Cannabinoid Receptor Type 1 (CB1) Binding Proteins and Their Uses

Abstract

The present specification provides isolated, engineered, non-naturally occurring CB1-binding proteins, comprising anti-CB1 antibodies or antigen-binding fragments thereof. The CB1-binding proteins have uses in the treatment and diagnosis of CB1-mediated conditions, diseases, and disorders.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. provisional application No. 62 / 664,882 filed on April 30, 2018, the full text of which is incorporated herein by reference.

[0003] Field of invention

[0004] The present invention relates to cannabinoid receptor type 1 (CB1) binding proteins and their uses.

[0005] Inclusion of reference materials

[0006] The contents of all cited references (including reference literature, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated into the references in their entirety for all purposes, as with the references cited herein. Unless otherwise indicated, this specification will use the ordinary techniques of immunology, molecular biology, cell biology, drug development, and drug delivery that are well known in the art. Background Technology

[0007] Background of the Invention

[0008] Cannabinoid receptor type 1 (CB1) is a 7-transmembrane receptor of the G protein-linked receptor superfamily that is expressed primarily in the brain, as well as locally in the lungs, liver, kidneys, and adipose tissue. CB1 is activated by naturally occurring cannabinoids within the body called endocannabinoids (e.g., eicosinoids), cannabinoids introduced into the body (e.g., cannabis), or related synthetic compounds. Cannabinoids bind to CB1 reversibly and stereoselectively. After CB1 binds, several intracellular signaling pathways are activated, resulting in inhibition of adenylyl cyclase and activation of mitogen-activated protein (MAP) kinase, inhibition of presynaptic N- and P / Q-type calcium channels and D-type outward potassium channels, and activation of inwardly rectifying and A-type outward potassium channels. The expression of CB1 is considered to regulate neurotransmitter release by preventing excessive neuronal activity, reducing pain and other inflammatory symptoms, as well as regulating food intake.

[0009] Abnormal CB1 activity is associated with obesity and related disorders, such as dyslipidemia, diabetes, fibrosis, liver diseases, such as hepatic steatosis, kidney disease, cardiovascular disease, and cancer.

[0010] Prader-Willi syndrome (PWS) is a genetic disorder resulting from the loss of a specific paternal gene, characterized by obesity, the slow development of type 2 diabetes, and muscle weakness. CB-1 has been validated as a target in PWS using the inverse agonist rimonabant (Motaghedi et al. (2011) Eur. J. Med. Genet. 54: 14-18). Rimonabant (also known as SR141716, Acomplia, and Zimulti) is an oral central CB1 antagonist and an appetite-suppressing anti-obesity drug developed and launched by Sanofi-Aventis. This product has been used in conjunction with diet and exercise to treat obese and overweight patients with associated risk factors, such as type 2 diabetes or dyslipidemia. In June 2006, the drug received approval from the EMEA for the treatment of obesity. In 2008, Sanofi-Aventis discontinued all development and marketing of the drug for all indications due to the risk of serious mental illness, including suicidal ideation. In January 2009, the EC withdrew the marketing authorization for the drug.

[0011] Merck investigated another inverse agonist, taranabant (MK-0364), but Phase 3 clinical 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., cannabigerol, ibipinabant, otenabant, surinabant, tetrahydrocannabivarin, and virodhamine) were studied, but were either in the early stages of research or downgraded to non-human studies due to CNS side effects.

[0012] A number of CB-1 inverse agonists / antagonists targeting primarily peripherally expressed CB1 are being developed by limiting the ability to cross the blood-brain barrier (BBB). For example, TM-38837 is a Phase 1 CB1 inverse agonist / antagonist being developed by 7TM Pharma A / S for the treatment of obesity and metabolic disorders. AM6545 is a peripherally selective silencing antagonist that is not yet in clinical practice. Peripherally selective CB-1 antagonism may be a safer and more effective method of targeting peripheral endocannabinoid activity in various tissues: (1) liver – reduced lipogenesis, fat storage, and glucose secretion; (2) muscle – increased glucose intake and oxidation; (3) adipocytes – reduced lipogenesis and fat storage; reduced adiponectin synthesis; And (4) gastrointestinal (GI) - increased satiation, GI transit and absorption (Kloet and Woods (2009) Endocrinol. 150: 2531-2536).

[0013] Biological molecules, such as antibodies and related binding proteins, provide a potentially safer and more effective method for delivering therapeutic agents and avoiding CNS-related side effects. Generally, only about 0.1% of circulating antibodies cross the 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 biological agents to the CNS provides an opportunity for them to form an exclusive relationship with CB-1 peripherally, thereby limiting side effects caused by the pharmacology of small molecules in the CNS.

[0014] Antibodies against CB1 have recently been described in the art, but their clinical benefits are not yet known. See US Patent Publications Nos. 20170210797 and 20160145333.

[0015] Therefore, in order to avoid pharmacological side effects associated with CB1 in the brain, there remains a need to identify safe and effective peripherally restricted anti-CB1 inverse agonists or antagonists that do not severely penetrate the BBB.

[0016] Summary of the Invention

[0017] The present invention provides binding proteins that bind to a cannabinoid type 1 receptor (CB1), such as antibodies and antigen-binding fragments thereof, which are useful for the treatment and diagnosis of diseases.

[0018] An isolated antibody or an antigen-binding fragment thereof that binds to a human cannabinoid type 1 receptor (CB1) (sequence identification number: 1) is provided, 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 sequence identification number: 329) or a modification of the aforementioned amino acid sequence by substitution of at least one amino acid residue, wherein G at position 1 is substituted with S; T at position 3 is substituted with E; T at position 5 is substituted with S or N; D at position 6 is substituted with R or Y; Y at position 7 is substituted with H; and W at position 8 is substituted with A or N; CDR-H2 has the amino acid sequence IYPYDGDT (residues 51-58 of sequence identification number: 329) or a modification of the aforementioned amino acid sequence by substitution of at least one amino acid residue, wherein I at position 1 is substituted with F; Y at position 2 is substituted with D, S, or T; P at position 3 is substituted with T; Y at position 4 is substituted with G, D, or S; D at position 5 is substituted with Y or S; G at position 6 is substituted with S; D at position 7 is substituted with E, G, or R; and T at position 8 is substituted with 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-Y (residues 98-113 of sequence identification number: 329) or a modification of the aforementioned amino acid sequence by substitution of at least one amino acid residue, wherein A at position 1 is substituted with S; G at position 3 is substituted with S; X1 at position 4 is Q, Y, K, R, or G, or is absent; X2 at position 5 is E, Y, L, or G, or is absent; X3 at position 6 is Y or P, or is absent; X4 at position 7 is Y, R, or E, or is absent; X5 at position 8 is G, or is absent; X6 at position 9 is T, or is absent; X7 at position 10 is N or D, or is absent; X8 at position 11 is Y, N, A, or G, or is absent; X9 at position 12 is N, Y, S, A, or R, or is absent; W at position 13 is substituted with Y, A, or P; X at position 14 10 is L, M, F, or G, or does not exist; X at position 15 11...is P, D, A, or T, or is absent; Y at position 16 is substituted with V; CDR-L1 has the amino acid sequence Q-X1-ISS-X2-Y (residues 27-33 of sequence identification number: 330) or a modification of the aforementioned amino acid sequence by substitution of at least one amino acid residue, wherein Q at position 1 is substituted with S or E; X1 at position 2 is E, S, T, N, G, or R; I at position 3 is substituted with V; S at position 4 is substituted with A, R, or G; S at position 5 is substituted with G, N, or T; X2 at position 6 is S, N, peptide FRYS, or is absent; and Y at position 7 is substituted with F, D, or N; CDR-L2 has the amino acid sequence: X1-TS (residues 51-53 of sequence identification number: 330) or a modification of the aforementioned amino acid sequence by substitution of at least one amino acid residue, wherein X1 at position 1 is A, Y, G, R, D, or S; T at position 2 is substituted with A; S at position 3 is substituted with R; and CDR-L3 has the amino acid sequence: QQY-X1-S-X2-PYT (residues 91-99 of sequence identification number: 330) or a modification of the aforementioned amino acid sequence by substitution of at least one amino acid residue, wherein Q at position 1 is substituted with L or H; Q at position 2 is substituted with H; Y at position 3 is substituted with S or G; X1 at position 4 is S, W, H, Y, N, or I; S at position 5 is substituted with E, R, G, T, or N; At position 6, X2 is Y, I, S, T, L, or W; and at position 8, Y is substituted with P, L, F, or is absent; and the aforementioned substitution, addition, or deletion of at least one amino acid residue does not inhibit the ability of the aforementioned antibody or its antigen-binding fragment to bind to human CB1.

[0019] Tables 5 and 6 provide the functions of exemplary anti-CB1 antibodies of the present invention and their antigen-binding fragments of the present invention.

[0020] An isolated antibody or an antigen-binding fragment thereof that binds to a human cannabinoid type 1 receptor (CB1) (sequence identification number: 1) is provided, wherein the antibody comprises CDRs of a variable heavy chain (VH) domain sequence and CDRs of a variable light chain (VL) domain sequence, wherein the VH domain sequence is selected from the group consisting of sequence identification numbers: 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, and / or wherein the VL domain is sequence identification numbers: 24, 36, 48, 60, It is selected from a group consisting of 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

[0021] An isolated antibody or its antigen-binding fragment is provided for binding to a human cannabinoid type 1 receptor (CB1) (sequence identification number: 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 selected from the group consisting of sequence identification numbers: 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, and / or wherein the VL domain is selected from sequence identification numbers: 24, 36, 48, 60, 72, 84, It is selected from a group consisting of 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

[0022] In a specific example, the antibody or its antigen-binding fragment is selected from the group consisting of sequence identification numbers: 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. It includes VH / VL pair heavy and light chain CDRs.

[0023] In a specific example, the antibody or its antigen-binding fragment is selected from the group consisting of sequence identification numbers: 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. It includes VH / VL pairs.

[0024] In a specific example, 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, 54); (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), selected from the HCDR set (HCDR1, HCDR2, 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, 196); (206, 207, 208); (218, 219, 220); (230, 231, 232); (242, 243, 244); (254, 255, 256); (266, 267, 268); It includes a set of LCDRs (LCDR1, LCDR2, LCDR3) selected from the group consisting of (278, 279, 280); (290, 291, 292); (302, 303, 304); (314, 315, 316); and (326, 327, 328).

[0025] In a specific example, the antibody or its antigen-binding fragment is sequence identification 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, 184); (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, 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 includes pairs of (HCDR set / LCDR set) selected from the group consisting of (320, 321, 322 / 326, 327, 328).

[0026] In a specific example, the antibody or its antigen-binding fragment is selected from the group consisting of sequence identification numbers: 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. Includes HC / LC pairs.

[0027] An isolated antibody or an antigen-binding fragment thereof that binds to a human cannabinoid type 1 receptor (CB1) (sequence identification number: 1) is provided, wherein the antibody comprises CDRs of a variable heavy chain (VH) domain sequence and CDRs of a variable light chain (VL) domain sequence, wherein the VH domain sequence has at least 95% identity with respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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, and / or the VL domain sequence is sequence identification numbers: 24, 36, It has at least 95% identity with respect to amino acid sequences selected from the group consisting of 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.

[0028] In a specific example, the VH is presented as sequence identification number: 114, and the VL is presented as sequence identification number: 120. In a specific example, the VH is presented as sequence identification number: 126, and the VL is presented as sequence identification number: 132. In a specific example, the VH is presented as sequence identification number: 138, and the VL is presented as sequence identification number: 144. In a specific example, the VH is presented as sequence identification number: 150, and the VL is presented as sequence identification number: 156. In a specific example, the VH is presented as sequence identification number: 162, and the VL is presented as sequence identification number: 168. In a specific example, the VH is presented as sequence identification number: 174, and the VL is presented as sequence identification number: 180. In a specific example, the VH is presented as sequence identification number: 186, and the VL is presented as sequence identification number: 192. In a specific example, the VH is presented as sequence identification number: 198, and the VL is presented as sequence identification number: 204. In a specific example, the VH is presented as sequence identification number: 210, and the VL is presented as sequence identification number: 216. In a specific example, the VH is presented as sequence identification number: 222, and the VL is presented as sequence identification number: 228. In a specific example, the VH is presented as sequence identification number: 234, and the VL is presented as sequence identification number: 240. In a specific example, the VH is presented as sequence identification number: 246, and the VL is presented as sequence identification number: 252. In a specific example, the VH is presented as sequence identification number: 258, and the VL is presented as sequence identification number: 264. In a specific example, the VH is presented as sequence identification number: 270, and the VL is presented as sequence identification number: 276. In a specific example, the VH is presented as sequence identification number: 282, and the VL is presented as sequence identification number: 288.In a specific example, the VH is presented as sequence identification number: 294, and the VL is presented as sequence identification number: 300. In a specific example, the VH is presented as sequence identification number: 306, and the VL is presented as sequence identification number: 312. In a specific example, the VH is presented as sequence identification number: 318, and the VL is presented as sequence identification number: 324.

[0029] In a specific example, the anti-CB1 antibody is a human or humanized antibody.

[0030] In a specific example, the anti-CB1 antigen-binding fragment comprises a Fab fragment, a Fab' fragment, an F(ab)2 fragment, or a scFv fragment.

[0031] In a specific example, the anti-CB1 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.

[0032] In a specific example, the anti-CB1 antibody or its antigen-binding fragment comprises 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.

[0033] In a specific example, the anti-CB1 antibody or its antigen-binding fragment inhibits CB1 signaling activity or is an antagonist of this activity.

[0034] In a specific example, the anti-CB1 antibody or its antigen-binding fragment enhances or activates CB1 signaling activity, or acts as an agonist thereof.

[0035] In a specific example, the anti-CB1 antibody or its antigen-binding fragment is an inverse agonist for CB1 signaling activity.

[0036] In a specific example, the anti-CB1 antibody or its antigen-binding fragment is a humanized antibody.

[0037] In a specific example, the anti-CB1 antibody or its antigen-binding fragment is a fully human antibody.

[0038] Anti-CB1 antibodies or their antigen-binding fragments are provided that specifically bind to an epitope of CB1 that is substantially identical to an isolated anti-human CB1 antibody or its antigen-binding fragment.

[0039] Anti-CB1 antibodies or their antigen-binding fragments that compete with isolated anti-human CB1 antibodies or their antigen-binding fragments for binding to CB1 are provided.

[0040] In a specific example, 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.

[0041] In a specific example, 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.

[0042] In a specific example, the anti-CB1 antibody or antigen-binding fragment exhibits reduced brain penetration compared to remonabant.

[0043] In a specific example, the anti-CB1 antibody or antigen-binding fragment inhibits CB1 signaling at least 2 times more than compared to rimonabant.

[0044] In a specific example, the anti-CB1 antibody or antigen-binding fragment exhibits reduced CNS side effects compared to rimonabant.

[0045] An isolated nucleic acid molecule encoding an anti-CB1 antibody or its antigen-binding fragment is provided.

[0046] An expression vector comprising a nucleic acid molecule encoding an anti-CB1 antibody or its antigen-binding fragment is provided.

[0047] A host cell comprising an expression vector containing a nucleic acid molecule encoding an anti-CB1 antibody or its antigen-binding fragment is provided.

[0048] A method for regulating CB1 signaling is provided, which includes contacting an anti-CB1 antibody or an antigen-binding fragment thereof to a cell expressing CB1.

[0049] A method for antagonizing CB1 is provided, which comprises contacting an anti-CB1 antibody or an antigen-binding fragment thereof to a cell expressing CB1.

[0050] A method for acting on CB1 is provided, which includes contacting an anti-CB1 antibody or an antigen-binding fragment thereof to a cell expressing CB1.

[0051] A method for reversing CB1 is provided, which comprises contacting an anti-CB1 antibody or an antigen-binding fragment thereof to a cell expressing CB1.

[0052] A pharmaceutical composition comprising an isolated anti-CB1 antibody or an antigen-binding fragment thereof is provided.

[0053] A method for inhibiting the biological activity of CB1 in a subject requiring inhibition of CB1 is provided, the method comprises administering an effective amount of a pharmaceutical composition containing an isolated anti-CB1 antibody or an antigen-binding fragment thereof to the subject, thereby inhibiting the activity of the CB1 protein in the subject.

[0054] A method for treating a disease associated with CB1 activity is provided, which comprises administering a pharmaceutical composition comprising the isolated anti-CB1 antibody or an antigen-binding fragment thereof to a subject suffering from the disease.

[0055] A method for treating a subject requiring treatment for a disease or disorder that is reactive to the regulation of CB1 signaling is provided, and the method comprises administering a pharmaceutical composition comprising the isolated anti-CB1 antibody or an antigen-binding fragment thereof.

[0056] A method for treating a disease or disorder requiring treatment in a subject who is antagonistic to or reactive to CB1 signaling is provided, and the method comprises administering to the subject a pharmaceutical composition comprising the isolated anti-CB1 antibody or an antigen-binding fragment thereof.

[0057] A method for treating a subject requiring treatment for a disease or disorder that is reactive to the inverse effect of CB1 signaling is provided, and the method comprises administering to the subject a pharmaceutical composition comprising the isolated anti-CB1 antibody or an antigen-binding fragment thereof.

[0058] A method for diagnosing a disease or disorder associated with CB1 activity is provided, which comprises contacting a cell with an anti-CB1 antibody or an antigen-binding fragment thereof.

[0059] In a specific example, the disease or disorder is selected from the group consisting of obesity, syndromic obesity including Prader-Willi syndrome (PWS), Alstrom syndrome, Bardet-Biedel syndrome (BBS), Albright hereditary osteogenesis imperfecta (AHO), and SIM1 deletion syndrome; diabetes mellitus and related complications; dyslipidemia; liver diseases such as, for example, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, and primary biliary cirrhosis; fibrosis, for example, renal fibrosis; chronic kidney disease; diabetic neuropathy, focal segmental glomerulosclerosis, kidney disease; metabolic disease, osteoporosis, atherosclerosis, inflammatory disease, cardiovascular disease, cancer, pain, systemic sclerosis, multiple sclerosis seizures, glaucoma, and nicotine addiction.

[0060] In a specific embodiment, the disease or disorder is a kidney disease (e.g., focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive renal disease, nephropathy syndrome, steroid-resistant nephropathy syndrome, minimal change disease, membranous nephropathy, idiopathic membranous nephropathy, membranoproliferative glomerulonephritis (MPGN), immune complex-mediated MPGN, complement-mediated MPGN, lupus nephritis, post-infectious glomerulonephritis, thin basement membrane disease, vascular interstitial hyperplasia glomerulonephritis, amyloidosis (primary), C1Q nephropathy, acute progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrotic sclerosis, IgA nephropathy, proteinuric renal disease, microalbuminuria, or macroalbuminuric renal disease), pulmonary arterial hypertension, pain (e.g., neuropathic pain or visceral pain), cancer (e.g., chemoresistant breast Carcinoma, adriamycin-resistant breast cancer, chemoresistant colorectal cancer, medulloblastoma, or tumor angiogenesis), anxiety, depression, transplant-associated FSGS, transplant-associated nephropathy syndrome, transplant-associated proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), obesity, insulin resistance, type II diabetes, pre-diabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), diabetic gastroparesis, or gastroparesis.

[0061] An antibody conjugate comprising an isolated anti-CB1 antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof is conjugated to a preparation selected from the group consisting of therapeutic agents, cytotoxic agents, immunoadhesive molecules, and imaging agents.

[0062] A kit is provided containing an isolated anti-CB1 antibody or its antigen-binding fragment and instructions for the use of said antibody in an immunoassay. Brief explanation of the drawing

[0063] The above features and other features and advantages of the present invention, as well as the invention itself, will be more fully understood from the following description of specific embodiments when read together with the accompanying drawings: Figure 1 shows the results of the cAMP assay. cAMP Hunter™ CHO-K1 CNR1 Gi cells were treated with a CB1 antibody, an isotype control, or the small molecule CB1 antagonist JD5037, followed by an agonist challenge with 30 nM CP-55,940 (indicated as "Plus CP") in the presence of forskolin. The antagonist was also tested without the addition of CP-55,940 to determine whether it possessed intrinsic activity. Key: CAB = Cell assay buffer, representing the 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, corresponding to ~EC80 for CP-55,940. Figure 2 shows the results of the p-ERK assay. The phospho / total ERK assay was performed using cAMP Hunter™ CHO-K1 CNR1 Gi cells. Cells were treated with a mouse anti-CB1 antibody or the small molecule CB1 antagonist JD5037, followed by an agonist challenge with 30 nM CP-55,940 in the presence of forskolin. At the end of the treatment, the plates were processed for p-ERK / total ERK using the MesoScale Discovery (MSD) kit. Results are expressed as the percentage of maximum response (max %). Figure 3A shows the binding of mouse anti-huCB1 antibodies to huCB1-CHO cells. The binding curves of purified murine anti-CB1 antibodies were titrated 3-fold starting from an antibody concentration of 200 nM. 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 shows the binding evaluation of purified anti-CB1 antibodies in the presence and absence of the agonist and antagonist CB1 small molecules, CP5990 and JD5037, respectively. Figure 5A shows the consensus sequence derived from the alignment of the amino acid sequences of the heavy chain variable region from the hybridoma antibody M1-M8. Figure 5B shows the concentration sequence derived from the alignment of the light chain variable region amino acid sequences from the hybridoma antibody M1-M8. Figure 6A shows the concentration sequence derived from the alignment of the heavy chain variable region amino acid sequences from humanized antibodies M7-H1 to M7-H16, M5-H1, and M5-H2. Figure 6B shows the concentration sequence derived from the alignment of the light chain variable region amino acid sequences from humanized antibodies M7-H1 to M7-H16, M5-H1, and M5-H2. Figure 7A illustrates the cell binding of a single concentration of 30 μg / mL antibody to humanized CB-1 antibody variants of clones M5 and M7 on CHO-huCB-1 and CHO paternal cells. Figure 7B illustrates the cellular binding of a single concentration of 30 μg / mL antibody to a humanized CB-1 antibody variant of clone M7 on CHO-huCB-1 and CHO paternal cells. Figure 8A shows the results of the cAMP assay described in Example 3. Figure 8A shows a side-by-side comparison between different backbones for the M5 antibody. Figure 8B shows the results of the cAMP assay described in Example 3. Figure 8B shows a side-by-side comparison between different backbones for the M7 antibody. Figure 9A shows the results of the p-ERK assay described in Example 4. Figure 9A shows a side-by-side comparison between different backbones for the M5 antibody. Figure 9B shows the results of the p-ERK assay described in Example 4. Figure 9B shows a side-by-side comparison between different backbones for the M7 antibody. Figure 10 shows the results of the cAMP assay for reverse action using a method similar to that described in Example 3. cAMP Hunter™ CHO-K1 CNR1 Gi cells were treated with a CB1 antibody or an isotype control, followed by the addition of forskolin. The purple dashed line represents the level of cAMP released upon forskolin treatment, representing the maximum amount of cAMP in the assay. The red line corresponds to the CB1 small molecule agonist CP-55,940 used as the control in the assay. Compounds exhibiting reverse action activity have a curve in the opposite direction to this agonist and at the top of the CAB / F line. This experiment demonstrates that all tested M7 variants possess reverse action activity. Specific details for implementing the invention

[0064] Detailed description of the invention

[0065] Unless otherwise defined, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Where there is potential ambiguity, the definitions provided herein take precedence over dictionary or external definitions. Unless required by the content, the singular includes the plural, and the copy includes the singular. The word "a" or "an" means "at least one" unless otherwise specified. The meaning of the phrase "at least one" is the same as the meaning of the phrase "one or more." The word "or" means "and / or" unless explicitly stated otherwise. As used herein, terms “include,” “containing,” “containing,” “having,” etc., may have the meanings given to them under U.S. patent law and may mean “contain,” “containing,” and similar things, and “essentially constituted” or “to be essentially constituted” likewise have the meanings prescribed by U.S. patent law, and such terms are open and allow for the existence of more than cited, provided that the underlying or new characteristics are not altered, but prior art embodiments are excluded. Unless specifically stated or evident in the context, the term “about” as used herein is understood to be within the normal tolerance range of the art, e.g., within two standard deviations of the mean. It may be approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless otherwise evident from the context, all figures provided herein are modified by the term "about."

[0066] The methods and techniques provided herein are practiced in accordance with conventional methods generally known in the art, unless otherwise noted, and are carried out as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature, laboratory procedures, and techniques described herein in cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry are well known and commonly used in the field. Standard techniques are used in enzymatic reactions and purification techniques, chemical synthesis, chemical analysis, pharmaceutical formulation, formulation, delivery, and patient treatment.

[0067] The ranges provided in this specification are understood to be abbreviations for all values ​​within the range. For example, the range from 1 to 50 is understood to include any number, combination of numbers, or sub-range in 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.

[0068] The term "cannabinoid receptor type 1" or "CB1" refers to a 7-membrane-sized cell membrane receptor encoded by the CNR1 gene and having the canonical amino acid sequence of sequence identification number: 1 (see https: / / www.uniprot.org / uniprot / P21554). Table 1 discloses this sequence and the domain of the said protein.

[0069]

[0070] The term "central CB1" refers to CB1 localized in any location in the body, including the brain and CNS.

[0071] The term "peripheral CB1" refers to CB1 that is not localized to the brain or CNS (e.g., peripherally localized CB1).

[0072] The term “antibody” means any antigen-binding molecule or molecular complex comprising at least one complementarity determining domain (CDR) that specifically binds to or interacts with a specific antigen. The term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, non-specific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, and grafted antibodies. For the purposes of this disclosure, unless otherwise modified by the term “intact,” as in “intact antibody,” the term “antibody” also encompasses Fab, F(ab’)2, Fv, scFv, Fd, dAb, and other antibody fragments possessing antigen-binding function, namely the ability to specifically bind to CB1. Typically, such fragments will comprise an antigen-binding domain. The term "antibody" comprises an immunoglobulin molecule comprising four polypeptide chains and a multimer thereof, each consisting of two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In a specific embodiment of a full-length antibody, each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). CH comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). CL comprises a single CL domain. The VH and VL may be further subdivided into supervariable regions called complementarity determining regions (CDRs), in which more conserved regions, named framework regions (FRs), are located. Generally, each VH and VL contains 3 CDRs and 4 FRs, which are arranged from amino-terminal to carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.In different embodiments of the present invention, the FRs of the anti-CB1 antibody may be identical to human germline sequences or may be naturally or artificially modified. The amino acid concentration sequence may be defined based on a 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.

[0073] The terms "HCDR set" and "LCDR set" refer to a group of three CDRs occurring in a single variable region of the heavy or light chain, in turn, capable of binding to an antigen. The term "(HCDR set / LCDR set) pair" refers to a pair of an HCDR set and an LCDR that provides six CDRs constituting the antigen binding site. The precise boundaries of these CDRs have been 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 defining three CDRs. These CDRs may be referred to as Kabat CDRs. The term “Kabat numbering” refers to a numbering system for amino acid residues that are more variable (e.g., supervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding fragment (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the supervariable region has a range of amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region has a range of amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3. Chothia and coworkers (Chothia and Lesk (1987) J. Mol. Biol.Chothia et al. (196: 901-917; Chothia et al. (1989) Nature 342: 877-883) discovered that certain sub-regions within Kabat CDRs adopt nearly identical peptide backbone forms despite exhibiting significant variability at the amino acid sequence level. These sub-regions were designated as L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs that have boundaries overlapping with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs were described by Padlan (1995) FASEB J. 9: 133-139 and Maccallum (1996) J. Mol. Biol. 262(5):732-45). While specific residues, groups of residues, or entire CDRs may be shortened or lengthened in light of other predicted or experimental findings where they do not significantly affect antigen-binding, other CDR boundary definitions may not strictly adhere to one of the systems of this invention, but will nevertheless overlap with Kabat CDRs. The compositions and methods described herein will utilize CDRs defined according to any of these systems.

[0074] The term "VH / VL pair" refers to a pair of VH and VL capable of binding to an antigen.

[0075] The term "HC / LC pair" refers to a pair of HC and LC that can bind to an antigen.

[0076] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that may be generated by papain degradation 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 comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. Substitution of amino acid residues in a portion of the Fc to modify antibody agent functions is known in the art (e.g., US Patent Nos. 5,648,260 and 5,624,821). The Fc region mediates several important agent functions, e.g., 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, this agent function is desirable for therapeutic immunoglobulins, but in other cases, it may be unnecessary or harmful depending on the therapeutic purpose.

[0077] The terms "antibody binding to CB1" and "anti-CB1 antibody" refer to soluble CB1 protein or a fragment thereof (e.g., part of the extracellular domain of CB1) and / or antibodies binding to cell surface-expressed CB1 and their antigen-binding fragments. The expression "cell surface-expressed CB1" refers to a CB1 protein or a part thereof expressed on the surface of a cell in vitro or in vivo, where at least a part of the CB1 protein is exposed to the extracellular side of the cell membrane, or the antigen-binding portion of the antibody is accessible.

[0078] The terms “CB1 binding protein” or “anti-CB1 binding protein” mean a protein that binds to CB1, comprising all or part of an antigen-binding fragment, and include proteins comprising alternative arrangements of typical antibody domains or frameworks, such as recombinant multivalent or multispecific immunoglobulins, as well as conjugate and fusion proteins. The CB1 binding proteins of the present invention, their variants and mutants 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.

[0079] With respect to binding proteins, such as antibodies, the terms “antigen-binding domain” and “antigen-binding fragment” mean a portion or fragment of an antibody, or a variant or mutant thereof, that possesses the ability to bind specifically to a target antigen of the antibody, and comprises any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein capable of specifically binding to an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from the whole antibody molecule using any suitable standard technique, such as proteolytic cleavage or recombinant genetic engineering techniques involving the regulation and expression of DNA encoding antibody variable and optionally constant domains. One or more variable and / or constant domains may be arranged in a suitable configuration, or arranged to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids. A number of fragment, mutant, or variant antibody formats containing antigen-binding fragments are known in the art. Non-limiting examples of antigen-binding fragments include: (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH1 domains; (ii) a bivalent fragment, F(ab')2 fragment, in which two Fab fragments are linked by a disulfide bridge at a hinge region; (iii) a Fd fragment containing VH and CH1 domains; (iv) a Fv fragment containing VL and VH domains of a single arm of an antibody; (v) a single-stranded Fv (scFv) molecule; (vi) a dAb fragment containing a single variable domain; and (vii) a minimal recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), e.g., a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, linear antibodies (including tandem Fv segment pairs; VH-CH1-VH-CH1 forming an antigen-binding site with a complementary light chain polypeptide), triabodidies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular immunotherapies (SMIPs), and shark variable IgNAR domains are also encompassed by the expression “antigen-binding fragment” as used herein. The term “its antigen-binding fragment” includes, but is not limited to, a fragment contained within a variant molecule that may have an additional or rearranged antibody region, and includes, for example, multispecific antibodies and antibody conjugates having the same antigen binding to a specific antigen.

[0080] The antigen-binding fragment of the antibody typically comprises at least one variable domain. The variable domain may be of any size or amino acid composition and generally comprises at least one CDR adjacent to or within the same framework sequence in the antigen-binding fragment having a VH domain associated with a VL domain, and the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, the variable domain may be dimeric and contains VH-VH, VH-VL, or VL-VL dimerics. Alternatively, the antigen-binding fragment of the antibody may contain a monomeric VH or VL domain.

[0081] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Exemplary arrangements of the variable domain and the constant domain that may be found in the antigen-binding fragment of the antibody of the present invention include, but are not limited to: (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 domains and constant domains comprising the exemplary coordinations listed above, the variable domains and constant domains may be directly connected to each other, or connected by a whole or part hinge or linker region. The hinge region consists of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids to form a soft or semi-soft linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may comprise a homodimer or heterodimer (or multimer) of any of the variable and constant domain coordinations listed above, which are non-covalently linked to each other and / or together with one or more monomeric VH or VL domains (e.g., by disulfide bond(s).

[0082] As with whole antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody typically comprises at least two different variable domains, each of which may specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody disclosed herein, may be used in the antigen-binding fragment content of the antibody of the present invention using conventional techniques available in the art.

[0083] The binding protein-related terms "specificity" or "specific to" refer to the ability of a binding protein to selectively bind to a target or antigen with a greater affinity (e.g., a lower Kd value) than to any other target or antigen.

[0084] In certain embodiments, the antibodies of the present invention may function via complement-dependent 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 response in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, thereby inducing lysis of the target cells. CDC and ADCC can be measured using assays well known and available in the art (see, e.g., US Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95: 652-656).

[0085] The term "mouse antibody" refers to an antibody having variable and constant regions derived from mouse germline immunoglobulin sequences. Mouse antibodies may contain amino acid residues that are not encoded by mouse germline immunoglobulin sequences, for example, in CDRs and specific CDR3s (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or somatic mutations in vivo). However, the meaning of the term "mouse antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as humans, are grafted onto mouse framework sequences.

[0086] The term “recombinant antibody” means an antibody prepared, expressed, produced, or isolated by a recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from recombinant, a complex antibody library, an antibody isolated from an animal (e.g., a mouse) that has been genetically inserted for an immunoglobulin gene, or an antibody prepared, expressed, produced, or isolated by any other means related to the grafting of an immunoglobulin gene sequence onto another DNA sequence. In certain embodiments, such recombinant antibodies may be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if a transgenic animal for the immunoglobulin sequence is used), and the amino acid sequences of the VH and VL regions of such recombinant antibodies may be derived from and related to human germline VH and VL sequences, but may not naturally exist in vivo within the said specific antibody germline repertoire.

[0087] The term "isolated antibody" means an antibody identified, separated, and / or recovered from at least one component of the natural environment. For example, an antibody separated or removed from an organism, tissue, or cell that is naturally present or naturally produced is an "isolated antibody" for the purposes of the present invention. The isolated antibody also contains antibodies in situ within recombinant cells. The isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, the isolated antibody is substantially free of other cellular material and / or chemicals.

[0088] The terms "neutralizing" or "blocking" antibody refer to an antibody whose binding to a ligand or antigen interferes with the biological activity of the ligand or antigen. In a specific example, the neutralizing binding protein binds to an antigen (e.g., 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 binding to CB1 interferes with (i) the interaction between CB1 or a CB1 fragment and a CB1 ligand (e.g., cannabinoid, etc.), and / or (ii) interferes with at least one biological function of CB1. The inhibition caused by the CB1 neutralizing antibody or blocking antibody does not need to be complete, as long as the antibody is detectable using an appropriate assay. Exemplary assays for detecting CB1 inhibition are described herein.

[0089] The term "affinity" refers to the strength of the interaction between a binding protein and its target antigen, and is determined by the CDRs sequences of said binding protein as well as the properties of the antigen and antibody, such as their size, shape, and / or charge. Binding proteins may be selected for an affinity that provides a desired therapeutic endpoint while minimizing adverse side effects. Affinity may be measured using methods known to those skilled in the art.

[0090] A "matured" antibody is an antibody having one or more alterations in one or more of its CDRs or FRs, which results in an improvement in the affinity of said antibody for its target antigen compared to an unaltered "paternal" antibody that does not possess these alteration(s). An exemplary matured antibody may have nanomolar or even picomolar affinity for a target antigen. Matured antibodies are prepared by methods known in the art. For example, Marks et al. (1992) BioTechnology 10: 779-783 describes maturation by VH- and VL-domain shuffling. Random mutagenesis of CDRs and / or framework residues is described by 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. Immunol. 155: 1994-2004; Jackson et al. (1995) J. Immunol. 154(7): 3310-9; Hawkins et al. (1992) J. Mol. Biol. 226:889-896, and a supermutation that contacts or enhances a mutant with an active enhancing amino acid residue at a selective mutagenic site is described in US Patent No. 6,914,128.

[0091] The term "CDR-grafted antibody" means an antibody comprising heavy and light chain variable region sequences in which one or more sequences of the CDR regions of VH and / or VL are replaced by the CDR sequences of another antibody. For example, the two antibodies may be of different species, such as antibodies having murine heavy and light chain variable regions in which one or more murine CDR sequences are replaced by human CDR sequences.

[0092] The term "humanized antibody" refers to an antibody from a non-human species that has been modified to be more similar to a human germline sequence. One type of humanized antibody is a CDR-grafted antibody, in which one or more CDR sequences are non-human, and the framework region (FR) sequence is human or substantially human (e.g., these 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 at least one, and typically two, variable domains, substantially both (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions correspond to those of a human immunoglobulin sequence. The humanized antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody also comprises at least a portion of the human immunoglobulin Fc region. In some embodiments, the humanized antibody comprises only the 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 at least the variable domain of the heavy chain as well as the light chain. In some embodiments, the humanized antibody comprises at least the variable domain of the light chain as well as the heavy chain.

[0093] The term "potency" refers to the ability of a binding protein to achieve a desired effect and is a measure of therapeutic efficacy. Potency can be evaluated using methods known to those skilled in the art.

[0094] The term "effective amount" refers to a dose or quantity sufficient to reduce CB1 activity to improve a patient's symptoms or achieve a desired biological outcome. Desired biological outcomes include, for example, a decrease or increase in CB1 activity.

[0095] The term "cross-reactive" refers to the ability of a binding protein to bind to a target antigen other than the one to which it was presented. Generally, a binding protein binds to a target antigen with appropriately high affinity, but may also bind to the same target antigen of a different species, or exhibit low affinity for a non-target antigen. Individual binding proteins are generally selected to meet two criteria: (1) tissue staining suitable for the known expression of the antibody target; and (2) similar staining patterns between those obtained from the same organ of humans and toxicological study species (e.g., mice and cynomolgus monkeys). These and other methods for evaluating cross-reactivity are known to those skilled in the art.

[0096] The term "biological function" refers to the specific in vitro or in vivo activity of a binding protein that is naturally occurring or made possible by recombinant means. Binding proteins can target various types of antigens and achieve desired therapeutic results through various mechanisms of action. Binding proteins can agonize, antagonize, or neutralize the activity of targets. Binding proteins can assist in the removal of the targets to which they bind, or induce cytotoxicity when they bind to cells. A portion of two or more antibodies may be incorporated in a multivalent format to acquire distinct functions from a single binding protein molecule. Biological activities include, but are not limited to, binding to receptors, induction of cell proliferation, inhibition of cell growth, induction of other cytokines, induction of apoptosis, and enzymatic activity. In vitro assays and biological models used to evaluate biological functions are known to those skilled in the art.

[0097] The term "stable" means the ability to maintain physical, chemical, and / or biological integrity or activity within a given period or storage conditions. Binding proteins that are stable in vitro at various temperatures over a long period are generally desired. Methods for stabilizing binding proteins and evaluating their stability at various temperatures are known to those skilled in the art.

[0098] The term "solubility" refers to the ability of a protein to remain dispersed in an aqueous solution. Since the solubility of a protein in aqueous formulations depends on the proper distribution of hydrophobic and hydrophilic amino acid residues, solubility can be related to the production of a properly folded protein. Those skilled in the art will be able to detect an increase or decrease in the solubility of a bound protein using routine HPLC techniques and methods known to them.

[0099] The term "immunogenicity" refers to the ability of a substance to induce an immune response. The administration of therapeutic binding proteins may result in the specific occurrence of an immune response. Methods for reducing the immunogenicity of antibodies and binding proteins are known to those skilled in the art.

[0100] The term "detectable label" refers to a moiety attached to a member of a specific binding pair, such as an antibody or its analyte, to make the reaction (e.g., binding) between the members of a specific binding pair detectable.

[0101] The labeled member of a specific binding pair is referred to as "detectable-labeled." Accordingly, the term "labeled binding protein" refers to a protein having a detectable label incorporated to identify said binding protein. In a specific embodiment, the detectable label may produce a detectable signal by visual or instrumental means, for example, by incorporating a radiolabeled amino acid or attaching to a biotinyl moiety of a polypeptide detectable by avidin to produce a detectable signal (e.g., a fluorescent marker detectable by optical or chromogenic methods or streptavidin containing enzymatic activity). Examples of detectable labels for polypeptides include, but are not limited to: radioisotopes or nuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153Sm); pigment sources, 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, binding sites for secondary antibodies, metal-binding domains, epitope tags); and magnetic materials, such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-generating moiety (e.g., acridinium compounds) and fluorescence-generating moiety (e.g., fluorescein). In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with another moiety.

[0102] The term “conjugate” refers to a binding protein, such as an antibody, chemically linked to another functional molecule or a second chemical moiety, such as a therapeutic agent, a cytotoxic agent, a cell proliferation inhibitor, or a contrast agent (see, for example, 7,850,962). The term “agent” includes chemical compounds, mixtures of chemical compounds, biological macromolecules such as peptides of proteins, or extracts made from biological materials. In specific examples, therapeutic agents or cytotoxic agents include, but are not limited to, anti-metabolites, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, anthracyclines, toxins, and apoptosis agents. Useful agents include, for example, pertussis toxin, taxol, cytocalcin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or homologues. Imaging agents useful for making anti-CB1 binding protein conjugates include, but are not limited to, radiolabels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, and biotin. When used in the context of an immunoassay, the conjugate antibody may be a detectably labeled antibody used as a detection antibody. Antibodies may be linked by chemical-crosslinking or recombinant methods. Antibodies may also be linked to one of various non-protein polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene, by the method presented in US Patent No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.Antibodies can be chemically modified by covalent conjugation to polymers, for example, to increase their circulating half-life. Exemplary polymers and methods of attaching them are also shown in US Patent Nos. 4,766,106; 4,179,337; 4,495,285, and 4,609,546.

[0103] The term "crystallized" refers to binding proteins that exist in a crystalline form. A crystal is a form of the solid state of a substance, distinguished from other forms such as the amorphous solid state or the liquid crystal state. Crystals consist of a regular, repetitive three-dimensional arrangement of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen / antibody complexes).

[0104] The term "vector" refers to a nucleic acid molecule capable of transporting another associated nucleic acid. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which another DNA segment can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome. Other vectors include RNA vectors. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial replication origin and episomal mammalian vectors). Other vectors, when introduced into a host cell, can be incorporated into the host cell's genome and thus replicate along with the host genome (e.g., non-episosome mammalian vectors). "Recombinant expression vectors" or "expression vectors" can direct the expression of genes to which they are operably associated. 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, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0105] The term "recombinant host cell" or "host cell" refers to a cell into which exogenous DNA or RNA has been introduced. These terms refer not only to specific target cells but also to the offspring of such cells. Because specific modifications may occur in subsequent generations due to mutations 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 specific embodiments, host cells include prokaryotic and eukaryotic cells. In specific embodiments, eukaryotic cells include protists, fungi, plant and animal cells. In another embodiment, host cells are prokaryotic cell lines such as Escherichia coli ( E. coli); mammalian cell lines CHO, HEK 293, COS, NSO, SP2, and PER.C6; insect cell line Sf9; and fungal cells Saccharomyces cerevisiae ( Saccharomyces cerevisiae Includes, but is not limited to.

[0106] The term "transfection" refers to various techniques commonly used to introduce exogenous nucleic acids into host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and similar methods.

[0107] The term “biological sample” means an amount of material derived from a living organism or a former living thing. 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.

[0108] The term "control" refers to a composition known not to contain a specific analyte ("negative control") or a composition known to contain a specific analyte ("positive control"). A positive control may contain a known concentration of the analyte, be used to establish analytical performance characteristics, and serve as a useful indicator of reagent integrity.

[0109] 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, an antibody and its antigen, biotin and avidin (or streptavidin), a carbohydrate and a lectin, complementary nucleotide sequences, an operator and a receptor molecule, an enzyme and an inhibitor and an enzyme, and fragments and their analogs that maintain specific binding. An example of a specific binding pair is the VH and VL regions of an antibody ("VH / VL").

[0110] The term "linker" means an amino acid residue or a polypeptide comprising two or more amino acid residues connected by a peptide bond used to link two polypeptides (e.g., two VH or two VL domains). Linkers are known in the art (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljaketal. (1994) Structure 2: 1121-1123).

[0111] The term "epitope" refers to an epitope that interacts with a specific antigen-binding site in a variable region of an antibody molecule known as a paratope. A single antigen may possess one or more epitopes. Therefore, different antibodies may bind to different regions on the antigen and possess different biological effects. Epitopes can be stereotyped or linear. Stereotyped epitopes are formed by amino acids spatially arranged side-by-side from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues of the polypeptide chain. Under certain circumstances, epitopes may contain moietyes of amino acids, saccharides, phosphoryl groups, or sulfonyl groups on the antigen and may possess specific three-dimensional structural characteristics and / or specific charge characteristics. Binding proteins "bind to the same epitope" if they bind to the same amino acid of the antigen, and may also engage in cross-competition (where one antibody interferes with the binding or regulatory effect of another antibody). In addition, the structural definitions of epitopes (overlapping, similar, identical) are informative; the functional definitions include structural (combining) and functional (coordination, competition) parameters.

[0112] The term "pharmacokinetics" refers to the process by which a drug is absorbed, distributed, metabolized, and excreted by an organism.

[0113] The term "bioavailability" refers to the amount of active drug that reaches the 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.

[0114] The term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biological interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore® system (BIAcore International AB, Uppsala, Sweden and Piscataway, NJ; Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26).

[0115] The terms "Kon," "association rate constant," and "Ka" refer to the association rate constant for the binding of a binding protein (e.g., an antibody) and an antigen to form a binding protein / antigen complex. This value represents the binding rate of the binding protein to the target antigen or the rate of complex formation between the binding protein and the antigen, as shown by the following equation:

[0116] Antibody ("Ab") + Antigen ("Ag") → "Ab-Ag"

[0117] 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 the target antigen, or the rate of separation from the Ab-Ag complex into free antibody and antigen over time, as shown by the following equation:

[0118] Ab + Ag ← Ab-Ag

[0119] The terms "Kd" and "equilibrium dissociation constant" refer to values ​​obtained from titration measurements at equilibrium, or the value obtained by dividing the dissociation constant (Koff) by the association constant (Kon). Methods for determining the association constant and the dissociation constant are well known in the art. Fluorescence-based techniques provide high-sensitivity capabilities for examining samples of physiological buffer at equilibrium. Other experimental methods and instruments, such as the BIAcore® assay (BIAcore international AB, Uppsala, Sweden) or the KinExA® assay (Sapidyne Instruments, Boise, Idaho), may be utilized.

[0120] The term "variant" refers to a polypeptide that differs from a given polypeptide in its amino acid sequence due to the addition, insertion, deletion, or conservative substitution of amino acids, but retains the biological activity of the given polypeptide (e.g., variant antibodies compete with the native antibody to bind to its target). Conservative substitution of amino acids—that is, replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of the charged region)—is recognized in the art as typically involving minor changes. As recognized in the art, such minor changes can be partially identified by considering the hydropathic index of the amino acid. The hydropathic index of an amino acid is based on the hydrophobicity and charge of that amino acid. In a protein, amino acids of similar numerical indices can be substituted, and the protein still retains its function. In one aspect, an amino acid with a numerical index of ±2 is substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that cause a protein to retain its biological function. In the context of peptides, the hydrophilicity of amino acids allows for the calculation of the peptide's maximum local mean hydrophilicity, which is a useful measure reported to correlate sufficiently with antigenicity and immunogenicity. Substitution of amino acids with similar hydrophilic values ​​can result in peptides that retain biological activity, e.g., immunogenicity, as understood in the field. In one aspect, substitution is performed with amino acids having hydrophilic values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilic value of an amino acid are influenced by the specific side chain of the amino acid. Consistent with these observations, amino acid substitutions compatible with biological function are understood to depend on the relevant similarity of the amino acids and, in particular, on the side chains of these amino acids, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties.The term “variant” also includes polypeptides or fragments thereof that have been differentially treated by proteolytic degradation, phosphorylation, or other post-translation modifications but retain biological activity or antigenicity. Unless otherwise defined, the term “variant” includes fragments of variants. Variants may have 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% identity with respect to the wild-type sequence.

[0121] The anti-CB1 antibody disclosed herein may include one or more amino acid substitutions, additions, insertions, and / or deletions in the CDR regions of the framework and / or heavy chain domain and the light chain variable domain when compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be easily identified by comparing the amino acid sequence disclosed herein with germline sequences available in public antibody sequence databases. The invention comprises an antibody and antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated with respect to corresponding residue(s) of the germline sequence from which the antibody is derived, or with respect to corresponding residue(s) of another human germline sequence, or with respect to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily make a number of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequence and light chain variable region sequence disclosed herein. In certain embodiments, all CDR residues within the framework and / or VH and / or VL domains are reverse-mutated to residues found in the original germline sequence from which the antibody is derived. In other embodiments, only specific residues are reverse-mutated to the original germline sequence, e.g., a mutated residue found only in the first 8 amino acid residues of FR1, or a mutated residue found only in the last 8 amino acids of FR4, or a mutated residue found only in CDR1, CDR2, or CDR3.In other embodiments, one or more framework and / or CDR residue(s) are mutated into corresponding residue(s) of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, such that, for example, specific individual residues are mutated into corresponding residues of a specific germline sequence, while specific other residues different from the original germline sequence are retained or mutated into corresponding residues of a different germline sequence. Once obtained, the antibody and the antigen-binding fragment containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as in some cases), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are incorporated into the present invention.

[0122] When referring to nucleic acids, the terms “substantially identical” and “substantially identical” indicate that when a nucleic acid having appropriate nucleotide insertions or deletions is optimally aligned with another nucleic acid (or its complementary counterpart), at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the nucleotide bases are identical when measured by any known algorithm for sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having an amino acid sequence identical to or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0123] When referring to polypeptides, the terms “substantially similar” and “substantially similar” 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 by programmed GAP or BESTFIT using default gap weights, for example. In specific examples, non-identical residue positions differ by conservative amino acid substitutions. The present invention also comprises an anti-CB1 antibody comprising any variant having one or more conservative substitutions in the VH, VL, and / or CDR amino acid sequences disclosed herein. For example, the present invention comprises an anti-CB1 antibody having a VH, VL, and / or CDR amino acid sequence having, for any of the VH, VL, and / or CDR amino acid sequences disclosed herein, conserved amino acid substitutions, such as 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. "Conserved amino acid substitution" is the substitution of an amino acid residue with another amino acid residue having a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions will not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or similarity may be upscaled to correct for the conserved nature of the substitutions. Means for such adjustment are well known to those skilled in the art. For example, Pearson (1994) Methods Mol. Biol. See 24: 307-331, incorporated into the references of this institution.Examples of amino acid groups with side chains having similar chemical properties are as follows: (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 are cysteine ​​and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, conservative substitutions are Gonnet et al. It is any change that has a positive value in the PAM250 log-like matrix disclosed in (1992) Science 256: 1443-1445 (incorporated into the references of this publication). "Usually conservative" substitution is any change that has a non-negative value in the PAM250 log-like matrix.

[0124] Sequence similarity for polypeptides, also known as sequence identity, is typically measured using sequencing software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which are used in conjunction with basic parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides of different organism species, or between a wild-type protein and its mutates. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared in FASTA using basic or recommended parameters, which are programs in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the region with the most overlap between the query and search sequences and the percentage of sequence identity (Pearson (2000) supra). When comparing the sequence of the present invention with a database containing multiple sequences from different organisms, another preferred algorithm is a computer program BLAST using basic parameters, in particular BLASTP or TBLASTN. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402, each incorporated into the references of this application.

[0125] Biological characteristics of antibodies

[0126] The present invention comprises an anti-CB1 antibody that binds to CB1 with high affinity and antigen-binding fragments thereof.

[0127] When measured by surface plasmon resonance, at least about 10 2 M -1 s -1; at least about 10 3 M -1 s -1 ; at least about 10 4 M -1 s -1 ; at least about 10 5 M -1 s -1 ; and at least about 10 6 M -1 s -1 The combined rate constant (K) for CB1 selected from the group composed of on An anti-CB1 antibody having ) and its antigen-binding fragments are provided.

[0128] When measured by surface plasmon resonance, at most about 10 -3 s -1 ; At most about 10 -4 s -1 ; At most about 10 -5 s -1 ; and at most about 10 -6 s -1 The dissociation rate constant (K) for the aforementioned target selected from the group composed of off An anti-CB1 antibody having ) and its antigen-binding fragments are provided.

[0129] At most about 10 -7 M; maximum about 10 -8 M; maximum about 10 -9 M; maximum about 10 -10 M; maximum about 10 -11 M; maximum about 10 -13 M; and as much as 10 -14The present invention provides an anti-CB1 antibody having a dissociation constant (KD) for the aforementioned target selected from the group consisting of M, and antigen-binding fragments thereof. The anti-CB1 antibody and its fragments may have binding affinity Kd values ​​for CB1 in the range of about 0.01 nM to about 500 nM, about 0.02 nM to about 250 nM, about 0.02 to about 200 nM, about 0.05 to about 100 nM, and about 0.05 to about 50 nM. The above antibodies and fragments thereof may have binding affinity Kd values ​​for CB1 in the range of about 500 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 50 pM or less, or about 10 pM or less. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind to CB1 with a Kd value of less than about 15 pM, less than about 10 pM, less than about 8 pM, less than about 6 pM, less than about 4 pM, less than about 2 pM, or less than about 1 pM.

[0130] In some embodiments, the anti-CB1 antibody or its antigen-binding fragment has at least as much efficacy as small molecule CB1 receptor modulators, such as, for example, limonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibifinabant, otenabant, surinabant, tetrahydrocannavivarin, and virodhamin, and AM6545. In some embodiments, the anti-CB1 antibody or its antigen-binding fragment possesses CB1 antagonist or inverse-agonist activity that is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 times greater than that of small molecule CB1 receptor modulators such as, for example, rimonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibifinabant, otenabant, surinabant, tetrahydrocannavivarin, and virodhamin. 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.

[0131] In some embodiments, the anti-CB1 antibody and its antigen-binding fragment have the advantage of reduced or absent BBB penetration or brain exposure. In some embodiments, the BBB penetration of the anti-CB1 antibody and its antigen-binding fragment exhibits reduced brain penetration compared to small molecule CB1 agonists, antagonists, or inverse agonists (e.g., rimonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibifinabant, otenabant, surinabant, tetrahydrocannavivarine, and virodhamin, and AM6545). In some embodiments, the anti-CB1 antibody and its antigen-binding fragment provided herein offer the therapeutic advantage of 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).

[0132] Epitope mapping and related technologies

[0133] The present invention comprises an anti-CB1 antibody that interacts with one or more amino acids found in the extracellular domain of human CB1 (e.g., in amino acids 1-116, and / or in the extracellular loop e1 (amino acids 176-187; sequence identification number: 6), e2 (amino acids 256-273; sequence identification number: 10), and / or e3 (amino acids 366-377; sequence identification number: 14)). The epitope to which the antibody binds may comprise three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) single adjacent sequences located within the extracellular domain of CB1. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within the extracellular domain of CB1. Additionally, the epitope to which the CB antibody binds may include a non-extracellular portion of CB1 due to conformational changes or exposure resulting from binding. The sequences and various domains of CB1 are presented in Table 1.

[0134] Using various techniques known to those skilled in the art, it is possible to determine whether an antibody interacts with "one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for instance, conventional cross-blocking assays, such as those described in *Antibodies, Harlow and Lane* (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation assays, peptide blot assays (Reineke (2004) Methods Mol. Biol. 248: 443-463), and peptide cleavage assays. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen may be used (Tomer (2000) Protein Science 9: 487-496). Another method that can be used to identify the amino acids within the polypeptide with which the antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding the corresponding antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After the antibody dissociates, the target protein undergoes protease cleavage and mass spectrometry analysis, which reveals the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochem. 267(2): 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

[0135] The present invention further comprises an anti-CB1 antibody that binds to the same epitope as any specifically exemplary antibody described herein (e.g., M1, M2, M3, M4, M5 (and its humanized variants), M6, M7 (and its humanized variants), and M8). Similarly, the present invention further comprises an anti-CB1 antibody that competes for binding to CB1 with any specifically exemplary antibody described herein (e.g., M1, M2, M3, M4, M5 (and its humanized variants), M6, M7 (and its humanized variants), and M8).

[0136] Using routine methods known in the art, it is easy to determine whether an antibody binds to the same epitope as a reference anti-CB1 antibody or competes for binding thereto. 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 allowed to bind to the CB1 protein (e.g., the soluble portion of the CB1 extracellular domain or CB1 expressed on the cell surface). Next, the ability of the test antibody to bind to the CB1 molecule is evaluated. If, after saturation binding with the reference anti-CB1 antibody, the test antibody is able to bind to CB1, it can be concluded that the test antibody binds to an epitope different from that of the reference anti-CB1 antibody. On the other hand, if, after saturation binding with the reference anti-CB1 antibody, the test antibody is unable to bind to the CB1 molecule, the test antibody may bind to the same epitope as that to which the reference anti-CB1 antibody of the present invention has bound. Additional routine experiments (e.g., peptide mutation and binding assays) can be performed to determine whether the observed absence of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or if the cause of the observed absence of binding is due to stereoblocking (or other phenomena). These types of experiments may be performed using ELISA, RIA, Biacore, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art. According to specific embodiments of the present invention, if, when measured by a competitive binding assay, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of another antibody by at least 50%, preferably 75%, 90%, or even 99%, then the two antibodies bind to the same (or overlapping) epitope (see, e.g., Junghans et al. (1990) Cancer Res. 50:1495-1502).Alternatively, if all amino acid mutations on an antigen that reduce or remove binding of one antibody essentially reduce or remove binding of another antibody, these two antibodies are considered to bind to the same epitope. If only some subset of amino acid mutations that reduce or remove binding of one antibody reduce or remove binding of another antibody, these two antibodies are considered to have an "overlapping epitope."

[0137] In some embodiments, the present invention provides an anti-CB1 antibody or an antigen-binding fragment thereof that can compete with the antibody or its antigen-binding fragment disclosed herein for binding to CB1. Such antibodies can be identified using conventional competitive binding assays. For example, to determine whether an antibody competes for binding with a reference anti-CB1 antibody, the binding method described above is 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 saturation 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 saturation conditions, and then the binding of the reference antibody to the CB1 molecule is evaluated. In both directions, if only the first (saturated) antibody is able to bind to the CB1 molecule, it is concluded that the test antibody and the reference antibody are competing for binding to CB1. Antibodies competing for binding to a reference antibody do not necessarily have to bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to, for example, overlapping or adjacent epitopes. Competition can be measured by ELISA, flow cytometry, or surface plasmon resonance (SPR) assays. Additionally, cross-competition and epitope binning assays can be performed using the Octet HTX system (Pall ForteBio LLC, Fremont, CA 94538).

[0138] In one embodiment, the anti-CB1 antibody or its antigen-binding fragment comprises 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 respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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 comprises 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 with respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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 comprises 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 with respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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 comprises 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 with respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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 comprises 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 with respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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 comprises 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 with respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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.

[0139] The heavy chain and light chain CDRs of the anti-CB1 antibody provided herein may be independently selected and may be matched to form an antibody or its antigen-binding fragment comprising any heavy chain CDR1, CDR2, and CDR3 of the antibody provided herein; and any light chain CDR1, CDR2, and CDR3. The heavy chain variable region and light chain variable region of the antibody provided herein may be independently selected and may be matched to form an antibody or its antigen-binding fragment comprising any heavy chain and light chain of the antibody provided herein.

[0140] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment comprises a variable heavy chain (VH) 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 respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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.

[0141] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment comprises a variable light chain (VL) 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 respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 24, 36, 48, 60, 72, 84, 84, 96, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, and 324.

[0142] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment comprises 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 respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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. In another embodiment, the anti-CB1 antibody or its antigen-binding fragment comprises 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 with respect to an amino acid sequence selected from the group consisting of sequence identification numbers: 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.

[0143] In certain embodiments, the anti-CB1 antibody or its antigen-binding fragments, CDRs, VH, VL, heavy chain and / or light chain comprises 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.

[0144] 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, 177, 178); (188, 189, 190); (200, 201, 202); It includes a set of VH CDRs having amino acid sequences selected from the group consisting of (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).

[0145] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment is sequence identification 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, 183, 184); (194, 195, 196); (206, 207, 208); It includes a set of VL CDRs having amino acid sequences selected from the group consisting of (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).

[0146] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment is composed of sequence identification numbers: 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 It includes a set of VH / VL having amino acid sequences selected from the group.

[0147] In another embodiment, the anti-CB1 antibody or its antigen-binding fragment is sequence identification number: 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 It includes a set of heavy chains and a set of light chains having amino acid sequences selected from the group consisting of 317 / 323.

[0148] In some embodiments, the anti-CB1 antibody or its antigen-binding fragment binds to CB1 and exhibits reduced agent functions, such as, e.g., C1q binding, complement-dependent 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 comprises one or more modifications of the Fc region that reduce, impair, or eliminate one or more agent functions. For example, in one embodiment, the anti-CB1 antibody and its antigen-binding fragment disclosed herein bind to CB1 but exhibit reduced, impaired C1q binding and / or CDC and / or ADCC, or none of these. The Fc modification may be an amino acid insertion, deletion, or substitution, or a chemical modification. For example, Fc region modifications may be made to increase or decrease complement binding, increase or decrease ADCC or CDC, or alter glycosylation. Various Fc modifications are known in the art, for example, 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 Fc modification known in the art may be applied to the exemplary CB1 antibodies disclosed herein to alter agent function.In a specific example, the anti-CB1 antibody or its antigen-binding fragment may possess specific mutations, e.g., L234A / L235A ("LALA"), S228P, A330S, P331S, E233P / L234V / L235A, A327G / A330S / P331S, L234F / L235E / P331S, and N297Q.

[0149] The binding proteins provided herein can be produced by any number of techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding CB1 binding proteins are transfected into host cells by standard techniques. Although it is possible to express the CB1 binding proteins provided herein in prokaryotic or eukaryotic host cells, mammalian host cells fold more appropriately than prokaryotic cells and are more likely to assemble and secrete immunologically active binding proteins.

[0150] In an exemplary system for the recombinant expression of CB1 binding proteins, a recombinant expression vector encoding both the heavy and light chains of a CB1 antibody is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the CB1 antibody heavy and light chain sequences are operably linked to a CMV enhancer and promoter regulatory element, respectively, to induce high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, which enables the selection of CHO cells transfected with the vector using methotrexate screening / amplification. Selected transfected host cells are cultured to allow the expression of the CB1 antibody heavy and light chains, and intact CB1 antibody proteins are recovered from the culture medium. Using standard molecular biology techniques, the recombinant expression vector is prepared, host cells are transfected, transfected, host cells are selected, and CB1 antibody proteins are recovered from the culture medium.

[0151] Bioequivalents

[0152] The anti-CB1 antibodies and antibody fragments thereof described herein comprise proteins having amino acid sequences that possess the ability to bind to human CB1, although these sequences are variable from those of the described antibody. These variant antibodies and antibody fragments include one or more additions, deletions, or substitutions of amino acids when compared to the parental sequence, but exhibit biological activity that is fundamentally equivalent to that of the described antibody. Similarly, the anti-CB1 antibody encoding the DNA sequence of the present invention encodes an anti-CB1 antibody or antibody fragment that is fundamentally biologically equivalent to the anti-CB1 antibody or antibody fragment of the present invention, although it includes one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence. Examples of such variant amino acids and DNA sequences are discussed above.

[0153] Two antigen-binding proteins or antibodies are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in absorption rate and degree of absorption when administered, for example, at the same molar dose (whether a single dose or multiple doses) under similar experimental conditions. Some antibodies will be considered equivalents or pharmaceutical substitutes if they have the same degree of absorption but different absorption rates, and if these differences in absorption rates are intentional and reflected in the labeling, and are not essential to reaching an effective body drug concentration in chronic use, for example, and are considered medically insignificant in the specific drug product being studied, and thus can be considered bioequivalent.

[0154] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in safety, purity, and efficacy. In one embodiment, two antigen-binding proteins are bioequivalent if, when switching one or more times between the reference product and the biological product, the switching can be made without an expected increase in the risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy in the patient, compared to receiving continuous treatment without such switching. In one embodiment, two antigen-binding proteins are bioequivalent if both act to a known degree through a common mechanism or mechanism of action for the conditions of use or conditions.

[0155] Bioequivalence may be demonstrated by in vivo and in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo tests in humans or other mammals, wherein the concentration of the antibody or one or more of its metabolites is measured in blood, plasma, serum, or other biological fluids as a function of time; (b) reasonably predictable in vitro tests related to human in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials demonstrating the safety, efficacy, bioavailability, or bioequivalence of the antibody.

[0156] Bioequivalent variants of anti-CB1 antibodies may be constructed, for example, by performing various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In another context, bioequivalent antibodies may include anti-CB1 antibody variants containing amino acid changes that modify the glycosylation properties of the antibody, for example, mutations that eliminate or remove glycosylation.

[0157] Species selectivity and species cross-reactivity

[0158] The present invention also comprises an anti-CB1 antibody that binds to human CB1 and binds to CB1 of one or more non-human species. For example, the anti-CB1 antibody of the present invention may bind to human CB1 and may bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomylgus monkey, marmoset, rhesus monkey, or chimpanzee CB1. According to specific embodiments of the present invention, the anti-CB1 antibody binds to human CB1 but does not bind to CB1 of other species.

[0159] Immunoconjugates

[0160] The present invention comprises therapeutic moiety ("immunoconjugate"), such as an anti-CB1 antibody conjugated to a cytotoxic agent, a chemotherapy drug, an immunosuppressant, or a radioisotope. Cytotoxic agents include any agent harmful to cells. Examples of cytotoxic agents and chemotherapy agents suitable for forming immunoconjugates are known in the art and are described herein.

[0161] Multispecific binding proteins

[0162] The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different epitopes of a single target polypeptide, or may contain antigen-binding domains specific to one or more target polypeptides. 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 may be linked to another functional molecule, for example, another peptide or protein, or co-expressed with them. For example, an antibody or a fragment thereof may be functionally linked to one or more other molecular entities, such as another antibody or antibody fragment (e.g., chemical binding, genetic fusion, non-covalent association, or others), to form a bispecific or multispecific antibody having a second binding specificity. For example, the present invention comprises a dual-specific antibody, wherein 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 conjugated to a therapeutic moiety.

[0163] Uses of binding proteins in various diseases

[0164] The antibodies and binding proteins of the present invention are useful for treating, preventing, or improving any disease or disorder associated with or mediated by CB1 expression or activity, or a disease or disorder treatable by blocking the interaction between CB1 and CB1 ligands (e.g., cannabinoids), or otherwise a disease or disorder treatable by inhibiting CB1 activity and / or signaling, and / or promoting receptor refractoryization and / or reducing the number of cell surface receptors. The term "disorder where CB1 activity is harmful" means a disorder or disease where the presence or activity of CB1 (e.g., abnormal or over-activated) in a subject suffering from the disorder causes the pathophysiology of the disorder or contributes to the exacerbation of the disease. Thus, a disorder where CB1 activity is harmful is a disorder where a reduction in CB1 activity is expected to alleviate the symptoms and / or progression of the disorder.

[0165] 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 harmful. For example, the binding molecules provided herein include any disease or condition characterized by the overexpression of CB1, upregulation or increased activity of its signaling, or failure of healthy homeostatic regulatory mechanisms resulting therefrom. Such diseases and conditions include obesity, syndromic obesity including Prader-Willi syndrome, Alstrom syndrome, Bardet-Biedel syndrome (BBS), Albright genetic osteogenesis imperfecta (AHO), and SIM1 deficiency syndrome; diabetes mellitus and related complications; dyslipidemia; liver diseases such as, for example, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, and primary biliary cirrhosis; fibrosis, for example, renal fibrosis; chronic kidney disease; kidney disease; It includes metabolic diseases, osteoporosis, atherosclerosis, inflammatory diseases, cardiovascular diseases, cancer, pain, systemic sclerosis, multiple sclerosis, seizures, glaucoma, and nicotine addiction.

[0166] Pharmaceutical composition

[0167] The present invention provides a pharmaceutical composition comprising the anti-CB1 binding protein, e.g., an antibody or an antigen-binding fragment thereof. The pharmaceutical composition of the present invention is formulated with suitable excipients, carriers, prophylactic agents, therapeutic agents, and other agents that improve the stability, delivery, tolerance, and efficacy of the anti-CB1 binding protein. Various suitable formulations can be found in prescription books known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, 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 carbowax. See also Powell et al. (1998) J. Pharm. Sci. Technol. 52: 238-311.

[0168] The pharmaceutical composition comprising the CB1 binding protein provided herein is intended for use in diagnosing, detecting, or monitoring a disorder, and in preventing, treating, managing, or improving one or more symptoms of the disorder or the disorder, and / or studying them, but is not limited thereto.

[0169] Various delivery systems are known and may be used to administer the pharmaceutical composition of the present invention, such as liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and encapsulation in receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). The composition may be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucosal walls (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biological active agents. Administration may be systemic or local.

[0170] The methods of administration of the prophylactic or therapeutic agents provided herein include, but are not limited to, non-oral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, intratumoral administration, mucosal administration (e.g., nasal and oral routes), and pulmonary administration (e.g., aerosolized compounds administered by an inhaler or nebulizer). Formulations of pharmaceutical compositions for specific routes of administration, and materials and technologies required for various methods of administration, are available and are known to those skilled in the art.

[0171] Dosage formulation may be adjusted to provide an 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 directed according to the urgency of the treatment situation. It is particularly desirable to formulate parenteral compositions in dosing unit forms for ease of administration and uniformity of dosage. The term “dosing unit form” means a unit that is physically discrete and suitable as a single dose for a 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. Specifications of dosing unit forms in this specification depend on (a) the unique characteristics of the active compound and the specific therapeutic or prophylactic effect to be achieved, and (b) limitations inherent in the field of formulating such active compounds for the treatment of individual sensitivities.

[0172] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, a pen delivery device is readily applicable for delivering the pharmaceutical composition of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally use a replaceable cartridge containing the pharmaceutical composition. When all the pharmaceutical composition in the cartridge is administered and the cartridge is empty, the empty cartridge is immediately discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0173] A number of reusable pens and automatic syringe delivery devices are applied to the subcutaneous delivery of the pharmaceutical composition 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™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices for use in the subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ automatic syringe (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA™ pen (AbbVie, Inc., Abbott Park, IL).

[0174] In certain situations, pharmaceutical compositions may be delivered to a controlled-release system. In one embodiment, a pump may be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: 201-240). In another embodiment, polymeric materials may 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 may be placed near the target of the composition, so only a portion of the systemic dose is required (Goodson (1984) in Medical Applications of Controlled Release, supra, 2: 115-138). Other controlled-release systems were discussed in a review in Langer (1990) Science 249: 1527-1533.

[0175] Preparations for injection may include administration forms for intravenous, subcutaneous, intradermal, and intramuscular injection, drip infusion, etc. Such preparations for injection may be prepared by known methods. Preparations for injection may be prepared, for example, by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous or oily medium commonly used for injection. Examples of aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), and non-ionic 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 injection solution prepared in this way is preferably filled into a suitable ampoule.

[0176] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a unit dose dosage form suitable for the dose of the active ingredient. Such dosage forms in unit dose 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 dosage form in a unit dose; particularly in the injection form, the antibody is about 5 to about 100 mg, and in other dosage forms, about 10 to about 250 mg.

[0177] The dose of the antibody administered to a patient may vary depending on the patient's age and physique, target disease, disease stage, gender, presence of medical complications, other medications, conditions, route of administration, etc. The preferred dose is generally calculated based on body weight or body surface area. When the antibody of the present invention is used to treat conditions or diseases associated with CB1 activity in adult patients, it may generally be advantageous to administer the antibody of the present invention intravenously at a single dose of about 0.01 to about 100 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. The effective dose and schedule for administering the anti-CB1 antibody may be determined empirically; for example, the patient's progress may be monitored through periodic evaluations, and the dose may be adjusted accordingly. Furthermore, interspecies scaling of the dose may 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 specific subject, a specific dosage regimen may be adjusted over time according to the personal needs and professional judgment of the person administering or managing the administration of the composition, and that the dosage ranges presented herein are merely illustrative and are not intended to limit the scope or practice of the claimed composition.

[0178] Combination Therapy

[0179] The binding protein provided herein may also be administered together with one or more additional therapeutic agents useful for the treatment of various diseases, and the additional agents are selected by those skilled in the art for the intended purpose. For example, the additional agents may be therapeutic agents recognized in the art as useful for the treatment of the disease or condition treated by the CB1 binding protein provided herein. Such combinations may also include one or more additional agents.

[0180] Non-limiting examples of these additional therapeutic active ingredients include other CB1 antagonists (e.g., CB1 second-generation anti-CB1 antibodies or small molecule inhibitors (e.g., rimonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibifinabant, otenabant, surinabant, tetrahydrocannavivarine, and virodhamin, and AM6545), and antagonists of other CB1 family members.

[0181] The present invention also comprises therapeutic combinations comprising any anti-CB1 antibody and additional inhibitors mentioned herein, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibodi, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, e.g., diabodies, triabodidies, testabodidies, minibodies, and minimal recognition units). The anti-CB1 antibody of the present invention may also be administered in combination with and / or co-formulated with additional therapeutic agents. Additional therapeutic active ingredient(s) may be administered immediately, concurrently, or immediately after the administration of the anti-CB1 antibody of the present invention; (for the purposes of this disclosure, such administration regimen is considered as the administration of the anti-CB1 antibody “combined” with additional therapeutic active ingredients). The present invention comprises a pharmaceutical composition in which the anti-CB1 antibody of the present invention is co-formulated with one or more of additional therapeutic active ingredients as described elsewhere in this invention.

[0182] The present invention also includes a composition and a method comprising a combination of an "antagonist antibody" and a "reverse-agonist antibody." An "antagonist anti-CB1 antibody" means an anti-CB1 antibody that inhibits, reduces, or prevents the signal transduction activity of a ligand (e.g., cannabinoid) for CB1. Non-limiting examples of the 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. "Reverse-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 receptor activity above baseline levels, a reverse-agonist decreases activity below baseline levels. Non-limiting examples of the reverse-agonist antibodies of the present invention include M7. The inventors have considered combining an antagonist antibody and a reverse-agonist antibody to enhance efficacy, either synergistically or otherwise. Accordingly, the present invention comprises a pharmaceutical composition comprising at least one antagonist antibody and at least one reverse-agonist antibody. The present invention also comprises a therapeutic method comprising administering a combination of an antagonist antibody and a reverse-agonist antibody to a subject (as a separate administration or as a co-formulation).

[0183] Combination therapy agents include, but are not limited to, antineoplastic agents, radiotherapy, DNA alkylating agents, cisplatin, carboplatin, anti-tubulin agents, 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), chemotherapy agents such as kinase inhibitors and siRNA.

[0184] Diagnostics

[0185] The disclosure of the present invention provides diagnostic applications including, but not limited to, a diagnostic test method, a diagnostic kit containing one or more CB1 binding proteins, and adaptations of the method and kit for use in automated and / or semi-automatic systems. The provided method, kit and adaptation may be used for the detection, monitoring, and / or treatment of diseases or disorders in individuals.

[0186] The anti-CB1 antibody of the present invention may 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 may be used to diagnose a pathology or disease characterized by abnormal expression of CB1 (e.g., over-expression, under-expression, lack of expression, etc.). An exemplary diagnostic assay for CB1 may include, for example, contacting the anti-CB1 antibody of the present invention with a sample obtained from a patient, wherein the anti-CB1 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-CB1 antibody may be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. Suitable detectable materials include various enzymes, prosthetic groups, fluorescent materials, chemiluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkali phosphatase, β-galactosidase, luciferase, and acetylcholinesterase; examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorothiazinylamine fluorescein, danyl chloride, and phycoerythin. An example of a luminescent material is luminol, and examples of suitable radioactive materials are, for instance, 3 H, 14 C, 32 P, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, and 153 Includes Sm.

[0187] The immunoassays provided by the present disclosure may include, among others, sandwich immunoassays, radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), competitive-inhibitory immunoassays, fluorescence polarization immunoassays (FPIA), enzyme multiplexed assay techniques (EMIT), bioluminescence resonance energy transfer (BRET), fluorescence-activated cell sorting (FACS), and homogeneous chemiluminescence assays.

[0188] A chemiluminescent microparticle immunoassay using the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, IL) can be used.

[0189] A method of using mass spectrometry is provided by the present invention and includes, but is not limited to, MALDI (matrix-assisted laser desorption / ionization) or SELDI (surface-enhanced laser desorption / ionization).

[0190] Methods for collecting, handling, processing, and analyzing biological test samples using immunoassay and mass spectrometry are well known to those skilled in the art.

[0191] Kit

[0192] A kit is also provided for analyzing a test sample for the presence, amount, or concentration of an analyte or a fragment thereof in the test sample. The kit comprises one or more components for analyzing a test sample for an analyte or a fragment thereof, and instructions for analyzing a test sample for an analyte or a fragment thereof. At least one component for testing a test sample for an analyte or a fragment thereof comprises, as described herein, a composition comprising a binding protein and / or an anti-analyte binding protein (or a fragment, variant, or fragment of a variant thereof), which are optionally immobilized on a solid.

[0193] Optionally, the kit may include a calibrator or a control, which may include an isolated or purified analyte. The kit may include at least one component for analyzing a test sample of the analyte by immunoassay and / or mass spectrometry. Kit components comprising the analyte, binding protein, and / or anti-analyte binding protein, or fragments thereof may optionally be labeled using any detectable label known in the art. The materials and methods for production provided for carrying out the present disclosure may be known to those skilled in the art.

[0194] The assay method described in the present invention, such as a kit (or its components) for measuring the presence, amount, or concentration of an analyte in a test sample using an immunoassay, as well as the method, may be adapted for use in various automated and semi-automated systems (those in which the solid phase contains microparticles), such as, for example, US Patent Nos. 5,089,424 and 5,006,309, and commercially available, for example, ARCHITECT® of Abbott Laboratories (Abbott Park, IL). Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (e.g., US Patent No. 5,294,404, PRISM®, EIA (Beads), and Quantum™ II. Additionally, the assays, kits, and kit components may be used in other forms, for example, in electrochemical or other portable or point-of-care assay systems. For example, this specification is applicable to commercial Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay systems that perform sandwich immunoassays. For example, an immunosensor and its manufacture and operation method in a disposable test device are described in US Patent Nos. 5,063,081, 7,419,821, 7,682,833, 7,723,099, and 9,035,027; and US Publication No. 20040018577 and is explained in 20060160164.

[0195] Other suitable modifications and adaptations of the methods described herein will be apparent to those skilled in the art, and it will also be apparent to those skilled in the art that such modifications and adaptations can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Although specific embodiments have been described in detail, the practice of the invention will be more fully understood from the following embodiments, which are presented herein merely for illustrative purposes and should not be construed as limiting the invention in any way.

[0196] Examples

[0197] Example 1: Generation and Screening of Anti-CB1 Antibodies for Functional Evaluation

[0198] To potentially stabilize the protein to provide it in an appropriate stereotype during immunization, CD2F1 mice were immunized with human CB-1 antigen in the presence or absence of the CB-1 antagonist JD5037 (Cayman Chemical, Ann Arbor, MI; catalog number 1205). Mice were immunized on both hocks with approximately 5 μg / 30 μl of antigen per hock using Titer Max Gold adjuvant (St. Loui, MO; Sigma Aldrich; catalog number T2684). Subsequently, mice were immunized twice a week for approximately 30 days with CpG (InvivoGen, San Diego, CA; catalog number ODN1826) and Alhydrogel (InvivoGen, San Diego, CA; catalog number vac-alu-250). Serum was harvested at 13 and 26 days to determine antibody titers and increases over time. Mice were euthanized at 30 days, popliteal and inguinal lymph nodes were collected for fusion, washed in Media B (a 1:1 mix of RPMI 1640 (Thermo Fisher Scientific-Gibco, San Diego, CA; catalog number 11879020) and IMDM (Lonza, Anaheim, CA; catalog number 12-722F) without the addition of other nutrients, and a single-cell suspension was prepared. P3Ag8.563 myeloma cells (ATCC, Manasas, VA; catalog number PTA-9393) were harvested from the culture and washed in Media B. Lymphoma cells and myeloma cells were mixed in a 1:1 ratio and fused using the BTX Harvard Apparatus ECM 2001 (BTX Harvard Apparatus, Holliston, MA; catalog number 45-0012).Recover the fused cells and resuspend them in Media C (Stem Cell Technologies, Seattle, WA; Catalog No. 03803), and freeze overnight at 37°C in T75 cm. 2 The cells were retrieved from the flask. The next day, the fused cells were collected and resuspended in hybridoma screening methylcellulose Media D (Stem Cell Technologies, Seattle, WA; Catalog No. 03804) containing anti-mouse IgGFITC clone detection (Molecular Devices, San Jose, CA; Catalog No. K8220). The cells were mixed, and 1 x 10⁶ cells were added per 10 mL of Media D. 6The cells were plated. The plated cells were incubated at 37°C for 7 days. Hybridoma colonies were selected based on colony size and the ability to display a potent FITC halo indicating IgG production, and were transferred to 96-well tissue culture plates containing hybridoma growth media E (Stem Cell Technologies, Seattle, WA; catalog number 03805) using clone Pix2 (Molecular Devices, San Jose, CA). When macroscopic colonies were observed, the supernatant was screened for cell binding using CHO paternal cells and CHO-huCB-1 overexpressing cells. For this primary screening, hybridoma paternal CHO cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Anaheim, CA; catalog number 34554) and mixed with non-CFSE-labeled CHO-huCB1 overexpressing cells to allow for efficient and simultaneous screening of both cell lines and to identify huCB-1 specific binders. Clones that specifically bound to CHO-huCB-1 overexpressing cells and not to paternal CHO cells were selected and advanced for confirmation screening. A total of 97 clones were selected to proceed with small-scale purification. A summary of the relevant fusion and primary screening is shown in Table 2.

[0199]

[0200] Example 2: Purification of a murine anti-huCB-1 specific binding clone

[0201] The hybridoma clone selected based on the primary screen for verification is T75 cm 2It was further extended in 50 mL of low Ig medium (1:1 IMDM (Lonza, Anaheim, CA; Catalog No. 12-722F): Ham's F12-K (Gibco, Anheim, CA; Catalog No. 21127022) medium containing 5 mL of 100 mM sodium pyruvate solution (Invitrogen, Grand Island, NY; Catalog No. 11360070), 5 mL of 100 mM non-essential amino acids (Invitrogen, Grand Island, NY; Catalog No. 11140050), and 5 mL of 100 mM glutamine (Invitrogen, Grand Island, NY; Catalog No. 35050061)) in flasks for 3-4 weeks. Using standard protein A purification methods, the supernatant was harvested and purified.

[0202] Example 3: Functional characterization of mouse anti-CB1 antibody in cAMP assay

[0203] The antagonist activity of the isolated mouse anti-huCB-1 antibodies was evaluated using the cAMP assay. The cAMP assay was performed using the cAMP Hunter™ CHO-K1 CNR1 Gi cell line (DiscoverX / Eurofins, Fremont, CA; catalog number 95-0071C2), which overexpresses naturally Gi-linked wild-type G-protein-linked receptors (GPCRs) and is designed to detect increases in intracellular cAMP levels in response to agonist stimulation of the corresponding receptors. cAMP Hunter™ CHO-K1 CNR1 Gi cells were treated with the CB1 antibody, an isotype control, or the small molecule CB1 antagonist JD5037, followed by an agonist challenge with 30 nM CP-55,940 (indicated as "Plus CP") in the presence of forskolin. The antagonist was also tested without the addition of CP-55,940 to determine whether it possessed intrinsic agonist activity. Forskolin activates the enzyme adenylate cyclase and increases intracellular levels of cAMP. In the case of Gi receptors, agonist binding inhibits intracellular cAMP accumulation induced by forskolin. Therefore, to measure Gi-linked receptors, the agonist compound CP-55,940 was added in the presence of forskolin. Consequently, Gi-linked receptors inhibit forskolin-induced production of cAMP, and as a result, the dose-response curve generated in the presence of the agonist + forskolin will have a negative slope. Briefly, cells were placed at 1.5 x 10⁶ per well in Cell Plating 2 Medium (DiscoverX / Eurofins; Fremont, CA; Catalog No. 93-0563R2A) in a 96-well plate (Costar, Fisher Scientific, San Diego, CA; Catalog No. 3909). 4Seed at the cell level and incubated overnight at 37℃ and 5% CO2. The following day, the culture medium was replaced with 30 μl of Cell Assay Buffer (CAB; 1x HBSS / 10 nM HEPES (ThermoFisher, Anaheim, CA; catalog numbers 14025134 and 15630080, respectively) and treated with the test antibody or isotype control (7.5 μl of 6x concentrated working dilution). The plates were incubated at 37°C, 5% CO2 for 30 minutes. 7.5 μl of challenge agent (0.18 μM CP 55,940 in CAB containing 90 μM forskolin) was added to each well, and the plates were incubated again at 37°C, 5% CO2 for 30 minutes. Following the manufacturer's instructions, HitHunter® cAMP Assay Detection Kit for Biologies (DiscoverX / Eurofins; Fremont, CA; catalog number Plates were processed for cAMP reading using 90-0075LM25. Initial evaluation of the isolated clones was performed at a single concentration of 30 μg / mL.

[0204] Of the 112 clones tested, only 8 clones exhibited antagonistic activity. 2 clones showed some degree of antagonism in the absence of the agonist, M3, and in the case of M1, at a lower level. The 8 antibodies M1, M2, M3, M4, M5, M6, M7, and M8 were further evaluated by titrating Abs concentrations to obtain dose-response curves and actual EC50 values ​​(Table 3 and Figure 1).

[0205] Example 4: Functional characterization of mouse anti-CB1 antibody in pERK assay

[0206] The eight antibodies acting as functional antagonists in the cAMP assay were further evaluated in the pERK phosphorylation assay performed using the cAMP Hunter™ CHO-K1 CNR1 Gi Cell Line (DiscoverX / Eurofins, Fremont, CA; Catalog No. 95-0071C2). Briefly, cells were cultured in 96-well plates at 2 x 10⁶ cells per well in Kit-107 Assay Complete Cell Culture Medium (DiscoverX / Eurofins, Fremont, CA; Catalog No. 92-3107G) + 800 μg / mL G418. 4 Cellular seeding was performed, and the plates were incubated at 37°C and 5% CO2. The following day, the culture medium was replaced with 100 μl of serum-free F-12K starvation medium per well (Invitrogen, Grand Island, NY; catalog number 11765054). The plates were incubated at 37°C and 5% CO2 for an additional day. On the day of treatment, the F-12K medium was replaced with 30 μl of freshly prepared F-12K medium per well. The test antibody or isotype control (7.5 μl of 6x concentrated working dilution) was added to the wells, and the plates were incubated at 37°C and 5% CO2 for 10 minutes. 7.5 μl of agent (a 6-fold working solution containing 90 μM forskolin and 0.18 μM CP 55,940 in CAB) was added to each well, and the plates were incubated again at 37°C, 5% CO2 for 10 minutes. The plates were treated for p-ERK / total ERK using the Meso Scale Discovery (MSD) kit (Meso Scale Discovery, Rockville, Maryland; catalog number K15107D) according to the manufacturer's instructions (Table 3 and Figure 2).

[0207]

[0208] Example 5: Mouse anti-CB-1 antibody bound to EC50 in CB-1 CHO overexpressing cells

[0209] To obtain binding curves and EC50 values, antibody binding was tested in a fluorescence-activated cell sorting (FACS)-based assay for the binding ability of mouse anti-CB1 antibodies to paternal CHO cells, human CB-1 overexpressing CHO cells, and mouse CB-1 overexpressing CHO cells. Three cell lines were collected, washed, and 1 x 10⁶ cells per well in 50 μl of FACS buffer (1X PBS / 2mM EDTA and 1% FBS (ThermoFisher Scientific, Anaheim, CA; Catalog No. 10438-026) in V-bottom 96-well polycarbonate FACS plates (Corning, Corning, NY; Catalog No. 3357). 5 The antibodies were distributed to the cells. Serial dilutions of the antibody were prepared starting at 200 nM and increasing to 2X concentrations, and were serially diluted three-fold. The titrated antibodies were added to plates containing three different cell lines (paternal, human, and mouse CB-1 CHO cells) and incubated at 4°C for 1 hour. The plates were washed 3X with FACS buffer. The cells were resuspended in 50 μl of 1 / 5,000 dilution goat anti-mouse IgG-HRP (Jackson Immuno Research, West Grove, PA; Catalog No. 115-035-003), incubated at 4°C for 30 minutes, washed 3X with FACS buffer, and data were collected using BD FACS Canto (BD Biosciences, San Jose, CA; Catalog No. 338962) and analyzed using FlowJo (FlowJo LLC, Ashland, OR) (Table 4). There was no mouse cross-reactivity among the 8 functional antibodies tested (Fig. 3).

[0210]

[0211] Example 6: EC50 Analysis and Stereotypic Binding Evaluation of Functional Anti-CB-1 Antibody

[0212] The purpose of this experiment was to determine whether anti-CB1 antagonist antibodies possess differential binding ability to CB1 in neutral, antagonist, or agonist states. Four different cell line preparations were used: CHO-huCB1 (in-house prepared), CHO-huCB1 pre-incubated with the inverse agonist JD5037 (Cayman Chemicals, Ann Arbor, MI; catalog number 1392116-14-1), CHO-huCB1 pre-incubated with the agonist CP-55,940 (TOCRIS, Minneapolis, MN; catalog number 0949), and paternal CHO-S cells (ThermoFisher Scientific, VA; catalog number R80007). 2 x 10⁶ 7 Paternal CHO-S cells and CHO-hu CB1 cells were kept separately in FACS buffer. Additionally, 2 x 10⁶ cells coated with the inverse agonist JD5037 or the agonist CP-55,940 7 CHO-huCB1 cells were incubated at 4°C for 1 hour. After incubation, two coated cell lines were washed 2x in FACS buffer and, in turn, 2 x 10⁶ in FACS buffer containing either an inverse agonist or an agonist molecule. 7They were resuspended. All four cell lines were plated on V-bottom FACS plates (Corning, Corning, NY; Catalog No. 3357), and pre-titrated anti-CB1 test antibodies were added to the cells to evaluate binding using BD FACS Canto (BD Biosciences, San Jose, CA; Catalog No. 338962). As shown in Figure 4, the antibodies did not bind to CHO paternal cells (blue curve), and the eight functional antibodies (M1, M2, M3, M4, M5, M6, M7, and M8) did not exhibit preferential binding in the presence of agonists or antagonists, as indicated by the binding observed under all conditions (red, purple, and green curves). Alternatively, in the cAMP or pERK assays, only two non-functional test antibodies showed preferential binding in the presence of an antagonist and in the absence of an agonist, respectively. This suggests that functional anti-CB1 antibodies may not be associated with the binding patterns that occur in the presence of known receptor agonists or antagonists.

[0213] Example 7: Sequence identification and analysis of mouse anti-CB1 antibody

[0214] Hybridomas of eight murine anti-huCB-1 antibodies were harvested 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; Catalog No. 37503). Four of the antibodies (M1, M3, M4, and M6) were IgG2a,K, and four antibodies (M2, M5, M7, and M8) were IgG2b,K. The pellets were processed for RNA and cDNA, and the cDNA was processed for sequencing using a SMARTER RACE Amplification kit (Clontech, Mountain View, CA; Catalog No. 634859). Using the isotypes of each antibody, reverse primers for the constant region of the heavy chain and forward primers for the light chain kappa constant region and SeqAmp polymerase (CloneTech, Mountain View, CA; Catalog No. 638504) were designed. MOPC21 PNA primers (synthesized based on the sequence) were included to prevent anomalous light chain amplification, which often appears during sequencing 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 provided in Table 5. The consensus sequences of the heavy and light chains of the hybridoma antibodies are provided in Figures 5A and 5B, respectively.

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] Example 8: Selection of mouse anti-CB1 antibodies for humanization

[0224] Based on functional data, mouse anti-huCB-1 clones M7 and M5 were selected for humanization using a predicted humanization tool derived from the PHEnon™-Software 5 package (Xoma, Berkley, CA) (US Patent No. 5,766,886). VH and VL sequences for each clone were submitted as queries, and output sequences were generated based on the closest human germline matches in the Kabat database. To evolve the VH and VL sequences toward human framework matches, a list of mutations in the framework region was created. The mutation risk of individual residues was evaluated through a series of criteria (US Patent No. 5,766,886). Cumulatively, mutations were grouped to form "low-risk" and "medium-risk" clone pools. The output sequences and introduced mutations were subjected to homology modeling in a virtual environment ( in silico) It was verified in [location]. 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 methods. The sequences of the humanized clones are provided in Table 6. The consensus sequences of the heavy and light chains of the humanized M7 and M5 antibodies are provided in Figures 6A and 6B, respectively.

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] Example 9: Re-evaluation of Humanized Anti-CB1 Antibody in Cell Binding and Functional Analysis

[0242] The humanized anti-CB1 variants were re-evaluated for their ability to bind to CHO-huCB-1 cells and compared with mouse paternal clones M5 and M7 to confirm whether binding was maintained (Figs. 7A and 7B). The assay conditions used were similar to those described in Example 5. After the binding assay, the test variants were evaluated for their function as antagonists in cAMP and pERK assays according to Examples 3 and 4 to confirm whether binding and activity were maintained after humanization (Figs. 8A, 8B, 9A, and 9B). All 10 clones maintained their binding and antagonist activities. Fig. 10 shows the inverse activity indicated by the humanized anti-CB1 Ab M7-H5 and IgG2b formats.

[0243] Equivalents

[0244] This specification may be embodied in other specific forms without departing from its spirit or essential nature. Therefore, the foregoing embodiments should be regarded as illustrative rather than limiting the invention in all aspects described in the specification. Accordingly, the scope of this specification is defined by the appended claims rather than by the foregoing description, and thus all modifications within the meaning and equivalent scope of the claims are intended to be included therein.

Claims

Claim 1 Six complementarity determining regions (CDRs): isolated antibodies or antigen-binding fragments thereof that bind to human cannabinoid type 1 receptor (CB1) (sequence identification number: 1), comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3: wherein CDR-H1 consists of the amino acid sequence SYTFTRYW (sequence identification number: 116); CDR-H2 consists of the amino acid sequence IDPYDSET (sequence identification number: 117); CDR-H3 consists of the amino acid sequence ARSQPRYYAMDY (sequence identification number: 118); CDR-L1 consists of the amino acid sequence QEISGF (sequence identification number: 122); and CDR-L2 consists of the amino acid sequence AAS (sequence identification number: 123); And CDR-L3 is composed of the amino acid sequence: LQYSSYPYT (sequence identification number: 124); or CDR-H1 is composed of the amino acid sequence GYEFSYYW (sequence identification number: 308); CDR-H2 is composed of the amino acid sequence IYPGDGDT (sequence identification number: 309); CDR-H3 is composed of the amino acid sequence ARGREAAWFAY (sequence identification number: 310); CDR-L1 is composed of the amino acid sequence QSVSSFRYSY (sequence identification number: 314); CDR-L2 is composed of the amino acid sequence YAS (sequence identification number: 315); and CDR-L3 is composed of the amino acid sequence QHSWEIPFT (sequence identification number: 316). Claim 2 The isolated antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises heavy chain and light chain CDRs of variable heavy chain (VH) / variable light chain (VL) pairs selected from the group consisting of sequence identification numbers: 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. Claim 3 In claim 1, the antibody or its antigen-binding fragment is sequence identification number: (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, 184); (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, 267, 268); (272, 273, 274 / 278, 279, 280); (284, 285, 286 / 290, 291, 292); Isolated antibodies or their antigen-binding fragments comprising pairs (HCDR set / LCDR set) selected from the group consisting of (296, 297, 298 / 302, 303, 304); (308, 309, 310 / 314, 315, 316); and (320, 321, 322 / 326, 327, 328). Claim 4 The isolated antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises an HC / LC pair selected from the group consisting of sequence identification numbers: 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, 293 / 299, 305 / 311, and 317 / 323. Claim 5 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 114 and VL is presented as sequence identification number:

120. Claim 6 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 126 and VL is presented as sequence identification number:

132. Claim 7 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 138 and VL is presented as sequence identification number:

144. Claim 8 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 150 and VL is presented as sequence identification number:

156. Claim 9 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 162 and VL is presented as sequence identification number:

168. Claim 10 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 174 and VL is presented as sequence identification number:

180. Claim 11 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 186 and VL is presented as sequence identification number:

192. Claim 12 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 198 and VL is presented as sequence identification number:

204. Claim 13 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 210 and VL is presented as sequence identification number:

216. Claim 14 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 222 and VL is presented as sequence identification number:

228. Claim 15 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 234 and VL is presented as sequence identification number:

240. Claim 16 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 246 and VL is presented as sequence identification number:

252. Claim 17 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 258 and VL is presented as sequence identification number:

264. Claim 18 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 270 and VL is presented as sequence identification number:

276. Claim 19 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 282 and VL is presented as sequence identification number:

288. Claim 20 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 294 and VL is presented as sequence identification number:

300. Claim 21 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 306 and VL is presented as sequence identification number:

312. Claim 22 In claim 2, the isolated antibody or its antigen-binding fragment, wherein VH is presented as sequence identification number: 318 and VL is presented as sequence identification number:

324. Claim 23 Any one of claims 1-22, wherein the antibody is an isolated antibody or an antigen-binding fragment thereof that is a human or humanized antibody. Claim 24 Any one of claims 1-22, wherein the fragment comprises an isolated antibody or its antigen-binding fragment comprising a Fab fragment, a Fab' fragment, an F(ab)2 fragment or a scFv fragment. Claim 25 Any one of claims 1-22, wherein the antibody or its antigen-binding fragment comprises an isolated antibody or its antigen-binding fragment comprising a human Fc region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM Fc. Claim 26 Any one of claims 1-22, wherein the antibody or its antigen-binding fragment comprises an isolated antibody or its antigen-binding fragment comprising a modified human Fc region. Claim 27 The isolated antibody or antigen-binding fragment of claim 26, wherein the antibody or its antigen-binding fragment comprises a modified human Fc region selected from the group consisting of L234A / L235A, S228P, A330S, P331S, E233P / L234V / L235A, A327G / A330S / P331S, L234F / L235E / P331S, and N297Q. Claim 28 A multispecific binding protein comprising an antigen-binding fragment according to any one of claims 1-22. Claim 29 Any one of claims 1-22, wherein the isolated antibody or antigen-binding fragment is a humanized antibody. Claim 30 An isolated nucleic acid molecule encoding an isolated antibody or an antigen-binding fragment according to any one of claims 1-22. Claim 31 An expression vector comprising the nucleic acid molecule of claim 30. Claim 32 A host cell comprising the expression vector of claim 31. Claim 33 A pharmaceutical composition for treating obesity, comprising an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1-22. Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete

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