Cannabinoid receptor type 1 (CB1) binding proteins and uses thereof

Abstract

To provide isolated, engineered, non-naturally occurring CB1 binding proteins, including anti-CB1 antibodies or antigen-binding fragment thereof, wherein the CB1 binding proteins find utility in the treatment and diagnosis of CB1 mediated conditions, diseases and disorders.SOLUTION: The present invention provides an isolated antibody or an antigen-binding fragment thereof that binds to the human cannabinoid receptor type 1 (CB1), including CDRs of the variable heavy (VH) domain sequence and CDRs of the variable light (VL) domain sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 664,882, filed April 30, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to cannabinoid receptor type 1 (CB1) binding proteins and uses thereof.

[0003] Incorporation by Reference The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated herein by reference in their entirety for all purposes, as are the references cited therein. This disclosure makes use of conventional techniques of immunology, molecular biology, cell biology, drug development and drug delivery, which are well known in the art, unless otherwise indicated. [Background technology]

[0004] Cannabinoid receptor type 1 (CB1) is a seven-transmembrane cell membrane receptor of the G protein-coupled receptor superfamily that is expressed primarily in the brain, as well as peripherally in the lungs, liver, kidneys, and adipose tissue. CB1 is activated by naturally occurring cannabinoids (e.g., eicosinoids), called endocannabinoids, or by introduced cannabinoids (e.g., cannabis) or related synthetic compounds. Cannabinoids bind to CB1 reversibly and stereoselectively. Upon engagement with CB1, multiple intracellular signaling pathways are activated, including inhibition of adenylyl cyclase and activation of mitogen-activated protein (MAP) kinases, inhibition of presynaptic N-type and P / Q-type calcium channels and D-type outward potassium channels, and activation of inwardly rectifying and A-type outward potassium channels. CB1 expression is thought to regulate neurotransmitter release to prevent excessive neuronal activity, reduce pain and other inflammatory symptoms, and even regulate food intake.

[0005] Abnormal CB1 activity has been implicated in several diseases, including obesity and related disorders such as dyslipidemia, diabetes, fibrosis, liver diseases such as fatty liver, kidney disease, cardiovascular disease, and cancer.

[0006] Prader-Willi syndrome (PWS) is a genetic disorder caused by a specific paternal gene defect and characterized by obesity, type 2 diabetes, growth retardation, and muscle weakness. CB-1 has been identified as the target of the inverse agonist rimonabant in PWS (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 developed and marketed by Sanofi-Aventis as an appetite-suppressing antiobesity drug. This product is indicated for the treatment of obese and overweight patients with associated risk factors, such as type 2 diabetes or dyslipidemia, in conjunction with diet and exercise. In June 2006, the drug was approved by the EMEA for obesity. In 2008, Sanofi-Aventis suspended all development and marketing of the drug for all indications due to the risk of serious psychiatric problems, including suicidal ideation. In January 2009, the EC suspended the drug's marketing authorization.

[0007] Another inverse agonist, taranabant (MK-0364), was studied by Merck, but its Phase 3 clinical trial was halted 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, sulinabant, tetrahydrocannabivarin, and virodamine) are being studied, but they are in early stages of research or have been downgraded to non-human studies due to CNS side effects.

[0008] Several CB-1 inverse agonists / antagonists are currently under development, targeting primarily peripherally expressed CB1 by limiting their 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 to treat obesity and metabolic disorders. Another peripherally selective silent antagonist that has not yet entered the clinic is AM6545. Peripherally selective CB-1 antagonism may be a safer and more effective way to target peripheral endocannabinoid actions in several tissues: (1) liver—decreased lipogenesis, fat accumulation, and glucose secretion; (2) muscle—increased glucose uptake and oxidation; (3) adipocytes—decreased lipogenesis and fat accumulation; decreased adiponectin synthesis; and (4) gastrointestinal (GI)—increased satiety, GI transit, and absorption (Kloet and Woods (2009) Endocrinol. 150:2531-2536).

[0009] Biomolecules such as antibodies and related binding proteins offer potentially safer and more effective ways to deliver therapeutic agents and avoid CNS damage and 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 biologics to the CNS provides an opportunity to engage CB-1 exclusively in the periphery, thereby limiting the adverse events caused by small molecule pharmacology in the CNS.

[0010] Antibodies against CB1 have recently been described in the art, but their clinical benefit is unknown, see U.S. Patent Application Publication Nos. 20170210797 and 20160145333.

[0011] Therefore, there is a need to identify safe, effective, peripherally restricted anti-CB1 inverse agonists or antagonists that do not significantly penetrate the BBB and avoid the adverse pharmacological effects of CB1 engagement in the brain. Summary of the Invention

[0012] The present invention provides binding proteins that bind to the cannabinoid type 1 receptor (CB1), such as antibodies and antigen-binding fragments thereof, that are useful in the treatment and diagnosis of disease.

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

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

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

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

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

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

[0019] In one embodiment, the antibody or antigen-binding fragment thereof is (20, 21, 22); (32, 33, 34); (44, 45, 46); (56, 57, 58); (68, 69, 70); (80, 81, 82); (92, 93, 94); (104, 105, 106); (116, 117, 118); (128, 129, 130); (140, 141, 142); (152, 153, 154); (164, 165, 166); (176, 177, 178 ; (188, 189, 190); (200, 201, 202); (212, 213, 214); (224, 225, 226); (236, 237, 238); (248, 249, 250); (260, 261, 262); (272, 273, 274); (284, 285, 286); (296, 297, 298); (308, 309, 310); and (320, 321, 322). R2, HCDR3) and (26, 27, 28); (38, 39, 40); (50, 51, 52); (62, 63, 64); (74, 75, 76); (86, 87, 88); (98, 99, 100); (110, 111, 112); (122, 123, 124); (134, 135, 136); (146, 147, 148); (158, 159, 160); (170, 171, 172); (182, 183, 184); (194, 195, 196, 197, 198, 199, 200, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 6); (206, 207, 208); (218, 219, 220); (230, 231, 232); (242, 243, 244); (254, 255, 256); (266, 267, 268); (278, 279, 280); (290, 291, 292); (302, 303, 304); (314, 315, 316); and (326, 327, 328).

[0020] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: (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) 4);(188, 189, 190 / 194, 195, 196);(200, 201, 202 / 206, 207, 208);(212, 213, 214 / 218, 219, 220);(224, 225, 226 / 230, 231, 232);(236, 237, 238 / 242, 243, 244);(248, 249, 250 / 254, 255, 256);(260, 261, 262 / 266 , 267, 268); (272, 273, 274 / 278, 279, 280); (284, 285, 286 / 290, 291, 292); (296, 297, 298 / 302, 303, 304); (308, 309, 310 / 314, 315, 316); and (320, 321, 322 / 326, 327, 328).

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

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

[0023] In one embodiment, the VH is set forth in SEQ ID NO: 114, and the VL is set forth in SEQ ID NO: 120. In one embodiment, the VH is set forth in SEQ ID NO: 126, and the VL is set forth in SEQ ID NO: 132. In one embodiment, the VH is set forth in SEQ ID NO: 138, and the VL is set forth in SEQ ID NO: 144. In one embodiment, the VH is set forth in SEQ ID NO: 150, and the VL is set forth in SEQ ID NO: 156. In one embodiment, the VH is set forth in SEQ ID NO: 162, and the VL is set forth in SEQ ID NO: 168. In one embodiment, the VH is set forth in SEQ ID NO: 174, and the VL is set forth in SEQ ID NO: 180. In one embodiment, the VH is set forth in SEQ ID NO: 186, and the VL is set forth in SEQ ID NO: 192. In one embodiment, the VH is set forth in SEQ ID NO: 198, and the VL is set forth in SEQ ID NO: 204. In one embodiment, the VH is set forth in SEQ ID NO: 210, and the VL is set forth in SEQ ID NO: 216. In one embodiment, the VH is represented by SEQ ID NO: 222 and the VL is represented by SEQ ID NO: 228. In one embodiment, the VH is represented by SEQ ID NO: 234 and the VL is represented by SEQ ID NO: 240. In one embodiment, the VH is represented by SEQ ID NO: 246 and the VL is represented by SEQ ID NO: 252. In one embodiment, the VH is represented by SEQ ID NO: 258 and the VL is represented by SEQ ID NO: 264. In one embodiment, the VH is represented by SEQ ID NO: 270 and the VL is represented by SEQ ID NO: 276. In one embodiment, the VH is represented by SEQ ID NO: 282 and the VL is represented by SEQ ID NO: 288. In one embodiment, the VH is represented by SEQ ID NO: 294 and the VL is represented by SEQ ID NO: 300. In one embodiment, the VH is represented by SEQ ID NO: 306 and the VL is represented by SEQ ID NO: 312. In one embodiment, the VH is represented by SEQ ID NO: 318 and the VL is represented by SEQ ID NO: 324.

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

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

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

[0027] In one embodiment, the anti-CB1 antibody or antigen-binding fragment thereof 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.

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

[0029] In one embodiment, the anti-CB1 antibody or antigen-binding fragment thereof enhances, activates, or is an agonist of CB1 signaling activity.

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

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

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

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

[0034] An anti-CB1 antibody or antigen-binding fragment thereof that competes with an isolated anti-human CB1 antibody or antigen-binding fragment thereof for binding to CB1 is provided.

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

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

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

[0038] In one embodiment, the anti-CB1 antibody or antigen-binding fragment inhibits CB1 signaling at least 2-fold more than rimonabant.

[0039] In one embodiment, the anti-CB1 antibody or antigen-binding fragment exhibits reduced CNS side effects compared to rimonabant.

[0040] An isolated nucleic acid molecule encoding an anti-CB1 antibody or antigen-binding fragment thereof is provided.

[0041] An expression vector comprising a nucleic acid molecule encoding an anti-CB1 antibody or antigen-binding fragment thereof is provided.

[0042] A host cell is provided that contains an expression vector that includes a nucleic acid molecule encoding an anti-CB1 antibody or antigen-binding fragment thereof.

[0043] A method of modulating CB1 signaling is provided, comprising contacting a cell expressing CB1 with an anti-CB1 antibody or antigen-binding fragment thereof.

[0044] A method of antagonizing CB1 is provided, comprising contacting a cell expressing CB1 with an anti-CB1 antibody or antigen-binding fragment thereof.

[0045] A method of agonizing CB1 is provided, comprising contacting a cell expressing CB1 with an anti-CB1 antibody or antigen-binding fragment thereof.

[0046] A method of inverse agonizing CB1 is provided, comprising contacting a cell expressing CB1 with an anti-CB1 antibody or antigen-binding fragment thereof.

[0047] Pharmaceutical compositions comprising isolated anti-CB1 antibodies or antigen-binding fragments thereof are provided.

[0048] Provided is a method for inhibiting the biological activity of CB1 in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an effective amount of an isolated anti-CB1 antibody or antigen-binding fragment thereof, thereby inhibiting the activity of CB1 protein in the subject.

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

[0050] Provided is a method for treating a disease or disorder responsive to modulation of CB1 signaling in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an isolated anti-CB1 antibody or antigen-binding fragment thereof.

[0051] Provided is a method for treating a disease or disorder responsive to antagonism or inverse agonism of CB1 signaling in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an isolated anti-CB1 antibody or antigen-binding fragment thereof.

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

[0053] A method for diagnosing a disease or disorder associated with CB1 is provided, comprising contacting a cell with an anti-CB1 antibody or antigen-binding fragment thereof.

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

[0055] In one embodiment, the disease or disorder is a kidney disease (focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive kidney disease, nephrotic syndrome, steroid-resistant nephrotic syndrome, minimal change nephrotic syndrome, membranous nephropathy, idiopathic membranous nephropathy, membranoproliferative glomerulonephritis (MPGN), immunoconjugate-mediated MPGN, complement-mediated MPGN, lupus nephritis, post-infectious glomerulonephritis, thin basement membrane disease, mesangial proliferative glomerulonephritis, amyloidosis (primary), clq nephropathy, rapidly progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, IgA nephropathy, proteinuric kidney disease, microalbuminuria, or microalbuminuric kidney disease), pulmonary arterial hypertension, pain (such as neuropathic pain or visceral pain), cancer (such as chemotherapy-resistant breast cancer, adriamycin-resistant breast cancer, chemotherapy-resistant colorectal cancer, medulloblastoma, or tumor angiogenesis), anxiety, depression, transplant-associated FSGS, transplant-associated nephrotic syndrome, transplant-associated proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), obesity, insulin resistance, type 2 diabetes, prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), diabetic gastroparesis, or gastroparesis.

[0056] Provided is an antibody conjugate comprising an isolated anti-CB1 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to an agent selected from the group consisting of a therapeutic agent, a cytotoxic agent, an immunoadhesion molecule, and an imaging agent.

[0057] Kits are provided that include an isolated anti-CB1 antibody or antigen-binding fragment thereof and instructions for using the antibody in an immunological assay.

[0058] These 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 the embodiments, when read in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0059] [Figure 1] Figure 1 shows the results of a cAMP assay. cAMP Hunter™ CHO-K1 CNR1 Gi cells were treated with CB1 antibody, 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. Antagonists were also tested without the addition of CP-55,940 to establish whether they possessed agonist activity on their own. Abbreviations: CAB = cell assay buffer (representing 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 approximately the EC80 for CP-55,940). [Figure 2] Figure 1 shows the results of a p-ERK assay. Phospho / total ERK assays were performed using cAMP Hunter™ CHO-K1 CNR1 Gi cells. Cells were treated with mouse anti-CB1 antibody or the small molecule CB1 antagonist JD5037, followed by agonist challenge with 30 nM CP-55,940 in the presence of forskolin. At the end of treatment, plates were processed for p-ERK / total ERK using the MesoScale Discovery (MSD) kit. Results are expressed as a percentage of the maximum response (% Max). [Figure 3] Figure 3A shows the binding of mouse anti-huCB1 antibodies to huCB1-CHO cells. Binding curves of purified mouse anti-CB1 antibodies were titrated 3-fold starting at 200 nM antibody concentration. All antibodies show specific binding to huCB1-CHO cells. Figure 3B shows the binding of mouse anti-huCB1 antibodies to moCB1-CHO cells. All antibodies show a lack of binding to moCB1-CHO cells. [Figure 4] 1 shows an assessment of the binding of purified anti-CB1 antibodies in the presence and absence of the agonist and antagonist CB1 small molecules CP5990 and JD5037, respectively. [Figure 5A]The consensus sequence obtained from the alignment of the heavy chain variable region amino acid sequences from hybridoma antibodies M1 to M8 is shown. [Figure 5B] The consensus sequence obtained from the alignment of the light chain variable region amino acid sequences from hybridoma antibodies M1 to M8 is shown. [Figure 6A] 1 shows the consensus sequence obtained from 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] 1 shows the consensus sequence obtained from 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] Figure 1 shows cell binding of humanized CB-1 antibody variants of clones M5 and M7 to CHO-huCB-1 and CHO parental cells at a single antibody concentration of 30 μg / mL. [Figure 7B] Figure 1 shows cell binding of humanized CB-1 antibody variants of clone M7 to CHO-huCB-1 and CHO parental cells at a single antibody concentration of 30 μg / mL. [Figure 8] Figure 8A shows the results of a cAMP assay as 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 a cAMP assay as described in Example 3. Figure 8B shows a side-by-side comparison between different backbones for the M7 antibody. [Figure 9] Figure 9A shows the results of a p-ERK assay as 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 a p-ERK assay as described in Example 4. Figure 9B shows a side-by-side comparison between different backbones for the M7 antibody. [Figure 10]This figure shows the results of a cAMP assay for inverse agonism using a method similar to that described in Example 3. cAMP Hunter™ CHO-K1 CNR1 Gi cells were treated with CB1 antibody or an isotype control, followed by the addition of forskolin. The purple dotted line represents the level of cAMP released upon treatment with forskolin, the maximum amount of cAMP in the assay. The red line corresponds to the CB1 small molecule agonist CP-55,940, used as a control in the assay. Compounds with inverse agonism activity show a curve that moves in the opposite direction to the agonist, toward the top of the CAB / F line. This experiment demonstrates that both M7 variants tested have inverse agonism activity. DETAILED DESCRIPTION OF THE INVENTION

[0060] Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the case of any potential ambiguity, the definitions set forth herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The word "a" or "an" means "at least one" unless otherwise indicated. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more." The word "or" means "and / or" unless otherwise indicated. As used herein, terms such as "comprise," "including," "containing," "having," and the like may have the meanings given to them in U.S. Patent Law and may mean "comprise," "comprising," and the like; "consisting essentially of" or "consisting essentially of" likewise have the meanings given to them in U.S. Patent Law, and the term is open-ended, allowing for the presence of more than what is stated but excluding prior art embodiments, so long as basic or novel characteristics are not altered. Unless specifically stated or clear from the context, the term "about," as used herein, is understood to mean within normal acceptance in the art, e.g., within two standard deviations of the mean. About may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​set forth herein are modified by the term about.

[0061] The methods and techniques described herein are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. The nomenclature, laboratory procedures and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization, analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used in enzymatic reactions and purification techniques, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

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

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

[0064] [Table 1]

[0065] The term "central CB1" refers to CB1 that is localized anywhere in the body, including the brain and CNS.

[0066] The term "peripheral CB1" refers to CB1 that is not localized in the brain or CNS (eg, peripherally restricted CB1).

[0067] The term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. This term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact," as in "intact antibody," for purposes of this disclosure, the term "antibody" also includes antibody fragments, such as Fab, F(ab'), Fv, scFv, Fd, dAb, and other antibody fragments, that retain antigen-binding function, i.e., the ability to specifically bind to CB1. Typically, such fragments will contain an antigen-binding domain. The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof. In one embodiment of a full-length antibody, each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The CL is composed of a single CL domain. VH and VL can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Generally, each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus 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. An amino acid consensus sequence can be defined based on side-by-side analysis of two or more CDRs and / or FRs. Antibody molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

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

[0069] The term "VH / VL pair" refers to a VH and VL that are paired and capable of binding to an antigen.

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

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

[0072] The terms "antibody that binds to CB1" and "anti-CB1 antibody" refer to antibodies that bind to soluble CB1 protein or fragments thereof (e.g., a portion of the extracellular domain of CB1) and / or cell surface-expressed CB1, and antigen-binding fragments thereof. The phrase "cell surface-expressed CB1" refers to a CB1 protein or portion thereof that is expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the CB1 protein is exposed extracellularly on the cell membrane and accessible to the antigen-binding portion of an antibody.

[0073] The term "CB1-binding protein" or "anti-CB1-binding protein" refers to proteins that bind to CB1, including all or a portion of an antigen-binding fragment, including proteins containing alternative arrangements of standard antibody domains or frameworks, such as recombinant multivalent or multispecific immunoglobulins, as well as conjugates and fusion proteins. CB1-binding proteins of the present invention, as well as variants and mutants thereof, may retain CB1 binding and function or may provide additional or alternative functions. Such CB1-binding proteins are within the scope of the present invention and are well known to those skilled in the art.

[0074] The terms "antigen-binding domain" and "antigen-binding fragment," with respect to a binding protein such as an antibody, refer to a portion or fragment of an antibody, or a variant or mutant thereof, that retains the ability to specifically bind to the antibody's target antigen, including any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained, for example, from intact antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. One or more variable and / or constant domains can be arranged in the appropriate position, or codons can be introduced, cysteine ​​residues can be created, amino acids can be modified, added, or deleted, etc. Numerous fragment, mutant, or variant antibody formats, including antigen-binding fragments, are known in the art. Non-limiting examples of antigen-binding fragments include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment comprising the VH and CH1 domains; (iv) an Fv fragment comprising the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv (scFv) molecule; (vi) a dAb fragment comprising 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) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, linear antibodies (comprising a pair of tandem Fv segments which, together with complementary light chain polypeptides, form a pair of antigen-binding sites; VH-CH1-VH-CH1), triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed within the scope of the expression "antigen-binding fragment" as used herein.The term "antigen-binding fragment thereof" is not meant to be limiting and includes fragments contained within variant molecules that may have additional or rearranged antibody regions, such as multispecific antibodies and antibody conjugates that retain the same antigen binding to a particular antigen.

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

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

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

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

[0079] 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 reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound on target cells, resulting in lysis of the target cells. CDC and ADCC can be measured using assays well known and available in the art (see, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656).

[0080] The term "murine antibody" refers to an antibody having variable and constant regions derived from murine germline immunoglobulin sequences. Murine antibodies may include amino acid residues, for example in the CDRs, particularly CDR3, that are not encoded by murine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "murine antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a human, have been grafted onto murine framework sequences.

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

[0082] The term "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from the tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

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

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

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

[0086] The term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences in which one or more of the VH and / or VL CDR regions have been replaced with CDR sequences from another antibody. The two antibodies may be derived from different species, such as an antibody having a murine heavy and light chain variable region in which one or more of the murine CDR sequences have been replaced with human CDR sequences.

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

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

[0089] The term "effective amount" refers to a dosage or amount sufficient to reduce the activity of CB1, resulting in symptomatic relief in a patient, or to achieve a desired biological result, including, for example, a decrease or increase in CB1 activity.

[0090] The term "cross-reactivity" refers to the ability of a binding protein to bind to a target antigen other than the antigen to which the antibody was raised. Generally, a binding protein should bind to its target antigen with appropriately high affinity, but may bind to the same target antigen in another species or may exhibit low affinity for non-target antigens. Individual binding proteins are generally selected to meet two criteria: (1) tissue staining appropriate for known expression of the antibody target; and (2) similar staining patterns in human tissues from the same organ and tissues from toxicity test species (e.g., mice and cynomolgus monkeys). These and other methods for assessing cross-reactivity are known to those skilled in the art.

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

[0092] The term "stable" means capable of retaining its physical, chemical, and / or biological integrity or activity over a given time or storage condition. Binding proteins that are stable in vitro at various temperatures over extended periods of time are generally desirable. Methods for stabilizing binding proteins and assessing their stability at various temperatures are known to those skilled in the art.

[0093] The term "solubility" refers to the ability of a protein to remain dispersed in an aqueous solution. Protein solubility in aqueous formulations depends on the proper distribution of hydrophobic and hydrophilic amino acid residues, and therefore solubility may be associated with the production of a correctly folded protein. Those skilled in the art will be able to detect an increase or decrease in the solubility of a binding protein using routine HPLC techniques and methods known to those skilled in the art.

[0094] The term "immunogenicity" refers to the ability of a substance to induce an immune response. Administration of a therapeutic binding protein may result in a certain incidence of an immune response. Methods for reducing the immunogenicity of antibodies and binding proteins are known to those skilled in the art.

[0095] The term "detectable label" refers to a moiety that is attached to a member of a specific binding pair, such as an antibody or its analyte, to render the reaction (e.g., binding) between the members of the specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein that has incorporated a detectable label that provides for the identification of the binding protein. In one embodiment, the detectable label can produce a signal that is detectable by visual or instrumental means, for example, the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to a polypeptide that can be detected by avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of detectable labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153fluorescent 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 agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. In this regard, the moiety itself need not be detectably labeled, but may become detectable upon reaction with another moiety.

[0096] The term "conjugate" refers to a binding protein, such as an antibody, chemically linked to another functional molecule or second chemical moiety, such as a therapeutic, cytotoxic, cytostatic, or imaging agent (see, e.g., US Pat. No. 7,850,962). The term "agent" includes a chemical compound, a mixture of chemical compounds, a biological macromolecule, such as a peptide of a protein, or an extract made from biological material. In one embodiment, therapeutic or cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, antibiotics, growth factors, cytokines, antiangiogenic agents, antimitotic agents, anthracyclines, toxins, and apoptotic agents. Useful agents include, for example, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or congeners thereof. Imaging agents useful in producing 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 immunoassays, the conjugated antibody may be a detectably labeled antibody used as a detection antibody. The antibody can be linked by chemical crosslinking or recombinant methods. Antibodies can also be linked to any of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by methods set forth in U.S. Patent Nos. 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 a polymer to, for example, extend their circulating half-life.Exemplary polymers and methods for attaching them are also set forth in US Pat. Nos. 4,766,106; 4,179,337; 4,495,285, and 4,609,546.

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

[0098] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA segment can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Other vectors include RNA vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). A "recombinant expression vector" or "expression vector" is capable of directing the expression of genes to which it is operably linked. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, are also included.

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

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

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

[0102] The term "control" refers to a composition known to contain either the analyte (a "negative control") or the analyte (a "positive control"). Positive controls may contain a known concentration of analyte or can be used to verify assay performance characteristics and are useful indicators of reagent integrity.

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

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

[0105] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include amino acid, sugar, phosphoryl, or sulfonyl moieties on an antigen and may have specific three-dimensional structural and / or charge characteristics. Binding proteins "bind to the same epitope" if they bind to the same amino acid on an antigen and may also cross-compete (one antibody may block the binding or regulatory action of another). In addition, the structural definition of the epitopes (overlapping, similar, identical) is informative; the functional definition encompasses structural (binding) and functional (modulatory, competitive) parameters.

[0106] The term "pharmacokinetics" refers to the process by which a drug is absorbed, distributed, metabolized, and excreted by an organism.

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

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

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

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

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

[0112] The term "variant" refers to a polypeptide whose amino acid sequence differs from that of a given polypeptide due to the addition, insertion, deletion, or conservative substitution of amino acids, but which retains the biological activity of the given polypeptide (e.g., a variant antibody can compete with the native antibody for binding to its target). Conservative amino acid substitutions replace one amino acid with another of similar properties (e.g., hydrophilicity and degree and distribution of charged regions), and are recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by examining the hydropathic index of an amino acid, as understood in the art. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted in a protein and the protein will still retain protein function. In one embodiment, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to identify substitutions that will result in a protein that retains biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the peptide's greatest local average hydrophilicity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. In one embodiment, substitutions are made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, and particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. The term "variant" also includes polypeptides or fragments thereof that have been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, but still retain biological activity or antigenic reactivity. The term "variant" encompasses fragments of variants unless otherwise defined.Variants can be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, or 75% identical to the wild-type sequence.

[0113] The anti-CB1 antibodies disclosed herein may contain one or more amino acid substitutions, additions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains when compared to the corresponding germline sequences from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues in the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived).Additionally, antibodies of the invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments 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 the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general approach are encompassed by the invention.

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

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

[0116] Sequence similarity in polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity 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 can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, e.g., GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) programs provide alignments and percentage sequence identity of the regions of optimal overlap between the query and search sequences (Pearson (2000) supra). When comparing the sequences of the present invention to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. 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 of which is incorporated herein by reference.

[0117] Biological characteristics of antibodies The present invention includes anti-CB1 antibodies and antigen-binding fragments thereof that bind to CB1 with high affinity.

[0118] at least about 10 for CB1 as measured by surface plasmon resonance 2 M -1 s -1 ; at least about 10 3 M -1 s -1 ; at least about 10 4 M -1s -1 ; at least about 10 5 M -1 s -1 and at least about 10 6 M -1 s -1 an on-rate constant (K on The present invention provides an anti-CB1 antibody and an antigen-binding fragment thereof, which have the following structure:

[0119] At most about 10 for the above targets as measured by surface plasmon resonance -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 an off-rate constant (K off The present invention provides an anti-CB1 antibody and an antigen-binding fragment thereof, which have the following structure:

[0120] At most, about 10 -7 M; at most about 10 -8 M; at most about 10 -9 M; at most about 10 -10 M; at most about 10 -11 M; at most about 10 -13 M; and at most 10 -14The present invention provides anti-CB1 antibodies and antigen-binding fragments thereof having a dissociation constant (KD) selected from the group consisting of M. The anti-CB1 antibodies and fragments thereof may have a binding affinity Kd value 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, or about 0.05 to about 50 nM. The antibodies and fragments thereof may have a binding affinity Kd value for CB1 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 invention bind to CB1 with a Kd 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.

[0121] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof is at least as potent as a small molecule CB1 receptor modulator, such as, for example, rimonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenabant, surinabant, tetrahydrocannabivarin, and virodamine, and AM6545. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof has CB1 antagonist or inverse agonist activity that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than that of a small molecule CB1 receptor modulator, such as rimonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenabant, surinabant, tetrahydrocannabivarin, virodamine, and AM6545. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof inhibits CB1 agonist-mediated signal transduction. In some embodiments, inhibition of CB1 agonist-mediated signal transduction is measured by determining intracellular cAMP levels and / or downstream ERK phosphorylation.

[0122] In some embodiments, the anti-CB1 antibodies and antigen-binding fragments thereof have the advantage of reduced or no BBB penetration or brain exposure. In some embodiments, the anti-CB1 antibodies and antigen-binding fragments thereof exhibit reduced BBB penetration compared to small molecule CB1 agonists, antagonists, or inverse agonists (e.g., rimonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenabant, sulinabant, tetrahydrocannabivarin, and virodamine, and AM6545). In some embodiments, the anti-CB1 antibodies and antigen-binding fragments thereof provided herein provide therapeutic efficacy with reduced CNS side effects compared to small molecule CB1 receptor agonists, antagonists, or inverse agonists. CNS side effects associated with the small molecule CB1 receptor antagonist rimonabant include, for example, anxiety, depression, agitation, eating disorders, irritability, aggression, and insomnia (Moreira (2009) Rev. Bras. Psiquiatr. 31(2):145-153).

[0123] Epitope mapping and related techniques The present invention includes anti-CB1 antibodies that interact with one or more amino acids found within the extracellular domain of human CB1 (e.g., amino acids 1-116, and / or extracellular loops e1 (amino acids 176-187; SEQ ID NO:6), e2 (amino acids 256-273; SEQ ID NO:10), and / or e3 (amino acids 366-377; SEQ ID NO:14)). The epitope to which such antibodies bind can consist of a single contiguous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) located within the extracellular domain of CB1. Alternatively, the epitope can consist of multiple noncontiguous amino acids (or amino acid sequences) located within the extracellular domain of CB1. Furthermore, the epitope to which CB antibodies bind can include portions of CB1 that are not extracellular due to conformational changes or exposure upon binding. The sequences of CB1 and its various domains are shown in Table 1.

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

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

[0126] 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 with it. For example, to determine whether a test antibody binds to the same epitope as a reference anti-CB1 antibody of the present invention, the reference antibody is bound to a CB1 protein (e.g., a soluble portion of the CB1 extracellular domain or CB1 expressed on the cell surface). The ability of the test antibody to bind to the CB1 molecule is then evaluated. If the test antibody can bind to CB1 after saturation binding with the reference anti-CB1 antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-CB1 antibody. On the other hand, if the test antibody cannot bind to CB1 after saturation binding with the reference anti-CB1 antibody, the test antibody may bind to the same epitope as the reference anti-CB1 antibody of the present invention. Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. In certain embodiments of the invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al. (1990) Cancer Res. 50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.Two antibodies are considered to have "overlapping epitopes" if only the subset of amino acid mutations that reduce or eliminate binding of one antibody reduces or eliminates binding of the other.

[0127] In some embodiments, the present invention provides anti-CB1 antibodies or antigen-binding fragments thereof that can compete with the antibodies or antigen-binding fragments thereof disclosed herein for binding to CB1. Such antibodies can be identified using routine competitive binding assays. For example, to determine whether an antibody competes with a reference anti-CB1 antibody for binding, the above-described binding method is performed in two ways: in the first way, the reference antibody is bound to a CB1 protein (e.g., a soluble portion of the CB1 extracellular domain or CB1 expressed on the cell surface) under saturating conditions, followed by assessing the binding of the test antibody to the CB1 molecule. In the second way, the test antibody is bound to a CB1 molecule under saturating conditions, followed by assessing the binding of the reference antibody to the CB1 molecule. If only the first (saturating) antibody can bind to the CB1 molecule in both ways, it is concluded that the test and reference antibodies compete for binding to CB1. An antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody, for example, by binding to an overlapping or adjacent epitope. 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).

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

[0129] The heavy chain CDRs and light chain CDRs of the anti-CB1 antibodies provided herein may be independently selected and matched to form antibodies or antigen-binding fragments thereof comprising any heavy chain CDR1, CDR2, and CDR3; and any light chain CDR1, CDR2, and CDR3 from the antibodies provided herein. The heavy and light chain variable regions of the antibodies provided herein may be independently selected and matched to form antibodies or antigen-binding fragments comprising any heavy and light chains from the antibodies provided herein.

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

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

[0132] In another embodiment, the anti-CB1 antibody or antigen-binding fragment thereof 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 161, 173, 185, 197, 209, 221, 233, 245, 257, 269, 281, 283, 305, and 317.

[0133] In another embodiment, the anti-CB1 antibody or antigen-binding fragment thereof 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 35, 47, 59, 71, 83, 95, 107, 119, 131, 143, 155, 167, 179, 191, 203, 215, 227, 239, 251, 263, 275, 287, 299, 311, and 323.

[0134] In certain embodiments, the anti-CB1 antibody or antigen-binding fragment thereof, CDR, 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% conservative variant amino acids.

[0135] In another embodiment, the anti-CB1 antibody or antigen-binding fragment thereof 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, 179, 180, 181, 182); (183, 184, 185, 186); (187, 188, 189, 190, 191, 192, 193, 194) (272, 273, 274); (284, 285, 286); (296, 297, 298); (308, 309, 310); and (320, 321, 322).

[0136] In another embodiment, the anti-CB1 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: (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); (278, 279, 280); (290, 291, 292); (302, 303, 304); (314, 315, 316); and (326, 327, 328).

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

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

[0139] In some embodiments, the anti-CB1 antibodies or antigen-binding fragments thereof bind to CB1 and exhibit reduced effector functions, such as, for example, 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 antibodies or antigen-binding fragments thereof bind to CB1 and contain one or more Fc region modifications that reduce, diminish, or eliminate one or more effector functions. For example, in one embodiment, the anti-CB1 antibodies and antigen-binding fragments thereof disclosed herein bind to CB1 but exhibit reduced, diminished, or absent C1q binding and / or CDC and / or ADCC. The Fc modifications may be amino acid insertions, deletions, or substitutions, or may be chemical modifications. For example, the Fc region can be modified to increase or decrease complement binding, increase or decrease ADCC or CDC, or modify glycosylation. Various Fc modifications are known in the art and are described, for example, in Labrijin et al. (2009) Nature Biotech. 27(8):767-771; Greenwood et al. (1993) Eur. J. Immunol. 23:1098-1104; Mueller et al. (1997) Mol. Immunol. 34:441-452; and Rother et al. (2007) Nature Biotechnol. 25:1256-1264. Any of the Fc modifications known in the art can be applied to the exemplary CB1 antibodies disclosed herein to alter effector function. In one embodiment, the anti-CB1 antibody or antigen-binding fragment thereof has certain mutations, such as L234A / L235A (“LALA”), S228P, A330S, P331S, E233P / L234V / L235A, A327G / A330S / P331S, L234F / L235E / P331S, and N297Q.

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

[0141] In an exemplary system for recombinant expression of CB1 binding proteins, a recombinant expression vector encoding both the CB1 antibody heavy and light chains is introduced into dhfr-CHO cells via calcium phosphate-mediated transfection. Within the recombinant expression vector, the CB1 antibody heavy and light chain sequences are operably linked to CMV enhancer and promoter regulatory elements, respectively, to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, allowing for the selection of CHO cells transfected with this vector using methotrexate selection / amplification. The selected transformed host cells are cultured to allow expression of the CB1 antibody heavy and light chains, and intact CB1 antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the CB1 antibody from the culture medium.

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

[0143] Two antigen-binding proteins, or antibodies, are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption, for example, when administered in single or repeated doses at the same molar dose under similar experimental conditions. Some antibodies would be considered equivalents or pharmaceutical substitutes if they were equivalent in their extent of absorption but not in their rate of absorption, and such differences in absorption rate may be considered bioequivalent because they are intentional, reflected in the label, and are not essential, for example, to achieving effective body drug concentrations with long-term use and are not medically significant for the particular drug product being studied.

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

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

[0146] Biologically equivalent variants of anti-CB1 antibodies can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences not required for biological activity. For example, cysteine ​​residues not essential for biological activity can be deleted or replaced with other amino acids to prevent unnecessary or incorrect intramolecular disulfide bridge formation during renaturation. In other situations, biologically equivalent antibodies can include anti-CB1 antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that remove or eliminate glycosylation.

[0147] Species selectivity and species cross-reactivity The present invention also includes anti-CB1 antibodies that bind to human CB1 and to CB1 from one or more non-human species. For example, the anti-CB1 antibodies of the present invention can bind to human CB1 and to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee CB1. In certain embodiments of the present invention, the anti-CB1 antibodies bind to human CB1 but do not bind to CB1 from other species.

[0148] Immunoconjugates The present invention encompasses anti-CB1 antibodies conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic agent, an immunosuppressant, or a radioisotope (an "immunoconjugate"). A cytotoxic agent includes any agent that is detrimental to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art and described herein.

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

[0150] Use of Binding Proteins in Various Diseases The antibodies and binding proteins of the invention are useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by CB1 expression or activity, or treatable by blocking the interaction between CB1 and CB1 ligands (e.g., cannabinoids), or otherwise by inhibiting CB1 activity and / or signal transduction, and / or by promoting receptor internalization and / or reducing the number of cell surface receptors. The term "a disorder in which CB1 activity is detrimental" refers to a disorder or disease in which the presence or activity (e.g., abnormal or excessive activity) of CB1 in a subject suffering from the disorder contributes to the pathophysiology of the disorder or disease or is a contributing factor in exacerbating the disorder or disease. Thus, a disorder in which CB1 activity is detrimental is one in which reducing CB1 activity is expected to alleviate the symptoms and / or progression of the disorder.

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

[0152] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising anti-CB1 binding proteins, e.g., antibodies or antigen-binding fragments thereof. The pharmaceutical compositions of the present invention are formulated with appropriate additives, carriers, prophylactic agents, therapeutic agents, and other agents that improve the stability, delivery, tolerability, and efficacy of the anti-CB1 binding proteins. Many suitable formulations can be found in formularies known to all pharmacists, such as 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, emulsions of carbowax (polyethylene glycols 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.

[0153] Pharmaceutical compositions comprising CB1 binding proteins provided herein are for use, including but not limited to, in the diagnosis, detection, or monitoring of a disorder, in the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof, and / or in research.

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

[0155] Methods of administering the prophylactic or therapeutic agents provided herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, intratumoral, mucosal (e.g., intranasal and buccal) and pulmonary administration (e.g., administration of an aerosolized compound using an inhaler or nebulizer). Formulation of pharmaceutical compositions for specific routes of administration, as well as the materials and techniques necessary for various administration methods, are available and known to those of skill in the art.

[0156] Dosage regimens can be adjusted to obtain the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, or multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage form" refers to a physically discrete unit adapted as a unitary dosage for the mammalian subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms provided herein are dictated by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds to treat susceptibility in individuals.

[0157] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily applied to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, in disposable pen delivery devices, a reservoir within the device is pre-filled with and holds the pharmaceutical composition. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

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

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

[0160] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying the antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injections include, for example, isotonic solutions containing saline, glucose, and other adjuvants, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injections prepared in this manner are preferably filled into appropriate ampoules.

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

[0162] The dose of an antibody administered to a patient may vary depending on the patient's age and size, target disease, disease stage, sex, presence of medical complications, other drug therapies, condition, route of administration, etc. A preferred dose is typically calculated based on body weight or body surface area. When the antibody of the present invention is used to treat a condition or disease associated with CB1 activity in an adult patient, it may be advantageous to administer the antibody of the present invention intravenously at a single dose of about 0.01 to about 100 mg / body weight. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective dosages and schedules for administering anti-CB1 antibodies can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al. (1991) Pharmaceut. Res. 8:1351-1359). It is further to be understood that for any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope and practice of the claimed compositions.

[0163] Combination therapy The binding proteins provided herein can also be administered with one or more additional therapeutic agents useful in treating various diseases, wherein the additional agent is selected by those skilled in the art for its intended purpose.For example, the additional agent can be a therapeutic agent recognized in the art as being useful for treating the disease or condition treated by the CB1 binding proteins provided herein.The combination can include more than one additional agent.

[0164] Non-limiting examples of such additional therapeutically active ingredients include other CB1 antagonists (e.g., a second anti-CB1 antibody or a small molecule inhibitor of CB1 (e.g., rimonabant, taranabant, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenabant, surinabant, tetrahydrocannabivarin, and virodamine, and AM6545), antagonists of another CB1 family member.

[0165] The present invention also includes therapeutic combinations comprising any of the anti-CB1 antibodies described herein and an additional inhibitor, where the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). The anti-CB1 antibodies of the present invention can also be administered in combination with and / or co-formulated with additional therapeutic agents. The additional therapeutically active ingredient(s) may be administered immediately before, simultaneously with, or immediately after administration of the anti-CB1 antibodies of the present invention; (For purposes of this disclosure, such administration regimens are considered administration of the anti-CB1 antibody "in combination" with the additional therapeutically active ingredient(s).) The present invention includes pharmaceutical compositions in which the anti-CB1 antibodies of the present invention are co-formulated with one or more additional therapeutically active ingredient(s) as described elsewhere herein.

[0166] The present invention also includes compositions and methods comprising a combination of an "antagonist antibody" and an "inverse agonist antibody." An "antagonist anti-CB1 antibody" refers to an anti-CB1 antibody that inhibits, diminishes, or blocks the signaling activity of a CB1 ligand (e.g., a cannabinoid). Non-limiting examples of antagonist antibodies of the present invention are M1, M2, M3, M4, M5, M6, M7, M8, M7-H1, M7-H2, M7-H3, M7-H4, M7-H5, M7-H6, M7-H7, M7-H8, M7-H9, M7-H10, M7-H11, M7-H12, M7-H13, M7-H14, M7-H15, M7-H16, M5-H1, and M5-H2. An "inverse agonist anti-CB1 antibody" refers to an anti-CB1 antibody that induces the opposite pharmacological response as an agonist. While an agonist increases receptor activity above its basal level, an inverse agonist reduces its activity below basal levels. Non-limiting examples of inverse agonist antibodies of the present invention include M7. The inventors contemplate combining antagonist and inverse agonist antibodies to synergistically or otherwise improve efficacy. Thus, the present invention includes pharmaceutical compositions comprising at least one antagonist antibody and at least one inverse agonist antibody. The present invention also includes methods of treatment comprising administering to a subject a combination of an antagonist antibody and an inverse agonist antibody (either separately or as a co-formulation).

[0167] Combination therapy agents include, but are not limited to, antineoplastic agents, radiation therapy, chemotherapeutic agents such as DNA alkylating agents, cisplatin, carboplatin, antitubulin 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), kinase inhibitors, and siRNA.

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

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

[0170] Immunoassays provided by the present disclosure may include sandwich immunoassays, radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), competitive-inhibition immunoassays, fluorescence polarization immunoassays (FPIA), enzyme-amplified immunoassay technology (EMIT), bioluminescence resonance energy transfer (BRET), fluorescence-activated cell sorting (FACS), and homogeneous chemiluminescence assays, among others.

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

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

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

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

[0175] Optionally, the kit may include a calibrator or control, which may include an isolated or purified analyte. The kit may include at least one component for assaying a test sample for an analyte by immunoassay and / or mass spectrometry. Kit components, including 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 of production provided in the practice of the present disclosure will be known to those of skill in the art.

[0176] The above-described kits (or components thereof), as well as methods for determining the presence, amount, or concentration of an analyte in a test sample by assays such as immunoassays described herein, can be adapted for use in a variety of automated and semi-automated systems (including those in which the solid phase comprises microparticles), such as those described in U.S. Pat. Nos. 5,089,424 and 5,006,309 and commercially available, for example, as ARCHITECT® by Abbott Laboratories (Abbott Park, IL). Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404), PRISM®, EIA (beads), and Quantum™ II, as well as other platforms. Additionally, the above-described assays, kits, and kit components can be used in other formats, such as electrochemical or other portable or point-of-care assay systems. The present disclosure is applicable, for example, to the commercially available Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay system, which performs sandwich immunoassays. Immunosensors and their methods of manufacture and operation in disposable devices are described, for example, in U.S. Patent Nos. 5,063,081, 7,419,821, 7,682,833, 7,723,099, and 9,035,027; and U.S. Patent Application Publication Nos. 20040018577 and 20060160164.

[0177] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein will be apparent and can be made, using appropriate equivalents, without departing from the scope of the embodiments disclosed herein. While certain embodiments have been described in detail, the practice of this invention will be more fully understood from the following examples, which are set forth herein for illustrative purposes only and should not be construed as limiting the invention in any way. [Example]

[0178] Example 1: Generation and selection of anti-CB1 antibodies for functional evaluation 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; Cat. No. 1205) to potentially stabilize the protein and ensure that it remains fully structured during immunization. Mice were immunized in both heel joints with approximately 5 μg of antigen per 30 μl per heel joint in Titer Max Gold adjuvant (St. Louis, MO; Sigma-Aldrich; Cat. No. T2684). Subsequently, mice were immunized twice weekly for approximately 30 days with CpG (InvivoGen, San Diego, CA; Cat. No. ODN1826) and Alhydrogel (InvivoGen, San Diego, CA; Cat. No. vac-alu-250). Serum was collected on days 13 and 26, and antibody titers and their progression over time were determined. On day 30, mice were euthanized, and popliteal and inguinal lymph nodes were collected for fusion. Single-cell suspensions were prepared by washing in Medium B (a 1:1 mixture of nutrient-free RPMI 1640 (Thermo Fisher Scientific-Gibco, San Diego, CA; Cat. No. 11879020) and IMDM (Lonza, Anaheim, CA; Cat. No. 12-722F)). P3Ag8.563 myeloma cells (ATCC, Manasas, VA; Cat. No. PTA-9393) were harvested from culture and washed in Medium B. Lymphocytes and myeloma cells were mixed at a 1:1 ratio and fused using an electrofusion BTX Harvard apparatus ECM2001 (BTX Harvard Apparatus, Holliston, MA; Cat. No. 45-0012). The fused cells were resuspended in Recovery Medium C (Stem Cell Technologies, Seattle, WA; Cat. No. 03803) and cultured in a 75cm 2The flasks were allowed to recover overnight at 37°C. The next day, the fused cells were harvested and resuspended in hybridoma selection methylcellulose medium D (Stem Cell Technologies, Seattle, WA; Cat. No. 03804) containing anti-mouse IgG FITC Clone Detect (Molecular Devices, San Jose, CA; Cat. No. K8220). The cells were mixed and resuspended at 1 x 10 per 10 mL of medium D. 6 The hybridoma cells were plated in 100% Fibroblast Growth Medium E (FGF-1) at 37°C. The plated cells were incubated at 37°C for 7 days. Hybridoma colonies were picked using Clone Pix2 (Molecular Devices, San Jose, CA) based on their size and their ability to display a strong FITC halo, indicative of IgG production, and transferred to 96-well tissue culture plates containing Hybridoma Growth Medium E (Stem Cell Technologies, Seattle, WA; Cat. No. 03805). When macroscopic colonies were observed, the supernatants were screened for cell binding using CHO parental cells and CHO-huCB-1-overexpressing cells. For this primary screen, hybridoma parental CHO cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Anaheim, CA; Cat. No. 34554) and mixed with non-CFSE-labeled CHO-huCB1-overexpressing cells to enable 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 but not to parental CHO cells were selected and carried forward for confirmation screening. A total of 97 clones were selected for small-scale purification. A summary of the relevant fusions and primary screening is shown in Table 2.

[0179] [Table 2]

[0180] Example 2: Purification of mouse anti-huCB-1 specific binding clones Hybridoma clones selected based on the confirmatory primary screening were further cultured in 50 mL of low-Ig medium (1:1 IMDM (Lonza, Anaheim, CA; Cat. No. 12-722F): Ham's F12-K (Gibco, Anaheim, CA; Cat. No. 21127022) medium containing 5 mL of 100 mM sodium pyruvate solution (Invitrogen, Grand Island, NY; Cat. No. 11360070), 5 mL of 100 mM non-essential amino acids (Invitrogen, Grand Island, NY; Cat. No. 11140050), and 5 mL of 100 mM glutamine (Invitrogen, Grand Island, NY; Cat. No. 35050061)) in a T75cm 2 The cells were expanded in flasks for 3-4 weeks, and the supernatant was harvested and purified using standard Protein A purification methods.

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

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

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

[0184] [Table 3]

[0185] Example 5: EC50 binding of mouse anti-CB-1 antibodies to CB-1 CHO overexpressing cells Antibody binding was tested in a fluorescence-activated cell sorting (FACS)-based assay for the ability of mouse anti-CB1 antibodies to bind to parental, human CB-1-overexpressing, and mouse CB-1-overexpressing CHO cells to generate binding curves and EC50 values. The three cell lines were harvested, washed, and plated at 1 x 10 per well of a v-bottom 96-well polycarbonate FACS plate (Corning, Corning, NY, Cat. No. 3357). 5 Cells were dispensed into 50 μl of FACS buffer (1x PBS / 2 mM EDTA and 1% FBS (ThermoFisher Scientific, Anaheim, CA; Cat. No. 10438-026). Serial dilutions of antibodies were prepared starting at 200 nM at 2-fold concentration and serially diluted 3-fold. The titrated antibodies were added to plates containing three different cell lines (parental, human, and mouse CB-1 CHO cells) and incubated for 1 hour at 4°C. The plates were washed three times with FACS buffer. Cells were resuspended in 50 μl of 1 / 5,000 dilution of goat anti-mouse IgG-HRP (Jackson Immuno Research, West Grove, PA; Cat. No. 115-035-003), incubated for 30 minutes at 4°C, washed three times with FACS buffer, and data were analyzed using a BD FACS Canto (BD Biosciences, San Jose, CA). Jose, CA; Cat. No. 338962) and analyzed using FlowJo (FlowJo LLC, Ashland, OR) (Table 4). None of the eight functional antibodies tested were mouse cross-reactive (Figure 3).

[0186] [Table 4]

[0187] Example 6: EC50 analysis and conformational binding evaluation of functional anti-CB-1 antibodies The purpose of this experiment was to determine whether anti-CB1 antagonist antibodies have differential binding abilities for CB1, confirming their neutral, antagonist, or agonist status. Four different cell line preparations were used: CHO-huCB1 (generated in-house), CHO-huCB1 preincubated with the inverse agonist JD5037 (Cayman Chemicals, Ann Arbor, MI; Cat. No. 1392116-14-1), CHO-huCB1 preincubated with the agonist CP-55,940 (TOCRIS, Minneapolis, MN; Cat. No. 0949), and parental CHO-S cells (ThermoFisher Scientific, VA; Cat. No. R80007). 2 × 10 7 Parental CHO-S and CHO-hu CB1 cells were left in FACS buffer. In addition, 2 × 10 cells coated with the inverse agonist JD5037 or the agonist CP-55,940 were added. 7 CHO-huCB1 cells were incubated for 1 hour at 4°C. After incubation, the two coated cell lines were washed twice with FACS buffer and 2 x 10 7The cells were resuspended in FACS buffer containing the inverse agonist or agonist molecules, respectively. All four cell lines were plated in v-bottom FACS plates (Corning, Corning, NY; Cat. No. 3357), and pre-titrated anti-CB1 test antibodies were added to the cells and assessed for binding using a BD FACS Canto (BD Biosciences, San Jose, CA; Cat. No. 338962). As shown in Figure 4, the antibodies did not bind to the CHO parental cells (blue curve), and the eight functional Abs (M1, M2, M3, M4, M5, M6, M7, and M8) did not exhibit preferential binding in the presence of agonist or antagonist, as indicated by the binding observed under all conditions (red, purple, and green curves). Only two tested antibodies that were otherwise non-functional in the cAMP or pERK assays showed preferential binding in the presence of antagonist and in the absence of agonist, respectively, suggesting that functional anti-CB1 antibodies may not associate with the binding conformation that occurs in the presence of known receptor agonists or antagonists.

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

[0189] [Table 5-1]

[0190] [Table 5-2]

[0191] [Table 5-3]

[0192] [Table 5-4]

[0193] [Table 5-5]

[0194] [Table 5-6]

[0195] [Table 5-7]

[0196] [Table 5-8]

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

[0198] [Table 6-1]

[0199] [Table 6-2]

[0200] [Table 6-3]

[0201] [Table 6-4]

[0202] Table 6-5

[0203] Table 6-6

[0204] Table 6-7

[0205] Table 6-8

[0206] Table 6-9

[0207] Table 6-10

[0208] Table 6-11

[0209] Table 6-12

[0210] Table 6-13

[0211] Table 6-14

[0212] [Table 6-15]

[0213] [Table 6-16]

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

[0215] equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the above-described embodiments should be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that binds to the human cannabinoid type 1 receptor (CB1) (SEQ ID NO: 1), wherein the antibody comprises a variable heavy (VH) domain sequence and a variable light (VL) domain sequence, and the sequence pair of the VH domain sequence and the VL domain sequence (VH / VL pair) is selected from the group consisting of SEQ ID NOs: 18 / 24, 30 / 36, 42 / 48, 54 / 60, 66 / 72, 78 / 84, 90 / 96, 102 / 108, 114 20. An isolated antibody or antigen-binding fragment thereof, wherein the VH / VL pair is selected from the group consisting of: 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.

2. The fragment may be a Fab fragment, a Fab' fragment, a F(ab) fragment, or a ２ 2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising a fragment or scFv fragment.

3. 3. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment thereof comprises a human Fc region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM Fc.

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, comprising a modified human Fc region.

5. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising a modified human Fc region comprising mutations selected from the group consisting of L234A / L235A, S228P, A330S, P331S, E233P / L234V / L235A, A327G / A330S / P331S, L234F / L235E / P331S, and N297Q.

6. A multispecific binding protein comprising the antigen-binding fragment of any one of claims 1 to 5.

7. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which inhibits CB1 signaling activity.

8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is an inverse agonist of CB1 signaling activity.

9. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or 7 to 8, which is a humanized antibody.

10. 10. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-5 or 7-9, which exhibits reduced brain penetration when compared to rimonabant.

11. 10. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5 or 7 to 9, which inhibits CB1 signaling at least two-fold more than rimonabant.

12. 10. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-5 or 7-9, which exhibits reduced CNS side effects compared to rimonabant.

13. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or 7 to 12.

14. 14. An expression vector comprising the isolated nucleic acid molecule of claim 13.

15. A host cell comprising the expression vector of claim 14.

16. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5 or 7 to 12 and at least one pharmaceutically acceptable excipient.

17. 17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition is for inhibiting the biological activity of CB1 in a subject in need thereof, and wherein the inhibition is achieved by administering an effective amount of the pharmaceutical composition of claim 16 to the subject, thereby inhibiting the activity of CB1 protein in the subject.

18. 17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition is for the treatment of a disease associated with CB1 activity, and the treatment is achieved by administering the pharmaceutical composition of claim 16 to a subject suffering from the disease.

19. 17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition is for the treatment of a disease or disorder responsive to antagonism or inverse agonism of CB1 signaling in a subject in need thereof, wherein the treatment is achieved by administering to the subject the pharmaceutical composition of claim 16.

20. 13. An antibody conjugate comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5 or 7-12, wherein the antibody or antigen-binding fragment thereof is conjugated to an agent selected from the group consisting of a therapeutic agent, a cytotoxic agent, an immunoadhesion molecule, and an imaging agent.

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