1 stably expressing CHO cells and do not bind to negative control cells are defined as primary hit antibodies.

[0354] [Example 3] Screening of antibodies based on the evaluation of the inhibition of intracellular cAMP signal transduction

[0355] According to the culture supernatant corresponding to the primary hit antibody in Example 2, a purified antibody was prepared by Protein A affinity purification according to a conventional method and used for the following evaluation. The intracellular cyclic adenosine monophosphate (cAMP) concentration was measured using a LANCE Ultra cAMP Kit (PERKINELMER).

[0356] Culture human LPA1 stably expressing CHO cells and mouse LPA1 stably expressing CHO cells in Ham's F-12 medium without FBS containing 0.5% BSA, 100 units / mL penicillin, and 100 μg / mL streptomycin for 24 hours. Recover the cells from the cell culture dish using enzyme-free cell dissociation buffer (ThermoFisher Scientific) and wash the cells with Hanks’ Balanced Salt Solution (HBSS). Suspend the cells with Stimulation buffer (5 mM HEPES, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), HBSS containing 0.1% BSA) to make it 2×10 5 / mL, 12.5 μL (2,500 cells) of cells were dispensed per well in an OptiPlate-96 well (manufactured by PERKINELMER). 6.25 μL of an antibody diluted to 400 nM with Stimulation buffer (final concentration 100 nM) was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes. Subsequently, 6.25 μL of a mixed solution of 12 μM forskolin (final concentration 3 μM) and 200 nM oleoyl-L-α-lysophosphatidic acid sodium salt (Merck) (final concentration 50 nM) was added, and the mixture was allowed to stand at room temperature for 30 minutes. Using the cAMP Detection Buffer included in the kit, 12.5 μL each of the Eu-cAMP tracer solution and ULight-anti-cAMP antibody solution diluted according to the instruction manual were added. After allowing the mixture to stand at room temperature for 1 hour, TR-FRET was measured using an EnVision 2104 microplate reader (PERKINELMER). Regarding the inhibition rate (%), the measured value when forskolin and lysophosphatidic acid were added was taken as an inhibition rate of 0%, and the measured value when only forskolin was added was taken as an inhibition rate of 100% and normalized to calculate the inhibition rate (%) of the relative intracellular cAMP signal transduction.

[0357] Among the primary screening hits, 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 the lysophosphatidic acid-induced intracellular cAMP signal transduction in human LPA1-expressing cells and mouse LPA1-expressing cells, were selected as antibodies having the activity of blocking LPA1-dependent cellular functions.

[0358] For the 11 antibodies, the types of antigens at the time of acquisition and the inhibition rates of the lysophosphatidic acid-induced intracellular cAMP signal transduction in human and mouse LPA1-expressing cells are shown in Table 1.

[0359] [Table 1]

[0360] Table 1

[0361]

[0362] [Example 4] Screening of Antibodies for Evaluation of Binding to Lysophosphatidic Acid Receptors Based on Flow Cytometry

[0363] To evaluate the binding specificity to lysophosphatidic acid (LPA) receptor subtypes LPA1, LPA2, and LPA3, artificial genes with a FLAG tag sequence added to the 5'-end of each gene were synthesized based on human LPA1 (SEQ ID NO.111), mouse LPA1 (SEQ ID NO.112), 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. Using Lipofectamine 2000, 10 μg of the expression vector into which each synthetic gene was introduced was transfected into HEK293FT cells to prepare cells transfected with the human LPA1 gene, cells transfected with the mouse LPA1 gene, cells transfected with the human LPA2 gene, cells transfected with the mouse LPA2 gene, cells transfected with the human LPA3 gene, and cells transfected with the mouse LPA3 gene.

[0364] The next day, the cells were washed with PBS, detached from the cell culture dish using an enzyme-free cell dissociation buffer (ThermoFisher Scientific), washed with PBS, and then recovered by centrifugation. Goat serum (ThermoFisher Scientific) was diluted 2-fold with FACS buffer (PBS containing 1% FBS) to a concentration of 1×10 7 / mL and suspended. The suspension was incubated at 4°C for 30 minutes for blocking. The cells were collected by centrifugation and resuspended in FACS buffer at a concentration of 4×10 6 / mL. Then, 25 μL (1×10 5 cells) of the cell suspension was aliquoted into each well of a V-bottom 96-well plate (ThermoFisher Scientific).

[0365] Add 25 μL each of the 11 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) at 20 μg / mL, anti-FLAG mouse IgG antibody (from Sigma-Aldrich) at 20 μg / mL, or mouse isotype control antibody (from FUJIFILM Wako Pure Chemical Corporation), and leave to stand at 4°C for 1 hour. Collect the cells by centrifugation and wash the cells with 120 μL of FACS buffer. Perform this washing operation twice. Dispense 25 μL each of the secondary antibody, Goat Anti-Mouse IgG H&L (DyLight 650) (manufactured by Abcam), diluted 500-fold with FACS buffer, onto the cell pellet, suspend, and leave to stand at 4°C for 1 hour. After washing the cells twice, suspend them in 50 μL of FACS buffer, transfer the entire volume to a V-bottom 96-well plate (manufactured by Greiner), and measure the binding to the antibody using a flow cytometer (Agilent Novocyte).

[0366] As a representative example, the histogram of the flow cytometer for 210309-4-A is shown in Figure 1. Compared with the case of using non-gene-transfected cells, it was investigated whether there was a right shift in the histogram in the case of using gene-transfected cells. First, in the case of using anti-FLAG mouse IgG antibody as the primary antibody, a right shift was observed in any of the human LPA1 gene-transfected cells, human LPA2 gene-transfected cells, human LPA3 gene-transfected cells, mouse LPA1 gene-transfected cells, mouse LPA2 gene-transfected cells, and mouse LPA3 gene-transfected cells. On the other hand, when the mouse isotype control antibody was used as the primary antibody or only the secondary antibody, no right shift was observed in any of the gene-transfected cells. From this, it was confirmed that any of the receptors were expressed on the cell surface.

[0367] On the other hand, in the case of using 210309-4-A as the primary antibody, a right shift in the histogram was observed only in the human LPA1 gene-transfected cells and mouse LPA1 gene-transfected cells. From this, it was observed 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, and mouse LPA3.

[0368] For antibodies other than 210309-4-A, the same results as in Figure 1 were also obtained.

[0369] The binding specificities of 11 antibodies other than 210309-4-A are shown in Table 2. The situation where a right shift of the histogram was observed was indicated as "+", and the situation where it was not observed was indicated as "-". It was confirmed that all 11 antibodies specifically bound to the extracellular domains of human LPA1 and mouse LPA, did not bind to the extracellular domains of human LPA2, human LPA3, mouse LPA2, and mouse LPA3, and were antibodies that specifically bound to the extracellular domain of human LP A1 and did not specifically bind to the extracellular domain of human LPA2 or the extracellular domain of human LPA3.

[0370] [Table 2]

[0371] Table 2

[0372]

[0373] [Example 5] Determination of CDR sequences of antibody heavy chain variable region and antibody light chain variable region

[0374] Using the 11 antibody genes 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 selected in Example 3, cloning into the pET vector was performed 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). In addition, in order to analyze the sequence of the cloned antibody heavy chain variable region (VH region), sequencing 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), sequencing 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. Regarding the determination of CDR, a combined analysis of the kabat numbering method and the IMGT numbering method was performed, and the region that inclusively included the regions determined by the two numbering methods was determined as CDR.

[0375] The amino acid sequences (AA) of the heavy chain variable region, heavy chain CDR1-3, light chain variable region, and light chain CDR1-3 of each antibody, as well as the base sequences of the DNAs encoding the heavy chain variable region and light chain variable region, are summarized in Tables 3-1 to 3-11.

[0376] [Table 3-1]

[0377] Table 3-1 210309-4 -A

[0378]

[0379] [Table 3-2]

[0380] Table 3-2 210309-1-C

[0381]

[0382] [Table 3-3]

[0383] Table 3-3 210309-1-G

[0384]

[0385] [Table 3-4]

[0386] Table 3-4 210309-2-A

[0387]

[0388] [Table 3-5]

[0389] Table 3-5 210309-2-D

[0390]

[0391] [Table 3-6]

[0392] Table 3-6 210310-1-D

[0393]

[0394] [Table 3-7]

[0395] Table 3-7 210420-4-E

[0396]

[0397] [Table 3-8]

[0398] Table 3-8 210420-1-H

[0399]

[0400] [Table 3-9]

[0401] Table 3-9 210420-3-E

[0402]

[0403] [Table 3-10]

[0404] Table 3-10 211222-1-G

[0405]

[0406] [Table 3-11]

[0407] Table 3-11 211222-1-A

[0408]

[0409] [Example 6] Evaluation of Binding to LPA1 by Flow Cytometry

[0410] Synthesize artificial genes in which 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 are linked to the heavy chain constant region and κ chain constant region of mouse IgG1. Introduce the antibody gene into CHO cells, and produce and purify the antibody from the culture solution by affinity chromatography for the following tests. When checking the purity of any of the purified antibodies by SDS-PAGE, it was confirmed to be 90% or more.

[0411] Wash human LPA1 stable expression CHO cells and mouse LPA1 stable expression CHO cells with PBS respectively, strip them from the cell culture dish with enzyme-free cell dissociation buffer (ThermoFisher Scientific), wash with PBS, and recover by centrifugation. Human LPA1 stable expression CHO cells and mouse LPA1 stable expression CHO cells were suspended at 1×10 7 / mL in Fc Block (Cytek Biosciences) diluted 500-fold with FACS buffer and left standing at 4°C for 30 minutes for blocking. Collect the cells by centrifugation to make it 4×10 6Suspend the cells in FACS buffer at a concentration of / mL, and dispense 25 μL (1×10 5 cells) into each well of a V-bottom 96-well plate (ThermoFisher Scientific). As the primary antibody, serially dilute 11 kinds of purified LPA1 antibodies produced by the described method and a mouse isotype control antibody (FUJIFILM Wako Pure Chemical Corporation), add 25 μL of each, and leave to stand at 4°C for 1 hour. Collect the cells by centrifugation and wash the cells with 120 μL of FACS buffer. Perform this washing operation twice. Dispense 25 μL of the secondary antibody, Goat Anti-Mouse IgG H&L (DyLight 650) (manufactured by Abcam), diluted 500-fold with FACS Buffer, into the cell pellet, suspend, and leave to stand at 4°C for 1 hour. After washing the cells twice, suspend them in 50 μL of FACS buffer and measure the binding to the antibody using a flow cytometer (Agilent Novocyte).

[0412] Plot a graph with the antibody concentration on the horizontal axis and the median fluorescence intensity of the histogram of the flow cytometer on the vertical axis. It was confirmed that all 11 antibodies bind to human LPA1 stably expressing CHO cells and mouse LPA1 stably expressing CHO cells in a dose-dependent manner. The results of the binding of each antibody to human LPA1 stably expressing CHO cells are as Figures 2A - 2K shown, and the results of the binding of each antibody to mouse LPA1 stably expressing CHO cells are as Figures 3A - 3K shown. Calculate the 50% binding concentration (EC50) and summarize in Table 4.

[0413] [Table 4]

[0414] Table 4

[0415]

[0416] Next, evaluate the binding of the LPA1 antibody to endogenously expressed LPA1 on the cell surface. Use a human lung-derived fibroblast cell line (IMR-90) in which the gene expression of human LPA1 has been confirmed for verification. According to the same procedure as described above, measure the binding of 10 μg / mL of the purified LPA1 antibody and the mouse isotype control antibody using a flow cytometer (Agilent Novocyte). As a result, it was confirmed that all 11 antibodies specifically bind to IMR-90. The histogram comparing the 11 antibodies with the mouse isotype control antibody is shown in Figure 4 .

[0417] [Example 7] Evaluation of Inhibition of Intracellular cAMP Signal Transduction

[0418] Subject the antibody produced in Example 6 to the following test.

[0419] The change in the intracellular cyclic adenosine monophosphate (cAMP) concentration was measured using the LANCE Ultra cAMP Kit (manufactured by PERKINELMER) based on the method of Example 3. In 12.5 μL of human LPA1-stably expressing CHO cells and mouse LPA1-stably expressing CHO cells at a cell concentration of 2×10 5 / mL, 6.25 μL of serially diluted purified LPA1 antibody or LPA1 small molecule antagonist BMS-986278 (manufactured by MedKoo) was added, and the mixture was allowed to stand at room temperature for 15 minutes. Further, 6.25 μL of a mixture of 12 μM forskolin (manufactured by Merck) (final concentration 3 μM) and 200 nM sodium oleoyl-L-α-lysophosphatidate (final concentration 50 nM) was added, and after allowing to stand at room temperature for 30 minutes, the cAMP concentration was measured based on the method of Example 3. For the inhibition rate (%), the measured value when forskolin and oleoyl-L-α-lysophosphatidic acid were added was taken as the inhibition rate of 0%, and the measured value when only forskolin was added was taken as the inhibition rate of 100% and standardized to calculate the inhibition rate (%) of the relative intracellular cAMP signal transduction. The horizontal axis represents the concentration of each antibody, and the vertical axis represents the inhibition rate (%) and is plotted. The dose-dependence of the inhibitory activity of each antibody on human LPA1 is as Figures 5A - 5K shown, and the dose-dependence of the inhibitory activity of each antibody on mouse LPA1 is as Figures 6A - 6K shown. The results showed that all 11 antibodies were confirmed to dose-dependently inhibit lysophosphatidic acid-induced intracellular cAMP signal transduction in human and mouse LPA1-expressing CHO cells and had the activity of blocking LPA1-dependent cellular functions.

[0420] The 50% inhibitory concentration (IC 50 ) of three independent intracellular cAMP signal transduction inhibition assays was calculated, and the average and standard deviation are shown in Table 5. In addition, the dose-dependence of the inhibition of lysophosphatidic acid-induced intracellular cAMP signal transduction in human and mouse LPA1-expressing CHO cells by BMS-986278 is as Figure 7A and Figure 7B shown. The IC 50 values of BMS-986278 for human and mouse LPA1 were 83 nM and 232 nM, respectively. The results showed that all 11 antibodies were equivalent to or more excellent than BMS-986278 in terms of the IC 50 value for the activity of blocking human LPA1-dependent cellular functions.

[0421] [Table 5]

[0422] Table 5

[0423]

[0424] [Example 8] Verification of the inverse agonist effect of the LPA1 antibody on intracellular cAMP signal transduction

[0425] Using human LPA1 stably expressing CHO cells, the effect of 210309-4-A on the change in intracellular cAMP concentration was evaluated in the absence of the LPA1 ligand. That is, in 12.5 μL of human LPA1 stably expressing CHO cells at a cell concentration of 2×10 5 / mL, 6.25 μL of 210309-4-A used in Example 6 diluted stepwise was added and allowed to stand at room temperature for 15 minutes. In addition, 6.25 μL (final concentration 3 μM) of 12 μM forskolin was added and allowed to stand at room temperature for 30 minutes, and then the cAMP concentration was measured. The graph with the antibody concentration on the horizontal axis and the cAMP concentration (nM) on the vertical axis is shown in Figure 8 . In Figure 8 , the dotted line represents the cAMP concentration (2.9 nM) stimulated by forskolin alone. That is, 210309-4-A increased the cAMP level in a dose-dependent manner, thus showing the characteristics of an inverse agonist. The same test was also carried out on 10 other LPA1 antibodies, and the results showed that the cAMP level increased in a dose-dependent manner, showing the characteristics of an inverse agonist. It was shown that this antibody is an antibody that can block LPA1 receptor-dependent cellular functions independently of the LPA1 ligand.

[0426] [Example 9] Verification of the inhibitory mode of the LPA1 antibody on intracellular cAMP signal transduction

[0427] In addition, using human LPA1 stably expressing CHO cells, the effect of various concentrations of 210309-4-A on the dose-response curve of lysophosphatidic acid as the LPA1 ligand was evaluated. That is, in 12.5 μL of human LPA1 stably expressing CHO cells at a cell concentration of 2×10 5 / mL, 6.25 μL of 210309-4-A used in Example 6 at a certain concentration (final concentrations of 0, 5, 7.5, 10, 100 nM) was added and allowed to stand at room temperature for 15 minutes. Further, 6.25 μL of a mixture of 12 μM forskolin (final concentration 3 μM) and sodium oleoyl-L-α-lysophosphatidate (final concentration 0.1 - 10000 nM) was added and allowed to stand at room temperature for 30 minutes, and then the cAMP concentration was measured. The dose-response curves of lysophosphatidic acid at each antibody concentration are as shown in Figure 9 . That is, when the antibody concentration increased, the dose-response curve shifted to the right, thus showing the characteristics of competitive inhibition.

[0428] [Example 10] Preparation of a humanized anti-LPA1 antibody

[0429] Illustrate the case of 210309-4-A. To prepare a humanized antibody transplanted with the CDR of 210309-4-A, a human framework was selected based on the identity between 210309-4-A and human germline VH and VK genes. Based on computer modeling, multiple antibodies with mutations added to the sequences of the selected human germline VH and VL were designed in a way that could support the predicted antibody's three-dimensional structure, namely, "h309-4-A-BP1", "h309-4-A-SS1", and "h309-4-A-SG1". 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 as shown in Figure 10A below. 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 as shown in Figure 10B below.

[0430] Synthesize the following artificial genes: artificial genes in which the heavy chain constant region of human IgG1 (including the LALA mutation) (SEQ ID NO.129) is linked to the VH regions of h309-4-A-BP1, h309-4-A-SS1, and h309-4-A-SG1, and the kappa chain constant region (SEQ ID NO.130) is linked to the VL regions. In addition, as a comparison object, an artificial gene of a chimeric anti-LPA1 antibody "309-4-A-Chimera" in which the heavy chain constant region of human IgG1 (including the LALA mutation) is linked to the VH region of 210309-4-A and the kappa chain constant region is linked to the VL region was also synthesized. The artificial genes were introduced into CHO cells, and purified antibodies were prepared from the culture solution by affinity chromatography for the following tests. When the purity of any of the purified antibodies was examined by SDS-PAGE, it was confirmed to be 90% or more.

[0431] Next, using human LPA1 stably expressing CHO cells, the binding activity to human LPA1 was measured according to the method of Example 6. A graph was plotted with the antibody concentration on the horizontal axis and the median fluorescence intensity of the histogram of the flow cytometer on the vertical axis. The results of the binding of each antibody to human LPA1 stably expressing CHO cells are as shown in Figures 11A - 11EAs shown, the calculated 50% binding concentration (EC50) is shown in Table 6. In addition, the inhibitory activity of intracellular cAMP signal transduction was measured according to the method of Example 7. A graph was plotted with the concentration of each antibody on the horizontal axis and the inhibition rate (%) on the vertical axis. The dose-dependence of the inhibitory activity of each antibody against human LPA1 is as Figures 12A - 12E shown, and the calculated 50% inhibitory concentration (IC 50 ) is shown in Table 7.

[0432] As shown by these results, the binding activity and inhibitory activity of 309-4-A-Chimera were equivalent to those of 210309-4-A. Thus, it was confirmed that the conversion of the human antibody to the constant region did not affect the binding activity and inhibitory activity. In addition, regarding the binding activity and inhibitory activity of h309-4-A-BP1, h309-4-A-SS1, and h309-4-A-SG1 designed as humanized LPA1 antibodies, they were equivalent to those of 309-4-A-Chimera. Thus, it was confirmed that the binding activity and inhibitory activity were maintained after humanization in all designs.

[0433] [Table 6]

[0434] Table 6

[0435]

[0436] [Table 7]

[0437] Table 7

[0438]

[0439] The amino acid sequences of SEQ ID NOs. 123 to 130 are shown in Table 8.

[0440] [Table 8]

[0441] Table 8

[0442]

Claims

1. 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.

2. The antibody according to claim 1, which has the activity of blocking LPA1-dependent cellular functions.

3. The antibody according to claim 1 or 2, which has: 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; 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; 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.

4. The antibody according to claim 1 or 2, which satisfies any one of the following (A1) to (A11): (A1) having: 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) having: 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) having: 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) having: 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) having: 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) having: 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) having: 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) having: 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) having: 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) having: 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.

5. The antibody according to claim 1 or 2, which satisfies any one of the following (B1) to (B11): (B1) having: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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.

6. The antibody according to claim 1 or 2, which satisfies any one of the following (AB1) to (AB11): (AB1) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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.

7. The antibody according to claim 1 or 2, which satisfies any one of the following (C1) to (C11): (C1) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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) has: 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.

8. An antibody that specifically binds to the extracellular domain of human LPA1 and does not specifically bind to the extracellular domain of human LPA2 Does not specifically bind to the extracellular domain of human LPA3, The antibody satisfies any one of the following (C1') to (C11'): (C1') has: 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 with 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') has: 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 with 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 with the amino acid sequence represented by SEQ ID NO. 19, (C3') has: 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 with 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 with the amino acid sequence represented by SEQ ID NO. 29, (C4') has: 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 with 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 with the amino acid sequence represented by SEQ ID NO. 39, (C5') has: 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 with the amino acid sequence represented by SEQ ID NO. 47; and a light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 49, (C6’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 57; and a light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 59, (C7’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 67; and a light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 69, (C8’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 77; and a light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 79, (C9’) having: a heavy chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 87; and a light chain variable region having 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 with the amino acid sequence represented by SEQ ID NO. 89, (C10’) has: 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 with 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 with the amino acid sequence represented by SEQ ID NO. 99, (C11’) has: 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 with 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 with the amino acid sequence represented by SEQ ID NO.

109.

9. The antibody according to claim 8, which has an activity of blocking LPA1-dependent cell functions.

10. 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 first antibody to the receptor, wherein the first antibody is the antibody according to claim 7.

11. The second antibody according to claim 10, which has an activity of blocking LPA1-dependent cell functions.

12. The antibody according to 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 conjugated with other molecules.

15. According to 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 according to claim 16.

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

19. The drug according to claim 18, which is used for the treatment of tissue fibrosis, cell proliferative diseases, pain, inflammatory diseases, autoimmune diseases, metabolic diseases, cardiovascular disorders, urinary system diseases or ophthalmic diseases.

20. The drug according to claim 19, which is used for the treatment of tissue fibrosis, wherein the tissue fibrosis is liver fibrosis, kidney fibrosis, lung fibrosis, skin fibrosis, cardiovascular fibrosis or digestive tract fibrosis.

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