Cannabinoid receptor type 1 (CB1) binding protein and its use

Abstract

This invention provides antibodies and their antigen-binding fragments, as well as binding proteins that bind to the cannabinoid type 1 receptor (CB1), which are useful in the treatment and diagnosis of diseases. [Solution] This disclosure provides an isolated, modified CB1-binding protein that does not exist in nature, comprising an anti-CB1 antibody or its antigen-binding fragment. The CB1-binding protein has utility in the treatment and diagnosis of CB1-mediated conditions, diseases, and disorders.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 664,882 filed April 30, 2018, the entirety of which is incorporated herein by reference.

[0002] This invention relates to a cannabinoid receptor type 1 (CB1) binding protein and its use.

[0003] Built-in by reference The contents of all cited references (including references, patents, patent applications, and websites) that may be cited throughout this application are incorporated herein by reference in their entirety for all purposes, as are the references cited therein. Unless otherwise indicated, this disclosure utilizes the prior art of immunology, molecular biology, cell biology, drug development, and drug delivery. [Background technology]

[0004] Cannabinoid receptor type 1 (CB1) is a seven-transmembrane cell membrane receptor of the G-protein-coupled receptor superfamily that is mainly expressed in the brain and also in the periphery in the lungs, liver, kidneys, and adipose tissue. CB1 is activated by endogenously produced cannabinoids (such as eicosinoids) in the body called endocannabinoids, or cannabinoids introduced into the body (such as cannabis), or related synthetic compounds. Cannabinoids bind reversibly and stereoselectively to CB1. After engaging with CB1, multiple intracellular signaling pathways are activated, resulting in inhibition of adenylyl cyclase and activation of mitogen-activated protein (MAP) kinase, inhibition of presynaptic N-type and P / Q-type calcium channels and D-type outward potassium channels, and activation of inward rectifier and A-type outward potassium channels. The expression of CB1 is thought to regulate neurotransmitter release so as to prevent the occurrence of excessive neuronal activity, reduce pain and other inflammatory symptoms, and further regulate food intake.

[0005] Abnormal CB1 activity is associated with several diseases, including obesity and related disorders such as dyslipidemia, diabetes, fibrosis, liver diseases such as fatty liver, kidney diseases, cardiovascular diseases, and cancer.

[0006] Prader-Willi syndrome (PWS) is a genetic disorder caused by the deletion of certain paternally inherited genes and is characterized by obesity, type 2 diabetes, growth retardation, and reduced muscle strength. CB-1 has been identified as a target for the inverse agonist rimonabant in PWS (Motaghedi et al. (2011) Eur. J. Med. Genet. 54:14-18). Rimonabant (also called SR141716, Acomplia, and Zimulti) was an appetite suppressant anti-obesity drug developed and marketed by Sanofi-Aventis as an oral central CB1 antagonist. This product was indicated for the treatment of obesity and overweight patients with associated risk factors such as type 2 diabetes or dyslipidemia in combination with diet and exercise. In June 2006, this drug was approved by the EMEA for obesity. In 2008, Sanofi-Aventis discontinued all development and marketing of this drug for all indications due to the risk of serious psychiatric problems including suicidal thoughts. In January 2009, the EC suspended the marketing authorization of this drug.

[0007] Another inverse agonist, taranabant (MK-0364), was studied by Merck, but its phase 3 trials were discontinued due to high levels of side effects including depression and anxiety. Several other CB1 inverse agonists (e.g., AM251, AM1387, and AM4113) and antagonists (e.g., cannabidiol, ibipinabant, otenabant, sirinabant, tetrahydrocannabinol, and virudamine) are being studied, but they are either in the early stages of research or have been downgraded to non-human studies due to CNS side effects.

[0008] Several CB-1 inverse agonists / antagonists are under development that target CB1, primarily expressed in the periphery, by limiting the ability of CB-1 inverse agonists / antagonists to cross the blood-brain barrier (BBB). For example, TM-38837 is a Phase 1 CB1 inverse agonist / antagonist under development by 7TM Pharma A / S to treat obesity and metabolic disorders. Another peripherally selective silent antagonist not yet in clinical practice is AM6545. Peripherally selective CB-1 antagonistism may be a safer and more effective method for targeting peripheral endocannabinoid activity in several tissues, namely: (1) liver - decreased lipid synthesis, fat storage, and glucose secretion; (2) muscle - increased glucose uptake and oxidation; (3) adipocytes - decreased lipid synthesis and fat storage; decreased adiponectin synthesis; and (4) gastrointestinal tract (GI) - increased satiety, GI transit, and absorption (Kloet and Woods (2009) Endocrinol. 150:2531-2536).

[0009] Biomolecules such as antibodies and associated binding proteins offer potentially safer and more effective methods for delivering therapeutic agents while avoiding CNS damage and side effects. Generally, only about 0.1% of circulating antibodies exceed intact BBB (Poduslo et al. (1994) Proc. Natl. Acad. Sci. USA 91:5705-5709; Yu and Watts (2013) Neurotherapeut. 10:459-472). Therefore, the inherently low exposure of functional anti-CB1 biologics to the CNS offers an opportunity to engage CB-1 primarily peripherally, thereby limiting adverse events driven by the pharmacology of small molecules in the CNS.

[0010] Recently, antibodies against CB1 have been described in this art, but their clinical benefits are unknown. See U.S. Patent Publications 20170210797 and 20160145333.

[0011] Therefore, there is a need to identify safe, effective, and peripherally limited anti-CB1 inverse agonists or antagonists that do not significantly permeate the blood-brain barrier and avoid the harmful pharmacological effects of CB1 engagement in the brain. [Overview of the project]

[0012] The present invention provides cannabinoid receptor 1 (CB1) binding proteins, such as antibodies and their antigen-binding fragments, which are useful in the treatment and diagnosis of diseases.

[0013] An isolated antibody or antigen-binding fragment thereof that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the binding protein comprises six complementarity-determining regions (CDRs): CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 has the amino acid sequence GYTFTDYW (residues 26-33 of SEQ ID NO: 329) or a modified form of the above amino acid sequence by substitution of at least one amino acid residue, wherein the substitution of G at position 1 is S; the substitution of T at position 3 is E; the substitution of T at position 5 is S or N; the substitution of D at position 6 is R or Y; the substitution of Y at position 7 is H; and 8 A substitution of W at position A or N; CDR-H2 has the amino acid sequence IYPYDGDT (residues 51-58 of SEQ ID NO: 329) or a modified form of the above amino acid sequence by substitution of at least one amino acid residue, in which case a substitution of I at position 1 is F; a substitution of Y at position 2 is D, S, or T; a substitution of P at position 3 is T; a substitution of Y at position 4 is G, D, or S; a substitution of D at position 5 is Y or S; a substitution of G at position 6 is S; a substitution of D at position 7 is E, G, or R; and a substitution of T at position 8 is A, S, or I; CDR-H3 has the amino acid sequence ARG-X1-X2-X3-X4-X5-X6-X7-X8-X9-WX 10 -X 11-Having a modified form of the above amino acid sequence by substitution of Y (residues 98-113 of SEQ ID NO: 329) or at least one amino acid residue, in which case A substitution at position 1 is S; G substitution at position 3 is S; X1 at position 4 is Q, Y, K, R, or G, or absent; X2 at position 5 is E, Y, L, or G, or absent; X3 at position 6 is Y or P, or absent; X4 at position 7 is Y, R, or E, or absent; X5 at position 8 is G, or absent; X6 at position 9 is T, or absent; X7 at position 10 is N or D, or absent; X8 at position 11 is Y, N, A, or G, or absent; X9 at position 12 is N, Y, S, A, or R, or absent; W substitution at position 13 is Y, A, or P; X at position 14 10 is L, M, F, or G, or does not exist; X in 15th place 11is P, D, A, or T, or absent; a substitution of Y at position 16 is V; CDR-L1 has the amino acid sequence Q-X1-ISS-X2-Y (residues 27-33 of SEQ ID NO: 330) or a modified form of the above amino acid sequence by substitution of at least one amino acid residue, in which case a substitution of Q at position 1 is S or E; X1 at position 2 is E, S, T, N, G, or R; a substitution of I at position 3 is V; a substitution of S at position 4 is A or R , or G; substitution of S at position 5 is G, N, or T; X2 at position 6 is S, N, peptide FRYS, or absent; and substitution of Y at position 7 is F, D, or N; CDR-L2 has the amino acid sequence: X1-TS (residues 51-53 of SEQ ID NO: 330) or a modified form of the above amino acid sequence by substitution of at least one amino acid residue, in which case X1 at position 1 is A, Y, G, R, D, or S; and T at position 2 The substitution is A; the substitution of S at position 3 is R; and CDR-L3 has the amino acid sequence: QQY-X1-S-X2-PYT (residues 91-99 of SEQ ID NO: 330) or a modified form of the above amino acid sequence by substitution of at least one amino acid residue, in which case the substitution of Q at position 1 is L or H; the substitution of Q at position 2 is H; the substitution of Y at position 3 is S or G; X1 at position 4 is S, W, H, Y, N, or I; the substitution of S at position 5 The present invention provides an isolated antibody or antigen-binding fragment that binds to human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the substitution is E, R, G, T, or N; X2 at position 6 is Y, I, S, T, L, or W; and the substitution of Y at position 8 is P, L, F, or absent; and in which case the above substitution, addition, or deletion of at least one amino acid residue does not inhibit the ability of the antibody or antigen-binding fragment to bind to human CB1.

[0014] Tables 5 and 6 show exemplary anti-CB1 antibodies of the present invention and their functional antigen-binding fragments.

[0015] An isolated antibody or its antigen-binding fragment that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the antibody comprises a variable heavy chain (VH) domain sequence CDR and a variable light chain (VL) domain sequence CDR, wherein the VH domain sequence is one of the following: SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 270, 282, 29 Selected from the group consisting of 4, 306, and 318, and / or the above VL domain is selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324, the provides an isolated antibody or antigen-binding fragment thereof that binds to human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1).

[0016] An isolated antibody or antigen-binding fragment thereof that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the antibody comprises a variable heavy chain (VH) domain sequence and a variable light chain (VL) domain sequence, wherein the VH domain sequence is one of the following: SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 270, 282, 294, 30 Selected from the group consisting of 6 and 318, and / or the above VL domain provides an isolated antibody or antigen-binding fragment thereof that binds to human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

[0017] In one embodiment, the antibody or its antigen-binding fragment comprises a VH / VL pair of heavy chain CDRs and light chain CDRs selected from the group consisting of SEQ ID NOs: 18 / 24, 30 / 36, 42 / 48, 54 / 60, 66 / 72, 78 / 84, 90 / 96, 102 / 108, 114 / 120, 126 / 132, 138 / 144, 150 / 156, 162 / 168, 174 / 180, 186 / 192, 198 / 204, 210 / 216, 222 / 228, 234 / 240, 246 / 252, 258 / 264, 270 / 276, 282 / 288, 294 / 300, 306 / 312, and 318 / 324.

[0018] In one embodiment, the antibody or its antigen-binding fragment comprises a VH / VL pair selected from the group consisting of SEQ ID NOs: 18 / 24, 30 / 36, 42 / 48, 54 / 60, 66 / 72, 78 / 84, 90 / 96, 102 / 108, 114 / 120, 126 / 132, 138 / 144, 150 / 156, 162 / 168, 174 / 180, 186 / 192, 198 / 204, 210 / 216, 222 / 228, 234 / 240, 246 / 252, 258 / 264, 270 / 276, 282 / 288, 294 / 300, 306 / 312, and 318 / 324.

[0019] In one embodiment, the antibody or its antigen-binding fragment is (20, 21, 22); (32, 33, 34); (44, 45, 46); (56, 57, 58); (68, 69, 70); (80, 81, 82); (92, 93, 94); (104, 105, 106); (116, 117, 118); (128, 129, 130); (140, 141, 142); (152, 153, 154); (164, 165, 166); (176, 177, 178 );(188, 189, 190);(200, 201, 202);(212, 213, 214);(224, 225, 226);(236, 237, 238);(248, 249, 250);(260, 261, 262);(272, 273, 274);(284, 285, 286);(296, 297, 298);(308, 309, 310); and (320, 321, 322) are selected from the group HCDR set (HCDR1, HCD R2, HCDR3) and (26, 27, 28); (38, 39, 40); (50, 51, 52); (62, 63, 64); (74, 75, 76); (86, 87, 88); (98, 99, 100); (110, 111, 112); (122, 123, 124); (134, 135, 136); (146, 147, 148); (158, 159, 160); (170, 171, 172); (182, 183, 184); (194, 195, 19 Includes an LCDR set (LCDR1, LCDR2, LCDR3) selected from the group consisting of 6);(206, 207, 208);(218, 219, 220);(230, 231, 232);(242, 243, 244);(254, 255, 256);(266, 267, 268);(278, 279, 280);(290, 291, 292);(302, 303, 304);(314, 315, 316); and (326, 327, 328).

[0020] In one embodiment, the antibody or its antigen-binding fragment is sequence number (20, 21, 22 / 26, 27, 28); (32, 33, 34 / 38, 39, 40); (44, 45, 46 / 50, 51, 52); (56, 57, 58 / 62, 63, 64); (68, 69, 70 / 74, 75, 76); (80, 81, 82 / 86, 87, 88); (92, 93, 94 / 98, 99, 100); (104 ,105,106 / 110,111,112);(116,117,118 / 122,123,124);(128,129,130 / 134,135,136);(140,141,142 / 146,147,148);(152,153,154 / 158,159,160);(164,165,166 / 170,171,172);(176,177,178 / 182,183,18 4);(188, 189, 190 / 194, 195, 196);(200, 201, 202 / 206, 207, 208);(212, 213, 214 / 218, 219, 220);(224, 225, 226 / 230, 231, 232);(236, 237, 238 / 242, 243, 244);(248, 249, 250 / 254, 255, 256);(260, 261, 262 / 266 Includes (HCDR set / LCDR set) pairs selected from the group consisting of , 267, 268); (272, 273, 274 / 278, 279, 280); (284, 285, 286 / 290, 291, 292); (296, 297, 298 / 302, 303, 304); (308, 309, 310 / 314, 315, 316); and (320, 321, 322 / 326, 327, 328).

[0021] In one embodiment, the antibody or its antigen-binding fragment comprises an HC / LC pair selected from the group consisting of SEQ ID NOs: 17 / 23, 29 / 35, 41 / 47, 53 / 59, 65 / 71, 77 / 83, 89 / 95, 101 / 107, 113 / 119, 125 / 131, 137 / 143, 149 / 155, 161 / 167, 173 / 179, 185 / 191, 197 / 203, 209 / 215, 221 / 227, 233 / 239, 245 / 251, 257 / 263, 269 / 275, 281 / 287, 283 / 289, 305 / 311, and 317 / 323.

[0022] An isolated antibody or its antigen-binding fragment that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the antibody comprises a variable heavy chain (VH) domain sequence CDR and a variable light chain (VL) domain sequence CDR, wherein the VH domain sequence is an amino acid selected from the group consisting of SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 270, 282, 294, 306, and 318. The present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), having at least 95% identity to the sequence, and / or the VL domain sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

[0023] In one embodiment, VH is represented by sequence number 114 and VL is represented by sequence number 120. In one embodiment, VH is represented by sequence number 126 and VL is represented by sequence number 132. In one embodiment, VH is represented by sequence number 138 and VL is represented by sequence number 144. In one embodiment, VH is represented by sequence number 150 and VL is represented by sequence number 156. In one embodiment, VH is represented by sequence number 162 and VL is represented by sequence number 168. In one embodiment, VH is represented by sequence number 174 and VL is represented by sequence number 180. In one embodiment, VH is represented by sequence number 186 and VL is represented by sequence number 192. In one embodiment, VH is represented by sequence number 198 and VL is represented by sequence number 204. In one embodiment, VH is represented by sequence number 210 and VL is represented by sequence number 216. In one embodiment, VH is represented by sequence number 222 and VL is represented by sequence number 228. In one embodiment, VH is represented by sequence number 234 and VL is represented by sequence number 240. In one embodiment, VH is represented by sequence number 246 and VL is represented by sequence number 252. In one embodiment, VH is represented by sequence number 258 and VL is represented by sequence number 264. In one embodiment, VH is represented by sequence number 270 and VL is represented by sequence number 276. In one embodiment, VH is represented by sequence number 282 and VL is represented by sequence number 288. In one embodiment, VH is represented by sequence number 294 and VL is represented by sequence number 300. In one embodiment, VH is represented by sequence number 306 and VL is represented by sequence number 312. In one embodiment, VH is represented by sequence number 318 and VL is represented by sequence number 324.

[0024] In one embodiment, the anti-CB1 antibody is a human or humanized antibody.

[0025] In one embodiment, the anti-CB1 antigen-binding fragment includes a Fab fragment, a Fab' fragment, an F(ab)2 fragment, or an scFv fragment.

[0026] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment includes a human Fc region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM Fc.

[0027] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment includes a modified human Fc region selected from the group consisting of L234A / L235A ("LALA"), S228P, A330S, P331S, E233P / L234V / L235A, A327G / A330S / P331S, L234F / L235E / P331S, and N297Q.

[0028] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment inhibits CB1 signaling activity or acts as an antagonist thereof.

[0029] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment enhances or activates CB1 signaling activity, or acts as an agonist against it.

[0030] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment is an inverse agonist for CB1 signaling activity.

[0031] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment is a humanized antibody.

[0032] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment is a fully human antibody.

[0033] The present invention provides an anti-CB1 antibody or its antigen-binding fragment that specifically binds to substantially the same CB1 epitope as an isolated anti-human CB1 antibody or its antigen-binding fragment.

[0034] This invention provides an anti-CB1 antibody or its antigen-binding fragment that competes with isolated anti-human CB1 antibodies or their antigen-binding fragments for binding to CB1.

[0035] In one embodiment, the isolated antibody or its antigen-binding fragment that binds to CB1 has a binding affinity Kd of about 1 μM or less for CB1.

[0036] In one embodiment, the isolated antibody or its antigen-binding fragment that binds to CB1 has a binding affinity Kd of about 100 nM or less for CB1.

[0037] In one embodiment, the anti-CB1 antibody or antigen-binding fragment exhibits reduced brain permeability compared to rimonabant.

[0038] In one embodiment, an anti-CB1 antibody or antigen-binding fragment inhibits CB1 signaling at least twice as effectively as limonabant.

[0039] In one embodiment, an anti-CB1 antibody or antigen-binding fragment shows a reduction in CNS side effects compared to limonabant.

[0040] This invention provides an isolated nucleic acid molecule encoding an anti-CB1 antibody or its antigen-binding fragment.

[0041] This invention provides an expression vector containing a nucleic acid molecule encoding an anti-CB1 antibody or its antigen-binding fragment.

[0042] The present invention provides host cells containing an expression vector comprising a nucleic acid molecule encoding an anti-CB1 antibody or its antigen-binding fragment.

[0043] The present invention provides a method for regulating CB1 signaling, comprising contacting cells expressing CB1 with an anti-CB1 antibody or an antigen-binding fragment thereof.

[0044] The present invention provides a method for antagonistizing CB1, comprising contacting cells expressing CB1 with an anti-CB1 antibody or an antigen-binding fragment thereof.

[0045] The present invention provides a method for agonizing CB1, comprising contacting cells expressing CB1 with an anti-CB1 antibody or an antigen-binding fragment thereof.

[0046] The present invention provides a method for inverse agonizing CB1, comprising contacting cells expressing CB1 with an anti-CB1 antibody or an antigen-binding fragment thereof.

[0047] The present invention provides a pharmaceutical composition comprising an isolated anti-CB1 antibody or its antigen-binding fragment.

[0048] The present invention provides a method for inhibiting the biological activity of CB1 in a subject requiring such activity, comprising administering a pharmaceutical composition containing an effective amount of isolated anti-CB1 antibody or an antigen-binding fragment thereof to the subject, thereby inhibiting the activity of the CB1 protein in the subject.

[0049] The present invention provides a method for treating a disease associated with CB1 activity, comprising administering a pharmaceutical composition containing an isolated anti-CB1 antibody or an antigen-binding fragment thereof to a subject suffering from the disease.

[0050] The present invention provides a method for treating a disease or disorder responsive to the modulation of CB1 signaling in a subject requiring such treatment, the method comprising administering a pharmaceutical composition comprising an isolated anti-CB1 antibody or an antigen-binding fragment thereof.

[0051] A method is provided for treating a disease or disorder responsive to CB1 signaling antagonistism or inverse agonism in a subject requiring such treatment, the method comprising administering to the subject a pharmaceutical composition comprising an isolated anti-CB1 antibody or an antigen-binding fragment thereof.

[0052] A method is provided for treating a disease or disorder responsive to CB1 signaling agonism in a subject requiring such treatment, the method comprising administering to the subject a pharmaceutical composition comprising an isolated anti-CB1 antibody or an antigen-binding fragment thereof.

[0053] The present invention provides a method for diagnosing a disease or disorder associated with CB1, comprising contacting cells with an anti-CB1 antibody or an antigen-binding fragment thereof.

[0054] In one embodiment, the disease or disorder is selected from the group consisting of obesity, symptomatic obesity including Prader-Willi syndrome (PWS), Alström syndrome, Valde-Beedl syndrome (BBS), Albride hereditary osteodystrophy (AHO), and SIM1 deletion syndrome; diabetes and related complications; dyslipidemia; liver diseases such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, and primary biliary cirrhosis; fibrosis, such as renal fibrosis; chronic kidney disease; diabetic neuropathy, focal segmental glomerulosclerosis, kidney disease; metabolic diseases, osteoporosis, atherosclerosis, inflammatory diseases, cardiovascular diseases, cancer, pain, systemic sclerosis, multiple sclerosis spasticity, glaucoma, and nicotine addiction.

[0055] In one embodiment, the above-mentioned disease or disorder is a kidney disease (focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive kidney disease, nephrotic syndrome, steroid-resistant nephrotic syndrome, minimal change nephrotic syndrome, membranous nephropathy, idiopathic membranous nephropathy, membranoproliferative 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, Other conditions include microalbuminuria (or other kidney diseases), pulmonary hypertension, pain (neuropathic pain or visceral pain, etc.), cancer (chemotherapy-resistant breast cancer, adriamycin-resistant breast cancer, chemotherapy-resistant colorectal cancer, medulloblastoma, or tumor angiogenesis, etc.), anxiety, depression, transplant-related FSGS, transplant-related nephrotic syndrome, transplant-related proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), obesity, insulin resistance, type II diabetes mellitus, prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), diabetic gastroparesis, or gastroparesis.

[0056] The present invention provides an antibody conjugate comprising an isolated anti-CB1 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is conjugated with a drug selected from the group consisting of therapeutic agents, cytotoxic agents, immunoadhesion molecules, and imaging agents.

[0057] The present invention provides a kit comprising an isolated anti-CB1 antibody or its antigen-binding fragment, and instructions for using the antibody in an immunological assay.

[0058] The following description of embodiments, in conjunction with the accompanying drawings, will provide a more complete understanding of the above-mentioned and other features and advantages of the present invention, as well as the invention itself. [Brief explanation of the drawing]

[0059] [Figure 1] The results of the cAMP assay are shown. cAMP Hunter™ CHO-K1 CNR1 Gi cells were treated with CB1 antibody, isotype control, or small molecule CB1 antagonist JD5037, followed by agonist attack with 30 nM CP-55,940 (indicated as "Plus CP") in the presence of forskolin. Antagonists were also tested without the addition of CP-55,940 to demonstrate whether they possess agonist activity on their own. Abbreviations: CAB = Cell assay buffer (indicating assay background); CAB / F = CAB + 15 μM forskolin (representing the maximum amount of cAMP in the assay); CAB / F / CP = CAB / F + 30 nM CP-55,940 (approximately corresponding to EC80 at CP-55,940). [Figure 2] The results of the p-ERK assay are shown. The phospho / total ERK assay was performed using cAMP Hunter® CHO-K1 CNR1 Gi cells. Cells were treated with mouse anti-CB1 antibody or the small molecule CB1 antagonist JD5037, followed by agonist attack with 30 nM CP-55,940 in the presence of forskolin. At the end of the treatment, plates were treated for p-ERK / total ERK using the MesoScale Discovery (MSD) kit. Results are expressed as the percentage of maximum response (maximum %). [Figure 3] Figure 3A shows the binding of mouse anti-huCB1 antibodies to huCB1-CHO cells. The binding curves of purified mouse anti-CB1 antibodies were titrated 3-fold, starting from a 200 nM antibody concentration. All antibodies show specific binding to huCB1-CHO cells. Figure 3B shows the binding of mouse anti-huCB1 antibodies to moCB1-CHO cells. All antibodies show a lack of binding to moCB1-CHO cells. [Figure 4] This shows the evaluation of the binding of purified anti-CB1 antibodies in the presence and absence of the agonist and antagonist CB1 small molecules CP5990 and JD5037, respectively. [Figure 5A]This shows the consensus sequence obtained from the alignment of heavy chain variable region amino acid sequences from hybridoma antibodies M1-M8. [Figure 5B] This shows the consensus sequence obtained from the alignment of the light chain variable region amino acid sequences from hybridoma antibodies M1-M8. [Figure 6A] The consensus sequences obtained from the alignment of heavy chain variable region amino acid sequences from humanized antibodies M7-H1~M7-H16, M5-H1, and M5-H2 are shown. [Figure 6B] The consensus sequences obtained from the alignment of light chain variable region amino acid sequences from humanized antibodies M7-H1~M7-H16, M5-H1, and M5-H2 are shown. [Figure 7A] This shows the cell binding of humanized CB-1 antibody variants of clones M5 and M7 to CHO-huCB-1 and CHO parental cells at a single antibody concentration of 30 μg / mL. [Figure 7B] This shows the cell binding of the humanized CB-1 antibody variant of clone M7 to CHO-huCB-1 and CHO parental cells at a single antibody concentration of 30 μg / mL. [Figure 8] Figure 8A shows the results of the cAMP assay as described in Example 3. Figure 8A shows a side-by-side comparison between different mainchains using the M5 antibody. Figure 8B shows the results of the cAMP assay as described in Example 3. Figure 8B shows a side-by-side comparison between different mainchains using the M7 antibody. [Figure 9] Figure 9A shows the results of the p-ERK assay as described in Example 4. Figure 9A shows a side-by-side comparison between different mainchains using the M5 antibody. Figure 9B shows the results of the p-ERK assay as described in Example 4. Figure 9B shows a side-by-side comparison between different mainchains using the M7 antibody. [Figure 10]The results of a cAMP assay using an inverse agonism method similar to that described in Example 3 are shown. cAMP Hunter® CHO-K1 CNR1 Gi cells were treated with CB1 antibody or an isotype control, followed by the addition of forskolin. The purple dotted line represents the level of cAMP released upon treatment with forskolin, representing the maximum amount of cAMP in the assay. The red line corresponds to the CB1 small molecule agonist CP-55,940 used as a control in the assay. Compounds with inverse agonism activity show a curve that progresses in the opposite direction to the agonist, towards the top of the CAB / F line. This experiment demonstrates that both M7 variants tested possess inverse agonism activity. [Modes for carrying out the invention]

[0060] Unless otherwise defined, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The words “a” or “an” mean “at least one” unless otherwise indicated. The meaning of the phrase “at least one” is equivalent to the meaning of the phrase “one or more.” The word “or” means “and / or” unless otherwise indicated. As used herein, terms such as “contain,” “include,” “contain,” and “have” may have the meanings given to them under U.S. patent law, and may also mean “encompass,” “encompass,” etc.; “essentially from” or “essentially” also have the meanings given to them under U.S. patent law, and this term is open and allows for more than described existence as long as the fundamental or novel characteristics do not change, but excludes prior art embodiments. Unless otherwise specifically stated or evident from the context, as used herein, the term “about” is understood to be within the normal tolerances of the art, e.g., within two standard deviations of the mean. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the described value. Unless otherwise evident from the context, all numerical values ​​shown herein are modified by the term “about.”

[0061] The methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art and, unless otherwise indicated, as described in the various general and more specific references cited and discussed herein. The nomenclature, laboratory procedures and techniques for cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization, analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques are used in enzymatic reactions and purification techniques, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and patient care.

[0062] In this specification, ranges are understood to be abbreviations for all values ​​within that range. For example, the range from 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0063] The term "cannabinoid receptor type 1" or "CB1" refers to a seven-transmembrane cell receptor encoded by the CNR1 gene and having the canonical amino acid sequence of Sequence ID No. 1 (see https: / / www.uniprot.org / uniprot / P21554). Table 1 discloses this sequence, as well as each of the domains of this protein.

[0064] [Table 1]

[0065] The term "central CB1" refers to CB1 that is localized in either the brain or the CNS (central nervous system).

[0066] The term "peripheral CB1" refers to CB1 that is not localized to the brain or CNS (e.g., peripherally limited CB1).

[0067] The term “antibody” means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. This term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, mutant, and transplantable antibodies. For this purpose of the Disclosure, unless otherwise modified by the term “intact,” as in “intact antibody,” the term “antibody” also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind to CB1. Typically, such fragments would contain an antigen-binding domain. The term “antibody” also includes immunoglobulin molecules, and even their polymers, that contain four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains linked together by disulfide bonds. In one embodiment of a full-length antibody, each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The CL consists of a single CL domain. The VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Generally, each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present invention, the FRs of an anti-CB1 antibody may be identical to the human germline sequence, or they may be naturally or artificially modified. The amino acid consensus sequence can be defined based on side-by-side analysis of two or more CDRs and / or FRs. The antibody molecule may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0068] The terms "HCDR set" and "LCDR set" refer to a group of three CDRs that arise in a single variable region of either the heavy or light chain, respectively, capable of binding to an antigen. The term "(HCDR set / LCDR set) pair" refers to a pair of HCDR sets and LCDRs, representing the six CDRs that constitute the antigen-binding site. The precise boundaries of these CDRs are defined differently depending on the system. The system described by Kabat (Kabat et al. (1987 and (1991)) provides not only a clear residue numbering system applicable to any variable region of an antibody, but also precise residue boundaries that define the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. The term "Kabat numbering" refers to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of antibodies or their antigen-binding fragments (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region is in the range of amino acid positions 31-35 in CDR1, amino acid positions 50-65 in CDR2, and amino acid positions 95-102 in CDR3. In the light chain variable region, the hypervariable region is in the range of amino acid positions 24-34 in CDR1, amino acid positions 50-56 in CDR2, and amino acid positions 89-97 in CDR3. Chothia and collaborators (Chothia and Lesk (1987) J.Mol.Biol.196:901-917; Chothia et al. (1989) Nature 342:877-883) found that certain sub-regions within Kabat CDRs adopt nearly identical peptide backbone structures despite exhibiting significant diversity at the amino acid sequence level.These sub-regions are named L1, L2, and L3 or H1, H2, and H3, where "L" and "H" indicate the light chain region and heavy chain region, respectively. These regions are sometimes referred to as the Chothia CDR, which has boundaries that overlap with the Kabat CDR. Other boundaries defining CDRs that overlap with the Kabat CDR are described in Padlan (1995) FASEB J.9:133-139 and Maccallum (1996) J.Mol.Biol.262(5):732-45). Further definitions of other CDR boundaries may not strictly follow one of the systems described above, but will still overlap with the Kabat CDR; however, they may be shortened or lengthened in consideration of predictions or experimental findings that specific residues or groups of residues or even the entire CDR will not have a significant effect on antigen binding. The compositions and methods described herein may utilize CDRs defined according to any of these systems.

[0069] The term "VH / VL pair" refers to a paired VH and VL that can bind to an antigen.

[0070] The term "HC / LC pair" refers to HC and LC that are paired and capable of binding to an antigen.

[0071] The term “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that can be produced by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally contains two constant domains, one CH2 domain and one CH3 domain, and optionally one CH4 domain. Substitutions of amino acid residues in the Fc region to alter antibody effector function are known in the art (e.g., U.S. Patents 5,648,260 and 5,624,821). The Fc region mediates several important effector functions, such as cytokine induction, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance rate of antibodies and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic immunoglobulins, but in other cases, depending on the therapeutic goal, they may be unnecessary or even harmful.

[0072] The terms “CB1-binding antibody” and “anti-CB1 antibody” mean an antibody that binds to the soluble CB1 protein or a fragment thereof (e.g., a portion of the extracellular domain of CB1) and / or CB1 expressed on the cell surface, and its antigen-binding fragment. The expression “CB1 expressed on the cell surface” means the CB1 protein or a portion thereof that is expressed on the cell surface in vitro or in vivo so that at least a portion of the CB1 protein is exposed extracellularly on the cell membrane and accessible to the antigen-binding portion of the antibody.

[0073] The terms “CB1-binding protein” or “anti-CB1-binding protein” refer to proteins that bind to CB1, including all or part of an antigen-binding fragment, and include proteins that include alternative arrangements of typical antibody domains or frameworks, such as recombinant polyvalent or multispecific immunoglobulins, as well as conjugates and fusion proteins. The CB1-binding proteins of the present invention, as well as their variants and variants, may retain CB1 binding and function, or provide additional or alternative functions. Such CB1-binding proteins are within the scope of the present invention and are well known to those skilled in the art.

[0074] The terms “antigen-binding domain” and “antigen-binding fragment” in relation to binding proteins such as antibodies mean a portion or fragment of an antibody, or a variant or mutant thereof, that retains the ability to specifically bind to the antibody’s target antigen, and include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained, for example, from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable domain and optionally the constant domain. One or more variable and / or constant domains can be arranged to a suitable position, or codons can be introduced, cysteine ​​residues can be created, amino acids can be modified, added, or deleted, etc. Numerous fragment, variant, or variant antibody forms containing antigen-binding fragments are known in the art. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, which are monovalent fragments consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide crosslink at a hinge region; (iii) Fd fragments containing the VH and CH1 domains; (iv) Fv fragments containing the VL and VH domains of a single arm of the antibody; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments containing a single variable domain; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of the antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR-transplant antibodies, diabodies, linear antibodies (including pairs of tandem Fv segments that form a pair of antigen-binding sites with complementary light chain polypeptides; including VH-CH1-VH-CH1), triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular immunotherapy drugs (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included within the scope of the expression “antigen-binding fragment” as used herein.The term “antigen-binding fragment” is not limited to fragments contained within variant molecules that may have additional or rearranged antibody regions, such as multispecific antibodies and antibody conjugates that retain the same antigen-binding to a particular antigen.

[0075] Antibody antigen-binding fragments typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be located relative to each other in any suitable configuration. For example, the variable region may be a dimer, including a VH-VH dimer, a VH-VL dimer, or a VL-VL dimer. Alternatively, antibody antigen-binding fragments may contain a monomeric VH domain or a VL domain.

[0076] In certain embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains that may be found within the antigen-binding fragment of the antibody of the present invention include: (i)VH-CH1; (ii)VH-CH2; (iii)VH-CH3; (iv)VH-CH1-CH2; (v)VH-CH1-CH2-CH3; (vi)VH-CH2-CH3; (vii)VH-CL; (viii)VL-CH1; (ix)VL-CH2; (x)VL-CH3; (xi)VL-CH1-CH2; (xii)VL-CH1-CH2-CH3; (xiii)VL-CH2-CH3; and (xiv)VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may comprise homodimers or heterodimers (or other polymers) of any of the variable and constant domain configurations listed above, both with each other and / or in non-covalent association with one or more monomeric VH or VL domains (e.g., by disulfide bonds).

[0077] When a complete antibody molecule is present, the antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically contain at least two distinct variable domains, each capable of specifically binding to a different antigen or a different epitope on the same antigen. Any multispecific antibody form, including the exemplary bispecific antibody forms disclosed herein, can be employed for use in the context of the antibody antigen-binding fragment of the present invention using routine techniques available in the art.

[0078] The terms "specific" or "specific to" in relation to binding proteins refer to the ability of a binding protein to selectively bind to a target or antigen with higher affinity (i.e., lower Kd value) than, for example, any other target or antigen.

[0079] In certain embodiments, the antibodies of the present invention may function via complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). CDC refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, resulting in the lysis of target cells. CDC and ADCC can be measured using assays well known and available in the art (see, for example, U.S. Patents 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656).

[0080] The term "mouse antibody" refers to an antibody having a variable region and a constant region derived from a mouse germline immunoglobulin sequence. A mouse antibody may contain amino acid residues not encoded by the mouse germline immunoglobulin sequence (e.g., mutations introduced in vitro by random or site-directed mutagenesis, or in vivo by somatic mutation) in, for example, the CDR, particularly CDR3. However, the term "mouse antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as humans, has been transplanted into the mouse framework sequence.

[0081] The term "recombinant antibody" means an antibody prepared, expressed, produced, or isolated by recombinant means, including antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combinatorial antibody library, antibodies isolated from an animal transgenic for an immunoglobulin gene (e.g., a mouse), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of an immunoglobulin gene sequence to another DNA sequence. In certain embodiments, such recombinant antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic for an immunoglobulin sequence is used, in vivo somatic mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from germline VH and VL sequences and, in relation thereto, sequences that cannot naturally exist in vivo within a particular antibody germline repertoire.

[0082] The term “isolated antibody” means an antibody identified, isolated, and / or recovered from at least one component of its natural environment. For example, an antibody isolated or removed from at least one component of an organism, or from a tissue or cell in which antibodies are naturally present or naturally produced, is an “isolated antibody” for the purposes of this invention. Isolated antibodies also include antibodies found in situ within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may substantially contain no other cellular material and / or chemical substances.

[0083] The terms “neutralizing” or “blocking” antibody refer to an antibody whose binding to its ligand or antigen interferes with the biological activity of the ligand or antigen. In one embodiment, a neutralizing-binding protein binds to an antigen (e.g., a cytokine) and reduces its biological activity by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. A neutralizing antibody that binds to CB1: (i) interferes with the interaction between CB1 or a CB1 fragment and a CB1 ligand (e.g., a cannabinoid), and / or (ii) results in the inhibition of at least one biological function of CB1. The inhibition resulting from a CB1 neutralizing or blocking antibody does not need to be complete, as long as it is detectable using a suitable assay. Exemplary assays for detecting CB1 inhibition are described herein.

[0084] The term "affinity" refers to the strength of the interaction between a binding protein and its target antigen, and is determined by the CDR sequence of the binding protein, as well as by the properties of the antigen and antibody, such as their size, shape, and / or charge. Binding proteins can be selected for their affinity, which brings about the desired therapeutic endpoint while minimizing negative side effects. Affinity can be measured using methods known to those skilled in the art.

[0085] The term "affinity-mature antibody" means that one or more modifications have been made in one or more CDRs or FRs, resulting in improved antibody affinity for its target antigen compared to an unmodified "parent" antibody without those modifications. Exemplary affinity-mature antibodies have nanomolar or even picomolar affinity for their target antigen. Affinity-mature antibodies are produced by procedures known in the art. For example, Marks et al. (1992) BioTechnology 10:779-783 describes affinity maturation by VH domain and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues is described in Barbas et al. (1994) Proc. Nat. Acad. Sci. USA 91:3809-3813; Schier et al. (1995) Gene 169:147-155; Yelton et al. (1995) J. lmmunol. 155:1994-2004; Jackson et al. (1995) J. lmmunol. 154(7):3310-9; Hawkins et al. (1992) J. Mol. Biol. 226:889-896, and mutations at selective mutagenesis sites, contact or hypermutation sites with activity-enhancing amino acid residues are described in U.S. Patent No. 6,914,128.

[0086] The term "CDR-transplanted antibody" refers to an antibody containing heavy chain variable region sequences and light chain variable region sequences in which one or more sequences of the VH and / or VL CDR regions are replaced by the CDR sequences of another antibody. For example, the two antibodies may originate from different species, such as an antibody having a mouse heavy chain variable region and a mouse light chain variable region in which one or more of the mouse CDR sequences are replaced by human CDR sequences.

[0087] The term "humanized antibody" refers to an antibody from a non-human species that has been modified to more closely resemble a human germline sequence. One type of humanized antibody is a CDR-transplanted antibody in which one or more CDR sequences are non-human and the framework region (FR) sequences are human or substantially human (e.g., they are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a human antibody). A humanized antibody may contain substantially all, at least one, typically two variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all sequences of the CDR region correspond to those of a non-human immunoglobulin and all or substantially all sequences of the FR region correspond to those of a human immunoglobulin. A humanized antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In one embodiment, a humanized antibody also contains at least a portion of the human immunoglobulin Fc region. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody comprises only the humanized heavy chain. In some embodiments, the humanized antibody comprises only the humanized variable domain of the light chain and / or the humanized variable domain of the heavy chain. In some embodiments, the humanized antibody comprises the variable domain of the light chain, and even more specifically, the variable domain of the heavy chain. In some embodiments, the humanized antibody comprises the variable domain of the heavy chain, and even more specifically, the variable domain of the light chain.

[0088] The term "efficacy" refers to the ability of a binding protein to achieve a desired effect and is a measure of its therapeutic efficacy. Efficacy can be evaluated using methods known to those skilled in the art.

[0089] The term "effective dose" refers to a dosage or amount sufficient to reduce CB1 activity, thereby resulting in symptom remission in a patient or achieving a desired biological outcome. Desired biological outcomes include, for example, a decrease or increase in CB1 activity.

[0090] The term "cross-reactivity" refers to the ability of a binding protein to bind to a target antigen other than the antigen that produced the antibody. Generally, a binding protein should bind to its target antigen with appropriate high affinity, but it may also bind to the same target antigen in a different species, or it may exhibit low affinity for a non-target antigen. Individual binding proteins are generally selected to satisfy two criteria: (1) tissue staining suitable for the known expression of the antibody target; and (2) similar staining patterns in human tissue from the same organ and in tissue from toxicity test species (e.g., mouse and cynomolgus monkey). These and other methods for evaluating cross-reactivity are known to those skilled in the art.

[0091] The term "biological function" refers to the specific in vitro or in vivo activity of a binding protein, whether naturally occurring or made possible by recombinant means. Binding proteins may target multiple classes of antigens and achieve desired therapeutic outcomes through multiple mechanisms of action. Binding proteins may agonize, antagonize, or neutralize the activity of their targets. Binding proteins may assist in the clearance of the target to which they bind, or, if they bind to cells, may cause cytotoxicity. Two or more antibody moieties may be incorporated into a single polyvalent form to achieve distinct functions in a single binding protein molecule. Biological activity includes, but is not limited to, receptor binding, induction of cell proliferation, inhibition of cell growth, induction of other cytokines, induction of apoptosis, and enzymatic activity. In vitro assays and in vivo models used to assess biological function are known to those skilled in the art.

[0092] The term "stable" means that its physical, chemical, and / or biological integrity or activity can be maintained within a given time or storage conditions. In vitro stable binding proteins at various temperatures over long periods are generally desirable. Methods for stabilizing binding proteins and for evaluating their stability at various temperatures are known to those skilled in the art.

[0093] The term "solubility" means the ability of a protein to remain dispersed in an aqueous solution. The solubility of a protein in an aqueous formulation depends on the proper distribution of hydrophobic and hydrophilic amino acid residues, and thus solubility can be involved in the production of correctly folded proteins. One of ordinary skill in the art will be able to detect an increase or decrease in the solubility of a binding protein using routine HPLC techniques and methods known to one of ordinary skill in the art.

[0094] The term "immunogenicity" means the ability of a substance to induce an immune response. Administration of a therapeutic binding protein can result in a certain incidence of an immune response. Methods for reducing the immunogenicity of antibodies and binding proteins are known to one of ordinary skill in the art.

[0095] The term "detectable label" means a moiety that attaches to a member of a specific binding pair, such as an antibody or its analyte, and enables the detection of a reaction (e.g., binding) between members of that specific binding pair. A labeled member of a specific binding pair is referred to as "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a detectable label that provides for the identification of the binding protein has been incorporated. In one embodiment, the detectable label can produce a signal detectable by visual or instrumental means, for example, incorporation of a radiolabeled amino acid, or attachment of a biotinyl moiety to a polypeptide that can be detected by avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity that can be detected by optical or colorimetric methods). Examples of detectable labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153These include Sm); pigments; fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors); enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Typical examples of labels commonly used in immunoassays include photoluminescent moieties, e.g., acridinium compounds, and fluorescent moieties, e.g., fluorescein. In this regard, the moiety itself does not need to be detectably labeled, but may become detectable when reacting with another moiety.

[0096] The term “conjugate” refers to a binding protein, such as an antibody, that is chemically linked to another functional molecule or a second chemical part, such as a therapeutic agent, cytotoxic agent, cell proliferation inhibitor, or imaging agent (see, for example, Patent No. 7,850,962). The term “drug” includes chemical compounds, mixtures of chemical compounds, biomacromolecules such as protein peptides, or extracts made from biomaterials. In one embodiment, therapeutic agents or cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, anthracyclines, toxins, and apoptotic agents. Useful agents include, for example, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or congeners. Useful imaging agents in the preparation of anti-CB1 binding protein conjugates include, but are not limited to, radiolabeling, enzymes, fluorescent labeling, luminescence labeling, bioluminescence labeling, magnetic labeling, and biotin. When used in the context of immunoassays, the conjugate antibody may be a detectably labeled antibody used as the detection antibody. Antibodies can be linked by chemical crosslinking or recombinant methods. Antibodies can also be conjugated to various non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described in U.S. Patents No. 4,640,835; No. 4,496,689; No. 4,301,144; No. 4,670,417; No. 4,791,192; or No. 4,179,337. Antibodies can be chemically modified by covalent conjugation to polymers to extend their cyclic half-lives, for example.Exemplary polymers and methods for bonding them are also shown in U.S. Patents 4,766,106; 4,179,337; 4,495,285; and 4,609,546.

[0097] The term "crystallized" refers to a binding protein that exists in crystalline form. A crystal is one form of solid state of matter, distinct from other forms such as amorphous solid or liquid crystal states. A crystal consists of a regular, repeating three-dimensional array of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen / antibody complexes).

[0098] The term "vector" refers to a nucleic acid molecule capable of transporting another ligated nucleic acid. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA segment can be ligated. Another type of vector is a viral vector, in which case an additional DNA segment can be ligated into the viral genome. Other vectors include RNA vectors. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome (e.g., non-episomal mammalian vectors). A "recombinant expression vector" or "expression vector" can direct the expression of a operably ligated gene. In this specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, other forms of expression vectors that provide equivalent function are also included, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0099] The terms “recombinant host cell” or “host cell” refer to a cell into which exogenous DNA or RNA has been introduced. Such terms refer not only to a specific target cell but also to the offspring of such a cell. Since certain modifications may occur in later generations due to either mutation or environmental influences, such offspring may not actually be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In one embodiment, host cells include prokaryotic cells and eukaryotic cells. In one embodiment, eukaryotic cells include protist cells, fungal cells, plant cells and animal cells. In another embodiment, host cells include, but are not limited to, prokaryotic cell lines E. coli; mammalian cell lines CHO, HEK293, COS, NSO, SP2 and PER.C6; insect cell line Sf9; and fungal cells Saccharomyces cerevisiae.

[0100] The term "transfection" refers to various techniques commonly used to introduce exogenous nucleic acids into host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection.

[0101] The term “biological sample” means any quantity of material from a living or dead organism. Such material includes, but is not limited to, blood, plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0102] The term "control" refers to a composition that is known to either contain the analyte ("negative control") or contain the analyte ("positive control"). A positive control may contain a known concentration of the analyte or can be used to demonstrate assay performance characteristics and is a useful indicator of reagent integrity.

[0103] The term "specific binding pair" refers to two different molecules that bind specifically to each other through chemical or physical means. Specific binding pairs include, for example, antibodies and their antigens, biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, as well as their fragments and analogues that maintain specific binding. An example of a specific binding pair is the VH and VL regions of an antibody ("VH / VL").

[0104] The term "linker" refers to a polypeptide containing one or more amino acid residues joined by a peptide bond, used to link two polypeptides (e.g., two VH domains or two VL domains). Linkers are well known in the art (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0105] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either three-dimensional or linear. Three-dimensional epitopes are generated by spatially parallel amino acids from various segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include portions of amino acids, sugars, phosphoryl groups, or sulfonyl groups on an antigen and may have specific three-dimensional structural properties and / or specific charge properties. Binding proteins "bind to the same epitope" if they bind to the same amino acid on an antigen, and they can cross-compete (one antibody may inhibit the binding or regulatory effect of another). In addition, the structural characteristics of an epitope (overlap, similarity, identity) are informative; and the functional characteristics include structural (coupling) and functional (regulation, competition) parameters.

[0106] The term "pharmacokinetics" refers to the process by which a drug is absorbed, dispersed, metabolized, and eliminated by living organisms.

[0107] The term "bioavailability" refers to the amount of active drug that reaches its target after administration. Bioavailability is a function of several properties, including stability, solubility, immunogenicity, and pharmacokinetics, and can be evaluated using methods known to those skilled in the art.

[0108] The term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore® system (BIAcore International AB, Uppsala, Sweden and Piscataway, NJ; Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26).

[0109] "Kon," "Association Rate Constant," and "Ka" refer to the on-rate constant for the association of a binding protein (e.g., an antibody) to an antigen for the formation of a binding protein / antigen complex. This value indicates the binding rate between the binding protein and its target antigen, or the rate of complex formation between the binding protein and the antigen, as shown by the following formula: Antibody (“Ab”) + Antigen (“Ag”) → “Ab-Ag”

[0110] The terms "Koff" and "dissociation rate constant" refer to the off-rate constant for the dissociation of a binding protein (e.g., an antibody) from a binding protein / antigen complex. This value represents the rate of dissociation of the binding protein from its target antigen, or the separation of the Ab-Ag complex into free antibody and antigen over time, as shown by the following formula: Ab+Ag ← Ab-Ag

[0111] The terms "Kd" and "equilibrium dissociation constant" refer to values ​​obtained by titration measurements at equilibrium or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). Methods for determining the association and dissociation rate constants are well known in the art. Fluorescence-based techniques offer high sensitivity and capability for examining samples in physiological buffers at equilibrium. Other experimental approaches and instruments, such as the BIAcore® assay (BIAcore international AB, Uppsala, Sweden) or the KinExA® assay (Sapidyne Instruments, Boise, Idaho), can also be used.

[0112] The term "variant" refers to a polypeptide whose amino acid sequence differs from a given polypeptide due to the addition, insertion, deletion, or conservative substitution of amino acids, but which retains the biological activity of the given polypeptide (for example, a variant antibody may compete with a native antibody for binding to its target). Conservative amino acid substitutions are understood in the art to involve replacing one amino acid with a different amino acid having similar properties (e.g., hydrophilicity and degree and distribution of charged regions), and typically result in only slight changes. These slight changes can in some cases be identified by examining the hydroxyl index of amino acids, as understood in the art. The hydroxyl index of amino acids is based on the examination of their hydrophobicity and charge. Amino acids with similar hydroxyl indices can be substituted in a protein, and the protein still retains its protein function. In one embodiment, amino acids with hydroxyl indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that would result in a protein that retains its biological function. Considering the hydrophilicity of amino acids in the context of peptides allows for the calculation of the peptide's maximum local mean hydrophilicity, which is a useful measure reported to correlate well with antigenicity and immunogenicity. Substitutions of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. In one embodiment, substitutions are made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are biologically functional and compatible depend on the relative similarity of the amino acids, and in particular, their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. The term “variant” also includes polypeptides or fragments thereof that have been processed differently by proteolysis, phosphorylation, or other posttranslational modifications, but still retain biological activity or antigenic reactivity. The term “variant” encompasses variant fragments unless otherwise defined.The variant may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, or 75% identical to the wild-type sequence.

[0113] The anti-CB1 antibodies disclosed herein may contain one or more amino acid substitutions, additions, insertions, and / or deletions in the framework and / or CDR region of the heavy chain variable domain and light chain variable domain when compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from public antibody sequence databases. The present invention includes antibodies derived from any of the amino acid sequences disclosed herein, and antigen-binding fragments thereof, wherein one or more amino acids in one or more frameworks and / or CDR regions are mutated to the corresponding residue(possibly) of the germline sequence from which the antibody is derived, or to the corresponding residue(possibly) of another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue(possibly) (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can easily generate a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy-chain and light-chain variable region sequences disclosed herein. In certain embodiments, all of the framework and / or CDR residues in the VH domain and / or VL domain are backmutated to residues found in the original germline sequence from which the antibody originates. In other embodiments, only certain residues are backmutated to the original germline sequence, for example, only mutant residues found in the first eight amino acids of FR1, or in the last eight amino acids of FR4, or only mutant residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originates).Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, in which certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues that are different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (if applicable), or decreased immunogenicity. Antibodies and antigen-binding fragments obtained by this general method are included in the present invention.

[0114] In the context of nucleic acids, the terms “substantially identical” and “substantially identical” indicate that, when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, nucleotide sequence identity exists in at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may, under certain circumstances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0115] In the context of polypeptides, the terms “substantially similar” and “substantially identical” mean that two polypeptide sequences share at least about 95% sequence identity, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity when optimally aligned using a GAP program or BESTFIT program with default gap weights. In one embodiment, the non-identical residue positions differ due to conserved amino acid substitutions. The present invention also includes anti-CB1 antibodies comprising variants of any of the VH, VL, and / or CDR amino acid sequences disclosed herein having one or more conserved substitutions. For example, the present invention includes anti-CB1 antibodies having VH, VL, and / or CDR amino acid sequences that include, for example, 10 or fewer, 8 or fewer, 6 or fewer, and 4 or fewer conserved amino acid substitutions compared to any of the VH, VL, and / or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions should not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other due to a conservative substitution, the conservative nature of the substitution can be modified by adjusting the sequence identity percentage or degree of similarity upwards. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acids with side chains having similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine.Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A “moderately conservative” substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix disclosed in 1443-1445.

[0116] Sequence similarity in polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutant proteins. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; the GCG version 6.1.FASTA program (e.g., FASTA2 and FASTA3) provides alignment of the best overlap region and sequence identity percentage between the query sequence and the search sequence (Pearson (2000), cited above). When comparing the sequences of the present invention with a database containing numerous sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. For example, see Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402, which are incorporated herein by reference, respectively.

[0117] Biological characteristics of antibodies The present invention comprises an anti-CB1 antibody that binds to CB1 with high affinity and an antigen-binding fragment thereof.

[0118] When measured by surface plasmon resonance, at least about 10 for CB1 2 M -1 s -1 ; at least about 10 3 M -1 s -1 ; at least about 10 4 M -1s -1 ; at least about 10 5 M -1 s -1 ; and at least about 10 6 M -1 s -1 On-rate constant (K) selected from the group consisting of the following on The present invention provides an anti-CB1 antibody and its antigen-binding fragment, which have the following properties:

[0119] When measured by surface plasmon resonance, the value for the above target is at most about 10 -3 s -1 Even at the highest, about 10 -4 s -1 Even at the highest, about 10 -5 s -1 ; and at most about 10 -6 s -1 The off-rate constant (K) is selected from the group consisting of the following: off The present invention provides an anti-CB1 antibody and its antigen-binding fragment, which have the following properties:

[0120] At most about 10 against the above target -7 M; at most about 10 -8 M; at most about 10 -9 M; at most about 10 -10 M; at most about 10 -11 M; at most about 10 -13 M; and at most 10 -14The present invention provides an anti-CB1 antibody and its antigen-binding fragment having a dissociation constant (KD) selected from the group consisting of M. The anti-CB1 antibody and its fragment may have binding affinity Kd values ​​for CB1 in the ranges of approximately 0.01 nM to approximately 500 nM, approximately 0.02 nM to approximately 250 nM, approximately 0.02 to approximately 200 nM, approximately 0.05 to approximately 100 nM, and approximately 0.05 to approximately 50 nM. The antibodies and their fragments may have binding affinity Kd values ​​of approximately 500 nM or less, approximately 250 nM or less, approximately 200 nM or less, approximately 150 nM or less, approximately 100 nM or less, approximately 75 nM or less, approximately 50 nM or less, approximately 25 nM or less, approximately 10 nM or less, approximately 5 nM or less, approximately 1 nM or less, approximately 500 pM or less, approximately 250 pM or less, approximately 100 pM or less, approximately 50 pM or less, or approximately 10 pM or less with respect to CB1. In certain embodiments, the antibody or antigen-binding fragment of the present invention binds to CB1 with a Kd of less than approximately 15 pM, less than approximately 10 pM, less than approximately 8 pM, less than approximately 6 pM, less than approximately 4 pM, less than approximately 2 pM, or less than approximately 1 pM.

[0121] In some embodiments, the anti-CB1 antibody or its antigen-binding fragment has at least the same potency as small molecule CB1 receptor modulators such as limonabant, taranaban, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenaban, surinabant, tetrahydrocannabivarin, virodamine, and AM6545. In some embodiments, the anti-CB1 antibody or its antigen-binding fragment has at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or at least 20-fold higher CB1 antagonist or inverse agonist activity than small molecule CB1 receptor modulators such as limonabant, taranaban, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenaban, surinabant, tetrahydrocannabivarin, virodamine, and AM6545. In some embodiments, the anti-CB1 antibody or its antigen-binding fragment inhibits CB1 agonist-mediated signaling. In some embodiments, inhibition of CB1 agonist-mediated signaling is measured by determining intracellular cAMP levels and / or downstream ERK phosphorylation.

[0122] In some embodiments, the anti-CB1 antibody and its antigen-binding fragment have the advantage of reduced or absent BBB permeability or brain exposure. In some embodiments, the BBB permeability of the anti-CB1 antibody and its antigen-binding fragment shows reduced brain permeability compared to small molecule CB1 agonists, antagonists, or inverse agonists (e.g., rimonabant, taranaban, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenaban, surinabant, tetrahydrocannabivarin, and virodamine, and AM6545). In some embodiments, the anti-CB1 antibody and its antigen-binding fragment provided herein provide therapeutic effects along with reduced CNS side effects compared to small molecule CB1 receptor agonists, antagonists, or inverse agonists. CNS side effects associated with the small molecule CB1 receptor antagonist rimonabant include, for example, anxiety, depression, agitation, eating disorders, irritability, aggression, and insomnia (Moreira (2009) Rev. Bras. Psiquiatr. 31(2):145-153).

[0123] Epitope mapping and related technologies The present invention comprises an anti-CB1 antibody that interacts with one or more amino acids found within the extracellular domain of human CB1 (e.g., amino acids 1-116, and / or within the extracellular loops e1 (amino acids 176-187; SEQ ID NO: 6), e2 (amino acids 256-273; SEQ ID NO: 10), and / or e3 (amino acids 366-377; SEQ ID NO: 14)). The epitope to which the antibody binds may consist of a single continuous sequence of three or more amino acids located within the extracellular domain of CB1 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). Alternatively, the epitope may consist of multiple discontinuous amino acids (or amino acid sequences) located within the extracellular domain of CB1. Furthermore, the epitope to which the CB antibody binds may include portions of CB1 that are not extracellular due to structural changes or exposure by binding. Table 1 shows the sequences of CB1 and its various domains.

[0124] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and antigen chemical modification can be used (Tomer (2000) Protein Science). 9:487-496). Another method that can be used to identify amino acids in polypeptides that antibodies interact with is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuterizing the protein of interest, followed by binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (maintaining the deuterium label). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to reveal the deuterium-labeled residues corresponding to the specific amino acids that the antibody interacts with. See, for example, Ehring (1999) Analytical Biochem. 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0125] The present invention further includes anti-CB1 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., M1, M2, M3, M4, M5 (and their humanized variants), M6, M7 (and their humanized variants), and M8). Similarly, the present invention also includes anti-CB1 antibodies that compete with any of the specific exemplary antibodies described herein (e.g., M1, M2, M3, M4, M5 (and their humanized variants), M6, M7 (and their humanized variants), and M8) for binding to CB1.

[0126] By using routine methods known in the art, it is possible to easily determine whether an antibody binds to the same epitope as a reference anti-CB1 antibody, or whether it competes for binding with it. For example, to determine whether a test antibody binds to the same epitope as the reference anti-CB1 antibody of the present invention, the reference antibody is bound to the CB1 protein (e.g., the soluble portion of the CB1 extracellular domain or CB1 expressed on the cell surface). The ability of the test antibody to bind to the CB1 molecule is then evaluated. If the test antibody can bind to CB1 after saturation binding with the reference anti-CB1 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-CB1 antibody. On the other hand, if the test antibody cannot bind to CB1 after saturation binding with the reference anti-CB1 antibody, then the test antibody may bind to the same epitope as the reference anti-CB1 antibody of the present invention. Subsequently, additional routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the failure to observe binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blockage (or another phenomenon) is the cause of the failure to observe binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. In certain embodiments of the present invention, if a competitive binding assay measures, for example, that one antibody in 1x, 5x, 10x, 20x, or 100x excess inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, then the two antibodies bind to the same (or overlapping) epitopes (see, for example, Junghans et al. (1990) Cancer Res. 50:1495-1502). Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then those two antibodies are thought to bind to the same epitope.If a subset of amino acid mutations reduces or eliminates the binding of one antibody, while the same subset reduces or eliminates the binding of the other antibody, then those two antibodies are considered to have a "duplicate epitope."

[0127] In some embodiments, the present invention provides an anti-CB1 antibody or its antigen-binding fragment that can compete with the antibodies or antigen-binding fragments disclosed herein for binding to CB1. Such antibodies can be identified using routine competitive binding assays. For example, to determine whether an antibody competes with a reference anti-CB1 antibody for binding, the binding method can be performed in two directions: In the first direction, the reference antibody is bound to the CB1 protein (e.g., the soluble portion of the CB1 extracellular domain or CB1 expressed on the cell surface) under saturated conditions, and then the binding of the test antibody to the CB1 molecule is evaluated. In the second direction, the test antibody is bound to the CB1 molecule under saturated conditions, and then the binding of the reference antibody to the CB1 molecule is evaluated. In both directions, if only the first (saturated) antibody can bind to the CB1 molecule, it is concluded that the test antibody and the reference antibody compete for binding to CB1. An antibody that competes with a reference antibody for binding does not necessarily have to bind to the same epitope as the reference antibody, but the binding of the reference antibody can be sterically blocked, for example, by overlapping or binding to an adjacent epitope. Competition can be measured by ELISA, flow cytometry, or surface plasmon resonance (SPR) assays. Furthermore, cross-competition and epitope binning assays can be performed using the Octet HTX System (Pall ForteBio LLC, Fremont, CA 94538).

[0128] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a heavy chain CDR1 sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 164, 176, 188, 200, 212, 224, 236, 248, 260, 272, 284, 296, 308, and 320. In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a heavy chain CDR2 sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153, 165, 177, 189, 201, 213, 225, 237, 249, 261, 273, 285, 297, 309, and 321. In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a heavy chain CDR3 sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 34, 46, 58, 70, 82, 94, 106, 118, 130, 142, 154, 166, 178, 190, 202, 214, 226, 238, 250, 262, 274, 286, 298, 310, and 322. In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a light chain CDR1 sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 158, 170, 182, 194, 206, 218, 230, 242, 254, 266, 278, 290, 302, 314, and 326.In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a light chain CDR2 sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 159, 171, 183, 195, 207, 219, 231, 243, 255, 267, 279, 291, 303, 315, and 327. In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a light chain CDR3 sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 160, 172, 184, 196, 208, 220, 232, 244, 256, 268, 280, 292, 304, 316, and 328.

[0129] The heavy chain CDR and light chain CDR of the anti-CB1 antibody provided herein may be independently selected and matched to form an antibody or antigen-binding fragment thereof containing any heavy chain CDR1, CDR2, and CDR3 from the antibody provided herein; and any light chain CDR1, CDR2, and CDR3. The heavy chain and light chain variable regions of the antibody provided herein may be independently selected and matched to form an antibody or antigen-binding fragment thereof containing any heavy chain and light chain from the antibody provided herein.

[0130] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment includes a variable weight (VH) chain sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 270, 282, 294, 306, and 318.

[0131] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a variable light (VL) chain sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

[0132] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 161, 173, 185, 197, 209, 221, 233, 245, 257, 269, 281, 283, 305, and 317.

[0133] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment contains a light chain sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 35, 47, 59, 71, 83, 95, 107, 119, 131, 143, 155, 167, 179, 191, 203, 215, 227, 239, 251, 263, 275, 287, 299, 311, and 323.

[0134] In certain embodiments, the anti-CB1 antibody or its antigen-binding fragment, CDR, VH, VL, heavy chain and / or light chain contains at least about 20%, at least about 15%, at least about 10%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% of a conserved variant amino acid.

[0135] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment is (20, 21, 22); (32, 33, 34); (44, 45, 46); (56, 57, 58); (68, 69, 70); (80, 81, 82); (92, 93, 94); (104, 105, 106); (116, 117, 118); (128, 129, 130); (140, 141, 142); (152, 153, 154); (164, 165, 166); (176, 17 The set includes a VH CDR having an amino acid sequence selected from the group consisting of 7, 178); (188, 189, 190); (200, 201, 202); (212, 213, 214); (224, 225, 226); (236, 237, 238); (248, 249, 250); (260, 261, 262); (272, 273, 274); (284, 285, 286); (296, 297, 298); (308, 309, 310); and (320, 321, 322).

[0136] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment is sequence number (26, 27, 28); (38, 39, 40); (50, 51, 52); (62, 63, 64); (74, 75, 76); (86, 87, 88); (98, 99, 100); (110, 111, 112); (122, 123, 124); (134, 135, 136); (146, 147, 148); (158, 159, 160); (170, 171, 172); (182, The set includes a VL CDR having an amino acid sequence selected from the group consisting of (183, 184); (194, 195, 196); (206, 207, 208); (218, 219, 220); (230, 231, 232); (242, 243, 244); (254, 255, 256); (266, 267, 268); (278, 279, 280); (290, 291, 292); (302, 303, 304); (314, 315, 316); and (326, 327, 328).

[0137] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment comprises a VH / VL set having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 / 24, 30 / 36, 42 / 48, 54 / 60, 66 / 72, 78 / 84, 90 / 96, 102 / 108, 114 / 120, 126 / 132, 138 / 144, 150 / 156, 162 / 168, 174 / 180, 186 / 192, 198 / 204, 210 / 216, 222 / 228, 234 / 240, 246 / 252, 258 / 264, 270 / 276, 282 / 288, 294 / 300, 306 / 312, and 318 / 324.

[0138] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment comprises a heavy chain set and a light chain set having amino acid sequences selected from the group consisting of SEQ ID NOs: 17 / 23, 29 / 35, 41 / 47, 53 / 59, 65 / 71, 77 / 83, 89 / 95, 101 / 107, 113 / 119, 125 / 131, 137 / 143, 149 / 155, 161 / 167, 173 / 179, 185 / 191, 197 / 203, 209 / 215, 221 / 227, 233 / 239, 245 / 251, 257 / 263, 269 / 275, 281 / 287, 283 / 289, 305 / 311, and 317 / 323.

[0139] In some embodiments, the anti-CB1 antibody or its antigen-binding fragment binds to CB1 and exhibits reduced effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, opsonization, and transcytosis. In one embodiment, the anti-CB1 antibody or its antigen-binding fragment binds to CB1 and includes modifications to one or more Fc regions that reduce, diminish, or eliminate one or more effector functions. For example, in one embodiment, the anti-CB1 antibody and its antigen-binding fragment disclosed herein bind to CB1 but exhibit reduced, diminished, or absent C1q binding and / or CDC and / or ADCC. The Fc modification may be an insertion, deletion, or substitution of amino acids, or it may be a chemical modification. For example, modifications to the Fc region may increase or decrease complement binding, increase or decrease ADCC or CDC, or modify glycosylation. Various Fc modifications are known in the art and are described, for example, in Labrijin et al. (2009) Nature Biotech. 27(8):767-771; Greenwood et al. (1993) Eur. J. Immunol. 23:1098-1104; Mueller et al. (1997) Mol. Immunol. 34:441-452; and Rother et al. (2007) Nature Biotechnol. 25:1256-1264. Any of the Fc modifications known in the art can be applied to the exemplary CB1 antibodies disclosed herein to alter the effector function. In one embodiment, the anti-CB1 antibody or its antigen-binding fragment has certain specific mutations, such as L234A / L235A ("LALA"), S228P, A330S, P331S, E233P / L234V / L235A, A327G / A330S / P331S, L234F / L235E / P331S, and N297Q.

[0140] The binding proteins provided herein can be generated by any of several techniques known in the art. For example, expression from host cells, in which case an expression vector(s) encoding the CB1 binding protein is transfected into the host cell using standard techniques. While the CB1 binding proteins provided herein can be expressed in either prokaryotic or eukaryotic host cells, mammalian host cells are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active binding proteins.

[0141] In an exemplary system for recombinant expression of CB1-binding protein, a recombinant expression vector encoding both the CB1 antibody heavy and light chains is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the CB1 antibody heavy chain and light chain sequences are operably ligated to CMV enhancer and promoter regulatory elements, respectively, to drive high levels of gene transcription. The recombinant expression vector also contains the DHFR gene, enabling selection of CHO cells transfected with this vector using methotrexate selection / amplification. Selected transformed host cells are cultured to enable expression of the CB1 antibody heavy and light chains, and intact CB1 antibody is recovered from the culture medium. Using standard molecular biology techniques, the recombinant expression vector is prepared, host cells are transfected, transformants are selected, host cells are cultured, and CB1 antibody is recovered from the culture medium.

[0142] biological equivalent The anti-CB1 antibodies and antibody fragments of this disclosure comprise proteins having amino acid sequences that are modified from those of the antibodies described above but retain the ability to bind to human CB1. Such variant antibodies and antibody fragments, compared to the parent sequence, contain one or more additions, deletions, or substitutions of amino acids, but exhibit biological activity that is essentially equivalent to the biological activity of the antibodies described above. Similarly, the anti-CB1 antibody coding DNA sequences of the present invention comprise sequences encoding anti-CB1 antibodies or antibody fragments that, compared to the sequences of the disclosure, contain one or more additions, deletions, or substitutions of nucleotides, but are essentially biologically equivalent to the anti-CB1 antibodies or antibody fragments of the present invention. Examples of such variant amino acid and DNA sequences are discussed above.

[0143] Two antigen-binding proteins or antibodies are considered bioequivalents or substitutes if, for example, when administered in the same molar dose under similar experimental conditions, either as a single or repeated dose, their absorption rates and extents do not show significant differences. Some antibodies may be considered equivalents or substitutes if their absorption rates are equivalent but not equivalent, and if such differences in absorption rates are intentional, reflected in labeling, and are not essential, for example, to achieving effective drug concentrations in the body with long-term use, and are not medically important to the specific drug product being studied, they may still be considered bioequivalent.

[0144] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency. In one embodiment, two antigen-binding proteins are bioequivalent if a patient can switch between a reference product and a biological product once or more times without predicting an increased risk of adverse effects, including a clinically significant difference in immunogenicity or a decrease in efficacy, compared to continuous treatment without such switching. In one embodiment, two antigen-binding proteins are bioequivalent if both act by one or more common mechanisms of action for one or more conditions of use, to the extent that such mechanisms are known.

[0145] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals measuring antibody concentrations or one or more of its metabolites as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with human in vivo bioavailability data and can be reasonably predicted; (c) in vivo studies in humans or other mammals measuring the appropriate acute pharmacological action of the antibody (or its target) as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0146] Bioequivalent variants of anti-CB1 antibodies can be constructed, for example, by performing various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not biologically essential. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent unnecessary or incorrect intramolecular disulfide crosslinking during regeneration. In other situations, bioequivalent antibodies may include anti-CB1 antibody variants that involve amino acid changes that alter the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

[0147] Species selectivity and species cross-reactivity The present invention also includes an anti-CB1 antibody that binds to human CB1 and to CB1 from one or more non-human species. For example, the anti-CB1 antibody of the present invention may bind to human CB1 and to one or more of the following species: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, crab-eating macaque, marmoset, rhesus macaque, or chimpanzee CB1. In certain embodiments of the present invention, the anti-CB1 antibody binds to human CB1 but not to CB1 from other species.

[0148] Immunoconjugate The present invention encompasses anti-CB1 antibodies conjugated to therapeutic moieties such as cytotoxins, chemotherapeutic agents, immunosuppressants, or radioisotopes ("immunoconjugates"). Cytotoxic agents include any agent harmful to cells. Examples of suitable cytotoxic and chemotherapeutic agents for forming immunoconjugates are known in the art and are described herein.

[0149] Multiple specificity binding proteins The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different epitopes of one target polypeptide, or they may contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al. (1991) J. Immunol. 147:60-69; Kufer et al. (2004) Trends Biotechnol. 22:238-244. The anti-CB1 antibodies of the present invention can be ligated to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment of an antibody can be operably ligated (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity. For example, the present invention includes a bispecific antibody in which one arm of the immunoglobulin is specific to human CB1 or a fragment thereof, and the other arm of the immunoglobulin is specific to a second therapeutic target or is conjugated to the therapeutic portion.

[0150] Use of binding proteins in various diseases The antibodies and binding proteins of the present invention are useful for treating, preventing, and / or relieving any disease or disorder that can be treated by blocking the interaction between CB1 and a CB1 ligand (e.g., cannabinoid), or otherwise by inhibiting CB1 activity and / or signaling, and / or by promoting receptor internalization and / or reducing the number of cell surface receptors, which are associated with or mediated by CB1 expression or activity. The term "disorder in which CB1 activity is harmful" means a disorder or disorder in which the presence or activity (e.g., abnormal or hyperactivity) of CB1 in the affected subject is the cause of the pathophysiology of the disorder or disease, or a factor that contributes to the exacerbation of the disorder or disease. Thus, a disorder in which CB1 activity is harmful is a disorder in which a decrease in CB1 activity is expected to alleviate the symptoms and / or progression of the disorder.

[0151] The binding protein molecules provided herein are useful, for example, as therapeutic molecules for treating various diseases or conditions in which the CB1 protein is detrimental. For example, the binding molecules provided herein include any disease or condition characterized by overexpression, upregulation, or increased activity or signaling of CB1, or any resulting dysfunction of health homeostatic regulatory mechanisms. Such diseases and conditions include obesity, symptomatic obesity including Prader-Willi syndrome (PWS), Alström syndrome, Valde-Beedl syndrome (BBS), Albride hereditary osteodystrophy (AHO), and SIM1 deletion syndrome; diabetes and related complications; dyslipidemia; liver diseases such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, and primary biliary cirrhosis; fibrosis, such as renal fibrosis; chronic kidney disease; kidney disease; metabolic diseases, osteoporosis, atherosclerosis, inflammatory diseases, cardiovascular diseases, cancer, pain, systemic sclerosis, multiple sclerosis spasticity, glaucoma, and nicotine addiction.

[0152] Pharmaceutical composition The present invention provides pharmaceutical compositions comprising an anti-CB1 binding protein, such as an antibody or an antigen-binding fragment thereof. The pharmaceutical compositions of the present invention are formulated with appropriate additives, carriers, prophylactic agents, therapeutic agents, and other agents that improve the stability, delivery, tolerability, and efficacy of the anti-CB1 binding protein. Numerous suitable formulations can be found in prescription collections known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. (1998) J.Pharm.Sci.Technol. 52:238-311.

[0153] Pharmaceutical compositions comprising CB1-binding proteins provided herein, but not limited to those provided herein, are intended for use in the diagnosis, detection, or monitoring of disorders, in the prevention, treatment, management, or remission of one or more symptoms of a disorder or its components, and / or in research.

[0154] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). The composition can be administered by any conventional route, for example, by infusion or large-volume injection, by absorption through the epithelial or mucocutaneous layer (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other biologically active agents. Administration may be systemic or topical.

[0155] Methods of administering prophylactic or therapeutic agents provided herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, intratumoral administration, mucosal administration (e.g., intranasal and oral administration), and pulmonary administration (e.g., administration of aerosolized compounds using inhalers or nebulizers). Formulations of pharmaceutical compositions for specific routes of administration, as well as the materials and techniques required for various methods of administration, are available and known to those skilled in the art.

[0156] The administration plan can be adjusted to obtain the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions into unit dosage forms. The term "unit dosage form" refers to a physically distinct unit adapted as a unit dose for the mammalian subject to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms provided herein are defined by and directly depend on (a) the inherent characteristics of the active compound and the specific therapeutic or prophylactic action to be achieved, and (b) the limitations inherent in the art when formulating such active compounds to treat susceptibility in an individual.

[0157] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable for the delivery of the pharmaceutical compositions of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, in disposable pen-type delivery devices, the pharmaceutical composition is pre-filled and held in a reservoir within the device. Once the pharmaceutical composition is emptied from the reservoir, the entire device is discarded.

[0158] Numerous reusable pen-type and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, CH), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN This includes STARLET® and OPTICLIK® (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices applied to the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR® pen (Sanofi-Aventis), FLEXPEN® (Novo Nordisk), and KWIKPEN® (Eli Lilly), SURECLICK® Autoinjector (Amgen, Thousand Oaks, CA), PENLET® (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA® Pen (AbbVie, Inc., Abbott Park, IL).

[0159] In certain circumstances, pharmaceutical compositions can be delivered by controlled-release systems. In one embodiment, a pump can be used (Sefton (1987) CRC Crit.Ref.Biomed.Eng.14:201-240). In another embodiment, a polymer material can be used (Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, FL). In yet another embodiment, the controlled-release system can be positioned near the target of the composition so that only a fraction of the systemic dose is required (Goodson (1984) in Medical Applications of Controlled Release, supra, 2:115-138). Other controlled-release systems are discussed in the overview in Langer (1990) Science 249:1527-1533.

[0160] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. These injectable formulations can be prepared by known methods. For example, an injectable formulation can be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injectable formulations. Examples of aqueous media for injectable formulations include isotonic solutions containing physiological saline, glucose, and other adjuvants, which may be used in combination with appropriate solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable formulation thus prepared is preferably filled into a suitable ampoule.

[0161] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared into a unit dose dosage form adapted to the dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per unit dose; particularly in the form of injection, it is preferable that the antibody is present in an amount of about 5 to about 100 mg, and in other dosage forms, about 10 to about 250 mg.

[0162] The dose of antibody administered to a patient may vary depending on the patient's age and size, target disease, disease stage, sex, presence of medical complications, other drug therapies, condition, and route of administration. The preferred dose is typically calculated by body weight or body surface area. When using the antibody of the present invention in adult patients to treat conditions or diseases associated with CB1 activity, it may be advantageous to administer the antibody intravenously in a single dose of approximately 0.01 to approximately 100 mg / body weight. The frequency and duration of treatment can be adjusted according to the severity of the condition. Effective dosages and schedules for administering anti-CB1 antibodies can be determined empirically; for example, the patient's progression can be monitored by periodic assessments, and the dose can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al. (1991) Pharmaceut. Res. 8:1351-1359). It should be further understood that for any particular subject, a specific administration plan may be adjusted over time in accordance with the individual needs and the professional judgment of the person administering or managing the administration of the composition, and that the dosage ranges shown herein are illustrative and not intended to limit the scope and implementation of the composition described in the claims.

[0163] Combination therapy The binding proteins provided herein can also be administered together with one or more additional therapeutic agents useful in treating a variety of diseases, the additional agents being selected by those skilled in the art for their intended purpose. For example, the additional agents could be therapeutic agents approved in the art as useful for treating the diseases or conditions treated by the CB1 binding proteins provided herein. The combination may include more than one additional agent.

[0164] Non-limiting examples of such additional therapeutically active ingredients include other CB1 antagonists (e.g., second anti-CB1 antibodies or small molecule inhibitors of CB1 (e.g., rimonabant, taranaban, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenaban, surinabant, tetrahydrocannabivarin, and virodamine, and AM6545)) and antagonists of other CB1 family members.

[0165] The present invention also includes therapeutic combinations comprising any of the anti-CB1 antibodies described herein and an additional inhibitor, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptide body, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other manipulated molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimum recognition units). The anti-CB1 antibodies of the present invention may also be administered in combination with and / or co-formulated with additional therapeutic agents. Additional therapeutic active ingredients(s) may be administered immediately before, concurrently with, or immediately after the administration of the anti-CB1 antibodies of the present invention; (for the purposes of this disclosure, such a dosing regimen is deemed to be the administration of the anti-CB1 antibody "in combination" with the additional therapeutic active ingredients). The present invention includes pharmaceutical compositions in which the anti-CB1 antibodies of the present invention are co-formulated with one or more additional therapeutic active ingredients(s) as described elsewhere herein.

[0166] The present invention also includes compositions and methods comprising a combination of an "antagonist antibody" and an "inverse agonist antibody." An "antagonist anti-CB1 antibody" means an anti-CB1 antibody that inhibits, reduces, or blocks the signaling activity of a ligand (e.g., cannabinoid) of CB1. Non-limiting examples of antagonist antibodies of the present invention are M1, M2, M3, M4, M5, M6, M7, M8, M7-H1, M7-H2, M7-H3, M7-H4, M7-H5, M7-H6, M7-H7, M7-H8, M7-H9, M7-H10, M7-H11, M7-H12, M7-H13, M7-H14, M7-H15, M7-H16, M5-H1, and M5-H2. An "inverse agonist anti-CB1 antibody" refers to an anti-CB1 antibody that induces a pharmacological response opposite to that of an agonist. While an agonist increases the receptor's activity above its basal level, an inverse agonist reduces its activity below the basal level. A non-limiting example of an inverse agonist antibody in the present invention is M7. The inventors are considering combining an antagonist antibody and an inverse agonist antibody to synergistically or otherwise improve efficacy. Accordingly, the present invention includes a pharmaceutical composition comprising at least one antagonist antibody and at least one inverse agonist antibody. The present invention also includes a therapeutic method comprising administering a combination of an antagonist antibody and an inverse agonist antibody (as separate doses or as a concurrent formulation) to a subject.

[0167] Combination therapy drugs include, but are not limited to, antineoplastic agents, radiotherapy, chemotherapeutic agents such as DNA alkylating agents, cisplatin, carboplatin, antitubulins, paclitaxel, docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines, adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinase inhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g., celecoxib), kinase inhibitors, and siRNAs.

[0168] diagnosis This disclosure provides diagnostic applications, including but not limited to diagnostic assay methods, diagnostic kits comprising one or more CB1-binding proteins, and adaptations of methods and kits for use in automated and / or semi-automated systems. The methods, kits, and adaptations provided can be used for the detection, monitoring, and / or treatment of diseases or disorders in individuals.

[0169] The anti-CB1 antibody of the present invention can also be used, for example, to detect and / or measure CB1 or CB1-expressing cells in a sample for diagnostic purposes. For example, the anti-CB1 antibody, or a fragment thereof, can be used to diagnose a condition or disease characterized by abnormal expression of CB1 (e.g., overexpression, underexpression, absence of expression, etc.). An exemplary diagnostic assay for CB1 may, for example, involve contacting a sample obtained from a patient with the anti-CB1 antibody of the present invention, wherein the anti-CB1 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-CB1 antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, chemiluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. An example of a luminescent substance is luminol, and an example of a suitable radioactive substance is (for example, 3 H, 14 C, 32 P, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I,177 Lu, 166 Ho, and 153 It contains Sm.

[0170] The immunoassays provided in this disclosure may include, among others, sandwich immunoassays, radioimmunoassays (RIAs), enzyme immunoassays (EIAs), enzyme-linked immunosorbent assays (ELISAs), competitive-inhibitory immunoassays, fluorescence-polarized immunoassays (FPIAs), enzyme amplification immunoassay techniques (EMITs), bioluminescent resonance energy transfer (BRETs), fluorescence-activated cell sorting (FACSs), and homogeneous chemiluminescence assays.

[0171] A chemiluminescent microparticle immunoassay can be used, which may be performed using an ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, IL).

[0172] This disclosure provides methods using mass spectrometry, which include, but are not limited to, MALDI (matrix-assisted laser desorption / ionization) or SELDI (surface-enhanced laser desorption / ionization).

[0173] Methods for collecting, handling, processing, and analyzing biological test samples using immunoassays and mass spectrometry are well known to those skilled in the art.

[0174] kit Kits are also provided for assaying a test sample for the presence, amount, or concentration of an analyte or fragment thereof in the test sample. The kit comprises at least one component for assaying a test sample for an analyte or fragment thereof, and instructions for assaying a test sample for an analyte or fragment thereof. The at least one component for assaying a test sample for an analyte or fragment thereof may optionally include a composition comprising a binding protein as disclosed herein, immobilized on a solid phase, and / or an anti-analyte binding protein (or a fragment, variant, or fragment of a variant thereof).

[0175] Optionally, the kit may include a calibration material or control which may contain an isolated or purified analyte. The kit may include at least one component for assaying a test sample for the analyte by immunoassay and / or mass spectrometry. Optionally, the kit components, including the analyte, binding protein, and / or anti-analyte binding protein, or fragments thereof, may be labeled with any detectable label known in the art. The substances and methods of production available in the course of this disclosure will be known to those skilled in the art.

[0176] Methods for determining the presence, quantity, or concentration of an analyte in a test sample by the above-described kit (or its components), and by assays such as the immunoassays described herein, can be adapted for use in various automated and semi-automated systems (including those with a solid phase containing microparticles), as described, for example, in U.S. Patent Nos. 5,089,424 and 5,006,309, and commercially available from Abbott Laboratories (Abbott Park, IL) as ARCHITECT®. Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, for example, U.S. Patent No. 5,294,404), PRISM®, EIA (Beads), and Quantum™ II, as well as other platforms. In addition, the above-described assays, kits, and kit components can be used in other forms, for example, in electrochemical or other portable or in-situ immediate assay systems. This disclosure is applicable, for example, to commercially available Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay systems for performing sandwich immunoassays. Immunosensors and methods for manufacturing and operating them as disposable devices are described, for example, in U.S. Patents No. 5,063,081, No. 7,419,821, No. 7,682,833, No. 7,723,099, and No. 9,035,027; and U.S. Patent Publications No. 20040018577 and No. 20060160164.

[0177] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein are obvious and can be done using suitable equivalents without departing from the scope of the embodiments disclosed herein. While certain embodiments have been described in detail, the implementation of the present invention will be more fully understood by the following embodiments, which are shown herein for illustrative purposes only and should not be construed as limiting the invention in any way. [Examples]

[0178] Example 1: Generation and selection of anti-CB1 antibodies for functional evaluation CD2F1 mice were immunized with human CB-1 antigen in or out of the presence of the CB-1 antagonist JD5037 (Cayman Chemical, Ann Arbor, MI; Cat. No. 1205) to potentially stabilize the protein and ensure it was structurally present during immunization. Mice were immunized at both heel joints with approximately 5 μg / 30 μl / heel joint of antigen containing Titer Max Gold adjuvant (St. Loui, MO; Sigma Aldrich; Cat. No. T2684). Subsequently, mice were immunized with CpG (InvivoGen, San Diego, CA; Cat. No. ODN1826) and Alhydrogel (InvivoGen, San Diego, CA; Cat. No. vac-alu-250) twice a week for approximately 30 days. Serum was collected on days 13 and 26, and antibody titers and their increase over time were determined. On day 30, mice were euthanized, and popliteal and inguinal lymph nodes were collected for fusion. These were washed in medium B (a 1:1 mixture of RPMI1640 (Thermo Fisher Scientific-Gibco, San Diego, CA; Cat. No. 11879020) and IMDM (Lonza, Anaheim, CA; Cat. No. 12-722F), which had no added nutrients, to prepare a single-cell suspension. P3Ag8.563 myeloma cells (ATCC, Manasas, VA; Cat. No. PTA-9393) were harvested from the culture and washed in medium B. Lymphocytes and myeloma cells were mixed in a 1:1 ratio and fused using the BTX Harvard ECM2001 electrocellular fusion apparatus (BTX Harvard Apparatus, Holliston, MA; Cat. No. 45-0012). The fused cells were resuspended in recovery medium C (Stem Cell Technologies, Seattle, WA; Cat. No. 03803) and heated to a temperature of 75 cm. 2The cells were allowed to recover overnight in a flask at 37°C. The following day, the fused cells were harvested and resuspended in hybridoma-selective methylcellulose medium D (Stem Cell Technologies, Seattle, WA; Cat. No. 03804) containing anti-mouse IgG FITC Clone Detect (Molecular Devices, San Jose, CA; Cat No. K8220). The cells were mixed and 1 × 10⁶ cells were added per 10 mL of medium D. 6 The cells were plated. The plated cells were incubated at 37°C for 7 days. Hybridoma colonies were isolated using Clone Pix2 (Molecular Devices, San Jose, CA) based on colony size and their ability to show a strong FITC halo indicating IgG production, and transferred to 96-well tissue culture plates containing Hybridoma Growth Medium E (Stem Cell Technologies, Seattle, WA; Cat. No. 03805). Where macroscopic colonies were observed, the supernatant was screened for cell binding using CHO parental cells and CHO-huCB-1 overexpressing cells. For this primary screening, hybridoma parental CHO cells were labeled with carboxyfluorescein succinimimidyl ester (CFSE) (Invitrogen, Anaheim, CA; Cat. No. 34554) and mixed with non-CFSE-labeled CHO-huCB1 overexpressing cells to enable efficient and simultaneous screening in both cell lines and to identify huCB-1 specific conjugates. Clones that specifically bound to CHO-huCB-1 overexpressing cells but not to parental CHO cells were selected and proceeded to confirmatory screening. A total of 97 clones were selected to proceed to small-scale purification. Table 2 shows an overview of related fusions and primary screening.

[0179] [Table 2]

[0180] Example 2: Purification of mouse anti-huCB-1 specific binding clones Based on the initial screening for confirmation, the selected hybridoma clones were further incubated in 50 mL of low Ig medium (containing 5 mL of 100 mM sodium pyruvate solution (Invitrogen, Grand Island, NY; Cat. No. 11360070), 5 mL of 100 mM non-essential amino acids (Invitrogen, Grand Island, NY; Cat. No. 11140050), and 5 mL of 100 mM glutamine (Invitrogen, Grand Island, NY; Cat. No. 35050061), in a 1:1 IMDM (Lonza, Anaheim, CA; Cat. No. 12-722F):5% low Ig serum (Invitrogen, Grand Island, NY; Cat. No. 1625007) medium (Gibco, Anheim, CA; Cat. No. 21127022) at a temperature of 75 cm. 2 The protein was grown in a flask for 3-4 weeks. The supernatant was collected and purified using a standard protein A purification method.

[0181] Example 3: Functional characterization of mouse anti-CB1 antibody in cAMP assay Isolated mouse anti-huCB-1 antibodies were evaluated for their antagonist activity in a cAMP assay. The cAMP assay was performed using the cAMP Hunter® CHO-K1 CNR1 Gi cell line (DiscoverX / Eurofins, Fremont, CA; Cat. No. 95-0071C2), which naturally overexpresses Gi-binding wild-type G protein-coupled receptors (GPCRs) and is designed to detect elevated intracellular cAMP levels in response to receptor agonist stimulation. cAMP Hunter® CHO-K1 CNR1 Gi cells were treated with CB1 antibodies, isotype controls, or the small molecule CB1 antagonist JD5037, followed by agonist attack with 30 nM CP-55,940 in the presence of forskolin (indicated as "Plus CP"). Antagonists were also tested without the addition of CP-55,940 to demonstrate their own agonist activity. Forskolin activates the enzyme adenylyl cyclase, increasing intracellular levels of cAMP. At the Gi receptor, agonist binding inhibits forskolin-induced intracellular cAMP accumulation. Therefore, to measure Gi-coupled receptor activity, the agonist compound CP-55,940 was added in the presence of forskolin. Thus, activation of the Gi-coupled receptor inhibits forskolin-induced cAMP production, and consequently, the dose-response curve in the presence of the agonist and forskolin should have a negative slope. Briefly, cells were subjected to 1.5 × 10⁶ cells in Cell Plating 2 Medium (DiscoverX / Eurofins; Fremont, CA; Cat. No. 93-0563R2A). 4Cells were seeded at a concentration of one cell per well into a 96-well plate (Costar, Fisher Scientific, San Diego, CA; Cat. No. 3909) and incubated overnight at 37°C with 5% CO2. The following day, the culture medium was replaced with 30 μl of Cell Assay Buffer (CAB; 1x HBSS / 10nM HEPES (ThermoFisher, Anaheim, CA; Cat.Nos. 14025134 and 15630080, respectively)) and treated with the test antibody or isotype control (7.5 μl of 6x concentrated working dilution). The plate was incubated at 37°C and 5% CO2 for 30 minutes. 7.5 μl of agonist attack (0.18 μM CP55,940 in CAB containing 90 μM forskolin) was added to each well, and the plate was incubated for another 30 minutes at 37°C and 5% CO2. HitHunter® cAMP Assay Detection Kit for Plates were treated for cAMP reading using Biologics (DiscoverX / Eurofins; Fremont, CA; Cat. No. 90-0075LM25) according to the manufacturer's instructions. Initial evaluation of isolated clones was performed at a single concentration of 30 μg / mL.

[0182] Of the 112 clones tested, only 8 exhibited antagonist activity. Two clones showed some degree of agonism in the absence of an agonist (M3 and, to a lesser extent, M1). These 8 antibodies, M1, M2, M3, M4, M5, M6, M7, and M8, were further evaluated by titrating the concentration of Ab to obtain dose-response curves and actual EC50 values ​​(Table 3 and Figure 1).

[0183] Example 4: Functional characterization of mouse anti-CB1 antibody in pERK assay Eight antibodies that were functional antagonists in the cAMP assay were further evaluated using a pERK phosphorylation assay performed with the cAMP Hunter® CHO-K1 CNR1 Gi Cell Line (DiscoverX / Eurofins, Fremont, CA; Cat. No. 95-0071C2). Briefly, cells were placed in a 96-well plate, 2 × 10⁶ cells. 4 Cells were seeded in Kit-107 Assay Complete Cell Culture Medium (DiscoverX / Eurofins, Fremont, CA; Cat. No. 92-3107G) containing 800 μg / mL G418 per well and incubated at 37°C and 5% CO2. The following day, the culture medium was replaced with 100 μl / well of serum-free F-12K starvation medium (Invitrogen, Grand Island, NY; Cat. No. 11765054). The plate was incubated for another day at 37°C and 5% CO2. On the day of treatment, the F-12K medium was replaced with 30 μl / well of fresh F-12K medium. The test antibody or isotype control (7.5 μl of 6-fold concentrated working dilution) was then added to the wells, and the plate was incubated at 37°C and 5% CO2 for 10 minutes. 7.5 μl of agonist (6x working solution containing 0.18 μM CP55,940 in CAB along with 90 μM forskolin) was added to each well, and the plates were incubated for a further 10 minutes at 37°C in 5% CO2. The plates were treated for p-ERK / total ERK using the Meso Scale Discovery (MSD) kit (Meso Scale Discovery, Rockville, Maryland; Cat. No. K15107D) according to the manufacturer's instructions (Table 3 and Figure 2).

[0184] [Table 3]

[0185] Example 5: EC50 binding of mouse anti-CB-1 antibody to CB-1 CHO overexpressing cells. Antibody binding was tested using a fluorescence-activated cell sorting (FACS)-based assay to assess the ability of a mouse anti-CB1 antibody to bind to parental CHO cells, human CB-1 overexpressing CHO cells, and mouse CB-1 overexpressing CHO cells. Binding curves and EC50 values ​​were obtained. The three cell lines were harvested, washed, and placed in a v-bottom 96-well polycarbonate FACS plate (Corning, Corning, NY, Cat. No. 3357) at a rate of 1 × 10⁶ cells per well. 5 Cells were partitioned into 50 μl of FACS buffer (1x PBS / 2 mM EDTA and 1% FBS (ThermoFisher Scientific, Anaheim, CA; Cat. No. 10438-026)). Antibody series dilutions were prepared starting from 200 nM at 2x concentration and then 3x series dilutions. The titrated antibodies were added to plates containing three different cell lines (parental, human, and mouse CB-1 CHO cells) and incubated at 4°C for 1 hour. The plates were washed three times with FACS buffer. Cells were resuspended in 50 μl of 1 / 5,000 dilution of goat anti-mouse IgG-HRP (Jackson Immuno Research, West Grove, PA; Cat. No. 115-035-003), incubated at 4°C for 30 minutes, washed three times with FACS buffer, and the data were collected. (BD FACS Canto (BD Biosciences, San)) The samples were collected at Jose (CA; Cat. No. 338962) and analyzed using FlowJo (FlowJo LLC, Ashland, OR) (Table 4). None of the eight functional antibodies tested showed mouse cross-reactivity (Figure 3).

[0186] [Table 4]

[0187] Example 6: EC50 analysis and evaluation of conformation of functional anti-CB-1 antibody The objective of this experiment was to determine whether anti-CB1 antagonist antibodies exhibited different binding aptitudes for CB1 in determining whether they were neutral, antagonist, or agonist. Four different cell line preparations were used: CHO-huCB1 (in-house generated), CHO-huCB1 pre-incubated with inverse agonist JD5037 (Cayman Chemicals, Ann Arbor, MI; Cat. No. 1392116-14-1), CHO-huCB1 pre-incubated with agonist CP-55,940 (TOCRIS, Minneapolis, MN; Cat. No. 0949), and pedigree CHO-S cells (ThermoFisher Scientific, VA; Cat. No. R80007). 2 × 10⁻⁶ 7 The parental CHO-S and CHO-hu CB1 cells were left in FACS buffer. In addition, 2 × 10⁶ cells coated with inverse agonist JD5037 or agonist CP-55,940 were added. 7 The CHO-huCB1 cells were incubated at 4°C for 1 hour. After incubation, these two coated cell lines were washed twice with FACS buffer and 2 × 10⁶ cells were used. 7The cells were then resuspended in FACS buffers containing either an inverse agonist or an agonist molecule. All four cell lines were plated in v-bottom FACS plates (Corning, Corning, NY; Cat. No. 3357), and pre-titrated anti-CB1 test antibodies were added to the cells. Binding was evaluated using BD FACS Canto (BD Biosciences, San Jose, CA; Cat. No. 338962). As shown in Figure 4, the antibodies did not bind to CHO parent cells (blue curve), and the eight functional antibodies (M1, M2, M3, M4, M5, M6, M7, and M8) did not show preferential binding in the presence of agonists or antagonists, as indicated by the binding observed under all conditions (red, purple, and green curves). Only two test antibodies that were non-functional in the cAMP or pERK assays showed preferential binding in the presence of an antagonist and in the absence of an agonist, respectively. This suggests that functional anti-CB1 antibodies cannot associate with the binding conformations that occur in the presence of known receptor agonists or antagonists.

[0188] Example 7: Sequence identification and analysis of mouse anti-CB1 antibody Hybridomas of the eight mouse anti-huCB-1 antibodies listed above were collected as cell pellets, and the supernatant was used to determine the isotype of each hybridoma using a standard mouse isotyping ELISA kit (Pierce / ThermoFisher Scientific, San Diego, CA; Cat. No. 37503). Four of the antibodies (M1, M3, M4, and M6) were IgG2a,K, and four of the antibodies (M2, M5, M7, and M8) were IgG2b,K. The pellets were treated with RNA and cDNA, and the cDNA was treated for sequencing using the SMARTER RACE Amplification kit (Clontech, Mountain View, CA; Cat. No. 634859). Using each antibody isotype, SeqAmp polymerase (CloneTech, Mountain View, CA; Cat. No. 638504) was designed as reverse primers and forward primers for the constant regions of the heavy and light chain kappa constant regions. MOPC21 PNA primers (synthesized based on sequences) were included to prevent the amplification of anomalous light chains, which often appear during the sequencing process and can interfere with the identification of the actual light chain variable region sequence. A total of eight unique sequences and seven unique families were identified. The sequences of the eight clones are shown in Table 5. The consensus sequences of the heavy and light chains of the hybridoma antibodies are shown in Figures 5A and 5B, respectively.

[0189] [Table 5-1]

[0190] [Table 5-2]

[0191] [Table 5-3]

[0192] [Table 5-4]

[0193] [Table 5-5]

[0194] [Table 5-6]

[0195] [Table 5-7]

[0196] [Table 5-8]

[0197] Example 8: Selection of mouse anti-CB1 antibody for humanization Based on functional data, mouse anti-huCB-1 clones M7 and M5 were selected for humanization using a predictive human engineering tool (US Patent No. 5,766,886) derived from the PHEnon® software package (Xoma, Berkley, CA). The VH and VL sequences from each clone were presented as queries, and output sequences were generated based on the closest human germline match from the Kabat database. A list of mutations in the framework region was generated to develop the VH and VL sequences into human framework matches. The mutation risk of individual residues was assessed through a set of criteria (US Patent No. 5,766,886). Cumulatively, mutations were grouped to form "low-risk" and "intermediate-risk" clone pools. Output sequences and transmutations were confirmed in silico by homology modeling. The final humanized VH and VL antibody sequences were cloned into a human IgG1 vector backbone (TCAL DGV vector), expressed in CHO cells, and purified by protein A affinity chromatography according to standard procedures. The sequences of the humanized clones are shown in Table 6. The consensus sequences of the heavy and light chains of the humanized M7 antibody and humanized M5 antibody are shown in Figures 6A and 6B, respectively.

[0198] [Table 6-1]

[0199] [Table 6-2]

[0200] [Table 6-3]

[0201] [Table 6-4]

[0202] Table 6-5

[0203] Table 6-6

[0204] Table 6-7

[0205] Table 6-8

[0206] Table 6-9

[0207] Table 6-10

[0208] Table 6-11

[0209] Table 6-12

[0210] Table 6-13

[0211] Table 6-14

[0212] [Table 6-15]

[0213] [Table 6-16]

[0214] Example 9: Re-evaluation of humanized anti-CB1 antibodies in cell binding and functional assays. Humanized anti-CB1 variants were re-evaluated for their ability to bind to CHO-huCB-1 cells and compared with mouse parental clones M5 and M7 (Figures 7A and 7B) to confirm that binding was retained. The assay conditions used were the same as those described in Example 5. After binding analysis, the test variants were also evaluated for antagonist function in cAMP and pERK assays according to Examples 3 and 4 (Figures 8A, 8B, 9A, and 9B) to confirm that binding and activity were retained after humanization. All clones retained their binding and antagonist activity. Figure 10 shows inverse agonism represented by humanized anti-CB1 Ab M7-H5 and IgG2b form.

[0215] equivalent This disclosure can be implemented in other specific forms without departing from its intent or essential features. Therefore, the embodiments described above should be considered illustrative and not limiting in any way to this disclosure. Accordingly, the scope of this disclosure is indicated not by the foregoing but by the appended claims, and all changes that fall within the meaning and scope of equivalence of the claims are intended to be incorporated into this disclosure.

Claims

1. An isolated antibody or its antigen-binding fragment that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the binding protein comprises six complementarity-determining regions (CDRs): CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, in which case, CDR-H1 has the amino acid sequence G-Y-T-F-T-D-Y-W (residues 26-33 of SEQ ID NO: 329) or a modified form of the amino acid sequence by substitution of at least one amino acid residue, wherein the substitution of G at position 1 is S; the substitution of T at position 3 is E; the substitution of T at position 5 is S or N; the substitution of D at position 6 is R or Y; the substitution of Y at position 7 is H; and the substitution of W at position 8 is A or N; CDR-H2 has the amino acid sequence I-Y-P-Y-D-G-D-T (residues 51-58 of SEQ ID NO: 329) or a modified form of the amino acid sequence by substitution of at least one amino acid residue, wherein the substitution of I at position 1 is F; the substitution of Y at position 2 is D, S, or T; the substitution of P at position 3 is T; the substitution of Y at position 4 is G, D, or S; the substitution of D at position 5 is Y or S; the substitution of G at position 6 is S; the substitution of D at position 7 is E, G, or R; and the substitution of T at position 8 is A, S, or I; CDR-H3 has the amino acid sequence A-R-G-X １ -X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -X ８ -X ９ -W-X １０ -X １１ -Y (residues 98-113 of SEQ ID NO: 329) or a modified form of the said amino acid sequence by substitution of at least one amino acid residue, wherein the substitution of A at position 1 is S; the substitution of G at position 3 is S; X １ at position 4 is Q, Y, K, R, or G, or does not exist; X ２ at position 5 is E, Y, L or G, or does not exist; X ３ at position 6 is Y or P, or does not exist; X ４ at position 7 is Y, R, or E, or does not exist; X ５ at position 8 is G, or does not exist; X ６ at position 9 is T, or does not exist; X ７ at position 10 is N or D, or does not exist; X ８ at position 11 is Y, N, A, or G, or does not exist; X ９ at position 12 is N, Y, S, A, or R, or does not exist; the substitution of W at position 13 is Y, A, or P; X １０ at position 14 is L, M, F, or G, or does not exist; X １１ at position 15 is P, D, A, or T, or does not exist; the substitution of Y at position 16 is V; CDR-L1 has the amino acid sequence Q-X １ -I-S-S-X ２ - The amino acid sequence has been modified by substitution of Y (residues 27-33 of SEQ ID NO: 330) or at least one amino acid residue, in which case the substitution of Q at position 1 is S or E; and X at position 2. １ is E, S, T, N, G, or R; the substitution of I at position 3 is V; the substitution of S at position 4 is A, R, or G; the substitution of S at position 5 is G, N, or T; and X at position 6 ２ is either S, N, peptide F-R-Y-S, or absent; and the substitution of Y at position 7 is F, D, or N; CDR-L2 has the amino acid sequence: X １ -T-S (residues 51-53 of SEQ ID NO: 330) or the modified amino acid sequence by substitution of at least one amino acid residue, in which case X at position 1 １ is A, Y, G, R, D, or S; the substitution of T at position 2 is A; the substitution of S at position 3 is R; and CDR-L3 has the amino acid sequence: Q-Q-Y-X １ -S-X ２ The amino acid sequence has been modified by the substitution of -P-Y-T (residues 91-99 of SEQ ID NO: 330) or at least one amino acid residue, in which case the substitution of Q at position 1 is L or H; the substitution of Q at position 2 is H; the substitution of Y at position 3 is S or G; and the substitution of X at position 4. １ is S, W, H, Y, N, or I; the substitutions of S at position 5 are E, R, G, T, or N; and X at position 6. ２ The isolated antibody or antigen-binding fragment thereof is Y, I, S, T, L, or W; and the substitution of Y at position 8 is P, L, F, or absent; and in such case, the said substitution, addition, or deletion of at least one amino acid residue does not inhibit the ability of the antibody or antigen-binding fragment thereof to bind to human CB1.

2. An isolated antibody or antigen-binding fragment thereof that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the antibody comprises a variable weight (VH) domain sequence CDR and a variable light (VL) domain sequence CDR, wherein the VH domain sequence is one of the following: SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 24 The isolated antibody or its antigen-binding fragment, selected from the group consisting of 6, 258, 270, 282, 294, 306, and 318, and / or the VL domain is selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

3. An isolated antibody or antigen-binding fragment thereof that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the antibody comprises a variable weight (VH) domain sequence and a variable light (VL) domain sequence, wherein the VH domain sequence is one of the following: SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 25 The isolated antibody or its antigen-binding fragment, selected from the group consisting of 8, 270, 282, 294, 306, and 318, and / or the VL domain is selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

4. The isolated antibody or its antigen-binding fragment according to claim 3, comprising a VH / VL pair heavy chain CDR and a light chain CDR selected from the group consisting of SEQ ID NOs: 18 / 24, 30 / 36, 42 / 48, 54 / 60, 66 / 72, 78 / 84, 90 / 96, 102 / 108, 114 / 120, 126 / 132, 138 / 144, 150 / 156, 162 / 168, 174 / 180, 186 / 192, 198 / 204, 210 / 216, 222 / 228, 234 / 240, 246 / 252, 258 / 264, 270 / 276, 282 / 288, 294 / 300, 306 / 312, and 318 / 324.

5. The isolated antibody or its antigen-binding fragment according to claim 4, comprising a VH / VL pair selected from the group consisting of SEQ ID NOs: 18 / 24, 30 / 36, 42 / 48, 54 / 60, 66 / 72, 78 / 84, 90 / 96, 102 / 108, 114 / 120, 126 / 132, 138 / 144, 150 / 156, 162 / 168, 174 / 180, 186 / 192, 198 / 204, 210 / 216, 222 / 228, 234 / 240, 246 / 252, 258 / 264, 270 / 276, 282 / 288, 294 / 300, 306 / 312, and 318 / 324.

6. (20, 21, 22); (32, 33, 34); (44, 45, 46); (56, 57, 58); (68, 69, 70); (80, 81, 82); (92, 93, 94); (104, 105, 106); (116, 117, 118); (128, 129, 130); (140, 141, 142); (152, 153, 154); (164, 165, 166); (176, 177, 178); (188, 189, 190); (200, 201, 2 02); (212, 213, 214); (224, 225, 226); (236, 237, 238); (248, 249, 250); (260, 261, 262); (272, 273, 274); (284, 285, 286); (296, 297, 298); (308, 309, 310); and (320, 321, 322) selected from the group consisting of HCDR sets (HCDR1, HCDR2, HCDR3) and (26, 27, 28); (3 8, 39, 40); (50, 51, 52); (62, 63, 64); (74, 75, 76); (86, 87, 88); (98, 99, 100); (110, 111, 112); (122, 123, 124); (134, 135, 136); (146, 147, 148); (158, 159, 160); (170, 171, 172); (182, 183, 184); (194, 195, 196); (206, 207, 208); (218, 219 The isolated antibody or its antigen-binding fragment according to claim 3, comprising an LCDR set (LCDR1, LCDR2, LCDR3) selected from the group consisting of (220); (230, 231, 232); (242, 243, 244); (254, 255, 256); (266, 267, 268); (278, 279, 280); (290, 291, 292); (302, 303, 304); (314, 315, 316); and (326, 327, 328).

7. Sequence IDs (20, 21, 22 / 26, 27, 28); (32, 33, 34 / 38, 39, 40); (44, 45, 46 / 50, 51, 52); (56, 57, 58 / 62, 63, 64); (68, 69, 70 / 74, 75, 76); (80, 81, 82 / 86, 87, 88); (92, 93, 94 / 98, 99, 100); (104, 105, 106 / 110, 111, 112); (11 6, 117, 118 / 122, 123, 124); (128, 129, 130 / 134, 135, 136); (140, 141, 142 / 146, 147, 148); (152, 153, 154 / 158, 159, 160); (164, 165, 166 / 170, 171, 172); (176, 177, 178 / 182, 183, 184); (188, 189, 190 / 194, 195, 1 96); (200, 201, 202 / 206, 207, 208); (212, 213, 214 / 218, 219, 220); (224, 225, 226 / 230, 231, 232); (236, 237, 238 / 242, 243, 244); (248, 249, 250 / 254, 255, 256); (260, 261, 262 / 266, 267, 268); (272, 273, 274 / 27 The isolated antibody or its antigen-binding fragment according to claim 6, comprising a (HCDR set / LCDR set) pair selected from the group consisting of (8, 279, 280); (284, 285, 286 / 290, 291, 292); (296, 297, 298 / 302, 303, 304); (308, 309, 310 / 314, 315, 316); and (320, 321, 322 / 326, 327, 328).

8. The isolated antibody or its antigen-binding fragment according to claim 7, comprising an HC / LC pair selected from the group consisting of SEQ ID NOs: 17 / 23, 29 / 35, 41 / 47, 53 / 59, 65 / 71, 77 / 83, 89 / 95, 101 / 107, 113 / 119, 125 / 131, 137 / 143, 149 / 155, 161 / 167, 173 / 179, 185 / 191, 197 / 203, 209 / 215, 221 / 227, 233 / 239, 245 / 251, 257 / 263, 269 / 275, 281 / 287, 283 / 289, 305 / 311, and 317 / 323.

9. An isolated antibody or antigen-binding fragment thereof that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the antibody comprises a variable weight (VH) domain sequence CDR and a variable light (VL) domain sequence CDR, wherein the VH domain sequence is one of the SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 270, 282, 294, 306, and 318 The isolated antibody or its antigen-binding fragment, wherein the VL domain sequence has at least 95% identity to an amino acid sequence selected from the group consisting of, and / or has at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

10. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 114 and the VL is indicated by SEQ ID NO:

120.

11. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 126 and the VL is indicated by SEQ ID NO:

132.

12. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 138 and the VL is indicated by SEQ ID NO:

144.

13. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 150 and the VL is indicated by SEQ ID NO:

156.

14. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 162 and the VL is indicated by SEQ ID NO:

168.

15. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 174 and the VL is indicated by SEQ ID NO:

180.

16. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 186 and the VL is indicated by SEQ ID NO:

192.

17. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is represented by SEQ ID NO: 198 and the VL is represented by SEQ ID NO:

204.

18. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 210 and the VL is indicated by SEQ ID NO:

216.

19. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 222 and the VL is indicated by SEQ ID NO:

228.

20. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 234 and the VL is indicated by SEQ ID NO:

240.

21. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 246 and the VL is indicated by SEQ ID NO:

252.

22. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 258 and the VL is indicated by SEQ ID NO:

264.

23. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 270 and the VL is indicated by SEQ ID NO:

276.

24. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 282 and the VL is indicated by SEQ ID NO:

288.

25. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 294 and the VL is indicated by SEQ ID NO:

300.

26. The isolated antibody or its antigen-binding fragment according to claim 9, wherein the VH is indicated by SEQ ID NO: 306 and the VL is indicated by SEQ ID NO:

312.

27. The isolated antibody or its antigen-binding fragment according to claim 1, wherein the VH is indicated by SEQ ID NO: 318 and the VL is indicated by SEQ ID NO:

324.

28. The isolated antibody or its antigen-binding fragment according to any one of claims 1 to 27, wherein the antibody is human or a humanized antibody.

29. The aforementioned fragments are Fab fragment, Fab' fragment, F(ab) ２ An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 28, comprising a fragment or an scFv fragment.

30. The isolated antibody or antigen-binding fragment according to any one of claims 1 to 29, wherein the antibody or its antigen-binding fragment comprises a human Fc region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM Fc.

31. An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 29, comprising a modified human Fc region.

32. An isolated antibody or its antigen-binding fragment according to claim 31, comprising a modified human Fc region containing a mutation selected from the group consisting of L234A / L235A, S228P, A330S, P331S, E233P / L234V / L235A, A327G / A330S / P331S, L234F / L235E / P331S, and N297Q.

33. A multispecific binding protein comprising an antigen-binding fragment according to any one of claims 1 to 32.

34. An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 33, which inhibits CB1 signaling activity.

35. An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 33, which enhances or activates CB1 signaling activity.

36. An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 33, which is an inverse agonist for CB1 signaling activity.

37. An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 36, which is a humanized antibody.

38. An isolated antibody or its antigen-binding fragment according to any one of claims 1 to 36, which is a fully human antibody.

39. An isolated antibody or its antigen-binding fragment that competes with the isolated anti-human CB1 antibody or its antigen-binding fragment described in any one of claims 1 to 38 for binding to CB1.

40. The isolated antibody or antigen-binding fragment according to claim 39, wherein the antibody or antigen-binding fragment that competes for binding is a human or humanized antibody.

41. An antibody or an antigen-binding fragment thereof that specifically binds to substantially the same CB1 epitope as the antibody or antigen-binding fragment described in any one of claims 1 to 38.

42. The isolated antibody or antigen-binding fragment according to claim 41, wherein the antibody or antigen-binding fragment thereof that specifically binds to substantially the same CB1 epitope is a human or humanized antibody.

43. An isolated antibody or its antigen-binding fragment that binds to the cannabinoid type 1 receptor (CB1), having a binding affinity Kd of approximately 1 μM or less for CB1.

44. The isolated antibody or antigen-binding fragment according to claim 43, having a binding affinity Kd of approximately 100 nM or less for CB1.

45. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 44, which exhibits reduced brain permeability compared to limonabant.

46. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 44, which inhibits CB1 signaling at least twice as much as limonabant.

47. An isolated antibody or antigen-binding fragment according to any one of claims 1 to 44, which shows a reduction in CNS side effects compared to limonabant.

48. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 47.

49. An expression vector comprising the nucleic acid molecule described in claim 48.

50. A host cell comprising the expression vector described in claim 49.

51. A method for regulating CB1 signaling, comprising contacting a cell expressing CB1 with an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 47.

52. A method for antagonistizing CB1, comprising contacting a cell expressing CB1 with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 47.

53. A method for agonizing CB1, comprising contacting a cell expressing CB1 with an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 47.

54. A method for inverse agonizing CB1, comprising contacting a cell expressing CB1 with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 47.

55. A pharmaceutical composition comprising an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1 to 47, and at least one pharmaceutically acceptable additive.

56. A method for inhibiting the biological activity of CB1 in a subject requiring such activity, comprising administering an effective amount of the pharmaceutical composition according to claim 55 to the subject, thereby inhibiting the activity of the CB1 protein in the subject.

57. A method for treating a disease associated with CB1 activity, comprising administering the pharmaceutical composition according to claim 55 to a subject suffering from the disease.

58. A method for treating a disease or disorder that responds to the modulation of CB1 signaling in a subject requiring such treatment, comprising administering the pharmaceutical composition according to claim 55.

59. A method for treating a disease or disorder in which the subject is in need of such treatment, comprising administering to the subject the pharmaceutical composition according to claim 55.

60. A method for treating a disease or disorder in which the agonism of CB1 signaling is required, comprising administering the pharmaceutical composition according to claim 55 to the subject.

61. A method for diagnosing a disease or disorder associated with CB1, comprising contacting cells with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 47.

62. The method according to claim 61, wherein the disease or disorder is selected from the group consisting of obesity, symptomatic obesity including Prader-Willi syndrome (PWS), Alström syndrome, Baldew-Beedl syndrome (BBS), Albride hereditary osteodystrophy (AHO), and SIM1 deletion syndrome; diabetes and related complications; dyslipidemia; liver diseases such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, and primary biliary cirrhosis; fibrosis, such as renal fibrosis; chronic kidney disease; diabetic neuropathy, focal segmental glomerulosclerosis, kidney disease; metabolic diseases, osteoporosis, atherosclerosis, inflammatory diseases, cardiovascular diseases, cancer, pain, systemic sclerosis, multiple sclerosis spasticity, glaucoma, and nicotine addiction.

63. An antibody conjugate comprising an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1 to 47, wherein the antibody or antigen-binding fragment is conjugated to a drug selected from the group consisting of therapeutic agents, cytotoxic agents, immunoadhesion molecules, and imaging agents.

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