Antibodies binding to cannabinoid type 1 receptor

Abstract

The purpose of the present invention is to provide a novel antibody or antibody fragment thereof that binds to the cannabinoid type 1 (CB1) receptor. Disclosed is a CB1 receptor antibody or an antibody fragment thereof, which has six specific CDRs (CDR1-CDR3 of a heavy chain and CDR1-CDR3 of a light chain) or a specific heavy chain variable region / light chain variable region. The antibody or the like can be used in the treatment or prevention of obesity, diabetes, non-alcoholic steatohepatitis and the like.

Description

[Technical field]

[0001] The present invention relates to an antibody or an antibody fragment thereof that binds to a novel cannabinoid type 1 (CB1) receptor. More specifically, it relates to an antibody or an antibody fragment thereof that specifically binds to a CB1 receptor. Still more specifically, it relates to an antibody or an antibody fragment thereof that selectively inhibits a CB1 receptor, a pharmaceutical composition containing the same, or a CB1 receptor detection kit. [Background technology]

[0002] The CB1 receptor is a type of G protein-coupled receptor (GPCR) family expressed mainly in the central nervous system, liver, kidney, lung, and adipose tissue, and mediates reactions with Δ9-tetrahydrocannabinol, which is an endogenous cannabinoid or a plant phytocannabinoid. When the signal transduction via the CB1 receptor is activated by endogenous cannabinoids, etc., the interaction with Gi / O proteins causes inhibition of acetyl cyclase, activation of mitogen-activated protein kinase (MAPK), inhibition of potential-dependent Ca ion channels, influence on NO signal transduction, etc. (Non-patent Document 1).

[0003] CB1 receptors are associated with a variety of diseases such as obesity, diabetes, pain, neurodegenerative diseases, fibrosis, liver disease, cardiovascular disease, etc. In fact, antagonist therapeutic drugs for CB1 receptors such as rimonabant show anti-obesity effects and metabolic improvement effects such as decreased appetite or reduction of neutral fat values. On the one hand, low molecular antagonists such as rimonabant are known to cause central side effects such as mental effects, depression, suicidal desire, etc. (Non-patent Document 2).

[0004] It is known that low-molecular-weight CB1 receptor antagonists with low brain mobility that minimize the risk of central side effects have shown improvement effects on insulin resistance, dyslipidemia, and hyperuricemia in mice (Non-patent Document 3), and peripheral CB1 receptor inhibitors with low brain mobility are believed to have the same effect as systemic antagonists. Therefore, CB1 receptor antagonists or CB1 receptor agonists that are not accompanied by central side effects are useful drugs that act widely on the periphery.

[0005] Patent Document 1 discloses an antibody against the CB1 receptor as an example of a compound capable of regulating CB1 receptor signaling.

[0006] Patent Document 2 discloses antibodies that bind to an epitope within the EC2 domain of the human CB1 receptor. Although examples of evaluating the antagonist activity or neutralizing activity of these antibodies in vitro are described, there is no description of in vivo evaluation.

[0007] Patent Documents 3 and 4 disclose antibodies that are inverse agonists of the CB1 receptor and describe in vitro and in vivo evaluations of the CB1 antibodies. On the other hand, Patent Documents 3

[0361] to

[0363] and Patent Documents 4

[0367] to

[0369] describe that administration of P1C4-h2-IgG4 to primates reduces triglyceride levels and other cardiovascular risk factors and improves insulin sensitivity, but no specific data are disclosed.

[0008] Patent Document 5 discloses an antibody that binds to a CB1 receptor. Although examples of evaluating in vitro antagonist activity or neutralizing activity are described, there is no description of in vivo evaluation.

[0009] That is, in any prior art document, there is no disclosure of the CDR sequences, light chain variable regions, heavy chain variable regions, etc. possessed by the antibody of the present invention.

[0010] [Prior art literature]

[0011] [Patent Literature]

[0012] [Patent Document 1] WO2005 / 023232

[0013] [Patent Document 2] WO2014 / 210205

[0014] [Patent Document 3] WO2015 / 148984

[0015] [Patent Document 4] WO2017 / 058771

[0016] [Patent Document 5] WO2019 / 211665

[0017]

Non-patent literature

[0018]

Non-patent document 1

[0019] [Non-patent document 2] Psychopharmacology volume 205, pages 171-174 (2009)

[0020]

Non-patent document 3

[0021] [Summary of the Invention]

[0022] [Problems to be solved by the invention]

[0023] The purpose of the present invention is to provide a novel antibody or antibody fragment thereof that binds to a CB1 receptor. A novel CB1 receptor antibody or antibody fragment thereof that can be used as a therapeutic drug for obesity, diabetes, etc. is also provided. As a result of intensive research, the inventors have found an antibody or antibody fragment thereof that specifically binds to one or more extracellular epitopes on the CB1 receptor and regulates CB1 receptor signal transduction. Furthermore, it was found that the antibody of the present invention has an improvement in small intestinal transport energy or an anti-obesity effect, and is useful for the treatment of obesity, diabetes, non-alcoholic steatohepatitis (NASH), etc.

[0024] That is, the present invention relates to the following technical solutions.

[0025] (1-1) An antibody or an antibody fragment thereof that binds to human cannabinoid type 1 (CB1) receptor, comprising:

[0026] The light chain variable region comprises:

[0027] A CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 44: Xaa1-ASSSVS-Xaa2-Xaa3-YLH (wherein Xaa1 is arginine or lysine, Xaa2 is threonine or serine, and Xaa3 is arginine or serine),

[0028] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 45: Xaa4-TSN-Xaa5-AS (wherein Xaa4 is glycine or serine, and Xaa5 is isoleucine or leucine), and

[0029] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 46: QQY-Xaa6-Xaa7-FPLT (wherein Xaa6 is threonine or serine, and Xaa7 is alanine or glycine), and

[0030] The heavy chain variable region comprises:

[0031] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0032] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 47: QIYPGD-Xaa8-DN-Xaa9-YN-Xaa10-KFKG (wherein Xaa8 is glycine or alanine, Xaa9 is lysine or arginine, and Xaa10 is glycine or alanine), and

[0033] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 26;

[0034] (ie, heavy chain variable regions and light chain variable regions comprising the CDRs of mIML1-2, m20C3, hIML1-2, hIML1-2-OP1 or hIML1-2-2 of the Examples or variants thereof).

[0035] (1-2) An antibody or an antibody fragment thereof that binds to human cannabinoid type 1 (CB1) receptor, comprising:

[0036] (a1) a light chain variable region comprising:

[0037] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 44,

[0038] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 45, and

[0039] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 46, and

[0040] (a2) a heavy chain variable region comprising:

[0041] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0042] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 27, and

[0043] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0044] (b1) a light chain variable region comprising:

[0045] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 44,

[0046] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 45, and

[0047] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 21, and

[0048] (b2) a heavy chain variable region comprising:

[0049] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0050] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 47, and

[0051] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0052] (c1) a light chain variable region comprising:

[0053] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 44,

[0054] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 20, and

[0055] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 46, and

[0056] (c2) a heavy chain variable region comprising:

[0057] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0058] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 47, and

[0059] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0060] (d1) a light chain variable region comprising:

[0061] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 19,

[0062] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 45, and

[0063] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 46, and

[0064] (d2) a heavy chain variable region comprising:

[0065] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0066] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 47, and

[0067] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26; or

[0068] (e1) a light chain variable region comprising:

[0069] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 44,

[0070] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 45, and

[0071] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 46, and

[0072] (e2) a heavy chain variable region comprising:

[0073] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0074] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 25, and

[0075] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0076] (ie, a heavy chain variable region and a light chain variable region comprising the CDRs of mIML1-2 or hIML1-2 of the embodiment or a variant thereof).

[0077] (1-3) An antibody or an antibody fragment thereof that binds to human cannabinoid type 1 (CB1) receptor, comprising:

[0078] (a1) a light chain variable region comprising:

[0079] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 44,

[0080] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 45, and

[0081] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 46, and

[0082] (a2) a heavy chain variable region comprising:

[0083] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0084] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 36, and

[0085] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0086] (b1) a light chain variable region comprising:

[0087] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 44,

[0088] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 45, and

[0089] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 35, and

[0090] (b2) a heavy chain variable region comprising:

[0091] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0092] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 47, and

[0093] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0094] (c1) a light chain variable region comprising:

[0095] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 44,

[0096] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 34, and

[0097] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 46, and

[0098] (c2) a heavy chain variable region comprising:

[0099] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0100] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 47, and

[0101] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26; or

[0102] (d1) a light chain variable region comprising:

[0103] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 33,

[0104] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 45, and

[0105] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 46, and

[0106] (d2) a heavy chain variable region comprising:

[0107] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0108] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 47, and

[0109] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0110] (ie, a heavy chain variable region and a light chain variable region comprising the CDRs of hIML1-2-OP1 of the embodiment or a variant thereof).

[0111] (2) An antibody or an antibody fragment thereof that binds to a human cannabinoid type 1 (CB1) receptor, comprising:

[0112] (a1) a light chain variable region comprising:

[0113] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 33,

[0114] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 34, and

[0115] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 35, and

[0116] (a2) a heavy chain variable region comprising:

[0117] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0118] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 36, and

[0119] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0120] (i.e., a heavy chain variable region and a light chain variable region containing the CDR of hIML1-2-OP1 of the embodiment or a variant thereof)

[0121] (b1) a light chain variable region comprising:

[0122] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 40,

[0123] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 20, and

[0124] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 23, and

[0125] (b2) a heavy chain variable region comprising:

[0126] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0127] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 28, and

[0128] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 29;

[0129] (i.e., a heavy chain variable region and a light chain variable region comprising the CDRs of m2F8 or h2F8 of the embodiment or a variant thereof)

[0130] (c1) a light chain variable region comprising:

[0131] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 19,

[0132] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 20, and

[0133] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 21, and

[0134] The heavy chain variable region comprises:

[0135] (c2-1)

[0136] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0137] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 27, and

[0138] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 26, or

[0139] (c2-2)

[0140] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0141] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 25, and

[0142] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26;

[0143] (i.e., a heavy chain variable region and a light chain variable region comprising the CDRs of m20C3, mIML1-2hIML1-2 or hIML1-2-2 of the embodiments or variants thereof); or

[0144] (d1) a light chain variable region comprising:

[0145] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 22,

[0146] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 20, and

[0147] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 23, and

[0148] The heavy chain variable region comprises:

[0149] (d2-1)

[0150] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0151] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 28, and

[0152] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 30,

[0153] (d2-2)

[0154] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0155] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 28, and

[0156] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 31, or

[0157] (d2-3)

[0158] CDR1 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 24,

[0159] A CDR2 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 28, and

[0160] A CDR3 consisting of an amino acid sequence in which one or two amino acids are optionally deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 32;

[0161] (i.e., heavy chain variable regions and light chain variable regions comprising the CDRs of m4D7, m24C4 or m26C7 of the embodiments or variants thereof).

[0162] (3-1) The antibody or antibody fragment thereof according to any one of (1-1) to (1-3) or (2), comprising:

[0163] A light chain variable region consisting of any one of the amino acid sequences of SEQ ID NOs: 13 to 15, or an amino acid sequence having 95% or more identity with any one of the amino acid sequences of SEQ ID NOs: 13 to 15, and

[0164] A heavy chain variable region consisting of any one of the amino acid sequences of SEQ ID NOs: 16 to 18, and 52, or an amino acid sequence having 95% or more identity with any one of the amino acid sequences of SEQ ID NOs: 16 to 18, and 52.

[0165] (3-2) The antibody or antibody fragment thereof according to any one of (1-1) to (1-3) or (2), comprising:

[0166] A light chain variable region consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 4, or an amino acid sequence having 95% or more identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 4, and

[0167] A heavy chain variable region consisting of any one of the amino acid sequences of SEQ ID NOs: 7 to 12, or an amino acid sequence having 95% or more identity with any one of the amino acid sequences of SEQ ID NOs: 7 to 12.

[0168] (4) The antibody or antibody fragment thereof according to any one of (1-1) to (1-3), (2) or (3-1), comprising:

[0169] (a1) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 14, and

[0170] a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17;

[0171] (a2) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 15, and

[0172] a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18;

[0173] (a3) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and

[0174] A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16; or

[0175] (a4) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and

[0176] The heavy chain variable region consists of the amino acid sequence of SEQ ID NO:52.

[0177] (5) The antibody or antibody fragment thereof described in any one of (1-1) to (1-3), (2), (3-1), (3-2) or (4), which further comprises: a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 37 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 38.

[0178] (6) A pharmaceutical composition comprising the antibody or antibody fragment thereof according to any one of (1-1) to (1-3), (2), (3-1), (3-2), (4) or (5).

[0179] (7) The pharmaceutical composition according to (6), which is a therapeutic and / or preventive agent for obesity, diabetes, and / or non-alcoholic steatohepatitis (NASH).

[0180] (8) a polynucleotide,

[0181] encoding the heavy chain variable region of the antibody described in (3-1), (3-2) or (4),

[0182] Optionally, it also encodes the heavy chain constant region of the antibody described in (5).

[0183] (9) a polynucleotide,

[0184] encoding the light chain variable region of the antibody described in (3-1), (3-2) or (4),

[0185] Optionally, it also encodes the light chain constant region of the antibody described in (5).

[0186] (10) An expression vector comprising the polynucleotide described in (8) or (9).

[0187] (11) An antibody or an antibody fragment thereof that binds to human cannabinoid type 1 (CB1) receptor, comprising:

[0188] (a1) a light chain variable region comprising:

[0189] CDR1 consisting of the amino acid sequence of SEQ ID NO: 39: Xaa11-ASSSVS-Xaa12-Xaa13-YLH (wherein Xaa11 is threonine, isoleucine, arginine or lysine, Xaa12 is serine or threonine, and Xaa13 is serine or arginine),

[0190] a CDR2 consisting of the amino acid sequence of SEQ ID NO:45: Xaa14-TSN-Xaa15-AS (wherein Xaa14 is serine or glycine, and Xaa15 is leucine or isoleucine), and

[0191] a CDR3 consisting of the amino acid sequence of SEQ ID NO:41: Xaa16-QY-Xaa17-Xaa18-Xaa19-P-Xaa20-T (wherein Xaa16 is histidine or glutamine, Xaa17 is histidine, threonine or serine, Xaa18 is arginine, alanine or glycine, Xaa19 is serine or phenylalanine, and Xaa20 is arginine or leucine), and

[0192] (a2) a heavy chain variable region comprising:

[0193] CDR1 consisting of the amino acid sequence of SEQ ID NO: 24,

[0194] a CDR2 consisting of the amino acid sequence of SEQ ID NO:42: QIYPGD-Xaa21-D-Xaa22-Xaa23-YN-Xaa24-KFKG (wherein Xaa21 is glycine or alanine, Xaa22 is threonine or asparagine, Xaa23 is asparagine, lysine or arginine, and Xaa24 is glycine or alanine), and

[0195] a CDR3 consisting of the amino acid sequence of SEQ ID NO:43: Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Y (wherein Xaa25 is serine or threonine, Xaa26 is histidine or glycine, Xaa27 is glycine or serine, Xaa28 is asparagine or glycine, Xaa29 is tyrosine or arginine, Xaa30 is methionine or phenylalanine, and Xaa31 is isoleucine, alanine, aspartic acid or histidine);

[0196] (i.e., heavy chain variable regions and light chain variable regions comprising the CDRs of mIML1-2, m20C3, m2F8, m4D7, m24C4, m26C7, hIML1-2, hIML1-2-OP1, hIML1-2-2 or h2F8 of the embodiments or variants thereof).

[0197] (12) An antibody or an antibody fragment thereof that binds to human cannabinoid type 1 (CB1) receptor, comprising:

[0198] (a1) a light chain variable region comprising:

[0199] CDR1 consisting of the amino acid sequence of SEQ ID NO: 5: Xaa32-ASSSVS-Xaa33-Xaa34-YLH (wherein Xaa32 is isoleucine, lysine or arginine, Xaa33 is serine or threonine, and Xaa34 is serine or arginine),

[0200] CDR2 consisting of the amino acid sequence of SEQ ID NO: 45, and

[0201] a CDR3 consisting of the amino acid sequence of SEQ ID NO: 41, and

[0202] (a2) a heavy chain variable region comprising:

[0203] CDR1 consisting of the amino acid sequence of SEQ ID NO: 24,

[0204] CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and

[0205] A CDR3 consisting of the amino acid sequence of SEQ ID NO:6: Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Y (wherein Xaa35 is serine or threonine, Xaa36 is histidine or glycine, Xaa37 is glycine or serine, Xaa38 is asparagine or glycine, Xaa39 is tyrosine or arginine, Xaa40 is methionine or phenylalanine, and Xaa41 is isoleucine or alanine);

[0206] (i.e., a heavy chain variable region and a light chain variable region comprising the CDRs of hIML1-2, hIML1-2-OP1, hIML1-2-2 or h2F8 of the Examples or variants thereof)

[0207] (b1) a light chain variable region comprising:

[0208] CDR1 consisting of the amino acid sequence of SEQ ID NO: 48: Xaa42-ASSSVSSSYLH (wherein Xaa42 is threonine, isoleucine or arginine),

[0209] CDR2 consisting of the amino acid sequence of SEQ ID NO: 20, and

[0210] a CDR3 consisting of the amino acid sequence of SEQ ID NO:49: Xaa43-QY-Xaa44-Xaa45-Xaa46-P-Xaa47-T (wherein Xaa43 is histidine or glutamine, Xaa44 is histidine or serine, Xaa45 is arginine or glycine, Xaa46 is serine or phenylalanine, and Xaa47 is arginine or leucine), and

[0211] (b2) a heavy chain variable region comprising:

[0212] CDR1 consisting of the amino acid sequence of SEQ ID NO: 24,

[0213] a CDR2 consisting of the amino acid sequence of SEQ ID NO:50: QIYPGD-Xaa48-D-Xaa49-Xaa50-YN-Xaa51-KFKG (Xaa48 is glycine or alanine, Xaa49 is threonine or asparagine, Xaa50 is asparagine or lysine, and Xaa51 is glycine or alanine), and

[0214] a CDR3 consisting of the amino acid sequence of SEQ ID NO: 43;

[0215] (i.e., heavy chain variable regions and light chain variable regions comprising the CDRs of mIML1-2, m20C3, m2F8, m4D7, m24C4 or m26C7 of the Examples or variants thereof).

[0216] (13) A polynucleotide encoding the antibody or antibody fragment described in any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12).

[0217] (14) An antibody or antibody fragment as described in any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12), wherein the antibody fragment is Fab, Fab', F(ab')2, scFv, dsFv or Diabody.

[0218] (15) A method for preventing or treating a CB1 receptor-associated disease, comprising administering the antibody or antibody fragment thereof described in any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12).

[0219] (16) The antibody or antibody fragment thereof according to any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12), for use in the manufacture of a therapeutic or preventive agent for a CB1 receptor-associated disease.

[0220] (17) The antibody or antibody fragment thereof according to any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12), for use in the treatment or prevention of a CB1 receptor-associated disease.

[0221] (18) The method described in (15) or the antibody or antibody fragment thereof described in (16) or (17), wherein the CB1 receptor-associated disease is obesity and / or diabetes.

[0222] (19) A diagnostic kit for a CB1 receptor-associated disease, comprising the antibody or antibody fragment thereof described in any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12).

[0223] (20) The antibody or antibody fragment thereof according to any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12), which is a humanized antibody or an antibody fragment thereof.

[0224] (21) The antibody or antibody fragment thereof described in any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12), which is a fully human antibody.

[0225] (22) A pharmaceutical composition for modulating CB1 receptor signal transduction activity, comprising the antibody or antibody fragment thereof described in any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12).

[0226] (23) A pharmaceutical composition which is an antagonist or inverse agonist of CB1 receptor signal transduction activity, comprising the antibody or antibody fragment thereof described in any one of (1-1) to (1-3), (2), (3-1), (3-2), (4), (5), (11) or (12).

[0227] [Effects of the Invention]

[0228] Since the antibody or antibody fragment of the present invention specifically binds to the CB1 receptor, it can be used to detect the CB1 receptor in a biological sample. Furthermore, since the antibody or antibody fragment of the present invention has the activity of regulating the signal transduction of the CB1 receptor, the pharmaceutical composition containing the antibody or antibody fragment of the present invention is very useful as a medicine, especially a medicine for the treatment or prevention of CB1 receptor-related diseases.

[0229] [Brief description of the attached drawings]

[0230]

【 Figure 1 Kabat numbering of the light chain variable region of mIML1-2 (SEQ ID NO: 1), the light chain variable region of m20C3 (SEQ ID NO: 2), and the light chain variable region of m2F8 (SEQ ID NO: 3) (LFR1 to LFR4 refer to light chain framework regions 1 to 4, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, in Figure 2 , 5 And 6 also have the same meaning.)

[0231]

【 Figure 2 Kabat numbering of the light chain variable regions of m4D7, m24C4 and m26C7 (SEQ ID NO: 4)

[0232]

【 Figure 3Kabat numbering of the heavy chain variable region of mIML1-2 (SEQ ID NO: 7), the heavy chain variable region of m20C3 (SEQ ID NO: 8), and the heavy chain variable region of m2F8 (SEQ ID NO: 9) (HFR1 to HFR4 refer to heavy chain framework regions 1 to 4, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, in Figure 4 , 7 And 8 also has the same meaning.)

[0233]

【 Figure 4 Kabat numbering of the heavy chain variable region of m4D7 (SEQ ID NO: 10), the heavy chain variable region of m24C4 (SEQ ID NO: 11), and the heavy chain variable region of m26C7 (SEQ ID NO: 12)

[0234]

【 Figure 5 Kabat numbering of the light chain variable region of h2F8 (SEQ ID NO: 15) and the light chain variable region of hIML1-2-OP1 (SEQ ID NO: 14)

[0235]

Figure 6

[0236]

【 Figure 7 Kabat numbering of the heavy chain variable region of h2F8 (SEQ ID NO: 18) and the heavy chain variable region of hIML1-2-OP1 (SEQ ID NO: 17)

[0237]

【 Figure 8 Kabat numbering of the heavy chain variable region of hIML1-2 (SEQ ID NO: 16) and the heavy chain variable region of hIML1-2-2 (SEQ ID NO: 52)

[0238]

【 Fig. 9 Evaluation of the in vivo antagonist effect (improvement of small intestinal transit capacity) of anti-human CB1 receptor antibodies

[0239]

【 Fig.10 Evaluation of the anti-obesity effect (weight change) of anti-human CB1 receptor antibodies

[0240]

【 Fig.11 Evaluation of the anti-obesity effect of anti-human CB1 receptor antibody (amount of triglycerides in liver)

[0241] The terms used in this specification are used with the meanings commonly used in the art, except when specifically mentioned. In the present invention, methods for preparing antibodies known in the art can be used. For example, methods described in Immunochemistry in Practice (Blackwell Scientific Publications) can be cited. In addition, methods for gene manipulation known in the art can be used. For example, methods described in Molecular Cloning, A Laboratory Manual, Forth Edition, Cold Spring Harbor Laboratory Press (2012), Current Protocols Essential Laboratory Techniques, Current Protocols (2012) can be cited.

[0242] The human cannabinoid (CB1) receptor is a protein composed of amino acids encoded by the gene CNR1 (UniProtKB / Swiss-Prot: P21554: SEQ ID NO: 51). The extracellular domain of the human CB1 receptor corresponds to the N-terminal region composed of amino acids 1 to 116, the loop 1 region (EC loop 1) composed of amino acids 176 to 187, the loop 2 region (EC loop 2) composed of amino acids 256 to 273, and the loop 3 region (EC loop 3) composed of amino acids 366 to 377. In addition, in this specification, the terms "CB1 receptor" and "CB1" both refer to cannabinoid receptor type 1.

[0243] The antibody or antibody fragment of the present invention is an antibody or a fragment of the antibody having the CDR or heavy chain variable region / light chain variable region described in this specification. The antibody or antibody fragment may be derived from any class (e.g., IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecules, for example, and may also be obtained from any species including mice, rats, sharks, rabbits, pigs, hamsters, camels, alpacas, goats or humans. As the antibody or antibody fragment, a humanized antibody or an antibody fragment of a humanized antibody is preferred.

[0244] In the present invention, the "antibody fragment" refers to a part of the antibody of the present invention, and refers to a fragment that specifically binds to the CB1 receptor and has the activity of regulating CB1 receptor signal transduction like the antibody.

[0245] Specifically, Fab (fragment of antigen binding), Fab', F(ab')2, single-chain antibody (single chain Fv; hereinafter referred to as scFv), disulfide stabilized antibody (disulfide stabilized Fv; hereinafter referred to as dsFv), divalent body V region fragment (hereinafter referred to as Diabody), CDR-containing peptides, etc. that specifically bind to the human CB1 receptor (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5, pp. 441-456, 1996).

[0246] Fab is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity, which is obtained by decomposing the peptide portion of the upper part of the two disulfide bonds (SS bonds) that cross-link the two H chains in the hinge region of IgG with the enzyme papain, and is composed of about half of the N-terminal side of the H chain and the entire L chain. The Fab used in the present invention can be obtained by treating the antibody of the present invention with papain. In addition, Fab can also be produced by inserting DNA encoding the Fab of the antibody of the present invention into a cell expression vector and expressing it by introducing the vector into the cell.

[0247] Fab' is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity, which is obtained by cutting the SS bond between the hinges of F(ab')2. The Fab' used in the present invention can be obtained by treating the F(ab')2 of the antibody of the present invention with a reducing agent, dithiothreitol. Alternatively, Fab' can be produced by inserting a DNA encoding the Fab' of the antibody of the present invention into a cell expression vector, and then introducing the vector into Escherichia coli, yeast or animal cells for expression.

[0248] F(ab')2 is an antibody fragment with antigen-binding activity having a molecular weight of about 100,000 and formed by decomposing the lower part of the two SS bonds of the hinge region of IgG with the enzyme pepsin, and two Fab' regions are bound via the hinge portion. The F(ab')2 used in the present invention can be obtained by treating the antibody of the present invention with pepsin. In addition, F(ab')2 can be produced by inserting a DNA encoding the F(ab')2 of the antibody of the present invention into a cell expression vector, and then introducing the vector into Escherichia coli, yeast or animal cells for expression.

[0249] scFv is an antibody fragment having antigenic activity in which one VH and one VL are connected by an appropriate peptide linker (hereinafter referred to as P) to form a VH-P-VL or VL-P-VH polypeptide. The VH and VL contained in the scFv used in the present invention can be any antibody of the present invention. The scFv used in the present invention can also be produced by constructing an scFv expression vector using cDNA encoding the VH and VL of the antibody of the present invention and introducing it into Escherichia coli, yeast or animal cells for expression.

[0250] dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine ​​residue and bonded via an SS bond. The amino acid residue substituted with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7, 697 (1994)). The VH or VL contained in the dsFv used in the present invention can be any antibody of the present invention. The dsFv used in the present invention can be constructed by inserting the cDNA encoding the VH and VL of the antibody of the present invention into an appropriate expression vector, and the expression vector is introduced into Escherichia coli, yeast or animal cells, and expressed to produce.

[0251] Diabody is an antibody fragment that forms a dimer of scFv with the same or different antigen binding specificities, and has bivalent antigen binding activity for the same antigen or two specific antigen binding activities for different antigens. For example, a bivalent Diabody that specifically reacts with the antibody of the present invention can be produced by constructing a DNA encoding an scFv having a peptide linker of 3 to 10 residues using cDNA encoding VH and VL of the antibody of the present invention, inserting the DNA into a cell expression vector, and introducing the expression vector into Escherichia coli, yeast or animal cells to express the Diabody.

[0252] The peptide containing CDR is composed of at least one region containing CDR of VH or VL. A plurality of CDRs can be combined directly or via an appropriate peptide linker. The peptide containing CDR used in the present invention can be constructed by using cDNA encoding VH and VL of the antibody of the present invention to encode the DNA encoding CDR, by inserting the DNA into an expression vector for animal cells, and introducing the vector into Escherichia coli, yeast or animal cells for expression to produce. In addition, the peptide containing CDR can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

[0253] The antibody or antibody fragment of the present invention is characterized by specifically binding to the CB1 receptor.

[0254] The specific binding may be at least about 1×10 -6The equilibrium dissociation constant M or its fraction (e.g., a smaller KD indicates a tighter binding) is used as a characteristic. The Kd value is preferably 1×10 -7 M or less, more preferably 1×10 -8 M or less, more preferably 1×10 -9 M or less. Methods for determining whether two molecules are specifically bound are well known in the art, and examples thereof include competitive binding ELISA, surface plasmon resonance, and the like. However, isolated antibodies that specifically bind to the CB1 receptor sometimes show cross-reactivity to target antigens other than the antigen with antibodies derived from other species, such as CB1 receptor molecules. Nevertheless, multispecific antibodies that bind to the CB1 receptor and one or more further antigens, or bispecific antibodies that bind to two different regions of the CB1 receptor (e.g., EC loop 1 and EC loop 2) are considered antibodies that "specifically bind" to the CB1 receptor.

[0255] The antibody or antibody fragment of the present invention is characterized by having the activity of regulating CB1 receptor signal transmission. The activity of regulating CB1 receptor signal transmission refers to the activity of inhibiting or antagonizing CB1 receptor signal transmission, the activity of reverse agonism, or the activity of enhancing or agonism. As the antibody or antibody fragment of the present invention, it is preferably an antibody or antibody fragment having CB1 receptor reverse agonist activity or CB1 receptor antagonist activity. CB1 receptor reverse agonist activity refers to the activity of reducing CB1 receptor activity to a level below the basal level, and CB1 receptor antagonist activity refers to the activity of inhibiting, reducing or preventing the signal transmission activity of the ligand of the CB1 receptor.

[0256] As a procedure for measuring the inverse agonist activity of the CB1 receptor, for example, CB1 receptor cells expressing human CB1 receptors can be used to measure the Ca2+ activity added by a CB1 receptor antagonist (CP55940, etc.). 2+ The IC50 value was determined by calculating the inhibition rate when the CB1 receptor antagonist was not added and the inhibition rate when the CB1 receptor antagonist was added and the antibody was not added (see Example 4). In addition, the cAMP assay described in Example 4, the aequorin assay or the GTP-Eu assay can be used to determine whether the signal transmission is inhibited.

[0257] The antibody or antibody fragment of the present invention has, in addition to the above, any one or all of the following excellent features.

[0258] (a) Shows strong anti-fibrotic effect.

[0259] (b) It has a strong inhibitory effect on small intestinal transit.

[0260] (c) It has a strong anti-obesity effect.

[0261] (d) Low immunogenicity in humans.

[0262] (e) It has high stability.

[0263] (f) Good dynamics and high stability in blood.

[0264] The antibodies of the present invention can be produced by established methods in the art using the CDRs or heavy chain variable regions / light chain variable regions described in the present specification.

[0265] The antibodies of the present invention also include monoclonal, single specific, single chain, chimeric, humanized, fully human, synthetic, recombinant, hybrid, variant, transplanted antibodies, antibody low molecular weight complexes (ADC) and bispecific antibodies. Humanized antibodies are useful for administration to humans for therapeutic purposes, etc., because their antigenicity in humans is reduced. Humanized antibodies are obtained by transplanting the complementarity determining region (CDR) of a mammal other than a human, such as a mouse antibody, into the framework region (FR) of a human antibody. Thus, the FR of the humanized antibody is derived from humans. Appropriate FRs can be selected with reference to the literature of Kabat EA, etc. As the FR at this time, a CDR that can form a good antigen binding site is selected. If necessary, the amino acids of the FR of the variable region of the antibody can be substituted in such a way that the CDR of the reconstructed humanized antibody forms a suitable antigen binding site (Sato, K. et al. Cancer Res. 1993, Vol. 53, p. 851). The proportion of the substituted FR amino acids can be 0 to 15% of the entire FR region, preferably 0 to 5%. Fully human antibodies specific for the CB1 receptor can be produced, for example, by immunizing transgenic mice containing human immunoglobulin genes with a CB1 receptor antigen, obtaining lymphocytes from the antibody-expressing mice, and then using hybridoma cells fused with myeloid cell lines and lymphocytes.

[0266] Furthermore, in the humanized antibody of the present invention, the constant region of a human antibody is used. As a preferred constant region of a human antibody, Cγ can be cited as a heavy chain, for example, Cγ1, Cγ2, Cγ3, Cγ4 are used, and as a light chain, Cκ, Cλ are used. In addition, in order to improve the stability of the antibody or its production, the C region of the human antibody can also be modified. The human antibody used in humanization can be a human antibody of any isotype such as IgG, IgM, IgA, IgE, IgD, etc., preferably IgG is used in the present invention, and more preferably IgG1 or IgG4.

[0267] The humanized antibody of the present invention preferably has a light chain constant region having the amino acid sequence of SEQ ID NO: 38 and a heavy chain constant region having the amino acid sequence of SEQ ID NO: 37. However, the C-terminal lysine or the C-terminal two amino acid residues (glycine-lysine) of SEQ ID NO: 37 may or may not be present.

[0268] Humanized antibodies can be made by general manufacturing methods (see, for example, Example 4 below, WO95 / 14041, WO96 / 02576, etc.). Specifically, first, a DNA sequence encoding a variable region designed to connect the CDR of a mouse antibody to the FR of a human antibody is synthesized by PCR from several oligonucleotides made to have a portion overlapping with the terminal portion (see WO98 / 13388). The obtained DNA is connected to the DNA encoding the constant region of the human antibody and then integrated into an expression vector. Alternatively, the DNA encoding the variable region of the antibody can also be integrated into an expression vector containing the DNA of the constant region of the antibody. To manufacture the antibodies used in the present invention, the antibody gene is integrated into the expression vector in a manner according to the expression control region, for example, the expression controlled by the enhancer / promoter. Next, the host cell can be transformed with this expression vector to express the antibody.

[0269] As host cells of the above-mentioned transformants, for example, vertebrate cells such as COS cells and CHO cells, prokaryotic cells, yeast, etc. can be cited. The transformant can be cultured according to a method known to those skilled in the art, and the antibody of the present invention is produced in or outside the transformant cells by the culture. As the culture medium used in the culture, various conventional ones corresponding to the host cells adopted can be appropriately selected. For example, in the case of COS cells, a culture medium such as RPMI-1640 culture medium or Dulbecco's modified Eagle's minimum essential medium (DMEM) can be used, and serum components such as fetal bovine serum (FBS) can be added as needed. The culture temperature during the culture of the transformant can be any temperature as long as it does not significantly reduce the protein synthesis ability in the cell, preferably 32 to 42°C, and most preferably cultured at 37°C. In addition, if necessary, it can be cultured in air containing 1 to 10% (v / v) of carbon dioxide gas.

[0270] The fractions containing the antibody of the present invention produced in or outside the cells of the transformant can be separated and purified by various known separation operations utilizing the physical properties or chemical properties of the protein. Specifically, for example, various chromatography methods such as treatment with a common protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof can be used. The antibody of the present invention can be easily produced with high yield and high purity by this method.

[0271] Furthermore, the antibody or antibody fragment thereof of the present invention may be modified with various molecules such as polyethylene glycol (PEG), radioactive substances, toxins, etc. The antibody modification method may be a method known in the art.

[0272] In addition, the antibody of the present invention may also be fused to other proteins at its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). The fused protein can be appropriately selected by those skilled in the art.

[0273] The pharmaceutical composition containing the antibody of the present invention or its antibody fragment (the pharmaceutical composition of the present invention) can be administered systemically or locally or orally or parenterally. For parenterally administered, for example, intravenous injection such as drip, intramuscular injection, epidural injection, intracerebroventricular injection, intraperitoneal injection, subcutaneous injection, intranasal administration, inhalation, etc. can be selected.

[0274] The pharmaceutical composition of the present invention is useful as a medicine for treating and / or preventing CB1 receptor-related diseases.

[0275] Examples of CB1 receptor-associated diseases include obesity, Alström's syndrome, Prader-Willi syndrome, Bardet-Biedl syndrome, Albright hereditary osteodystrophy, symptomatic obesity including SIM1 deficiency syndrome, diabetes and its associated complications, insulin resistance, type II diabetes, prediabetes, metabolic syndrome, dyslipidemia, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diabetic gastroparesis, gastroparesis, liver diseases such as primary biliary cirrhosis, renal fibrosis, chronic kidney disease, nested segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive renal disease, nephrotic syndrome, steroid-resistant nephrotic syndrome, minimal change nephrotic syndrome, membranous nephropathy, idiopathic membranous nephropathy, and symptomatic nephrotic syndrome. disease, membranous proliferative glomerulonephritis (MPGN), immunoconjugate-mediated MPGN, complement-mediated MPGN, lupus nephritis, post-infectious glomerulonephritis, thin basement membrane disease, mesangial proliferative glomerulonephritis, amyloidosis (primary), clq nephropathy, rapidly progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, IgA nephropathy, proteinuric kidney disease, microalbuminuria, microalbuminuria kidney disease, etc., diabetic neuropathy, osteoporosis, atherosclerosis, inflammatory diseases, cardiovascular diseases, cancer, pain, systemic sclerosis, multiple sclerosis spasm, glaucoma and nicotine poisoning, anxiety, depression, transplant-related FSGS, transplant-related nephrotic syndrome, transplant-related proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), etc. In particular, it is very useful as a medicine for the treatment and / or prevention of obesity, diabetes, and NASH.

[0276] The antibody or antibody fragment thereof or the pharmaceutical composition of the present invention is:

[0277] (1) Supplementing and / or enhancing the therapeutic effect of the pharmaceutical composition of the present invention,

[0278] (2) the performance of the pharmaceutical composition of the present invention, improved absorption, reduced dosage, and / or

[0279] (3) Reduction of side effects of the pharmaceutical composition of the present invention,

[0280] Therefore, the drug may be combined with other active ingredients or pharmaceutical agents containing the active ingredients and administered as a combination agent.

[0281] As the drug containing other active ingredients, for example, CB1 receptor antagonists such as CB1 receptor antibodies or low molecular weight CB1 receptor inhibitors other than the antibody or antibody fragment of the present invention can be mentioned. As low molecular weight CB1 receptor inhibitors, for example, rimonabant, tarenabant, AM251, AM1387, AM4113, cannabidiol, ibenabant, octenabant, ethinabant, tetrahydrocannabinol, virodhamine, AM6545, etc. can be mentioned.

[0282] The combination of the antibody or antibody fragment thereof of the present invention and other active ingredients can be administered in the form of a combination of two ingredients in one preparation, or can be administered as separate preparations (including the pharmaceutical composition of the present invention and the medicament of other active ingredients). When administered as such separate preparations, simultaneous administration and administration with a time difference are included. In addition, administration with a time difference may be performed by first administering the pharmaceutical composition of the present invention and then administering other medicaments, or by first administering other medicaments and then administering the pharmaceutical composition of the present invention, and the respective administration methods may be the same or different.

[0283] The effective dosage of the pharmaceutical composition of the present invention is selected from the range of 0.01 mg to 100 mg per kg of body weight at a time. Alternatively, a dosage of 5 to 5000 mg, preferably 10 to 500 mg, may be selected per patient. However, the pharmaceutical composition of the present invention is not limited to these dosages. In addition, the administration period may be appropriately selected according to the age and symptoms of the patient, and the dosage may be calibrated using methods known in the art (e.g., Mordenti et al. (1991) Pharmaceut. Res. 8: 1351-1359). The pharmaceutical composition of the present invention may also contain pharmaceutically acceptable carriers or additives in order of administration route. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate, water-soluble dextran, pectin, methylcellulose, ethylcellulose, casein, diglycerol, propylene glycol, polyethylene glycol, vaseline, human serum albumin (HSA), mannitol, sorbitol, lactose, and surfactants acceptable as pharmaceutical additives. The additives used are appropriately selected from the above or in combination according to the dosage form, but are not limited to these.

[0284] The present invention includes polynucleotides encoding the heavy chain variable region and / or light chain variable region of the antibody of the present invention. Furthermore, the polynucleotides encoding the heavy chain variable region of the antibody of the present invention may also encode the heavy chain constant region. Furthermore, the polynucleotides encoding the light chain variable region of the antibody of the present invention may also encode the light chain constant region. In addition, the present invention also includes an expression vector containing at least one of the polynucleotides.

[0285] The polynucleotide is not particularly limited as long as it encodes the light chain variable region or heavy chain variable region of the antibody of the present invention, and is a polymer composed of multiple nucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It may also contain bases other than natural ones. The polynucleotide of the present invention can be used to manufacture antibodies by genetic engineering methods. In addition, it can also be used as a probe to screen for antibodies with equivalent functions to the antibodies of the present invention. That is, the polynucleotide encoding the antibody of the present invention, or a part thereof, can be used as a probe, and hybridized with the polynucleotide under strict conditions by hybridization, gene amplification technology (such as PCR), etc., and DNA encoding antibodies with equivalent activity to the antibodies of the present invention is obtained. Such DNA is also contained in the polynucleotide of the present invention.

[0286] Hybridization technology (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is a technology well known to those skilled in the art. As conditions for hybridization, for example, low stringency conditions can be cited. Low stringency conditions refer to the conditions of 42°C, 0.1×SSC, 0.1% SDS in the washing after hybridization, preferably 50°C, 0.1×SSC, 0.1% SDS. As more preferred conditions for hybridization, high stringency conditions can be cited. High stringency conditions refer to conditions of 65°C, 5×SSC and 0.1% SDS, for example. Among these conditions, it can be expected that polynucleotides with high homogeneity can be efficiently obtained when the temperature is raised. However, as factors affecting the stringency of hybridization, multiple factors such as temperature and salt concentration are considered, and as long as a person skilled in the art can achieve the same stringency by appropriately selecting these factors.

[0287] The antibodies encoded by the polynucleotides obtained by these hybridization techniques or gene amplification techniques that are functionally equivalent to the antibodies of the present invention generally have high identity with these antibodies in terms of amino acid sequence. The antibodies of the present invention also include antibodies that are functionally equivalent to the antibodies of the present invention and have high identity with the amino acid sequence of the antibodies. High identity refers to an identity of at least 75% or more, preferably 85% or more, and more preferably 95% or more at the amino acid level. To determine the identity of polypeptides, the algorithm described in the literature (Wilbur, WJ and Lipman, DJ Proc. Natl. Acad. Sci. USA (1983) 80, 726-730) can be used.

[0288] Since the antibody or its antibody fragment of the present invention specifically binds to the CB1 receptor, it can be used to detect the CB1 receptor in a biological sample. Examples of biological samples include blood, plasma, serum, urine, organs, tissues, bone marrow, lymph nodes, etc. Therefore, a kit containing the antibody of the present invention can be used as a kit for detecting the CB1 receptor. The kit contains the antibody or its antibody fragment of the present invention, and further, may contain a labeled second antibody, a substrate necessary for the detection of the label, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, a purified CB1 receptor protein as a standard substance, instructions for use, etc. [Example]

[0289] Next, the present invention will be described in more detail with reference to examples of the present invention, but the present invention is not limited to these examples.

[0290] [Example 1: Preparation of anti-human CB1 receptor mouse antibodies by hybridoma method]

[0291] ICR female mice knocked out of the CB1 receptor gene were immunized with DNA using a gene encoding the full length of the human CB1 receptor (UniProtKB / Swiss-Prot: P21554) or a gene encoding the ΔN-human CB1 receptor (T210A) which is a human CB1 receptor variant lacking the extracellular N-terminal region of the human CB1 receptor (amino acids 1 to 116) and introducing the T210A mutation as an antigen. DNA immunization was repeated 2 or 3 times at intervals of 2 weeks, and Expi293 cells transiently expressing the human CB1 receptor (Thermo Fisher SCIENTIFIC) were intraperitoneally administered 1 week after the final immunization. The spleen was removed 3 days later, and spleen cells were fused with mouse myeloma cells (p3x6363-Ag8., Tokyo Cancer Research Institute) by the PEG method, and selection was performed in a medium containing hypoxanthine, aminopterin, and thymidine. In the screening of antibodies that specifically bind to human CB1 receptors, clones that confirmed specific signals for human CB1 receptors were selected by reacting with Expi293 cells transiently expressing human CB1 receptors and Expi293 cells transiently expressing human CB2 receptors in the hybridoma culture supernatant, and detecting with MirrorBall using Alexa488-labeled anti-mouse IgG antibody (Thermo Fisher SCIENTIFIC). Anti-human CB1 receptor inhibitory antibodies were selected by cAMP measurement using HitHunter (registered trademark) cAMP kit (DiscoverX). 1×10 4CHO cells stably expressing human CB1 receptors (hCB1 receptor / CHO) were inoculated into 384-well plates at 37°C and 5% CO2 saturated water vapor pressure for O / N culture, and then the culture supernatant of the hybridoma was added and reacted at room temperature for 3 hours. After washing twice with 20mM Hepes-HBSS (pH7.4), CP55940 (CB1 receptor agonist) was adjusted to a final concentration of 10nM, and Forskolin was adjusted to a final concentration of 10μM and reacted at room temperature for 30 minutes. Thereafter, cAMP antibody reagent and cAMP working detection solution were added and the reaction was performed at room temperature for 1 hour in the dark. Thereafter, cAMP solutionA was added, and the reaction was performed at room temperature in the dark for 3 hours, and then the luminescence was detected. In the control where only the culture medium was reacted instead of the hybridoma supernatant, the signal value of CP55940+ and Forskolin+ was set to a suppression rate of 0%, and the signal value of CP55940- and Forskolin+ was set to a suppression rate of 100%. The suppression rate was calculated, and clones showing a suppression rate of 50% or more were selected as human CB1 receptor inhibitory antibodies.

[0292] [Example 2: Preparation of anti-human CB1 receptor mouse antibodies from immune phage antibody library]

[0293] About 1 / 20 (about 1×10 7 cells) were used for the preparation of immune phage antibody library. mRNA was extracted from spleen cells by Trizol and Dynabeads mRNA purification kit (Thermo Fisher SCIENTIFIC), and cDNA library was synthesized by SuperscriptIII first-strand cDNA synthesis kit (Thermo Fisher SCIENTIFIC). Primer mixtures encoding the N-terminal region and J chain of the mouse antibody germline registered in the IMGT (registered trademark) database were designed as forward primer set and reverse primer set, respectively. Using these, the variable regions of K chain and H chain were amplified respectively by KOD plus DNA polymerase (TOYOBO). These were connected by assembly PCR with G4S×3 linkers as scFv antibody gene library, and inserted into phagemid vector by restriction endonuclease treatment. It was transformed into Escherichia coli TG1 strain by electroporation and cultured O / N on LB agar medium to obtain a vector with about 10 9The phage antibody library was prepared by amplifying the E. coli in LB liquid medium to the logarithmic growth phase, infecting M13KO7 helper phage, inducing the supernatant of LB liquid medium culture at 26°C, O / N, and concentrating with PEG-NaCl. The phage antibody library was first reacted with Expi293 cells, and the operation (subtraction) of recovering the non-binding phage group as "phage that does not non-specifically bind to cells" was repeated 5 times. This supernatant was reacted with Expi293 cells transiently expressing human CB1 receptors, washed 10 times with PBS, and the phages that bound to the human CB1 receptor on the cell surface were eluted with 0.1M glycine-HCl. It was neutralized with 1 / 10 amount of Tris-HCl (pH8.0) and infected with E. coli TG1 strain to concentrate human CB1 receptor binding clones. The operation (panning) of concentrating the human CB1 receptor binding clones was repeated 3 cycles. The concentrated phage antibody library was infected with TG1 and monocloned in a 96-well plate. Anti-human CB1 receptor specific antibodies and anti-human CB1 receptor inhibitory antibodies were selected in the same manner as described in Example 1 using the scFv protein induced in the culture supernatant.

[0294] [Example 3: Determination of antibody sequence]

[0295] The amino acid sequences of the light chain variable region and heavy chain variable region of the antibody were determined by conventional methods from the hybridoma cells identified as human CB1 receptor inhibitory antibodies in Example 1 and the phagemid vector derived from the phage antibody identified as human CB1 receptor inhibitory antibodies in Example 2. The identified sequences are shown in Table 1.

[0296]

Table 1

[0297]

[0298] [Example 4: Neutralization Activity Evaluation]

[0299] The established hybridoma was cultured in a serum-free medium, and the culture supernatant was purified by affinity purification using a Protein G column and gel filtration to obtain a purified antibody. The purified antibody was analyzed by cAMP activity and Ca 2+ The neutralization activity was measured by the following method as an index. cAMP activity was evaluated by HitHunter cAMP kit. 3hCB1 receptor / CHO described in Example 1 was inoculated into cells / well, cultured in O / N at 37°C, 5% CO2 saturated water vapor pressure, and antibodies diluted with 0.1% BSA / 20mM HEPES-HBSS (pH7.4) were added and reacted at room temperature for 1.5 hours. After washing twice with 20mM HEPES-HBSS (pH7.4), CP55940 was made into a final concentration of 3nM, and Forskolin was made into a final concentration of 10μM and reacted at room temperature for 30 minutes. Thereafter, cAMP antibody reagent and cAMP working detection solution were added, and the reaction was further shielded from light for 1 hour at room temperature. cAMP solutionA was added, and the reaction was shielded from light at room temperature for 3 hours, and then the luminescence was detected. In the control without antibody addition, the inhibition rate was calculated by setting the signal value of CP55940+ and Forskolin+ to 0% inhibition rate, and the signal value of CP55940- and Forskolin+ to 100% inhibition rate. The IC50 (nM) of the antibody concentration showing 50% inhibition was calculated and expressed as the mean ± SD (Table 2). 2+ In advance, for collecting Ca 2+ The antibody solution diluted in the medium was added to the Gα16 CHO cells expressing the human CB1 receptor as the indicator, and the cells were reacted in an incubator at 37°C and 5% CO2 for 60 minutes. After washing twice with 20 mM HEPES-HBSS (pH 7.4), Ca was added to the cells at a final concentration of 10 μM CP55940. 2+ The inhibition rate was calculated by setting the signal when CP55940 was not added as 100%, and the signal when CP55940 was added and the antibody was not added as 0%, and the antibody concentration showing 50% inhibition rate was taken as IC50. At least 3 evaluations were performed, and IC50 was expressed as Average±SD (Table 2). As a result, in any evaluation system, it had a much stronger human CB1 receptor inhibitory activity than rimonabant, a low molecular inhibitor used as a positive control, showing an inverse agonist-like effect.

[0300]

Table 2

[0301] Antibody cAMP assay IC50 (nM) <![CDATA[Ca 2+ Influx assay IC50 (nM)]]> mIML1-2 5.9±2.0 1.77±0.96 m2F 4.9±1.4 0.90±0.58 m4D7 3.9±0.6 1.29±0.58 m24C4 5.0±1.0 1.22±0.63 m26C7 6.5±2.7 1.85±0.80 Reorganization 15.8±6.6 3629±1147

[0302] [Example 5: Humanization of Antibodies]

[0303] For mIML1-2 and m2F8, humanization was performed using the following method. The antibody sequence analysis software abYsis was used to identify the numbering according to Kabat's definition (Kabat numbering (Wu, TT and Kabat, EA, J Exp. Med. Aug 1; 132 (2): 211-50. (1970)) and CDR. Next, the human germline receptor sequence with high sequence identity to the variable region of the heavy chain and light chain of the mouse antibody amino acid sequence was searched using the sequence analysis software abYsis. The mouse antibody heavy chain CDR1, CDR2, CDR3 and the mouse antibody light chain CDR1, CDR2, CDR3 were transplanted into the human framework sequence for humanization. In addition, for the J chain region, the sequence with high sequence identity to the J chain region of the mouse antibody was searched using IMG. T (http: / / www.imgt.org / ) search and select. As a result, humanized monoclonal antibodies (hIML1-2, hIML1-2-2, h2F8) showing human CB1 receptor inhibitory activity roughly equivalent to that of mouse antibodies were obtained. In addition, variants of hIML1-2-2 were prepared by introducing single mutations into the framework and CDR, and mutations with improved affinity for human CB1 receptors were identified. Next, by overlapping their mutations, a humanized monoclonal antibody (hIML1-2-OP1) with improved human CB1 receptor inhibitory activity was obtained. These sequences are shown in Table 3.

[0304]

Table 3

[0305]

[0306]

[0307] In addition, the same method as in Example 4 was used to measure cAMP activity and Ca 2+ Influx was used as an indicator to evaluate the inhibitory activity of human CB1 receptors. For comparison with previous antibodies, Nimacimab (BIRD ROCK BIO) and M5 and M7 described in Patent Document 5 (WO2019211665) were prepared using a method using ExpiCHO cells. cAMP activity was measured under conditions where CP55940 was at a final concentration of 10 nM and Forskolin was at a final concentration of 10 μM, Ca 2+ The influx evaluation was conducted by comparatively evaluating the human CB1 receptor inhibitory activity under the condition that the final concentration of CP55940 was 10 μM. The results showed that the antibodies described in the present invention, as shown in Table 4, showed very strong human CB1 receptor inhibitory activity that was several to dozens of times higher, and had superior inhibitory activity to that of previous antibodies.

[0308]

Table 4

[0309] Antibody cAMP assay IC50 (nM) <![CDATA[Ca 2+ Influx assay IC50 (nM)]]> hIML1-2-OP1 8.5±2.4 0.55±0.24 HkDJ 5.9±2.9 2.03±0.68 Nimacimab 28.6±10.0 2.62±0.92 M5 117.0±40.6 4.02±2.27 M7 23.0±22.4 21.69±9.61 Reorganization 53.4±14.2 3629±1147

[0310] [Example 6: Single-administration POM test (small intestinal transport ability evaluation) in human CB1 receptor knock-in mice (hereinafter, hCB1 receptor-KI (KI / KI) mice)]

[0311] (1) Generation of hCB1 receptor-KI (KI / KI) mice

[0312] The mouse CB1 receptor gene (Gene ID: 12801) is composed of multiple exons, and the final exon contains the full length of the ORF (open reading frame). The full length of the ORF (CCDS ID: 18025.1) of the mouse CB1 receptor gene was removed, and the full length of the ORF (CCDS ID: 5015.1) of the human CB1 receptor gene with a DYKDDDDK (SEQ ID NO: 53) tag added to the C-terminus was inserted to produce hCB1 receptor-KI (KI / KI) mice that express completely humanized CB1 receptor proteins. The DNA fragment used as the homologous recombination arm was obtained by PCR amplification of mouse genomic sequences of about 2 kb (kilobase pairs) upstream and downstream of the ORF of the mouse CB1 receptor gene, and the mouse-derived genomic sequence was designed in a manner that only the ORF was removed. A targeting vector was prepared by seamlessly connecting homologous recombination arms before and after the full-length ORF sequence of the human CB1 receptor gene with a DYKDDDDK (SEQ ID NO: 53) tag added to the C-terminus, and subjected to homologous recombination to produce hCB1 receptor-KI (KI / +) C57 / BL6 mice. It was confirmed by PCR and DNA base sequence analysis that the full-length ORF of the mouse CB1 receptor gene and the full-length ORF of the human CB1 receptor gene in the produced hCB1 receptor-KI (KI / +) C57 / BL6 mice were correctly replaced, and there was no insertion or deletion of other sequences. hCB1 receptor-KI (KI / KI) C57 / BL6 mice were produced by mating hCB1 receptor-KI (KI / +) mice by normal mating.

[0313] (2) hCB1 receptor single administration POM test in hCB1 receptor-KI (KI / KI) mice

[0314] 13-week-old hCB1 receptor KI (KI / KI) mice were subcutaneously administered a single dose of hIML1-2-OP1 (0.3 mg / kg, 1 mg / kg), or Saline, as a humanized antibody against human CB1 receptor as shown in Example 5, and fasted. The next day, CB1 receptor agonist CP55940 (0.05 mg / kg) was intraperitoneally administered, and 30 minutes later, 10% charcoal suspension was orally administered at 0.1 mL / mouse. 30 minutes after the administration of the charcoal suspension, the small intestine was removed, and the small intestinal transport capacity was evaluated using the movement distance of the charcoal suspension as an indicator. As a result, a significant decrease in small intestinal transport capacity was confirmed by the administration of CP55940, and hIML1-2-OP1 showed significant inhibitory activity at 0.3 mg / kg and 1.0 mg / kg for this decrease ( Fig. 9 -i). In addition, using the same method, hIML1-2-OP1 (1 mg / kg), isotype control antibody (1 mg / kg), Nimacimab (BIRD ROCKBIO) (1 mg / kg) known as a previous antibody, M5 (1 mg / kg) and M7 (1 mg / kg) described in Patent Document 5 (WO2019211665) were also compared and evaluated. The results showed that a strong inhibitory effect was reproduced in hIML1-2-OP1. In addition, Nimacimab, M5 and M7, which are previous antibodies, did not show significant inhibition at 1 mg / kg. The antibodies described in the present invention also have excellent inhibitory activity in vivo compared to known antibodies ( Fig. 9 -ii).

[0315] [Example 7: Anti-obesity effect in hCB1 receptor-KI (KI / KI) mouse DIO model]

[0316] 7-week-old hCB1 receptor KI (KI / KI) mice were loaded with 60% high-fat diet (58Y1) and divided into PBS administration group and antibody administration group according to body weight 5 weeks after the start of loading. Thereafter, hIML1-2-OP1 and IsotypeControl antibodies, which are humanized antibodies against human CB1 receptors prepared in Example 5, were repeatedly administered subcutaneously at 10 mg / kg or 30 mg / kg once a week for 6 times. Body weight and food intake were monitored once a week to evaluate the anti-obesity effect. In addition, an autopsy was performed about 6 weeks after administration (Day 44), and the fatty liver improvement effect was evaluated by quantifying the amount of triglycerides in the liver. In addition, the same method was used for h2F8, with 5 subcutaneous administrations, and the endpoint was about 5 weeks later (Day 36), to evaluate the anti-obesity effect and fatty liver improvement effect. As a result, in the hIML1-2-OP1 and h2F8 administration groups, an inhibitory effect was observed on the weight gain caused by a high-fat diet, and a dose-dependent anti-obesity effect was observed. The weight loss effect on the PBS group about 5 weeks after the start of antibody administration was -5.0% and -7.6% in the hIML1-2-OP1 10 mg / kg administration group and 30 mg / kg administration group, respectively, which were significant effects ( Fig.10 -i). In addition, for h2F8, the 30 mg / kg administration group was -7.4%, showing a significant effect ( Fig.10 -ii). On the one hand, no significant changes were observed in the food intake during the antibody administration period compared with the PBS administration group, confirming the anti-obesity effect without food intake suppression. In addition, when the triglyceride content in the liver on Day 44 (hIML1-2-OP1) and Day 36 (h2F8) set as the endpoints was quantified, a decrease of 38% and 51% was observed in the 10 mg / kg and 30 mg / kg administration groups of hIML1-2-OP1, respectively, compared with the PBS group ( Fig.11 -i), the h2F8 10 mg / kg administration group and 30 mg / kg administration group showed a 30% and 43% decrease, respectively, compared with the PBS group ( Fig.11 -ii), showed a strong effect of reducing the amount of fat in the liver.

[0317] [Possibility of Industrial Application]

[0318] The antibody or antibody fragment of the present invention can be used to detect CB1 receptors in biological samples. Furthermore, a pharmaceutical composition containing the antibody or antibody fragment of the present invention is very useful as a medicine for treating or preventing CB1 receptor-related diseases.

Claims

1. An antibody or antibody fragment thereof that binds to a human cannabinoid type 1 (CB1) receptor, comprising: a light chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, CDR2 consisting of the amino acid sequence of SEQ ID NO: 45, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and The heavy chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 47, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

26.

2. An antibody or antibody fragment thereof that binds to a human cannabinoid type 1 (CB1) receptor, comprising: (a1) a light chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, CDR2 consisting of the amino acid sequence of SEQ ID NO: 34, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 35, and (a2) a heavy chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 26; (b1) a light chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 40, CDR2 consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 23, and (b2) a heavy chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 28, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 29; (c1) a light chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 19, CDR2 consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 21, and The heavy chain variable region comprises: (c2-1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 26, or (c2-2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 25, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 26; or (d1) a light chain variable region comprising: CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, CDR2 consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 23, and The heavy chain variable region comprises: (d2-1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 30, (d2-2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 28, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 31, or (d2-3) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

32.

3. The antibody or antibody fragment thereof according to claim 1 or 2, comprising: a light chain variable region consisting of the amino acid sequence of SEQ ID NOs: 13 to 15, or an amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NOs: 13 to 15, and The heavy chain variable region consists of the amino acid sequence of SEQ ID NOs: 16 to 18, 52, or an amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NOs: 16 to 18, 52.

4. The antibody or antibody fragment thereof according to any one of claims 1 to 3, comprising: (a1) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 14, and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17; (a2) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18; (a3) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16; or (a4) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and The heavy chain variable region consists of the amino acid sequence of SEQ ID NO:

52.

5. The antibody or antibody fragment thereof according to any one of claims 1 to 4, further comprising: a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 37, and A light chain constant region consisting of the amino acid sequence of SEQ ID NO:

38.

6. A pharmaceutical composition comprising the antibody or antibody fragment thereof according to any one of claims 1 to 5. 7 . The pharmaceutical composition according to claim 6 , which is a therapeutic and / or preventive agent for obesity, diabetes, and / or non-alcoholic steatohepatitis (NASH).

8. A polynucleotide, encoding the heavy chain variable region of the antibody according to claim 3 or 4, Optionally, it also encodes the heavy chain constant region of the antibody according to claim 5.

9. A polynucleotide, encoding the light chain variable region of the antibody according to claim 3 or 4, Optionally, it also encodes the light chain constant region of the antibody according to claim 5.

10. An expression vector comprising the polynucleotide according to claim 8 or 9.

Images