Cannabinoid Receptor Type 1 Binding Protein and Its Use

Abstract

As used herein, anti-CB1 (e.g., huCB1) antibodies and antigen-binding fragments thereof, compositions containing the same, and their use for the treatment of various disorders or diseases such as obesity and its co-existing diseases, chronic kidney disease, liver disease (e.g., NAFLD), and NASH are described.

Description

Technical Field

[0001] The present disclosure relates to a cannabinoid receptor type 1 (CB1) binding protein and its use for the treatment of diseases or disorders.

[0002] Incorporation by reference of electronically submitted materials The following amino acid sequence listing, identified as follows and submitted concurrently with this specification, is hereby incorporated by reference in its entirety: the 7027 kilobyte XML document named "10129-WO01-SEC Sequence Listing.xml," created on July 20, 2023.

[0003] Cross-reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 392,891, filed on July 28, 2022.

Background Art

[0004] Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor (GPCR) expressed in the central nervous system (CNS) as well as peripheral organs such as the liver, skeletal muscle, adipose tissue, and endocrine pancreas (Cinar R. et al., Pharmacology & Therapeutics 208 (2020) 107477). The CB1 receptor is expressed at lower levels in peripheral organs compared to the CNS, and the receptor is involved in the regulation of metabolic homeostasis in both systems. Due to its role in energy homeostasis, efforts have been made for many years in the development of CB1 receptor antagonists and / or inverse agonists for the treatment of obesity and its comorbidities. (See, for example, Cinar R. et al., Pharmacology & Therapeutics 208 (2020) 107477; Thomas Murphy and Bernard Le Foll, Biomolecules 2020, 10, 855).

[0005] In clinical trials, the small molecule CB1 receptor antagonist / inverse agonist rimonabant (also known as SR141716) reduced the weight of overweight individuals and significantly improved multiple cardiometabolic parameters, such as waist circumference, hemoglobin A1c, HDL, plasma cholesterol, and triglycerides. Rimonabant was approved as an anti-obesity drug in Europe in 2006. However, the CNS penetration of rimonabant also induced severe mental side effects such as anxiety, depression, and suicide, leading to its discontinuation in 2008. Further efforts have been made in the development of peripherally restricted small molecule CB1 receptor antagonists (e.g., Tam et al, J. Clin. Invest. (2010) 120:2953-66; US2011 / 0144157), but success in this area has yet to be seen.

[0006] Since antibodies do not cross the blood-brain barrier well enough to penetrate the CNS, scientists hypothesized that CB1 antagonist antibodies with rimonabant-like potency could provide beneficial metabolic effects (e.g., treatment of obesity and its comorbidities) without CNS-related side effects. However, the discovery and manipulation of potent antibody antagonists of CB1 have proven difficult in the art. For example, the potencies of antagonist CB1 antibodies described in the art are in the two- or three-digit nanomolar range (see, e.g., WO 2014 / 210025, WO 2015 / 148948, and WO 2019 / 211665); in comparison, the potency of rimonabant is in the single-digit nanomolar range. (See, e.g., Congy C. et al., FEBS Letters 350 (1994) 240-244; Bauer M. et al., The Journal of Biological Chemistry, 287(44)(2016)36944-36967). In addition, the binding affinity of those antibodies for CB1 (e.g., huCB1) is inferior to that of rimonabant.

[0007] Several factors are thought to contribute to this difficulty. In general, it has been difficult to produce antibodies against GPCRs with small extracellular loops such as CB1 via immunization (see, for example, Jo Migyeong and Jung Sang Take, Experimental & Molecular Medicine (2016) 48, e207). Furthermore, many CB1 antibodies produced in animal campaigns tend not to function. Additionally, the titers and / or affinities of antagonist antibodies isolated from animal campaigns typically are not high enough to meet the titer and / or affinity requirements (e.g., equivalent to or better than rimonabant). Affinity maturation can be used to improve the titer of antagonist antibodies, but it has been difficult to use this strategy for CB1 antibodies due to the lack of a stable and high-quality preparation of the structurally related CB1 protein.

[0008] There remains a need in the art to identify CB1 antagonist antibodies having rimonabant-like titers and affinities that can achieve a therapeutic effect while avoiding CNS-related adverse effects.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Means for Solving the Problems

[0011] This application relates to antagonists or inverse agonists anti-CB1 (e.g., huCB1) antibodies and antigen-binding fragments having a high titer (e.g., a titer equal to or better than rimonabant). The antibodies disclosed herein have a much higher titer compared to previously known anti-CB1 antibodies and are suitable for the safe and effective treatment of various diseases such as obesity and its comorbidities, chronic kidney disease, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). Based on the disclosure provided herein, those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of the invention described herein by mere routine experimentation. Such equivalents are intended to be encompassed by the following embodiment (E).

[0012] An isolated antibody that binds to E1.huCB1, wherein the antibody comprises an HV and an LV, a) the HV comprises HCDR1, HCDR2, and HCDR3, where HCDR1 comprises the amino acid sequence of RGGDYWX1 (SEQ ID NO: 530) [wherein X1 is A, S, or G]; HCDR2 comprises the amino acid sequence of HX2YX3X4GX5TX6YNPX7X8X9X10 (SEQ ID NO: 531) [wherein X2 is I or V; X3 is H, Y, or Q; X4 is E, T, or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; HCDR3 comprises the amino acid sequence of X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO: 532) [wherein X11 is D, E, or N; X12 is A, I, P, T, or V; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y]; b) the LV is of the VK1 / O2 / JK4, VK2 / A19 / JK1, or VK3 / A27 / JK1 germline.

[0013] E2. a) HCDR1 comprises the amino acid sequence of RGGDYWX1 (SEQ ID NO: 530) [wherein X1 is A or S]; HCDR2 comprises the amino acid sequence of HX2YX3X4GX5TX6YNPX7X8X9X10 (SEQ ID NO: 531) [wherein X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; HCDR3 comprises the amino acid sequence of X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO: 532) [wherein X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is S or T; X15 is H or Y]; b) the LV is of the VK2 / A19 / JK1 germline, the antibody according to E1.

[0014] E3. The antibody according to E2, wherein X4 is T and X15 is Y.

[0015] E4.b) The LV comprises LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the amino acid sequence RSSQSLLX16SX17GX18NYX19D (SEQ ID NO: 533) [wherein X16 is H, S, or T; X17 is S, T, or Y; X18 is A, N, I, or Y; X19 is L or V]; LCDR2 comprises the amino acid sequence of X20GSNRA (SEQ ID NO: 534) [wherein X20 is L or Q]; LCDR3 comprises the amino acid sequence of X21QAX22X23X24PRT (SEQ ID NO: 535) [wherein X21 is M or R; X22 is L, I, R, or V; X23 is Q, E, T, A, or G; X24 is T, I, L, or Q], the antibody according to any one of E1 to E3.

[0016] E5. The antibody according to E4, wherein X24 is L.

[0017] E6.a) HCDR1 comprises the amino acid sequence of RGGDYWX1 (SEQ ID NO: 530) [wherein X1 is A, G, or S]; HCDR2 comprises the amino acid sequence of HX2YX3X4GX5TX6YNPX7X8X9X10 (SEQ ID NO: 531) [wherein X2 is I or V; X3 is H, Y, or Q; X4 is T or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; HCDR3 comprises the amino acid sequence of X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO: 532) [wherein X11 is D or N; X12 is A, I, or T; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y]; b) The LV is of the VK1 / O2 / JK4 or VK3 / A27 / JK1 germline, the antibody according to E1.

[0018] E7. The antibody according to E6, wherein a) X1 is A or G; X3 is Q or Y, X4 is T, X8 is F, X14 is T; X15 is Y.

[0019] The antibody according to E6 or E7, wherein the LV is of the VK1 / O2 / JK4 germline.

[0020] E9.b) The LV is of the VK1 / O2 / JK4 germline and comprises light chain LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO: 150), or RASQSISSYLN (SEQ ID NO: 186), LCDR2 comprises the amino acid sequence of AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151), and LCDR3 comprises the amino acid sequence of QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); or b) The antibody according to E6 or E7, wherein the LV is of the VK3 / A27 / JK1 germline and comprises light chain LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of RASQSVSSYLG (SEQ ID NO: 168), LCDR2 comprises the amino acid sequence of GASSRAT (SEQ ID NO: 169), and LCDR3 comprises the amino acid sequence of QQYGSSPRT (SEQ ID NO: 170).

[0021] E10. The antibody according to E9, wherein LCDR1 comprises the amino acid sequence of RASQSISNYLN (SEQ ID NO: 132), LCDR2 comprises the amino acid sequence of AASSLHS (SEQ ID NO: 133), and LCDR3 comprises the amino acid sequence of QQYQSYPLT (SEQ ID NO: 134).

[0022] E11.a) HCDR1 contains the amino acid sequence of RGGDYWX1 (SEQ ID NO: 530) [where X1 is A or G], HCDR2 contains the amino acid sequence of HX2YX3X4GX5TX6YNPX7X8X9X10 (SEQ ID NO: 531) [where X2 is I or V; X3 is Y or Q; X4 is T; X5 is S; X6 is K or N; X7 is S or R; X8 is F; X9 is K; X10 is G or D], and HCDR3 contains the amino acid sequence of X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO: 532) [where X11 is N; X12 is T; X13 is L or V; X14 is T; X15 is Y]. b) The antibody according to E6, wherein the LV is of the VK1 / O2 / JK4 germline.

[0023] E12.a) HCDR1 contains the amino acid sequence of RGGDYWA (SEQ ID NO: 414) or RGGDYWG (SEQ ID NO: 408); HCDR2 contains the amino acid sequence of HVYYTGSTKYNPSFKD (SEQ ID NO: 427), HVYYTGSTNYNPRFKD (SEQ ID NO: 445), HIYQTGSTNYNPRFKG (SEQ ID NO: 415), or HVYQTGSTKYNPSFKD (SEQ ID NO: 409); HCDR3 contains the amino acid sequence of NYDTLTGYSYYYYGMDV (SEQ ID NO: 410) or NYDTVTGYSYYYYGMDV (SEQ ID NO: 446). The antibody according to E11.

[0024] E13.b) The LV contains LCDR1, LCDR2, and LCDR3, where LCDR1 contains the sequence of RASQSISSYLN (SEQ ID NO: 405); LCDR2 contains the sequence of X39ARX40LX41S (SEQ ID NO: 536) [where X39 is N, S, K, or G; X40 is R, K, L, or A; X41 is A, G, or S]; LCDR3 contains the sequence of QQX42X43X44X45PX46T (SEQ ID NO: 537) [where X42 is Y or F; X43 is R, A, S, G, or Y; X44 is S, K, R, or H; X45 is S, L, F, Y, P, or M; X46 is L, I, or V]. The antibody according to E11 or E12.

[0025] The antibody according to E13, wherein E14.X42 is Y.

[0026] An isolated antibody that binds to E15.huCB1, wherein the antibody comprises HV and LV, and a) HV comprises CDRH1, CDRH2, and CDRH3, where CDRH1 comprises the amino acid sequence of RGGDYWS (SEQ ID NO: 1); CDRH2 comprises the amino acid sequence of HX25YX26X27GX28TX29YNPX30X31X32X33 (SEQ ID NO: 538) [wherein X25 is I or V; X26 is Y or Q; X27 is A, E, K, or T; X28 is S or Q; X29 is A, E, K, or T; X30 is N or S; X31 is F or L; X32 is K or R; X33 is G, S, or N]; CDRH3 comprises the amino acid sequence of X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539) [wherein X34 is D or G; X35 is A, I, T, or V; X36 is L or absent; X37 is S or T; X38 is M or L]; b) LV is of the VK2 / A19 / JK1 germline.

[0027] The antibody according to E15, wherein a) X26 is Y, X34 is G, X36 is absent, and X37 is S.

[0028] The antibody according to E15 or E16, wherein b) LV comprises LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the amino acid sequence of RSSQSLLHRSGYNYLD (SEQ ID NO: 257); LCDR2 comprises the amino acid sequence of LGSNRAS (SEQ ID NO: 258) or QGSNRAS (SEQ ID NO: 264); LCDR3 comprises the amino acid sequence of MQSLQTPRT (SEQ ID NO: 259), RQSVALPRT (SEQ ID NO: 271), or RQARALPRT (SEQ ID NO: 265).

[0029] The antibody according to any one of E1 to E14, wherein the antibody comprises any one of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, or a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of LIBC523596-1, LIBC523603-1, LIBC523661-1, LIBC523670-1, LIBC523748-1, LIBC523760-1, LIBC523797-1, LIBC523813-1, LIBC523815-1, LIBC523844-1, LIBC523857-1, LIBC523862-1, LIBC523868-1, LIBC523969-1, LIBC524049-1, LIBC527906-1, LIBC527814-1, LIBC527997-1, LIBC528141-1, LIBC527912-1, LIBC528116-1, LIBC527879-1, LIBC527919-1, LIBC527984-1, LIBC527968-1, LIBC528169-1, LIBC528131-1, LIBC527827-1, LIBC527869-1, and LIBC528148-1, LIBC680562-1, LIBC680574-1, LIBC680773-1, LIBC680800-1, LIBC680812-1, LIBC681593-1, LIBC681594-1, and LIBC681737-1.

[0030] The antibody according to any one of E15 to E17, wherein the antibody comprises any one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, or a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of LIBC673948-1, LIBC673952-1, LIBC673965-1, LIBC673982-1, LIBC673972-1, LIBC674024-1, LIBC674035-1, LIBC674043-1, LIBC674090-1, LIBC674002-1, LIBC674153-1, LIBC674200-1, LIBC674214-1, LIBC674216-1, LIBC674229-1, LIBC674235-1, LIBC674257-1, and LIBC674276-1.

[0031] The antibody according to E18 or E19, wherein the antibody comprises any one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, or a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of any one of LIBC680574, LIBC528116, LIBC673952, LIBC523797, LIBC527814, LIBC527997, LIBC527919, LIBC523661, and LIBC524049.

[0032] The antibody according to any one of E1 to E20, wherein the amino acid at position 83 of HV is N and the amino acid at position 85 of HV is Y.

[0033] The antibody according to E21, wherein the amino acid at position 94 of LV is S.

[0034] The antibody according to E19, wherein the amino acid at position 83 of HV is N, the amino acid at position 85 of HV is Y, and the amino acid at position 79 of HV is R.

[0035] The antibody according to E23, wherein the amino acid at position 94 of LV is S and the amino acid at position 76 of LV is D.

[0036] An antibody according to any one of E1 to E24, wherein the HCDR2 of the antibody contains 5 or fewer mutations, or 3 or fewer mutations, compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136).

[0037] An antibody according to any one of E1 to E24, wherein the HCDR2 of the antibody contains 5 or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8), and optionally, the HCDR3 of the antibody contains 4 or fewer mutations compared to DYDILTGYSYYYYGMDV (SEQ ID NO: 9).

[0038] The antibody according to any one of E1 to E26, comprising any one of the HVs and / or LVs of LIBC523596-1, LIBC523603-1, LIBC523661-1, LIBC523670-1, LIBC523748-1, LIBC523760-1, LIBC523797-1, LIBC523813-1, LIBC523815-1, LIBC523844-1, LIBC523857-1, LIBC523862-1, LIBC523868-1, LIBC523969-1, LIBC524049-1, LIBC527906-1, LIBC527814-1, LIBC527997-1, LIBC528141-1, LIBC527912-1, LIBC528116-1, LIBC527879-1, LIBC527919-1, LIBC527984-1, LIBC527968-1, LIBC528169-1, LIBC528131-1, LIBC527827-1, LIBC527869-1, and LIBC528148-1, LIBC680562-1, LIBC680574-1, LIBC680773-1, LIBC680800-1, LIBC680812-1, LIBC681593-1, LIBC681594-1, LIBC681737-1, LIBC673948-1, LIBC673952-1, LIBC673965-1, LIBC673982-1, LIBC673972-1, LIBC674024-1, LIBC674035-1, LIBC674043-1, LIBC674090-1, LIBC674002-1, LIBC674153-1, LIBC674200-1, LIBC674214-1, LIBC674216-1, LIBC674229-1, LIBC674235-1, LIBC674257-1, and LIBC674276-1.

[0039] The antibody according to E27, comprising any one of the HVs and / or LVs of LIBC680574, LIBC528116, LIBC673952, LIBC523797, LIBC527814, LIBC527997, LIBC527919, LIBC523661, and LIBC524049.

[0040] An antibody that binds to E29.huCB1 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of LIBC529593, LIBC560340, or LIBC560657, or a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0041] E30. The antibody according to E29, wherein the antibody comprises HV and / or LV of LIBC529593, LIBC560340, or LIBC560657.

[0042] E31. The antibody according to any one of E1 - E30, wherein the antibody is a monoclonal antibody, and optionally, the monoclonal antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0043] E32. The monoclonal antibody according to E31, wherein the antibody is an antagonist and / or inverse agonist antibody of huCB1.

[0044] E33. The antibody according to E31 or E32, wherein the antibody has a binding affinity for huCB1 that is at least 3 - fold higher compared to 10D10N35Y.

[0045] E34. The antibody according to E32 or E33, wherein the antibody has an IC50 of 10 nM or less, such as 5 nM or less, 3 nM or less, or 1 nM or less when measured using a cell - based cAMP assay.

[0046] E35. An isolated monoclonal antibody that binds to huCB1, wherein the antibody is an antagonist and / or inverse agonist antibody of huCB1 and has an IC50 of 10 nM or less, or 8 nM or less when measured using a cell - based cAMP assay.

[0047] E36. The antibody according to E35, wherein the antibody binds to extracellular loop 2 of huCB1 or to a region contained within extracellular loop 2 of huCB1.

[0048] The antibody according to any one of E34 to E36, wherein the cell line cAMP assay is a cell line cAMP assay using CP55,940.

[0049] The antibody according to any one of E31 to E37, wherein the monoclonal antibody is a human IgG1, IgG2, IgG3, or IgG4 antibody, preferably a human IgG1 antibody.

[0050] The antibody according to E38, wherein the antibody contains a mutation at amino acid position N297 such as N297G by EU numbering in its heavy chain.

[0051] The antibody according to E39, wherein the antibody further contains R292C and V302C mutations by EU numbering in its heavy chain.

[0052] The antibody according to any one of E37 to E40, wherein the antibody contains mutations at amino acid positions M252, S254, and T256, preferably M252Y, S254T, and T256E by EU numbering in its heavy chain.

[0053] A method for treating a subject in need of antagonizing or inverse agonizing a CB1 receptor, the method comprising administering to the subject an antibody according to any one of E1 to E41.

[0054] A method for treating a disease or disorder in a subject responsive to antagonizing or inverse agonizing a CB1 receptor, the method comprising administering to the subject an antibody according to any one of E1 to E41.

[0055] The method according to E42 or E43, wherein administration results in one or more of weight loss, reduced appetite, improved metabolic parameters, reduced blood glucose level, reduced insulin level, reduced triglyceride level, reduced kidney injury, reduced kidney fibrosis, reduced kidney inflammation, and improved kidney function.

[0056] The method according to E43, wherein the disease or disorder is selected from obesity, diabetes, dyslipidemia, metabolic diseases, liver diseases, fibrosis, non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis, kidney diseases, kidney fibrosis, chronic kidney diseases, IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular diseases, cancer, and inflammatory diseases.

[0057] The antibody according to any one of E1 to E41, for use in the treatment of a disease or disorder in a subject, wherein the disease or disorder is selected from obesity, diabetes, dyslipidemia, metabolic diseases, fibrosis, liver diseases, NASH, primary biliary cirrhosis, kidney diseases, kidney fibrosis, chronic kidney diseases, IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular diseases, cancer, and inflammatory diseases.

[0058] The antibody according to E46, which, upon use, results in one or more of weight loss, appetite reduction, improvement of metabolic parameters, reduction of blood glucose level, reduction of insulin level, reduction of triglyceride level, reduction of kidney injury, reduction of kidney fibrosis, reduction of kidney inflammation, and improvement of kidney function.

[0059] E48. The disease or disorder is obesity, and optionally, the subject has a BMI of at least 27 kg / m 2 or the subject has a BMI of at least 27 kg / m 2 The method according to E45 or the antibody according to E46.

[0060] E49. The method according to any one of E42 to E45 or the antibody according to E47 or E48, wherein the subject is human.

[0061] E50. An isolated polynucleotide encoding the antibody according to any one of E1 to E41.

[0062] E51. An expression vector comprising the polynucleotide according to E50.

[0063] E52. A host cell comprising the expression vector according to E51.

[0064] The antibody according to E5 or E15, wherein the 32 amino acids numbered by AHo in the heavy chain are R.

Brief Description of the Drawings

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[0066] Cannabinoid receptor 1 (CB1) is a G-protein coupled receptor for endogenous cannabinoids such as 2-arachidonoylglycerol (2-AG), and this receptor mediates many cannabinoid-induced effects (e.g., food intake). Activation of the receptor results in a decrease in intracellular cyclic AMP (cAMP) concentration and an increase in mitogen-activated protein kinase (MAP kinase) concentration. (Gerard C. et al., Biochem. J. 279:129-134 (1991)). The human CB1 (huCB1) receptor is a polypeptide of 472 amino acids (UniProtKB / Swiss-Prot: P21554) and is encoded by the CNR1 gene. The amino acid sequence of huCB1 is listed below (SEQ ID NO: 559). huCB1 has four extracellular regions (amino acid residues 1-116, 176-187, 256-273, and 366-377 of SEQ ID NO: 559), four cytoplasmic regions (amino acid residues 143-154, 213-232, 300-344, and 400-472 of SEQ ID NO: 559), and seven transmembrane domains (amino acid residues 117-142, 155-175, 188-212, 233-255, 274-299, 345-365, and 378-399 of SEQ ID NO: 559).

Chem.

[0067] The CB1 receptor is expressed in the CNS and peripheral systems and is involved in the regulation of energy homeostasis. This receptor has been a target for the development of drugs for the treatment of obesity and its comorbidities. Given the experience with rimonabant, the field has focused on the development of peripherally acting CB1 receptor antagonists or inverse agonists that confer beneficial metabolic effects without CNS-related side effects.

[0068] Antibodies against the CB1 receptor do not penetrate well enough to recognize the CNS and thus can function as peripherally acting antagonists or inverse agonists. In an effort to achieve a therapeutic effect without an associated side effect profile, efforts have been made to identify antagonist or inverse agonist anti-CB1 antibodies with rimonabant-like potency. The 10D10 antibody was a lead antagonist anti-CB1 antibody isolated from a Xenomouse antibody campaign, but its potency was significantly lower than that of rimonabant. (Pamphlet of International Publication No. WO 2014 / 210205, the disclosures of which are incorporated herein by reference in their entirety). To improve the potency of the antibody, multiple rounds of affinity maturation were performed. Some 10D10 affinity maturation variants isolated from an initial affinity maturation campaign had improved potencies compared to 10D10, but none of the variants met the design goal of rimonabant-like potency. The lack of a stable, high-quality preparation of the structurally related CB1 protein was thought to contribute to the inability to isolate anti-CB1 antibodies with rimonabant-like potency. In addition, the 10D10 antibody and its initial affinity maturation variants had suboptimal manufacturability (e.g., low expression levels and instability).

[0069] The evolution of technical sophistication in generating high-quality soluble preparations of complex membrane proteins has maximized the ability to formulate huCB1 into nanodiscs (NDs) or styrene maleic acid lipid particles (SMALPs) (Luna V. et al., European Polymer Journal 109 (2018) 483-488; Lavington S. and Watts A, Biophysical Reviews, vol. 12 (2020) 1287-1302). In contrast to detergent-solubilized cell lysates prepared from huCB1-expressing cells, both CB1-ND and -SMALP are stable after multiple freeze / thaw cycles and are purification reagents suitable for long-term storage. Furthermore, these can be added to binding experiments at user-defined concentrations of huCB1. The availability of CB1-ND and -SMALP has enabled the isolation of the high-potency antagonist or inverse agonist anti-CB1 antibodies disclosed herein.

[0070] This application relates to antagonist or inverse agonist anti-CB1 antibodies and antigen-binding fragments having a titer equal to or better than rimonabant, particularly anti-huCB1 antibodies and antigen-binding fragments. Antibodies that inhibit CB1 cause an increase in cAMP, which can be measured using in vitro cAMP assays such as cell-based cAMP assays. As exemplified in the Examples, the titers of the antibodies and antigen-binding fragments disclosed herein are in the single-digit nanomolar range; for example, they have an IC50 of less than about 10 nM in cell-based cAMP assays (see Examples 5, 8, and 9). As shown in the Examples, the high-titer antibodies and antigen-binding fragments disclosed herein reduce body weight, food intake, and multiple metabolic parameters in obese animal models (e.g., reducing body fat, insulin levels, and liver triglyceride levels, Example 7), and the increased energy consumption, lipid oxidation, and insulin sensitivity in animal models, although brain exposure was minimal, indicate that these may enable safe and effective therapeutic treatment of various diseases and disorders (e.g., obesity and its comorbidities, chronic kidney disease, NAFLD, NASH). In contrast, antagonist or inverse agonist antibodies having a titer value lower than rimonabant are not considered suitable for such therapeutic purposes. Furthermore, the anti-CB1 antibodies and antigen-binding fragments disclosed herein are stable and express well for manufacturing purposes. These high-titer anti-CB1 antibodies and antigen-binding fragments are described and exemplified herein.

[0071] Definitions The present invention provides an antagonist or inverse agonist antibody against human CB1. An "antibody" includes an antigen-binding fragment that specifically binds to an antigen, and is a protein that includes a backbone or framework portion that enables the antigen-binding fragment to adopt a conformation that promotes binding of the antibody to the antigen. As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein that includes two light chain polypeptides (each about 25 kDa) and two heavy chain polypeptides (each about 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide that includes, from the amino terminus to the carboxyl terminus, a single immunoglobulin light chain variable region (VL or LV) and a single immunoglobulin light chain constant domain (CL or LC). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide that includes, from the amino terminus to the carboxyl terminus, a single immunoglobulin heavy chain variable region (VH or HV), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (δ), gamma (γ), alpha (α), and epsilon (ε), and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG class antibodies and IgA class antibodies are further divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4 and IgA1 and IgA2, respectively. Heavy chains in IgG antibodies, IgA antibodies, and IgD antibodies have three domains (CH1, CH2, and CH3), whereas heavy chains in IgM antibodies and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain) and between the hinge regions of the two antibody heavy chains.

[0072] In a human antibody, CH1 refers to the region having an amino acid sequence at positions 118 to 215 of the EU index or EU numbering system based on the sequential numbering of the first human IgG1 (i.e., "EU antibody") to be sequenced (Edelman et al., Proc Natl Acad Sci USA, 63(1):78-85 (1969)). A highly flexible amino acid region called the "hinge region" exists between CH1 and CH2. CH2 represents the region having an amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region having an amino acid sequence at positions 341 to 446 of the EU index.

[0073] "CL" represents the constant region of the light chain. In the case of the κ chain of a human antibody, CL represents the region having an amino acid sequence at positions 108 to 214 of the EU index. In the case of the λ chain, CL represents the region having an amino acid sequence at positions 108 to 215.

[0074] "Variable domain" refers to either the variable region (VL or LV) of the antibody light chain or the variable region (VH or HV) of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) linked by three complementarity-determining regions (CDRs) and contribute to the formation of the antigen-binding site of the antibody. From the N-terminus to the C-terminus, both the naturally occurring light chain variable region and heavy chain variable region typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0075] The "complementary determining regions" (CDRs) of an antibody can be defined according to the Kabat, Chothia, combined Kabat and Chothia, AbM, contact, North, and / or conformation definitions, or any method for determining CDRs well known in the art. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structures). The AbM definition of CDRs is a compromise between Kabat and Chothia and uses Oxford Molecular's AbM antibody modeling software (Accelrys®). The identity of the amino acid residues of a particular antibody that make up the CDRs can be determined using methods well known in the art.

[0076] The term "antigen-binding fragment" refers to a molecule that is derived from an antibody and retains the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Examples of antigen-binding fragments include: (i) the Fab fragment, which is a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain; (ii) the F(ab’)2 fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment consisting of the VH domain and CH1 domain; (iv) the Fv fragment consisting of the VL domain and VH domain of a single arm of an antibody; and (v) the dAb fragment consisting of the VH domain (Ward et al., 1989 Nature 341:544-546). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv)); see, for example, Bird et al. Science 242:423-426 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0077] "About" or "approximately", when used in connection with a measurable numerical variable, refers to all values of the variable that are either within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or greater than the indicated value by up to ±10% of the indicated value. Numerical ranges include the numbers defining the range.

[0078] The term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, antibody or antigen-binding fragment) is, due to its origin or source of derivation, (1) not associated with the naturally associated components that accompany it in its natural state, (2) substantially free of other molecules from the same species, (3) expressed by cells from a different species, or (4) a molecule that does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cell line different from the cell in which it naturally occurs is "isolated" from its naturally associated components. Molecules can also be made substantially free of their naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be evaluated by many means well known in the art. For example, the purity of a polypeptide sample can be evaluated by using polyacrylamide gel electrophoresis, staining the gel using techniques well known in the art, and visualizing the polypeptide. For certain purposes, higher resolution can be provided by using HPLC or other purification means well known in the art.

[0079] Unless otherwise indicated, throughout this specification and the claims, the numbering of amino acid residues in immunoglobulin heavy or light chains follows the AHo numbering scheme as described in Honegger and Pluckthun, J. Mol. Biol. 309(3):657 - 670; 2001. In some embodiments, the EU numbering scheme as described in Edelman et al., Proc. Natl. Acad. USA, Vol. 63:78 - 85 (1969) is used when referring to the positions of amino acids having immunoglobulin constant regions. Such numbering schemes are well known in the art.

[0080] An "antagonist" binds to a protein (e.g., a receptor) and, inter alia, inactivates, desensitizes, or down-regulates the signaling activity of a ligand of the protein (e.g., an agonist of the receptor), either partially or completely. An inverse agonist is an agent that causes the opposite effect of an agonist; for example, it decreases the basal activity of a receptor.

[0081] Anti-CB1 antibodies and antigen-binding fragments Disclosed herein are antibodies and antigen-binding fragments to huCB1 (SEQ ID NO: 559), and antagonists and / or inverse agonists of huCB1. The titers of the antibodies and antigen-binding fragments are equal to or better than the titer of rimonabant, thereby making it possible to achieve a therapeutic effect (e.g., treatment of obesity, chronic kidney disease, liver disease) without CNS-related side effects. In some embodiments, the antibodies and antigen-binding fragments have an IC50 in the single-digit nanomolar range as measured by a cell-based cAMP assay. For example, in some embodiments, the IC50 of the antibodies and antigen-binding fragments is less than about 10 nM as measured by a cell-based cAMP assay. In some embodiments, the IC50 of the antibodies and antigen-binding fragments is less than about 5 nM as measured by a cell-based cAMP assay. In some embodiments, the IC50 of the antibodies and antigen-binding fragments is less than about 3 nM or less than about 1 nM as measured by a cell-based cAMP assay.

[0082] The anti-CB1 antibodies and antigen-binding fragments disclosed herein were identified from affinity maturation of 10D10 (e.g., Examples 1-4 and 9). The 10D10 antibody was isolated from a prior antibody campaign (WO 2014 / 210205 pamphlet), and the heavy and light chain CDR sequences and heavy and light chain variable region sequences of 10D10 are listed in Tables 7 and 8. The yeast display library design for affinity maturation was guided by next-generation sequencing, and the amino acids at specific HV and LV positions of 10D10 were optimized to improve binding affinity and titer. The 10D10 LC belongs to the Vκ2 germline, and as disclosed herein, only the HC of the antibody binds to the CB1 receptor (Examples 1 and 14). LC-swapping affinity matured antibodies (i.e., swapping the 10D10 LC with a light chain of the Vκ1 or Vκ3 germline with improved manufacturability) were also isolated. The heavy and light chain consensus CDR sequences of the affinity matured antibodies and antigen-binding fragments are summarized in Table 35. Thus, in one aspect, an antibody or antigen-binding fragment thereof that binds to huCB1 and comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein a) the HV comprises HCDR1, HCDR2, and HCDR3, where HCDR1 comprises the amino acid sequence of RGGDYWX1 (SEQ ID NO: 530) [wherein X1 is A, S, or G]; HCDR2 comprises the amino acid sequence of HX2YX3X4GX5TX6YNPX7X8X9X10 (SEQ ID NO: 531) [wherein X2 is I or V; X3 is H, Y, or Q; X4 is E, T, or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; HCDR3 comprises the amino acid sequence of X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO: 532) [wherein X11 is D, E, or N; X12 is A, I, P, T, or V; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y]; b) Antibodies or antigen-binding fragments thereof are disclosed herein, wherein the LV is of or derived from the VK1 / O2 / JK4, VK2 / A19 / JK1, or VK3 / A27 / JK1 germline.

[0083] As used herein, a "germline" or "germline-derived" variable region sequence (such as a variable region or CDR amino acid sequence) refers to a sequence having structural features characteristic of a particular germline, rather than another germline. For a host organism to produce an antibody that binds an antigen, the rearranged immunoglobulin germline genes (of the host organism's B cells) must first encode a primary antibody having a three-dimensional structure and biochemical properties capable of interacting with that antigen. Each host organism has a defined repertoire of immunoglobulin germline genes that can be rearranged to produce a primary antibody. Through the process of somatic hypermutation, the host organism can select mutations or a set of mutations in the genes encoding the antibody that further enhance the interaction with the antigen. Thus, an antibody sequence "of" or "derived from" a particular germline may have a variable region sequence that contains differences compared to that germline. However, the antibody sequence will contain structural features such as specific amino acids, CDR lengths, and / or conformational properties of a particular germline, indicating that the particular germline is the germline most closely related to the relevant sequence. This interaction may be similar to a hand (antigen) being fitted by a glove (germline antibody). The initial structure defined by the germline gene must be sufficient for the glove to fit the hand in the first place, and then it can be adjusted to fit more precisely. Since a particular combination of rearranged immunoglobulin germline genes encoding an antibody must specify a three-dimensional structure (glove) that interacts with the antigen (hand), the germline identity of an antibody will be understood to define the structural features of the paratope that interacts with the antigen. Since the light chain of the CB1 antibodies disclosed herein does not directly contact the antigen, it will be understood that the light chain germline identity defines the structural features of the light chain for pairing with the heavy chain for paratope interaction with the antigen.

[0084] The repertoire of germline genes typically used in antibody production, including Homo sapiens and Mus musculus, is a sequence and is known, and can be accessed, for example, from IMGT / GENE-DB (Giudicelli V., et al., "IMGT / GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes." Nucleic Acids Res., 33: D256-D261 (2005)). Antibody sequences can be aligned against the germline gene repertoire using an algorithm such as the Basic Local Alignment Search Tool (BLAST) under default settings, for example, to determine which host organism germline gene segment(s) the antibody is from.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A or S; an HCDR2 comprising the sequence of HX2YX3X4GX5TX6YNPX7X8X9X10 (SEQ ID NO: 531), wherein X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N; and an HCDR3 comprising the sequence of X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO: 532), wherein X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is S or T; X15 is H or Y. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530, wherein X1 is A or S; an HCDR2 comprising the sequence of SEQ ID NO: 531, wherein X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N; and an HCDR3 comprising the sequence of SEQ ID NO: 532, wherein X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is T; X15 is Y. In various embodiments, the HCDR1, HCDR2, and HCDR3 comprise the sequences defined above, and the LV is of or derived from the VK1 / O2 / JK4, VK2 / A19 / JK1, or VK3 / A27 / JK1 germline. In some embodiments, the LV is of or derived from the VK2 / A19 / JK1 germline.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or S]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is S or T; X15 is H or Y], and b) an LV comprising an LCDR1, an LCDR2, and an LCDR3, wherein the LCDR1 comprises the amino acid sequence RSSQSLLX16SX17GX18NYX19D (SEQ ID NO: 533) [wherein X16 is H, S, or T; X17 is S, T, or Y; X18 is A, N, I, or Y; X19 is L or V]; the LCDR2 comprises the amino acid sequence of X20GSNRA (SEQ ID NO: 534) [wherein X20 is L or Q]; and the LCDR3 comprises the amino acid sequence of X21QAX22X23X24PRT (SEQ ID NO: 535) [wherein X21 is M or R; X22 is L, I, R, or V; X23 is Q, E, T, A, or G; X24 is T, I, L, or Q].

[0087] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or S]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is T; X15 is Y]; and b) an LCDR1 comprising the sequence of SEQ ID NO: 533 [wherein X16 is H, S, or T; X17 is S, T, or Y; X18 is A, N, I, or Y; X19 is L or V]; an LCDR2 comprising the sequence of SEQ ID NO: 534 [wherein X20 is L or Q]; and an LCDR3 comprising the sequence of SEQ ID NO: 535 [wherein X21 is M or R; X22 is L, I, R, or V; X23 is Q, E, T, A, or G; X24 is T, I, L, or Q].

[0088] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or S]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is S or T; X15 is H or Y]; and b) an LCDR1 comprising SEQ ID NO: 533 [wherein X16 is H, S, or T; X17 is S, T, or Y; X18 is A, N, I, or Y; X19 is L or V]; an LCDR2 comprising the sequence of SEQ ID NO: 534 [wherein X20 is L or Q]; and an LCDR3 comprising the sequence of SEQ ID NO: 535 [wherein X21 is M or R; X22 is L, I, R, or V; X23 is Q, E, T, A, or G; X24 is L].

[0089] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or S]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is T; X15 is Y]; and b) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 533 [wherein X16 is H, S, or T; X17 is S, T, or Y; X18 is A, N, I, or Y; X19 is L or V]; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 534 [wherein X20 is L or Q]; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 535 [wherein X21 is M or R; X22 is L, I, R, or V; X23 is Q, E, T, A, or G; X24 is L].

[0090] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530, wherein X1 is A, G, or S; an HCDR2 comprising the sequence of SEQ ID NO: 531, wherein X2 is I or V; X3 is H, Y, or Q; X4 is T or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N; and an HCDR3 comprising the sequence of SEQ ID NO: 532, wherein X11 is D or N; X12 is A, I, or T; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530, wherein X1 is A or G; an HCDR2 comprising the sequence of SEQ ID NO: 531, wherein X2 is I or V; X3 is Y or Q; X4 is T; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F; X9 is E or K; X10 is G, D, S, or N; and an HCDR3 comprising the sequence of SEQ ID NO: 532, wherein X11 is D or N; X12 is A, I, or T; X13 is F, L or V; X14 is T; X15 is Y. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530, wherein X1 is A or G; an HCDR2 comprising the sequence of SEQ ID NO: 531, wherein X2 is I or V; X3 is Y or Q; X4 is T; X5 is S; X6 is K or N; X7 is S or R; X8 is F; X9 is K; X10 is G or D; and an HCDR3 comprising the sequence of SEQ ID NO: 532, wherein X11 is N; X12 is T; X13 is L or V; X14 is T; X15 is Y. In various embodiments, the HCDR1, HCDR2, and HCDR3 comprise the sequences defined above, and the LV is of or derived from the VK1 / O2 / JK4 or VK3 / A27 / JK1 germline. In some embodiments, the LV is of or derived from the VK1 / O2 / JK4 germline.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A, G, or S]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is H, Y, or Q; X4 is T or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D or N; X12 is A, I, or T; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y], and b) the LV is of or derived from VK1 / O2 / JK4 or VK3 / A27 / JK1, preferably the LV is of or derived from the VK1 / O2 / JK4 germline.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or G]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is T; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D or N; X12 is A, I, or T; X13 is F, L, or V; X14 is T; X15 is Y], and b) the LV is of or derived from the VK1 / O2 / JK4 germline.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A, G, or S]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is H, Y, or Q; X4 is T or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D or N; X12 is A, I, or T; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y]; and b) the LV is of or derived from VK1 / O2 / JK4 and comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO: 150), or RASQSISSYLN (SEQ ID NO: 186); LCDR2 comprises the amino acid sequence of AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151); LCDR3 comprises the amino acid sequence of QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); or the VL is of the VK3 / A27 / JK1 germline and comprises light chain LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of RASQSVSSYLG (SEQ ID NO: 168); LCDR2 comprises the amino acid sequence of GASSRAT (SEQ ID NO: 169); and LCDR3 comprises the amino acid sequence of QQYGSSPRT (SEQ ID NO: 170).

[0094] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A, G, or S]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is H, Y, or Q; X4 is T or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D or N; X12 is A, I, or T; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y]; and b) an LCDR1 comprising the sequence of RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO: 150), or RASQSISSYLN (SEQ ID NO: 186); an LCDR2 comprising the sequence of AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151); and an LCDR3 comprising the sequence of QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); preferably, b) an LCDR1 comprising the sequence of RASQSISNYLN (SEQ ID NO: 132); an LCDR2 comprising the sequence of AASSLHS (SEQ ID NO: 133); and an LCDR3 comprising the sequence of QQYQSYPLT (SEQ ID NO: 134).

[0095] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or G]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is T; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F; X9 is E or K; X10 is G, D, S, or N]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is D or N; X12 is A, I, or T; X13 comprises F, L, or V; X14 is T; X15 is Y]; and b) the LV is of or derived from the VK1 / O2 / JK4 germline and comprises an LCDR1, an LCDR2, and an LCDR3, wherein the LCDR1 comprises the amino acid sequence of RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO: 150), or RASQSISSYLN (SEQ ID NO: 186); the LCDR2 comprises the amino acid sequence of AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151); the LCDR3 comprises the amino acid sequence of QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); preferably, b) comprises an LCDR1 comprising the sequence of RASQSISNYLN (SEQ ID NO: 132); an LCDR2 comprising the sequence of AASSLHS (SEQ ID NO: 133); and an LCDR3 comprising the sequence of QQYQSYPLT (SEQ ID NO: 134).

[0096] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or G]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is T; X5 is S; X6 is K or N; X7 is S or R; X8 is F; X9 is K; X10 is G or D]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is N; X12 is T; X13 is L or V; X14 is T; X15 is Y], and b) the LV is of or derived from the VK1 / O2 / JK4 germline. In some embodiments, the antibody or antigen-binding fragment thereof comprises a) an HCDR1 comprising the amino acid sequence of RGGDYWA (SEQ ID NO: 414) or RGGDYWG (SEQ ID NO: 408); an HCDR2 comprising the amino acid sequence of HVYYTGSTKYNPSFKD (SEQ ID NO: 427), HVYYTGSTNYNPRFKD (SEQ ID NO: 445), HIYQTGSTNYNPRFKG (SEQ ID NO: 415), or HVYQTGSTKYNPSFKD (SEQ ID NO: 409); and an HCDR3 comprising the amino acid sequence of NYDTLTGYSYYYYGMDV (SEQ ID NO: 410) or NYDTVTGYSYYYYGMDV (SEQ ID NO: 446), and b) the LV is of or derived from the VK1 / O2 / JK4 germline.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 530 [wherein X1 is A or G]; an HCDR2 comprising the sequence of SEQ ID NO: 531 [wherein X2 is I or V; X3 is Y or Q; X4 is T; X5 is S; X6 is K or N; X7 is S or R; X8 is F; X9 is K; X10 is G or D]; and an HCDR3 comprising the sequence of SEQ ID NO: 532 [wherein X11 is N; X12 is T; X13 is L or V; X14 is T; X15 is Y]; and b) the LV is of or derived from the VK1 / O2 / JK4 germline lineage and comprises LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the sequence of RASQSISSYLN (SEQ ID NO: 405); LCDR2 comprises the sequence of X39ARX40LX41S (SEQ ID NO: 536) [wherein X39 is N, S, K, or G; X40 is R, K, L, or A; X41 is A, G, or S]; and LCDR3 comprises the sequence of QQX42X43X44X45PX46T (SEQ ID NO: 537) [wherein X42 is Y or F; X43 is R, A, S, G, or Y; X44 is S, K, R, or H; X45 is S, L, F, Y, P, or M; X46 is L, I, or V]. In some embodiments, X42 is Y and X1-X15, X39-X41, and X43-X44 are as defined above.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) an HCDR1 comprising the amino acid sequence of RGGDYWA (SEQ ID NO: 414) or RGGDYWG (SEQ ID NO: 408); an HCDR2 comprising the amino acid sequence of HVYYTGSTKYNPSFKD (SEQ ID NO: 427), HVYYTGSTNYNPRFKD (SEQ ID NO: 445), HIYQTGSTNYNPRFKG (SEQ ID NO: 415), or HVYQTGSTKYNPSFKD (SEQ ID NO: 409); and an HCDR3 comprising the amino acid sequence of NYDTLTGYSYYYYGMDV (SEQ ID NO: 410) or NYDTVTGYSYYYYGMDV (SEQ ID NO: 446), and b) an LCDR1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 405); an LCDR2 comprising the sequence of X39ARX40LX41S (SEQ ID NO: 536) [wherein X39 is N, S, K, or G; X40 is R, K, L, or A; X41 is A, G, or S]; and an LCDR3 comprising the sequence of QQX42X43X44X45PX46T (SEQ ID NO: 537) [wherein X42 is Y or F; X43 is R, A, S, G, or Y; X44 is S, K, R, or H; X45 is S, L, F, Y, P, or M; X46 is L, I, or V]. In some embodiments, X42 is Y and X39-X41 and X43-X44 are as defined above.

[0099] In certain embodiments, the anti-CB1 antibody or antigen-binding fragment thereof disclosed in the present invention comprises a heavy-chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light-chain variable region comprising LCDR1, LCDR2, and LCDR3, which are derived from any of the anti-CB1 antibodies described herein. Thus, in some embodiments, an anti-CB1 antibody or antigen-binding fragment thereof comprising any one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of LIBC523596-1, LIBC527906-1, LIBC523603-1, LIBC527814-1, LIBC523661-1, LIBC527997-1, LIBC523670-1, LIBC528141-1, LIBC523748-1, LIBC527912-1, LIBC523760-1, LIBC528116-1, LIBC523797-1, LIBC527879-1, LIBC523813-1, LIBC527919-1, LIBC523815-1, LIBC527984-1, LIBC523844-1, LIBC527968-1, LIBC523857-1, LIBC528169-1, LIBC523862-1, LIBC528131-1, LIBC523868-1, LIBC527827-1, LIBC523969-1, LIBC527869-1, LIBC524049-1, LIBC528148-1, LIBC680562-1, LIBC680574-1, LIBC680773-1, LIBC680800-1, LIBC680812-1, LIBC681593-1, LIBC681594-1, and LIBC681737-1 is described herein. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of any one of these antibodies. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of any one of LIBC523661-1, LIBC523797-1, LIBC524049-1, LIBC527814-1, LIBC527997-1, LIBC528116, LIBC527919-1, and LIBC680574-1.The HV and LV CDR sequences of these antibodies are listed in Tables 7, 10, and 26.

[0100] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HVs or LVs of LIBC523596-1, LIBC527906-1, LIBC523603-1, LIBC527814-1, LIBC523661-1, LIBC527997-1, LIBC523670-1, LIBC528141-1, LIBC523748-1, LIBC527912-1, LIBC523760-1, LIBC528116-1, LIBC523797-1, LIBC527879-1, LIBC523813-1, LIBC527919-1, LIBC523815-1, LIBC527984-1, LIBC523844-1, LIBC527968-1, LIBC523857-1, LIBC528169-1, LIBC523862-1, LIBC528131-1, LIBC523868-1, LIBC527827-1, LIBC523969-1, LIBC527869-1, LIBC524049-1, LIBC528148-1, LIBC680562-1, LIBC680574-1, LIBC680773-1, LIBC680800-1, LIBC680812-1, LIBC681593-1, LIBC681594-1, and LIBC681737-1.In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HVs and LVs of LIBC523596-1, LIBC527906-1, LIBC523603-1, LIBC527814-1, LIBC523661-1, LIBC527997-1, LIBC523670-1, LIBC528141-1, LIBC523748-1, LIBC527912-1, LIBC523760-1, LIBC528116-1, LIBC523797-1, LIBC527879-1, LIBC523813-1, LIBC527919-1, LIBC523815-1, LIBC527984-1, LIBC523844-1, LIBC527968-1, LIBC523857-1, LIBC528169-1, LIBC523862-1, LIBC528131-1, LIBC523868-1, LIBC527827-1, LIBC523969-1, LIBC527869-1, LIBC524049-1, LIBC528148-1, LIBC680562-1, LIBC680574-1, LIBC680773-1, LIBC680800-1, LIBC680812-1, LIBC681593-1, LIBC681594-1, and LIBC681737-1. The HV and LV sequences of these antibodies are listed in Tables 8, 11, and 27.

[0101] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HVs or LVs of LIBC523661-1, LIBC523797-1, LIBC524049-1, LIBC527814-1, LIBC527997-1, LIBC528116, LIBC527919-1, and LIBC680574-1. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HVs and LVs of LIBC523661-1, LIBC523797-1, LIBC524049-1, LIBC527814-1, LIBC527997-1, LIBC528116, LIBC527919-1, and LIBC680574-1.

[0102] Specific mutations in the HC_FR3 loop of 10D10 related to improved binding to CB1 were included in the affinity maturation library design (Example 1). Further, all test sequences identified after affinity maturation included a germline revertant of the R94S LC FR3 non-consensus mutation (Examples 2 and 3). Further, R32 (AHo numbering) adjacent to HCDR1 is also involved in epitope interaction. Thus, in some embodiments of the antibodies and antigen-binding fragments described above, the amino acid at HV position 83 (AHo numbering scheme) is N, and the amino acid at HV position 85 (AHo numbering scheme) is Y. In some embodiments of the antibodies and antigen-binding fragments described above, the amino acid at HV position 83 is N, the amino acid at HV position 85 is Y, and the amino acid at LV position 94 is S. In some embodiments, among the antibodies and antigen-binding fragments described above, the amino acid at HV position 32 is R.

[0103] In various embodiments, the antibodies and antigen-binding fragments disclosed herein are antagonists and / or inverse agonists of huCB1, e.g., the antibodies and binding fragments inhibit the signaling of huCB1. The titers of the antibodies and antigen-binding fragments are in the nanomolar or sub-nanomolar range, e.g., the antibodies and antigen-binding fragments have an IC50 of less than about 10 nM, less than about 5 nM, less than about 3 nM, or less than about 1 nM when measured in a cell-based cAMP assay. In some embodiments, the titers of the antibodies and antigen-binding fragments disclosed herein are equivalent to or better than the titer of rimonabant (e.g., at least 2-fold, 3-fold, 5-fold, 8-fold, or 10-fold better than rimonabant).

[0104] In another aspect, an antibody or antigen-binding fragment thereof that binds to huCB1 and comprises a heavy chain variable region (HV) and a light chain variable region (LV), a) VH comprises HCDR1, HCDH2, and HCDR3, where CDRH1 comprises the amino acid sequence of RGGDYWS (SEQ ID NO: 1); CDRH2 comprises the sequence of HX25YX26X27GX28TX29YNPX30X31X32X33 (SEQ ID NO: 538) [wherein X25 is I or V; X26 is Y or Q; X27 is A, E, K, or T; X28 is S or Q; X29 is A, E, K, or T; X30 is N or S; X31 is F or L; X32 is K or R; X33 is G, S, or N]; CDRH3 comprises the amino acid sequence of X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539) [wherein X34 is D or G; X35 is A, I, T, or V; X36 is L or absent; X37 is S or T; X38 is M or L]; b) Antibodies or antigen-binding fragments thereof are disclosed herein, wherein VL is of or derived from the VK2 / A19 / JK1 germline.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) CDRH1 comprising the sequence of RGGDYWS (SEQ ID NO: 1); CDRH2 comprising the sequence of HX25YX26X27GX28TX29YNPX30X31X32X33 (SEQ ID NO: 538) [wherein X25 is I or V; X26 is Y; X27 is A, E, K, or T; X28 is S or Q; X29 is A, E, K, or T; X30 is N or S; X31 is F or L; X32 is K or R; X33 is G, S, or N]; and CDRH3 comprising the sequence of X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539) [wherein X34 is G; X35 is A, I, T, or V; X36 is absent; X37 is S; X38 is M or L]; and b) VL is of or derived from the VK2 / A19 / JK1 germline.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) CDRH1 comprising the sequence of RGGDYWS (SEQ ID NO: 1); HX25YX26X27GX28TX29YNPX30X31X32X33 (SEQ ID NO: 538) [wherein X25 is I or V; X26 is Y or Q; X27 is A, E, K, or T; X28 is S or Q; X29 is A, E, K, or T; X30 is N or S; X31 is F or L; X32 is K or R; X33 is G, S, or N] for CDRH2; and X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539) [wherein X34 is D or G; X35 is A, I, T, or V; X36 is L or absent; X37 is S or T; X38 is M or L] for CDRH3; and b) VL is of or derived from the VK2 / A19 / JK1 germline and comprises LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the sequence of RSSQSLLHRSGYNYLD (SEQ ID NO: 257); LCDR2 comprises the sequence of X20GSNRA (SEQ ID NO: 534) [wherein X20 is L or Q]; and LCDR3 comprises X47QX48X49X50X51PRT (SEQ ID NO: 540) [wherein X47 is M or R; X48 is A or S; X49 is L, R, or V; X50 is Q or A; X51 is T or L].

[0107] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) CDRH1 comprising the sequence of RGGDYWS (SEQ ID NO: 1); CDRH2 comprising the sequence of HX25YX26X27GX28TX29YNPX30X31X32X33 (SEQ ID NO: 538) [wherein X25 is I or V; X26 is Y; X27 is A, E, K, or T; X28 is S or Q; X29 is A, E, K, or T; X30 is N or S; X31 is F or L; X32 is K or R; X33 is G, S, or N]; and CDRH3 comprising the sequence of X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539) [wherein X34 is G; X35 is A, I, T, or V; X36 is absent; X37 is S; X38 is M or L]; and b) LCDR1 comprising the sequence of RSSQSLLHRSGYNYLD (SEQ ID NO: 257); LCDR2 comprising the sequence of X20GSNRA (SEQ ID NO: 534) [wherein X20 is L or Q]; and LCDR3 comprising X47QX48X49X50X51PRT (SEQ ID NO: 540) [wherein X47 is M or R; X48 is A or S; X49 is L, R, or V; X50 is Q or A; X51 is T or L].

[0108] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) CDRH1 comprising the sequence of RGGDYWS (SEQ ID NO: 1); CDRH2 comprising the sequence of HX25YX26X27GX28TX29YNPX30X31X32X33 (SEQ ID NO: 538) [wherein X25 is I or V; X26 is Y or Q; X27 is A, E, K, or T; X28 is S or Q; X29 is A, E, K, or T; X30 is N or S; X31 is F or L; X32 is K or R; X33 is G, S, or N]; and CDRH3 comprising the sequence of X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539) [wherein X34 is D or G; X35 is A, I, T, or V; X36 is L or absent; X37 is S or T; X38 is M or L]; and b) LCDR1 comprising the amino acid sequence of RSSQSLLHRSGYNYLD (SEQ ID NO: 257); LCDR2 comprising the amino acid sequence of LGSNRAS (SEQ ID NO: 258) or QGSNRAS (SEQ ID NO: 264); and LCDR3 comprising the amino acid sequence of MQSLQTPRT (SEQ ID NO: 259), RQSVALPRT (SEQ ID NO: 271), or RQARALPRT (SEQ ID NO: 265).

[0109] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) CDRH1 comprising the sequence of RGGDYWS (SEQ ID NO: 1); CDRH2 comprising the sequence of HX25YX26X27GX28TX29YNPX30X31X32X33 (SEQ ID NO: 538) [wherein X25 is I or V; X26 is Y; X27 is A, E, K, or T; X28 is S or Q; X29 is A, E, K, or T; X30 is N or S; X31 is F or L; X32 is K or R; X33 is G, S, or N]; and CDRH3 comprising the sequence of X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539) [wherein X34 is G; X35 is A, I, T, or V; X36 is absent; X37 is S; X38 is M or L]; and b) LCDR1 comprising the amino acid sequence of RSSQSLLHRSGYNYLD (SEQ ID NO: 257); LCDR2 comprising the amino acid sequence of LGSNRAS (SEQ ID NO: 258) or QGSNRAS (SEQ ID NO: 264); and LCDR3 comprising the amino acid sequence of MQSLQTPRT (SEQ ID NO: 259), RQSVALPRT (SEQ ID NO: 271), or RQARALPRT (SEQ ID NO: 265).

[0110] In certain embodiments, the anti-CB1 antibody or antigen-binding fragment thereof disclosed in the present invention comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, which are derived from any of the anti-CB1 antibodies described herein. For example, in some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of LIBC673948-1, LIBC673952-1, LIBC673965-1, LIBC673982-1, LIBC673972-1, LIBC674024-1, LIBC674035-1, LIBC674043-1, LIBC674090-1, LIBC674002-1, LIBC674153-1, LIBC674200-1, LIBC674214-1, LIBC674216-1, LIBC674229-1, LIBC674235-1, LIBC674257-1, and LIBC674276-1. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of LIBC673948-1, LIBC673952-1, LIBC673965-1, LIBC673982-1, LIBC673972-1, LIBC674024-1, LIBC674035-1, LIBC674043-1, LIBC674090-1, LIBC674002-1, LIBC674153-1, LIBC674200-1, LIBC674214-1, LIBC674216-1, LIBC674229-1, LIBC674235-1, LIBC674257-1, and LIBC674276-1. The HV and LV CDR sequences of these antibodies are listed in Table 30.

[0111] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HVs or LVs of LIBC673948-1, LIBC673952-1, LIBC673965-1, LIBC673982-1, LIBC673972-1, LIBC674024-1, LIBC674035-1, LIBC674043-1, LIBC674090-1, LIBC674002-1, LIBC674153-1, LIBC674200-1, LIBC674214-1, LIBC674216-1, LIBC674229-1, LIBC674235-1, LIBC674257-1, and LIBC674276-1. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HVs and LVs of LIBC673948-1, LIBC673952-1, LIBC673965-1, LIBC673982-1, LIBC673972-1, LIBC674024-1, LIBC674035-1, LIBC674043-1, LIBC674090-1, LIBC674002-1, LIBC674153-1, LIBC674200-1, LIBC674214-1, LIBC674216-1, LIBC674229-1, LIBC674235-1, LIBC674257-1, and LIBC674276-1. The HV and LV sequences of these antibodies are listed in Table 31.

[0112] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of LIBC529593-1, LIBC560340-1, and LIBC560657-1. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of LIBC529593-1, LIBC560340-1, and LIBC560657-1. The HV and LV CDR sequences of the antibody are listed in Table 19. For example, the antibody or antigen-binding fragment thereof comprises a) an HCDR1 comprising the amino acid sequence of RGGDYWS (SEQ ID NO: 474) or RGGDYWN (SEQ ID NO: 480); an HCDR2 comprising the amino acid sequence of HIYYSGSTNYNPSLRS (SEQ ID NO: 475), HIYYSGSKNYNPSLKS (SEQ ID NO: 481), or HIYYTGTKYYNPSLKS (SEQ ID NO: 487); and an HCDR3 comprising the amino acid sequence of DYDILYGYSYYYYGLDV (SEQ ID NO: 476) or GYDSSGYSYYYYGMDV (SEQ ID NO: 475), and b) an LCDR1 comprising the amino acid sequence of RSSQSLLHRSGYNYLD (SEQ ID NO: 471), RSSQSLLYSNGHNFLD (SEQ ID NO: 477), or RSSQSLLYSNGHNYLD (SEQ ID NO: 483); an LCDR2 comprising the amino acid sequence of LGSNRAS (SEQ ID NO: 472) or LGSNRAP (SEQ ID NO: 484); and an LCDR3 comprising the amino acid sequence of MQSLQTPRT (SEQ ID NO: 473) or MQALQTPRT (SEQ ID NO: 479).

[0113] In some embodiments, the antibody or antigen-binding fragment thereof comprises any one of the HV or LV of LIBC529593-1, LIBC560340-1, and LIBC560657-1. In some embodiments, the antibody or antigen-binding fragment thereof comprises any one of the HV and LV of LIBC529593-1, LIBC560340-1, and LIBC560657-1. The HV and LV sequences of the antibody are listed in Table 20.

[0114] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises an amino acid mutation in the HV CDRs as compared to a reference antibody. For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises an HV comprising a) HCDR1, HCDR2, and HCDR3, wherein the HCDR2 of the antibody comprises five or fewer mutations as compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises four or fewer mutations as compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises five or fewer mutations as compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136), and the HCDR3 of the antibody or antigen-binding fragment thereof comprises three or fewer mutations as compared to NYDTVTGYSYYYYGMDV (SEQ ID NO: 137). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises four or fewer mutations as compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136), and the HCDR3 of the antibody or antigen-binding fragment thereof comprises three or fewer mutations as compared to NYDTVTGYSYYYYGMDV (SEQ ID NO: 137). In such embodiments, HCDR1 comprises the amino acid sequence of RGGDYWX1 [wherein X1 is A, S, or G, preferably A or S], and the LV of the antibody or antigen-binding fragment thereof is of or derived from the VK1 / O2 / JK4, VK2 / A19 / JK1, or VK3 / A27 / JK1 germline. Preferably, the LV is of or derived from the VK1 / O2 / JK4 or VK2 / A19 / JK1 germline, more preferably the VK1 / O2 / JK4 germline. Exemplary sequences of germline or germline-derived LV sequences are described above and in Tables 8, 11, 27, and 31.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy variable region (HV) comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR2 of the antibody or antigen-binding fragment thereof comprises five or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises four or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises five or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8), and the HCDR3 of the antibody or antigen-binding fragment thereof comprises three or fewer mutations compared to DYDILTGYSYYYYGMDV (SEQ ID NO: 9). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises four or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8), and the HCDR3 of the antibody or antigen-binding fragment thereof comprises three or fewer mutations compared to DYDILTGYSYYYYGMDV (SEQ ID NO: 9). In such embodiments, HCDR1 comprises the amino acid sequence of RGGDYWX1 (SEQ ID NO: 530) [wherein X1 is A, S, or G, preferably A or S], and the light chain variable region (LV) of the antibody or antigen-binding fragment thereof is of or derived from the VK1 / O2 / JK4 or VK2 / A19 / JK1 germline, preferably the VK2 / A19 / JK1 germline. Exemplary sequences of the germline or germline-derived LV sequences are described above and are set forth in Tables 8, 11, 20, 27, and 31.

[0116] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a sequence that is at least 90% identical, or at least 95% identical (e.g., at least 96%, at least 97%, or at least 98% identical) to the sequence of SEQ ID NO: 228. The LV of the antibody or antigen-binding fragment thereof is of, or derived from, the VK1 / O2 / JK4, VK2 / A19 / JK1, or VK3 / A27 / JK1 germline. Preferably, the LV is of, or derived from, the VK1 / O2 / JK4 or VK2 / A19 / JK1 germline, more preferably the VK1 / O2 / JK4 germline. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a sequence that is at least 90% identical, or at least 95% identical (e.g., at least 96%, at least 97%, or at least 98% identical) to the sequence of SEQ ID NO: 100. The LV of the antibody or antigen-binding fragment thereof is of, or derived from, the VK1 / O2 / JK4 or VK2 / A19 / JK1 germline, preferably the VK2 / A19 / JK1 germline. Exemplary sequences of germline or germline-derived LV sequences are described above and are set forth in Tables 8, 11, 20, 27, and 31.

[0117] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, which is determined by aligning and comparing the sequences. "Percent identity" as used herein means the percentage of identical residues between amino acids or nucleotides in the molecules being compared, and is calculated based on the smallest size of the molecules to be compared. For these calculations, gaps (if any) in the alignment must be addressed by specific mathematical models or computer programs (i.e., "algorithms"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include Computational Molecular Biology (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods commonly used to compare the similarity at the amino acid positions of two polypeptides.Using a computer program such as BLAST or FASTA, align two polypeptide sequences or two polynucleotide sequences so that their respective residues optimally match (aligning along the entire length of one or both sequences or along a predetermined portion of one or both sequences). The program provides default start penalties and default gap penalties and can use a scoring matrix such as PAM 250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3, 1978) or BLOSUM62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919) in conjunction with the computer program. Then, for example, the percent identity can be calculated as follows: multiply the total number of perfect matches by 100 and then divide by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequence to align the two sequences. In calculating the percent identity, the sequences being compared are aligned to maximize the matches between these sequences.

[0118] The GCG program package is a computer program that can be used to determine the percent identity. This package includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or two polynucleotides for which the percent sequence identity is to be determined. The sequences are arranged so that their respective amino acids or nucleotides are optimally matched (the "match span" determined by the algorithm). Along with this algorithm, a gap start penalty (calculated as 3× the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix used; the "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap start penalty) as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used. In certain embodiments, this algorithm also uses the standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345 - 352 for the PAM 250 comparison matrix; see Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915 - 10919 for the BLOSUM 62 comparison matrix).

[0119] The following are recommended parameters for determining the percent identity of a polypeptide sequence or nucleotide sequence using the GAP program: Algorithm: Needleman et al. 1970, J. Mol. Biol. 48:443 - 453; Comparison matrix: BLOSUM 62 of Henikoff et al., 1992 (supra); Gap penalty: 12 (however, no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0.

[0120] A specific alignment scheme for aligning two amino acid sequences can result in a match of only a short region of these two sequences, and this aligned small region can have a very high sequence identity even though there is no significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) can be adjusted as necessary to result in an alignment over at least 50 consecutive amino acids of the target polypeptide.

[0121] As described above, the anti-CB1 antibodies or antigen-binding fragments thereof disclosed herein may contain mutations in the HC_FR3 region as compared to 10D10. In the case of an antibody or an antigen-binding fragment thereof comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 538, and HCDR3 of SEQ ID NO: 539, in addition to the mutations at HV positions 83 and / or 85, the amino acid at HV position 79 can be R. In some embodiments, the amino acid at HV position 83 of the antibody is N, and the amino acid at HV position 85 of the antibody is Y. In some embodiments, the amino acid at position 79 of the antibody or an antigen-binding fragment thereof is R, the amino acid at HV position 83 of the antibody or an antigen-binding fragment thereof is N, and the amino acid at HV position 85 of the antibody or an antigen-binding fragment thereof is Y. In some embodiments of the above antibodies, the amino acid at HV position 83 of the antibody or an antigen-binding fragment thereof is N, the amino acid at HV position 85 of the antibody or an antigen-binding fragment thereof is Y, and the amino acid at LV position 94 of the antibody or an antigen-binding fragment thereof is S. In some embodiments, the amino acid at position 79 of the antibody or an antigen-binding fragment thereof is R, the amino acid at HV position 83 of the antibody or an antigen-binding fragment thereof is N, the amino acid at HV position 85 of the antibody or an antigen-binding fragment thereof is Y, and the amino acid at LV position 94 of the antibody or an antigen-binding fragment thereof is S.

[0122] In certain preferred embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of LIBC528116-1, LIBC680574-1, LIBC673952-1, LIBC523797-1, LIBC527814-1, LIBC527997-1, LIBC527919-1, LIBC523661-1, and LIBC524049-1. For example, the anti-CB1 antibody or antigen-binding fragment thereof comprises any one of the following sets of heavy and light chain CDRs: (a) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 135-137, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 138-140; (b) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 426-428, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 423-425; (c) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 266-268, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 263-265; (d) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 129-131, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 132-134; (e) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 159-161, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 162-164; (f) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 207-209, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 210-212; (g) HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of SEQ ID NOs: 1-3, and LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 4-6; and (h) HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of SEQ ID NOs: 55-57, and LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 58-60.

[0123] In some preferred embodiments, the antibody or its antigen-binding fragment contains an HV comprising an amino acid sequence selected from SEQ ID NO: 228, SEQ ID NO: 460, SEQ ID NO: 368, SEQ ID NO: 100, SEQ ID NO: 236, SEQ ID NO: 226, SEQ ID NO: 252, SEQ ID NO: 98, and SEQ ID NO: 116. In some preferred embodiments, the antibody or its antigen-binding fragment contains an LV comprising an amino acid sequence selected from SEQ ID NO: 227, SEQ ID NO: 459, SEQ ID NO: 367, SEQ ID NO: 99, SEQ ID NO: 235, SEQ ID NO: 225, SEQ ID NO: 251, SEQ ID NO: 97, and SEQ ID NO: 115. In some preferred embodiments, the antibody or its antigen-binding fragment contains an HV and an LV selected from any of the following: i) an HV comprising the amino acid sequence of SEQ ID NO: 228 and an LV comprising the amino acid sequence of SEQ ID NO: 227; ii) an HV comprising the amino acid sequence of SEQ ID NO: 460 and an LV comprising the amino acid sequence of SEQ ID NO: 459; iii) an HV comprising the amino acid sequence of SEQ ID NO: 368 and an LV comprising the amino acid sequence of SEQ ID NO: 367; iv) an HV comprising the amino acid sequence of SEQ ID NO: 100 and an LV comprising the amino acid sequence of SEQ ID NO: 99; v) an HV comprising the amino acid sequence of SEQ ID NO: 236 and an LV comprising the amino acid sequence of SEQ ID NO: 235; vi) an HV comprising the amino acid sequence of SEQ ID NO: 226 and an LV comprising the amino acid sequence of SEQ ID NO: 225; vii) an HV comprising the amino acid sequence of SEQ ID NO: 252 and an LV comprising the amino acid sequence of SEQ ID NO: 251; viii) an HV comprising the amino acid sequence of SEQ ID NO: 98 and an LV comprising the amino acid sequence of SEQ ID NO: 97; and ix) an HV comprising the amino acid sequence of SEQ ID NO: 116 and an LV comprising the amino acid sequence of SEQ ID NO: 115.

[0124] In various embodiments, the antibodies or antigen-binding fragments thereof disclosed herein have a higher binding affinity for CB1 (e.g., huCB1) compared to 10D10 LC N35Y (i.e., 10D10 containing a substitution from N to Y at LC amino acid position 35). In some embodiments, the antibody or antigen-binding fragment thereof has a binding affinity for CB1 that is at least 3-fold higher compared to 10D10 LC N35Y. In some embodiments, the antibody or antigen-binding fragment thereof has a binding affinity for CB1 that is at least 5-fold higher (e.g., at least 8-fold higher, at least 10-fold higher, at least 12-fold higher, at least 15-fold higher, or at least 20-fold higher) compared to 10D10 LC N35Y.

[0125] Affinity is determined using various techniques, and as an example, there is an affinity ELISA assay. In various embodiments, affinity is determined by surface plasmon resonance assays (e.g., BIAcore®-based assays). Using this methodology, the association rate constant (k a Unit: M -1 s -1 ) and the dissociation rate constant (k d Unit: s -1 ) can be measured. Next, the equilibrium dissociation constant (K D Unit: M) can be calculated from the ratio of the reaction rate constants (k d / k a ). In some embodiments, affinity is determined by kinetic methods such as the kinetic exclusion assay (KinExA) as described in Rathanaswami et al., Analytical Biochemistry, Vol. 373:52-60, 2008. Using the KinExA assay, the equilibrium dissociation constant (K D , M) and the association rate constant (k a , M -1 s -1 ) can be measured. From these values, the dissociation rate constant (k d , s -1 ) can be calculated (K D ×k a)。In other embodiments, the affinity is determined by biolayer interferometry as described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278:165-82, 2015 and used in the Octet® system (Pall ForteBio). The rate constants (k a and k d ) and the affinity constant (K D ) can be calculated in real time using biolayer interferometry. Binding to CB1 (e.g., huCB1) can be measured using these techniques and an antigen source (e.g., CB1-ND, CB1-SMALP, or CB1-expressing cells such as 293T cells).

[0126] An antibody or antigen-binding fragment that specifically binds to an antigen may have an equilibrium dissociation constant (K D ) ≤ 1 × 10 -6 M. An antibody or antigen-binding fragment specifically binds to an antigen with "high affinity" when K D is ≤ 3 × 10 -8 M. In some embodiments, the anti-CB1 antibody or antigen-binding fragment binds to a target antigen (e.g., huCB1) where K D is ≤ 100 nM (e.g., about 90 nM, 70 nM, 50 nM, 30 nM, 20 nM, less than 10 nM, or a range defined by any two of the foregoing values). In some embodiments, the anti-CB1 antibody or binding fragment has a dissociation constant (K D ) of about 70 nM to less than 10 nM (e.g., about 60 nM to 20 nM, or about 40 nM to 10 nM) for binding to CB1 (e.g., huCB1 such as huCB1-ND) as determined using the Octet® system and huCB1-ND. In some embodiments, the antibody or antigen-binding fragment binds to CB1 (e.g., hu-CB1) with a KD of ≤ 1 × 10 -8 M (e.g., ≤ 1 × 10 -9 M, ≤ 5 × 10 -10 M, or ≤ 1 × 10 -10 M) as determined using the KinExA assay and huCB1-SMAP or CB1-expressing cells.

[0127] In various embodiments, the anti-CB1 antibodies and antigen-binding fragments disclosed herein are antagonist and / or inverse agonist anti-huCB1 antibodies and antigen-binding fragments; for example, they inhibit the signaling of huCB1. The titers of the antibodies and antigen-binding fragments are in the single-digit nanomolar or sub-nanomolar range; for example, they have an IC50 of less than about 10 nM, less than about 5 nM, less than about 3 nM, or less than about 1 nM when measured in a cell-based cAMP assay (e.g., a cell-based cAMP assay in the presence of CP 55,940). In some embodiments, the anti-CB1 antibodies and antigen-binding fragments disclosed herein have titers comparable to that of rimonabant. In some embodiments, the anti-CB1 antibodies and antigen-binding fragments disclosed herein have titers higher (e.g., at least 2-fold, 3-fold, 5-fold, 8-fold, or more than 10-fold higher) than rimonabant. In various embodiments, the titers can be measured using a cell-based CMap assay.

[0128] The 10D10 antibody binds to the extracellular loop 2 region of huCB1 (i.e., amino acid residues 256-273 of SEQ ID NO: 559) (WO 2014 / 210205). The anti-CB1 antibodies and antigen-binding fragments disclosed herein bind to the same region on huCB1 because they are affinity matured variants of the 10D10 antibody. Thus, in some embodiments, the anti-CB1 antibodies and antigen-binding fragments disclosed herein bind to the extracellular loop 2 region of huCB1 and have an IC50 of less than about 10 nM, less than about 5 nM, less than about 3 nM, or less than about 1 nM when measured in a cell-based cAMP assay (e.g., a cell-based cAMP assay in the presence of CP 55,940).

[0129] The anti-CB1 antibody of the present invention can include any immunoglobulin constant region. As used herein in the same sense as "constant domain", the "constant region" refers to all domains of the antibody other than the variable region. The constant region is not directly involved in antigen binding but exhibits various effector functions. As described above, antibodies are divided into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) according to the amino acid sequence of the constant region of their heavy chains. The light chain constant region can be, for example, a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region, which are found in all five isotypes of antibodies. Examples of human immunoglobulin light chain constant region sequences are shown in Table 1.

[0130]

Table 1

[0131] The heavy chain constant region of the anti-CB1 antibody of the present invention can be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. In some embodiments, the anti-CB1 antibody includes a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin, such as human IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In one embodiment, the anti-CB1 antibody includes a heavy chain constant region derived from human IgG1 immunoglobulin. In such an embodiment, the human IgG1 immunoglobulin constant region can include one or more mutations that prevent glycosylation and / or the half-life of the antibody, as described in more detail herein. In another embodiment, the anti-CB1 antibody includes a heavy chain constant region derived from human IgG2 immunoglobulin. In yet another embodiment, the anti-CB1 antibody includes a heavy chain constant region derived from human IgG4 immunoglobulin. Examples of human IgG1, IgG2, and IgG4 heavy chain constant region sequences are shown in Table 2 below.

[0132]

Table 2

[0133]

Table 3

[0134] Each of the light chain variable regions, as well as each of the heavy chain variable regions disclosed herein (Tables 8, 11, 20, 27, and 31), can be combined with the above-described light chain constant region (Table 1) and heavy chain constant region (Table 2) to form the light chain and heavy chain of a complete antibody, respectively. In some embodiments, each of the light chain variable regions and each of the heavy chain variable regions disclosed in Tables 8, 11, 20, 27, and 31 are combined with SEQ ID NO: 518 or 519 and SEQ ID NO: 525, 526, or 527 to form a complete antibody light chain and heavy chain, respectively. In some embodiments, each of the light chain variable regions and each of the heavy chain variable regions disclosed in Tables 8, 11, 20, 27, and 31 are combined with SEQ ID NO: 518 and SEQ ID NO: 525, 526, or 527 to form a complete antibody light chain and heavy chain, respectively. In some embodiments, each of the light chain variable regions and each of the heavy chain variable regions disclosed in Tables 8, 11, 20, 27, and 31 are combined with SEQ ID NO: 518 and SEQ ID NO: 525 to form a complete antibody light chain and heavy chain, respectively. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 562 and a light chain having the amino acid sequence of SEQ ID NO: 563. In some embodiments, the monoclonal antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 562 with the C-terminal amino acid residue K deleted and a light chain having the amino acid sequence of SEQ ID NO: 563. Further, each of the heavy chain and light chain sequences thus generated can be combined to form a complete antibody structure. It should be understood that the heavy chain and light chain variable regions provided herein can also be combined with other constant domains having sequences different from the exemplary sequences listed above.

[0135] The anti-CB1 antibody or antigen-binding fragment of the present invention can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody or a multispecific antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-CB1 antibody is a monoclonal antibody. In such embodiments, the anti-CB1 antibody can be a chimeric antibody, a humanized antibody or a fully human antibody having a human immunoglobulin constant domain. In these and other embodiments, the anti-CB1 antibody is a human IgG1 (e.g., IgG1z), IgG2, IgG3 or IgG4 antibody. Thus, the anti-CB1 antibody can have a human IgG1, IgG2, IgG3 or IgG4 constant domain in some embodiments. In one embodiment, the anti-CB1 antibody is a monoclonal human IgG1 antibody, preferably a monoclonal human IgG1z antibody. In another embodiment, the anti-CB1 antibody is a monoclonal human IgG2 antibody. In yet another embodiment, the anti-CB1 antibody is a monoclonal human IgG4 antibody.

[0136] As used herein, the term "monoclonal antibody" (or "mAb") refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for naturally occurring variations that may be present in minor amounts. Monoclonal antibodies are highly specific and typically target an individual antigenic site or epitope, as opposed to polyclonal antibody preparations that typically include a variety of antibodies targeting a variety of epitopes. Monoclonal antibodies can be produced using any method known in the art, for example, by immortalizing spleen cells harvested from an animal after completion of an immunization schedule. The spleen cells can be immortalized using any technique known in the art, for example, by fusing them with myeloma cells to generate hybridomas. See, for example, Antibodies; Harlow and Lane, Cold Spring Harbor Laboratory Press, 1st Edition (e.g., from 1988) or 2nd Edition (e.g., from 2014). Myeloma cells for use in the hybridoma production fusion procedure preferably are non-antibody producing, have high fusion efficiency, and have an enzyme deficiency that renders their growth impossible in a specific selection medium that supports the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in fusions with mouse cells include, but are not limited to, Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XXO Bul. Examples of cell lines suitable for use in fusions with rat cells include, but are not limited to, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6. A further exemplary method for isolating monoclonal antibodies includes screening plasma B cells from an animal after completion of an immunization schedule.(See, for example, Pedrioli A. and Oxenius A., Trends in Immunology, 2021, 42(12):1148-1158). The properties of monoclonal antibodies isolated by these methods can be optimized using in vitro affinity maturation techniques such as yeast display-based affinity maturation known in the art (e.g., to improve binding). (See, for example, Cherf, G.M. and Cochran, J.R., Yeast Surface Display: Methods, Protocols, and Applications. 2015:155-175).

[0137] In some embodiments, the anti-CB1 antibodies or antigen-binding fragments of the invention are chimeric or humanized antibodies or antigen-binding fragments thereof based on the CDR and variable region sequences of the antibodies described herein. A chimeric antibody is an antibody composed of protein segments from different antibodies that covalently combine to produce a functional immunoglobulin light or heavy chain or binding fragments thereof. Generally, a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. Methods regarding chimeric antibodies can be found, for example, in U.S. Patent No. 4,816,567 and Morrison et al., 1985, Proc. Natl. Acad. Sci. USA 81:6851-6855. Both of these are hereby incorporated by reference in their entirety.

[0138] Generally, the aim in making a chimeric antibody is to make a chimera in which the number of amino acids from the intended species is maximized. One example is a "CDR grafted" antibody in which the antibody contains one or more CDRs that are from a particular species or belong to a particular antibody class or subclass, while the remainder of the antibody chain is identical or homologous to the corresponding sequences in an antibody from another species or belonging to another antibody class or subclass. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101. For use in humans, the variable region or selected CDRs from a rodent or rabbit antibody are often grafted onto a human antibody, replacing the naturally occurring variable region or CDRs of the human antibody.

[0139] One useful type of chimeric antibody is a "humanized" antibody. Generally, humanized antibodies are generated from monoclonal antibodies that were first produced in a non-human animal such as a rodent or rabbit. Certain amino acid residues within this monoclonal antibody, typically those derived from the non-antigen recognition portion of the antibody, are modified to be homologous to the corresponding residues in a human antibody of the corresponding isotype. Humanization can be performed, for example, by replacing at least a portion of the variable region of a rodent or rabbit with the corresponding region of a human antibody using various methods (see, for example, U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-1536).

[0140] In one aspect, the CDRs of the light and heavy chain variable regions of the antibodies presented herein (see Tables 7, 10, 19, 26, and 30) are grafted onto the framework regions (FRs) of antibodies from the same or different phylogenetic species. For example, the CDRs of the heavy and light chain variable regions listed in Tables 7, 10, 19, 26, and 30 can be grafted onto a consensus human FR. To create a consensus human FR, the FRs derived from several human heavy or light chain amino acid sequences can be aligned to identify a consensus amino acid sequence. Alternatively, the variable regions transplanted from one heavy or light chain can be used with a constant region that is different from the constant region of that particular heavy or light chain as disclosed herein.

[0141] In certain embodiments, the anti-CB1 antibodies or antigen-binding fragments of the invention are fully human antibodies or antigen-binding fragments thereof. A "fully human antibody" is an antibody that contains variable and constant regions that are derived from or represent human germline immunoglobulin sequences. One specific means provided for generating fully human antibodies is the "humanization" of the murine humoral immune system. Introducing a human immunoglobulin (Ig) locus into a mouse in which the endogenous Ig genes have been inactivated is one means of generating fully human monoclonal antibodies (mAbs) in a mouse, which is an animal that can be immunized with any desired antigen. The use of fully human antibodies can minimize the immunogenic and allergic responses that can occur upon administration of murine mAbs or mAbs derived from mice to humans as therapeutic agents.

[0142] The above-described transgenic mouse, called the "HuMab" mouse, contains human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg et al., 1994, Nature 368:856-859). Thus, the mouse shows decreased expression of mouse IgM and κ protein, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic hypermutation to generate high-affinity human IgGκ monoclonal antibodies (Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci. 764:536-546). The preparation of HuMab mice is described in detail in Taylor et al., 1992, Nucleic Acids Research 20:6287-6295; Chen et al., 1993, International Immunology 5:647-656; Tuaillon et al., 1994, J. Immunol. 152:2912-2920; Lonberg et al., 1994, Nature 368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113:49-101; Taylor et al., 1994, International Immunology 6:579-591; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci. 764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851, which are hereby incorporated by reference in their entirety.One particular transgenic mouse strain suitable for the production of fully human anti-CB1 antibodies is the XenoMouse® transgenic mouse strain described in U.S. Patent Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244; Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovitis, 1998, J. Ex. Med., 188:483-495; Green, 1999, Journal of Immunological Methods 231:11-23; Kellerman and Green, 2002, Current Opinion in Biotechnology 13,593-597 (each of which is incorporated herein by reference in its entirety). Also, human-derived antibodies can be produced using phage display technology. Phage display is described, for example, in International Publication No. WO 91 / 17271 by Dower et al., International Publication No. WO 92 / 01047 by McCafferty et al. and Caton and Koprowski, 1990, Proc. Natl. Acad. Sci. USA, 87:6450-6454, each of which is incorporated herein by reference in its entirety.

[0143] In certain embodiments, the anti-CB1 antibodies and antigen-binding fragments of the invention may include one or more mutations or modifications to the constant region. For example, the heavy chain constant region or Fc region of the anti-CB1 antibody may include one or more amino acid substitutions that affect the glycosylation, effector function, and / or Fcγ receptor binding of the antibody. The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that can be generated by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally includes two constant domains (CH2 domain and CH3 domain), and optionally includes a CH4 domain. In certain embodiments, the Fc region is an Fc region derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region includes the CH2 domain and CH3 domain derived from human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector functions and / or improve the half-life, as described in more detail below.

[0144] In some embodiments, the anti-CB1 antibodies of the invention include one or more amino acid substitutions in the Fc region that reduce effector function. An amino acid substitution in an amino acid sequence is herein typically indicated by the one-letter abbreviation of the amino acid residue at a particular position, followed by the number of the amino acid position relative to the original sequence of interest, followed by the one-letter abbreviation of the substituted amino acid residue. For example, "C220S" represents the substitution of a cysteine residue with a serine residue at amino acid position 220 relative to the original sequence of interest. Exemplary amino acid substitutions (according to the EU numbering scheme) that can reduce effector function include, but are not limited to, C220S, C226S, C229S, E233P, L234A, L234V, V234A, L234F, L235A, L235E, G237A, P238S, S267E, H268Q, N297A, N297G, V309L, E318A, L328F, A330S, A331S, P331S, or any combination of the foregoing.

[0145] Glycosylation can contribute to the effector functions of antibodies, particularly IgG1 antibodies. Thus, in some embodiments, the anti-CB1 antibodies of the present invention may include one or more amino acid substitutions that affect the level or type of glycosylation of the antibody. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the asparagine residue side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of carbohydrate moieties to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the linkage of one sugar of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0146] In some embodiments, the glycosylation of the anti-CB1 antibodies described herein is reduced or eliminated by removing one or more glycosylation sites, for example, from the Fc region of the antibody. In some embodiments, the anti-CB1 antibody is an aglycosylated human monoclonal antibody, such as an aglycosylated human IgG1 monoclonal antibody. Amino acid substitutions that eliminate or modify N-linked glycosylation sites can reduce or eliminate the N-linked glycosylation of the antibody. In certain embodiments, the anti-CB1 antibodies described herein include heavy chain mutations at the position of N297, such as N297Q, N297A, or N297G (according to the EU numbering scheme). In some embodiments, the anti-CB1 antibodies of the present invention include an Fc region derived from a human IgG1 antibody having a mutation at the position of N297. In one particular embodiment, the anti-CB1 antibody of the present invention includes an Fc region derived from a human IgG1 antibody having an N297G mutation. For example, in some embodiments, the anti-CB1 antibody of the present invention includes a heavy chain constant region comprising the sequence of SEQ ID NO: 524.

[0147] To improve the stability of molecules containing the N297 mutation, the Fc region of the anti-CB1 antibody can be further engineered. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine to promote disulfide bond formation in the dimeric state. Accordingly, the residues corresponding to V259, A287, R292, V302, L306, V323 or I332 (according to the EU numbering scheme) of the IgG1 Fc region can be substituted with cysteine. Preferably, specific pairs of residues are substituted with cysteine so as to preferentially form disulfide bonds with each other, thereby restricting or preventing scrambling of the disulfide bonds. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In certain embodiments, the anti-CB1 antibody described herein comprises an Fc region derived from a human IgG1 antibody having the mutations R292C and V302C. In such embodiments, the Fc region also includes an N297 mutation such as the N297G mutation. In some embodiments, the anti-CB1 antibody of the invention comprises a heavy chain constant region comprising the sequence of SEQ ID NO: 525.

[0148] Modification of the anti-CB1 antibody of the invention to extend serum half-life (e.g., in vivo half-life) may also be desirable. One approach to achieving an extended serum half-life is by amino acid mutations at specific positions in the constant domain such as those described in WO 2002 / 060919. Such mutations increase the affinity of the antibody for FcRn and extend its serum half-life. In some embodiments, the anti-CB1 antibody described herein comprises an Fc region derived from a human IgG1 antibody having one or more mutations at amino acid positions 251-256 (according to the EU numbering scheme). In some embodiments, the Fc region comprises one or more mutations at positions 252, 254, and 256. In some embodiments, the Fc region comprises M252Y, S254T, and T256E (according to the EU numbering scheme). In some embodiments, the anti-CB1 antibody of the invention comprises a heavy chain constant region comprising the sequence of SEQ ID NO: 527.

[0149] In some embodiments, the Fc region of the anti-CB1 antibodies described herein may include modifications that alter the glycosylation, stability, and serum half-life of the antibody. For example, in some embodiments, the anti-CB1 antibodies described herein include an Fc region derived from a human IgG1 antibody having N297 mutations such as the R292C and V302C mutations, the N297G (by EU numbering scheme) mutation, and may also include one or more mutations at positions 252, 254, and 256 such as M252Y, S254T, and T256E (by EU numbering scheme). In some embodiments, the anti-CB1 antibody of the invention includes a heavy chain constant region comprising the sequence of SEQ ID NO: 526.

[0150] The anti-CB1 antibodies and antigen-binding fragments described herein are expected not to cross-react (i.e., essentially not recognize or bind) with the human cannabinoid receptor type 2 (CB2). Human CB2 is a G protein-coupled receptor from the cannabinoid receptor family and is encoded by the CNR2 gene. Human CB2 contains 360 amino acids (UniProtKB - P34972, SEQ ID NO: 560) and shares approximately 44% sequence similarity with huCB1. The anti-CB1 antibodies and antigen-binding fragments described herein also do not cross-react with the mouse CB1 protein (UniProtKB - P47746, SEQ ID NO: 561).

[0151] The present invention includes one or more polynucleotides or nucleic acids encoding an anti-CB1 antibody or antigen-binding fragment described herein. In addition, the present invention includes vectors containing nucleic acids, host cells or cell lines containing nucleic acids, and methods for producing the anti-CB1 antibodies and antigen-binding fragments of the present invention. The nucleic acid may encode, for example, all or part of an antibody or antigen-binding fragment, such as one or both chains of an antibody of the present invention or a fragment, derivative or variant thereof, a polynucleotide for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, a PCR primer or sequencing primer, an antisense oligonucleotide for inhibiting the expression of a polynucleotide, and complementary sequences as described above. The nucleic acid can be of any length suitable for the desired use or function and can include one or more additional sequences, such as regulatory sequences, and / or a larger nucleic acid, such as part of a vector. The nucleic acid molecules of the present invention include both single-stranded and double-stranded forms of DNA and RNA and corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR-amplified DNA, and combinations thereof. The nucleic acid molecules of the present invention include combinations of full-length genes or cDNA molecules and fragments thereof. The nucleic acids of the present invention can be derived from human sources and non-human species.

[0152] The corresponding amino acid sequence from an immunoglobulin or region thereof (e.g., variable region, Fc region, etc.) or polypeptide of interest may be determined directly by protein sequence analysis methods, and a suitable coding nucleotide sequence can be designed according to the universal codon table. Alternatively, the genomic or cDNA encoding a monoclonal antibody or binding fragment thereof of the present invention can be isolated from cells producing such an antibody (e.g., by using an oligonucleotide probe capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody) and sequenced using conventional procedures.

[0153] The present invention also includes a vector comprising one or more nucleic acids encoding one or more components (e.g., variable regions, light chains, and heavy chains) of the antibody or antigen-binding fragment of the present invention. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of the coding sequence operably linked in a particular host cell. An expression vector can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome-binding site, and optionally other sequences. Eukaryotic cells are known to utilize promoters, enhancers, as well as termination and polyadenylation signals.

[0154] The secretion signal peptide sequence can also optionally be encoded by an expression vector and operably linked to the coding sequence of interest, such that the expressed polypeptide can be secreted by a recombinant host cell, if desired, to facilitate more readily isolation of the polypeptide of interest from the cell. For example, in some embodiments, the signal peptide sequence can be added / fused to the amino terminus of any of the variable region polypeptide sequences listed in Tables 8, 11, 20, 27, and 31. In certain embodiments, a signal peptide having the amino acid sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 541) is fused to the amino terminus of any of the variable region polypeptide sequences of Tables 8, 11, 20, 27, and 31. In other embodiments, a signal peptide having the amino acid sequence of MAWALLLLTLLTQGTGSWA (SEQ ID NO: 542) is fused to the amino terminus of any of the variable region polypeptide sequences of Tables 8, 11, 20, 27, and 31. In yet other embodiments, a signal peptide having the amino acid sequence of MTCSPLLLTLLIHCTGSWA (SEQ ID NO: 543) is fused to the amino terminus of any of the variable region polypeptide sequences of Tables 8, 11, 20, 27, and 31.Other suitable signal peptide sequences that can be fused to the amino terminus of the variable region polypeptide sequences described herein include MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 544), MEWTWRVLFLVAAATGAHS (SEQ ID NO: 545), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 546), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 547), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 548), MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 549), MDIRAPTQLLGLLLLWLPGAKC (SEQ ID NO: 550), MDIRAPTQLLGLLLLWLPGARC (SEQ ID NO: 551), MDTRAPTQLLGLLLLWLPGATF (SEQ ID NO: 552), MDTRAPTQLLGLLLLWLPGARC (SEQ ID NO: 553), METGLRWLLLVAVLKGVQC (SEQ ID NO: 554), METGLRWLLLVAVLKGVQCQE (SEQ ID NO: 555), MDMRAPTQLLGLLLLWLPGARC (SEQ ID NO: 556), MKILILGIFLFLCSTPAWA (SEQ ID NO: 557), and MRTLAILAAILLVALQAQA (SEQ ID NO: 558). Other signals or secretory peptides are known to those skilled in the art and can be fused to any of the variable region polypeptide chains listed in Tables 8, 11, 20, 27, and 31, for example, to facilitate or optimize expression in a particular host cell.

[0155] Typically, the expression vectors used in host cells to produce the anti-CB1 antibodies and antigen-binding fragments of the present invention include sequences for plasmid maintenance and sequences for the cloning and expression of exogenous nucleotide sequences encoding the components of the antibodies and antigen-binding fragments. In certain embodiments, such sequences, collectively referred to as "flanking sequences," typically include the following nucleotide sequences: a promoter (e.g., a promoter suitable for the host cell), one or more enhancer sequences (e.g., viral enhancer sequences), an origin of replication (e.g., a plasmid origin for bacteria or a viral origin for mammalian systems), a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a signal sequence for polypeptide secretion, a ribosome binding site (e.g., a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes)), a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding the expressed polypeptide, and one or more selectable marker elements (e.g., a sequence encoding a protein that confers resistance to an antibiotic or complements a nutritional requirement deficiency of the host cell). Flanking sequences and vectors are well known in the art. For example, expression vectors are commercially available. In addition, desired modifications can be made to commercially available expression vectors or they can be constructed using sequences known in the art. Nucleic acids encoding different components of the anti-CB1 antibody or binding fragment (e.g., HV and / or LV) can be inserted into appropriate sites of the same vector or different vectors for expression in host cells.

[0156] "Host cell" refers to a cell that has been transformed or can be transformed with a nucleic acid, thereby expressing a gene of interest. This term includes progeny of the parent cell, regardless of whether the morphological or genetic structure of the progeny of the parent cell is identical to that of the original parent cell, as long as the gene of interest is present. A host cell containing a nucleic acid of the present invention that is operably linked to at least one expression control sequence (e.g., a promoter or enhancer) is a "recombinant host cell". Methods for transforming or transfecting a host cell with a vector are well known in the art.

[0157] Suitable host cells include prokaryotic or eukaryotic cells, and also include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells and animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque or human.

[0158] Prokaryotic host cells include eubacteria, such as Gram-negative microorganisms or Gram-positive microorganisms, for example, the Enterobacteriaceae family, for example, the genus Escherichia, for example, Escherichia coli, the genus Enterobacter, the genus Erwinia, the genus Klebsiella, the genus Proteus, the genus Salmonella, for example, Salmonella typhimurium, the genus Serratia, for example, Serratia marcescens, and the genus Shigella, as well as the genus Bacillus, for example, Bacillus subtilis and B. licheniformis, the genus Pseudomonas, and the genus Streptomyces. Eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning hosts or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms.However, various other genera, species, and strains of hosts from the genera Pichia, such as P. pastoris, Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, Schwanniomyces, such as Schwanniomyces occidentalis, and filamentous fungi, such as the genera Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as A. nidulans and A. niger, are generally available and useful herein.

[0159] Host cells for the expression of glycosylated antibodies and antigen-binding fragments can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains and variants, and corresponding insect permissive host cells derived from hosts such as Spodoptera frugiperda (armyworm), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm) have been identified. Various virus strains for transfection of such cells, such as the L-1 variant of Autographa californica NPV, and the Bm-5 strain of Bombyx mori NPV, are publicly available.

[0160] Vertebrate host cells are also suitable hosts, and the recombinant production of antibodies and antigen-binding fragments derived from such cells is a conventional method. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, such as CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney strain (293 cells or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36:59, 1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells, and a plurality of other cell lines. In some embodiments, CHO cells are preferred host cells for the expression of the anti-CB1 antibodies and antigen-binding fragments of the present invention.

[0161] The host cell is transformed or transfected with the above nucleic acid or vector for the production of an anti-CB1 antibody or antigen-binding fragment and is cultured in a conventional nutrient medium that has been appropriately modified for induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence. Accordingly, the present invention provides a method for producing an anti-CB1 antibody or antigen-binding fragment described herein, the method comprising culturing a host cell comprising one or more expression vectors described herein in a culture medium under conditions that permit expression of the antibody or antigen-binding fragment encoded by the one or more expression vectors; and recovering the antibody or antigen-binding fragment from the culture medium or host cell.

[0162] When culturing the host cell, the antibody or antigen-binding fragment can be produced intracellularly, in the periplasmic cavity, or directly secreted into the culture medium. When the antibody or antigen-binding fragment is produced intracellularly, as a first step, debris of the microparticles, i.e., either the host cell or the lysed fragment, is removed, for example, by centrifugation or ultrafiltration. The antibody or antigen-binding fragment can be purified by methods known in the art, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, preferably affinity chromatography using the antigen of interest, protein A, or protein G as the affinity ligand. Protein A can be used to purify proteins containing polypeptides based on each heavy chain of human γ1, γ2, or γ4 (Lindmark et al., J. Immunol. Meth. 62:1-13, 1983). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567-1575, 1986). Although the matrix to which the affinity ligand binds is most often agarose, other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl) benzene can achieve a faster flow rate and shorter processing time than can be achieved with agarose. When the protein contains a CH3 domain, Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Depending on the specific antibody or antigen-binding fragment to be recovered, other techniques for protein purification such as ethanol precipitation, reverse-phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also possible.

[0163] Composition In addition, the present specification discloses a composition comprising an anti-CB1 antibody or an antigen-binding fragment. Preferred compositions include those comprising an anti-CB1 antibody or an antigen-binding fragment described and exemplified herein and one or more pharmaceutically acceptable excipients. "Pharmaceutically acceptable" refers to molecules and compounds that are non-toxic to human recipients at the dosages and concentrations used and / or do not cause allergic or adverse reactions when administered to humans. Examples of the pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0164] In some embodiments, the pharmaceutical composition may contain materials for modifying, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include buffers (such as acetate buffer, Tris-HCl, citrate buffer, phosphate buffer, or buffers containing other organic acids); bulking agents or fillers such as monosaccharides; disaccharides (e.g., sucrose); and other carbohydrates; proteins (such as serum albumin, gelatin, or immunoglobulins); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine), coloring agents and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); low molecular weight polypeptides; salt-forming counterions (such as sodium chloride, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); suspending agents; surfactants or wetting agents (Pluronic®, PEG, sorbitan esters, polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, thioxapal); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants, but are not limited thereto. Methods of formulating molecules for therapeutic use and suitable materials are known in the pharmaceutical art and are described, for example, in REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.

[0165] In some embodiments, the compositions disclosed herein are liquid compositions such as aqueous solutions. In some embodiments, the composition comprises a buffer, an isotonic agent or filler, a surfactant, and has a pH in the range of about 4.5 to 7.5. Optionally, the composition further comprises a salt and / or a preservative.

[0166] In some embodiments, the pH of the composition is within the range of about 4.5 to about 7.0, such as about 4.5 to about 6.5, or about 4.8 to about 5.5, or about 5.0. Examples of buffers suitable for this range of pH include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acid buffers. The buffer concentration can be, for example, about 1 mM to about 200 mM, or about 10 mM to about 60 mM, depending on the desired isotonicity of the buffer and the composition.

[0167] An isotonic agent capable of stabilizing the antibody or antigen-binding fragment can also be included in the composition. Exemplary isotonic agents include polyols such as sucrose, mannitol, or trehalose. Preferably, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. Exemplary concentrations of the polyol in the formulation can range from about 1 w / v% to about 15 w / v%.

[0168] A surfactant can also be added to the formulation to reduce aggregation of the formulated antibody or antigen-binding fragment, and / or minimize particle formation in the formulation, and / or reduce adsorption. Exemplary surfactants include nonionic surfactants such as polysorbate (e.g., polysorbate 20 or polysorbate 80) or poloxamer (e.g., poloxamer 188). Exemplary concentrations of the surfactant can range from about 0.001 weight / volume% to about 0.5 weight / volume%, or about 0.005 weight / volume% to about 0.2 weight / volume%, or about 0.004 weight / volume% to about 0.01% weight / volume.

[0169] In one embodiment, the composition contains the above-mentioned agents (i.e., antibody or antigen-binding fragment, buffer, polyol and surfactant), and is substantially free of one or more preservatives such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzethonium chloride. In another embodiment, the preservative may be included in the formulation at a concentration in the range of, for example, about 0.1% to about 2%, or about 0.5% to about 1%. In one embodiment, the composition may further contain a salt such as sodium chloride or one or more other pharmaceutically acceptable excipients. One or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company may be included in the formulation if they do not adversely affect the desired properties of the formulation.

[0170] Formulations used for in vivo administration must be sterile. The compositions of the present invention can be sterilized by conventional well-known sterilization techniques. For example, sterilization is readily achieved by filtration through a sterile filtration membrane. The resulting solution can be packaged for use or filtered and lyophilized under aseptic conditions. The lyophilized preparation is combined with a sterile solution prior to administration.

[0171] In some embodiments, a composition comprising an anti-CB1 antibody or antigen-binding fragment and one or more pharmaceutically acceptable excipients is a solid composition such as a lyophilized cake. The lyophilization process is often used to stabilize polypeptides for long-term storage, particularly when the polypeptide is relatively unstable in liquid compositions. A lyophilization cycle typically consists of three steps: freezing, primary drying, and secondary drying (see Williams and Polli, Journal of Parenteral Science and Technology, Volume 38, Number 2, pages 48-59, 1984). In the freezing step, the solution is cooled until it is fully frozen. Bulk water in the solution forms ice at this stage. The ice sublimes during the primary drying stage. This is accomplished using a vacuum to lower the chamber pressure below the vapor pressure of the ice. Finally, adsorbed or bound water is removed during the secondary drying stage at a reduced chamber pressure and elevated shelf temperature. This method produces a substance known as a lyophilized cake. The cake can then be redissolved prior to use. The standard method for reconstituting lyophilized materials is to add a quantity of pure water (usually equal to the amount removed during lyophilization), although a dilute solution of an antibacterial agent is sometimes used in the manufacture of pharmaceuticals for parenteral administration (see Chen, Drug Development and Industrial Pharmacy, Volume 18:1311-1354, 1992).

[0172] In some cases, excipients are known to act as stabilizers for lyophilized products (see Carpenter et al., Volume 74:225-239, 1991). For example, known excipients include polyols (including mannitol, sorbitol and glycerol), sugars (including glucose and sucrose), and amino acids (including alanine, glycine and glutamic acid). Furthermore, polyols and saccharides are also often used to protect polypeptides from damage induced by freezing and drying and to enhance stability during storage in the dry state. In general, sugars, particularly disaccharides, are effective both during the lyophilization process and during storage. Other classes of molecules, including monosaccharides and disaccharides as well as polymers such as PVP, have also been reported as stabilizers for lyophilized products.

[0173] The formulations of the present invention can be designed to be short-acting, immediate-release, long-acting, or sustained-release, as described herein. Thus, pharmaceutical formulations can also be formulated for controlled or sustained release. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing an antibody or antigen-binding fragment, which matrices have the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., Lupron Depot™ (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid can release molecules over 100 days or more, while certain hydrogels release proteins over a shorter period. When encapsulated polypeptides remain in the body for an extended period, they can denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational methods for stabilization according to the mechanisms involved can be considered. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation by intermolecular thio-disulfide exchange, stabilization can be achieved by modification of sulfhydryl residues, lyophilization from acidic solutions, control of moisture content, use of appropriate additives, and development of specific polymer matrix compositions. Excipients and methods for making sustained-release compositions are described, for example, in REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition, (A.R. Genrmo, ed.), 1990.

[0174] A composition comprising the above-mentioned anti-CB1 antibody or antigen-binding fragment and one or more pharmaceutically acceptable excipients can be administered by any suitable means including parenteral administration. Parenteral administration includes intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration. Preferably, the administration is given by injection, most preferably subcutaneous injection, intramuscular injection, or intravenous injection. Other methods of administration are contemplated, including topical administration, particularly transdermal, transmucosal, rectal, oral or local administration (e.g., by catheter placed near the desired site).

[0175] Use Also disclosed herein is the use of an anti-CB1 antibody or antigen-binding fragment or a composition comprising the same for inhibiting, reducing, or neutralizing the amount, activity, or signaling of a CB1 receptor (e.g., huCB1). The antibodies and antigen-binding fragments of the invention are useful for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by CB1 expression or activity, or for blocking the interaction between CB1 and a CB1 ligand (e.g., a cannabinoid), or otherwise inhibiting CB1 activity and / or signaling, and / or are treatable by promoting receptor internalization and / or decreasing the number of cell surface receptors. In various embodiments, the CB1 is a peripheral CB1.

[0176] Exemplary disorders, diseases, and conditions that can be treated by the anti-CB1 antibodies or antigen-binding fragments described herein include syndromes such as Prader-Willi syndrome, Alström syndrome, Bardet-Biedl syndrome (BBS), Albright hereditary osteodystrophy (AHO), and SIM1 deletion syndrome; obesity such as diabetes and related complications (e.g., abnormal plasma glucose, insulin, and / or residual lipid levels); dyslipidemia (e.g., abnormal HDL, plasma cholesterol, and / or triglyceride levels); liver diseases such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and primary biliary cirrhosis; fibrosis such as renal fibrosis; chronic kidney disease (CKD) such as CKD driven by metabolic abnormalities; IgA nephropathy; kidney diseases; metabolic diseases, osteoporosis, atherosclerosis, inflammatory diseases, cardiovascular diseases, cancer, pain, systemic sclerosis, multiple sclerosis spasticity, glaucoma, and nicotine addiction.

[0177] Obesity refers to an abnormal or excessive accumulation of fat that can potentially impair an individual's health and can be measured or classified by the body mass index (BMI). The BMI is defined as a person's weight (e.g., in kilograms) divided by the square of their height in meters (e.g., kg / m 2 )

[0178] Accordingly, in one aspect, disclosed herein is a method of treating a disease or disorder comprising administering the antibody or antigen-binding fragment thereof to a subject responsive to antagonizing or inverse agonizing a CB1 receptor (e.g., huCB1), or to a subject in need of antagonizing or inverse agonizing a CB1 receptor. In some embodiments, the antibody or antigen-binding fragment is a potent antagonist and / or inverse agonist of the CB1 receptor, e.g., they have a titer (IC50) of less than about 10 nM (e.g., less than about 8 nM, less than about 5 nM, less than about 3 nM, or less than about 1 nM) as measured in a cell-based cAMP assay. In some embodiments, the antibody or antigen-binding fragment has an IC50 of less than about 10 nM (e.g., less than about 5 nM, less than about 3 nM, or less than about 1 nM) as measured in a cell-based cAMP assay (e.g., a cell-based cAMP assay in the presence of CP 55,940). In some embodiments, the antibody or antigen-binding fragment comprises heavy chain and / or light chain CDRs as described above. CDR sequences of various exemplary antibodies are listed in Tables 7, 10, 19, 26, and 30. In some embodiments, the antibody or antigen-binding fragment comprises heavy chain and / or light chain variable regions as described above. In other embodiments, the antibody comprises heavy chain and / or light chain as described above. Heavy chain and light chain variable region sequences of exemplary antibodies are listed in Tables 8, 11, 20, 27, and 31. Exemplary heavy chain and light chain constant region sequences are listed in Tables 1 and 2. In various embodiments, the antibody or antigen-binding fragment is administered in an effective amount to treat the disorder or disease (e.g., to elicit a potential benefit or treatment effect). In various embodiments, the CB1 receptor is a peripheral CB1 receptor.

[0179] The terms "subject in need thereof" or "subject in need of treatment" include subjects suffering from the disorder or disease as well as subjects in which the disorder or disease has not yet manifested clinically. "Subject" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. In a preferred embodiment, the subject is a human.

[0180] In some embodiments, the disorder or disease is obesity, diabetes, dyslipidemia, metabolic disease, fibrosis, liver disease, such as non-alcoholic steatohepatitis (NASH) or NAFLD, primary biliary cirrhosis, kidney disease, kidney fibrosis, chronic kidney disease (e.g., chronic kidney disease caused by metabolic abnormalities), IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular disease, cancer, or an inflammatory disease. In various embodiments, the disorder or disease is obesity. In some embodiments, the subject being treated has a chronic kidney disease, such as chronic kidney disease caused by metabolic abnormalities or kidney injury. In some embodiments, the subject being treated has obesity with a BMI of 27 kg / m 2 or greater. In some embodiments, the subject being treated has obesity with a BMI of 30 kg / m 2 or greater. In other embodiments, the disorder or disease is liver disease (e.g., NASH).

[0181] In some embodiments, administration or treatment results in one or more of the following in the subject: weight loss, reduced appetite, improved metabolic parameters, reduced blood glucose levels (e.g., reduced fasting blood glucose levels), reduced insulin levels, reduced HbA1c levels, reduced blood lipid levels (e.g., reduced HDL, cholesterol, and / or triglyceride levels), reduced kidney injury, reduced kidney fibrosis, reduced kidney inflammation, and improved kidney function.

[0182] Kit In some embodiments, disclosed herein is a kit comprising a composition comprising an anti-CB1 antibody or antigen-binding fragment and one or more of the above-described pharmaceutically acceptable excipients. For example, such a kit may comprise a composition comprising an anti-CB1 antibody or antigen-binding fragment described herein, such as the aqueous or lyophilized composition above, packaged in a container such as a sealed bottle, container, single-use or multi-use vial, prefilled syringe, or prefilled injection device. For the lyophilized composition, a suitable vehicle or carrier such as water for injection or physiological saline solution may be provided. Other materials common in formulations for parenteral administration may also be included. In one aspect, the composition is packaged in a unit dosage form. The kit may further comprise an apparatus suitable for administering the formulation according to a particular route of administration. Preferably, the kit further contains a label describing the use of the antibody or antigen-binding fragment or the composition containing the same.

[0183] All references cited herein, including patents, patent applications, publications, etc., are hereby incorporated by reference in their entirety.

[0184] The following examples are presented merely to illustrate the present invention and are in no way intended to limit the scope of the present invention.

Examples

[0185] Example 1. Deep Mutational Scanning and Design of an Affinity-Matured Library Materials and methods: Preparation of CB1-ND. For nanodisc preparation, the CB1 construct described in Tian H. et al., Cell, 167(3)(2016)750-762 was expressed in Sf9 cells with a histidine purification tag and purified using affinity and size exclusion chromatography in HEPES buffer containing 0.05% DDM; 0.01% CH and 10 μM ligand 1981202#5. POPC lipids were solubilized with the help of 100 mM sodium cholate and mixed with MSP1D1 scaffold protein and CB1 at a molar ratio of 480:8:1. Nanodisc formation was achieved by incubating with SM2 BioBeads at 4 °C for 1 hour.

[0186] Binding to CB1-ND and empty ND. The growth and induction of yeast cells displaying antibodies for the binding experiment were described by Luna V. et al. (Luna V. et al., European Polymer Journal 109(2018)483-488). Yeast cells displaying at least Mg2×10 5 were removed and washed with 1× phosphate-buffered saline (without Mg 2+ , without Ca 2+ ) supplemented with 5 mg / mL bovine serum albumin (PBS-B). His-tagged huCB1-ND or empty ND was added to the desired concentration and incubated with yeast cells at 25 °C for 1 hour. The cells were washed with PBS-B and then incubated with allophycocyanin (APC)-conjugated anti-His antibody to detect binding and phycoerythrin (PE)-conjugated anti-huFc antibody to detect IgG surface display. After washing the cells with PBS-B, flow cytometry analysis was performed using a FACS-Canto (BD Biosciences). The APC and PE mean fluorescence intensities (MFI) were recorded.

[0187] Sorting of the FACS library. At least 10 7Individual cells were incubated with the above antigen. After fluorescent antibody staining, cells were sorted using a FACS Aria (BD Biosciences) with appropriate settings for allophycocyanin and phycoerythrin detection.

[0188] The anti-CB1 antibody 10D10 was previously generated by immunizing Xenomice with HEK293 cells expressing human CB1. (See WO 2014 / 210205 pamphlet). Affinity maturation is a common strategy used to improve the titer of antagonist antibodies, but previous efforts with 10D10 failed to generate an antibody with sufficient titer (e.g., titer in the single-digit nM range in a cell-based cAMP assay) to provide the desired therapeutic effect (e.g., reducing body weight) without CNS-related side effects. The lack of a high-quality soluble preparation of a complex membrane protein such as CB1 was thought to be the main factor in these previous failures. The development of CB1 nanodiscs (NDs) and CB1 styrene maleic acid lipid particles (SMALPs) has significantly improved the quality of the soluble preparation of CB1. (Luna V. et al., European Polymer Journal 109 (2018) 483-488). Both CB1-ND and -SMALP are purification reagents that are stable after multiple freeze / thaw cycles and are suitable for long-term storage. Furthermore, these reagents can be added to binding experiments with huCB1 at user-defined concentrations. The availability of these reagents has enabled further efforts at affinity maturation.

[0189] To inform the design of an affinity maturation library, deep mutational scanning of the antibody was performed. Deep mutational scanning requires the creation of a comprehensive library of point mutations, the performance of binding selections under various conditions, and the evaluation of changes in mutation frequencies after selection by high-throughput sequencing. (See, for example, Araya, C. L. and Fowler, D. M., Trends Biotechnol. 2011 Sep;29(9):435-442). Identification of a subset of mutations associated with improved specific huCB1 binding enables combinatorial library design focused on affinity maturation without the benefit of high-resolution antibody-antigen co-crystal structures.

[0190] Two positions, 53 and 37, within the 10D10 heavy chain (HC) and light chain (LC) were selected for single-site saturation mutagenesis, respectively. A point mutation IgG-yeast library was created by dividing a comprehensive set of 10D10 point mutations in the heavy and light chain CDRs and in the heavy chain (HC) and light chain FR3 loops into four libraries (two for the heavy chain and two for the light chain, respectively). Each library was sorted separately against huCB1-ND and against empty ND at two different stringencies using FACS for surface expression and to assess non-specific binding. The low-stringency huCB1-ND binding gate collected all cells showing at least 10D10-like binding (10-20% of the population), while the high-stringency gate collected only the top binders (1.5-6% of the population). After FACS, plasmids were recovered from the sorted pools using the Zymoprep II kit (Zymo Research), and amplicons were generated by minimal PCR amplification of the recovered plasmids.

[0191] Using Illumina MiSeq, 1710 mutations were ranked by comprehensively evaluating the change in the frequency of mutations within the sorting gates relative to their starting frequencies (freq) in the expression sorted reference pools. The binding enrichment (EF) coefficient for each mutation was calculated based on NGS analysis using the expression sorted pools as reference samples (EF = freq結合 / freq 参照(発現) )。Subsequently, using the enrichment factor, an array binding compatibility map for each mutation in CDR and FR3 loops was created. Mutations that resulted in better, equivalent, and worse binding to CB1 compared to the 10D10 antibody were designated as beneficial, neutral, and harmful mutations, respectively. Based on the compatibility map, many changes within HCDR1 and HCDR3 were harmful, indicating that these regions may already be sufficiently optimized and there may be little need and / or possibility for optimization. A number of mutations in HCDR2 and HC_FR3 loops seemed to drive the improvement of binding. Data from libraries containing LCDR1 and LCDR2 mutations showed the lack of beneficial mutations in these regions. Overall, the binding compatibility map suggested that the HCDR is a more promising region for diversification for affinity maturation to improve binding.

[0192] Based on experience, the ideal positions for diversification for affinity maturation typically include changes for both harmful and beneficial mutations. To identify the most promising mutations at a single position to include in combinatorial library design, each neutral or beneficial mutation in huCB1-ND binding was also evaluated for binding to empty ND. The most promising mutations were enriched for huCB1-ND binding and depleted for empty ND binding (relative to the 10D10 parent). Mutations showing a positive enrichment factor for both huCB1-ND and empty ND were excluded from consideration for library design.

[0193] Two NGS-derived HC yeast libraries were designed and constructed for affinity maturation. Each library was designed to explore distinct CDR loop regions and keep the combinatorial theoretical diversity manageable for routine yeast library transformation coverage. Tables 3-4 below summarize the diversification strategies of the HC libraries. By introducing multiple degenerate codons during library gene synthesis to construct an initial set of two yeast display IgG HC variant libraries paired with the parental LC of 10D10, the combinations of desired mutations were fully explored within distinct regions of 10D10 HC. Each degenerate codon at a specific position was selected to maximize the representation of desired mutations within the encoded theoretical diversity

[0194] [Table 4]

[0195] [Table 5]

[0196] One NGS-derived yeast library was designed and constructed for LC optimization to improve the productivity of 10D10. The long HCDR3 and LCDR1 were thought to contribute to the poor expression and aggregation tendency of the initial 10D10 variant, and the LC library design incorporated strategies to improve these properties (e.g., reduction of hydrophobicity at important solvent-exposed LC residues in LCDR1). Since the fitness map of LC mutations showed a lack of beneficial mutations, the LC library design included mutations that showed 10D10-like binding enrichment to CB1-ND and depletion to empty ND. Table 5 below summarizes the diversification strategies of the LC optimization library. By introducing multiple degenerate codons during library gene synthesis to construct a yeast-displayed IgG LC variant library paired with the parental 10D10 HC, the combination of desired mutations within distinct regions of 10D10 LC was fully explored. Each degenerate codon at a specific position was selected to maximize the representation of the desired set of mutations within the encoded theoretical diversity. This library was used in the cognate affinity maturation described below (Example 2).

[0197]

Table 6

[0198] The 10D10 LC belongs to the VK2 germline. It was known in the art that germline LCs such as VK1 or VK3 result in better biophysical properties and better manufacturability (e.g., Ewert S. et al., J. Mol. Biol., (2003) 325(3):531-53). In a second strategy for LC optimization, we explored swapping the 10D10 LC with LCs from different germlines (e.g., VK1 or VK3) predicted to have more favorable biophysical properties. Both the low-resolution X-ray structure and the CryoEM structure of the mAb / antigen complex showed that only the HC loop of 10D10 interacts with the CB1 receptor. These results indicated that it might be possible to actually swap the 10D10 VK2 LC with another LC belonging to a germline (e.g., VK1 or VK3) predicted to have excellent biophysical properties to improve manufacturability. For this purpose, the 10D10 HC was paired with a diverse human LC library and screened and selected for conjugates that retained binding equivalent to 10D10. Next, those having LCs related to the above-described 10D10 LC optimization design were removed from the positive clones. This process yielded yeast clones that showed CB1 binding equivalent to 10D10 but contained LCs from the VK1 and VK3 germlines, demonstrating the feasibility of the LC swap strategy. The top six clones obtained from the LC swap are listed in Table 6 below. The six LCs were then grown into a set of 22 LCs to improve germline derivation within the FR regions of four clones.

[0199]

Table 7

[0200] Example 2. Isologous Affinity Maturation Materials and Methods: GFP-fused huCB1-SMALP was prepared as described in Luna V. et al., European Polymer Journal 109 (2018) 483-488.

[0201] The preparation of yeast cells for the binding experiment and the measurement of binding to GFP-fused huCB1-SMALP by flow cytometry were performed as described in Luna V. et al., European Polymer Journal 109 (2018) 483-488, except that IgG surface display was detected using AlexaFluor647-conjugated anti-huFab antibody. The mean fluorescence intensities (MFI) of GFP (binding) and AlexaFluor647 (display) were recorded.

[0202] For the nanodisc binding experiment, the binding / display ratio of each yeast clone tested was calculated by dividing the APC MFI by the PE MFI. The binding / display ratio of each clone was further normalized to the binding / display ratio of the 10D10 LC N35Y control to calculate the "relative binding fold change" ratio that enables comparison of yeast clones. Specific binding to huCB1-ND relative to empty ND was evaluated by dividing the huCB1-ND binding / display ratio by the empty ND binding / display ratio. For the SMALP binding experiment, the GFP MFI of each clone after background subtraction was normalized to that of the 10D10 LC N35Y control to compare the binding of yeast clones.

[0203] Two HC libraries and one LC optimization library designed and constructed in Example 1 were used in the 10D10 germline affinity maturation. The two HC libraries were separately enriched by FACS for improved binders compared to 10D10 according to established methods (see, for example, Boder, E.T. and K.D. Wittrup, Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol, 2000. 328: p. 430-44; and Chao, G., et al., Isolating and engineering human antibodies using yeast surface display. Nat Protoc, 2006. 1(2): p. 755-68). The LC optimization library was enriched for cells showing 10D10-like binding signals. Enriched surviving HC and LC mutations were recovered from the yeast pool by zymoprep and PCR, and the pools of HCS and LCS were mixed by design during yeast transformation to construct a new set of shuffled HC / LC libraries (mutH1H3 / mutLC, mutH2 / mutLC and mutH1H2H3 / mutLC). Higher stringency FACS enrichment utilizing successively lower concentrations of huCB1-ND was performed on the chain-shuffled libraries. Clones from the enriched shuffled libraries were screened in two rounds for top clone selection. In the first round, approximately 600 clones from the three libraries were screened for clones that bound huCB1-ND better than the 10D10 LC N35Y control (10D10 having N35Y in LC).

[0204] In the second round of screening, 200 of the best binding clones from the first round were retested for higher stringency binding to 200 pM huCB1-ND, 100 nM GFP-tagged huCB1-SMALP, and for higher stringency binding to empty ND for non-specific binding. After ranking positive clones by CB1-specific binding, clones with N-linked glycosylation and unpaired Cys layer 1 hotspots were removed, and clones with deamidation and isomerization sites outside the predicted layer 1.5DT were prioritized. Additionally, positive clones with an R94S fix for LC FR3 consensus violation were favored. Positive clones with an HC D83K FR3 mutation, thought to be associated with enhanced anti-CB1 function, were also prioritized if they met a minimum threshold for binding improvement. Further, clones with decreased hydrophobicity at important residues in LCDR1 and LCDR2 were favored. Tables 7 and 8 below summarize the sequence information for the top 15 germline affinity matured 10D10 variants. Figures 1A and 1B show the flow cytometry binding profiles for the 15 variants; the binding data for the variants are shown in Table 9 below. All top clones showed significantly improved binding to huCB1-ND and huCB1-SMALP while maintaining minimal binding to empty ND (Table 9). Additionally, Y57H in HCDR2, and D83N and S85Y in the HC FR3 loop were observed in all top clones (Figure 3A). None of the top 15 binders contain predicted hotspots that pose the highest risk of chemical modification during the manufacture and storage of therapeutic antibodies.

[0205]

Table 8

[0206]

Table 9

[0207]

Table 10

[0208]

Table 11

[0209]

Table 12

[0210]

Table 13

[0211] Example 3. Light Chain Swapping Affinity Maturation In the parallel arm of affinity maturation, the 22 VK1 and VK3 LCs obtained in Example 1 were transformed with the 10D10 mutant HC gene or gene fragments extracted from yeast cells, showing improved binding variants obtained after FACS enrichment of H1H3 and H2 / loop libraries (Example 2). Three LC swapping libraries for affinity maturation (mutH1H3 / VK LC, mutH2 / VK LC, and mutH1H2H3 / VK LC) were enriched for the most improved binders using higher stringency FACS enrichment. Clones isolated from the LC swapping libraries were screened in two rounds for top clone selection. The screening and selection criteria for the LC swapping 10D10 affinity maturation variants were the same as those for the cognate affinity maturation described in Example 2, except that no prioritization of sequences with reduced hydrophobicity in LCDR1 and LCDR2 was used. A total of 600 positive clones entered the first round of screening, and 200 positive clones entered the second round. The 200 positive clones were then ranked for CB1-specific binding to obtain the final 15 top LC swapping binders. The CDR and variable region sequences of the top 15 LC swapping affinity maturation 10D10 variants are shown in Tables 10 and 11. Table 12 below summarizes the binding data of the top 15 LC swapping affinity maturation 10D10 variants. Figures 2A and 2B show the flow cytometry binding profiles of these variants. Similar to the cognate affinity maturation variants, all of the LC swapping top clones showed significantly improved binding to huCB1-ND and huCB1-SMALP, while maintaining minimal binding to empty ND compared to 10D10 LC N35Y (Table 12). Furthermore, the Y57H in HCDR2, and the D83N and S85Y mutations in the HC FR3 loop were observed in the top 15 LC swapping 10D10 affinity maturation variants (Figure 4A). Furthermore, VK1 LC was largely represented among the top LC swapping 10D10 variants, and only one variant had VK3 LC (Table 13). The R94S germline revertant was imposed on all VK LC sequences of the top variants if not present in the original sequence.

[0212]

Table 14

[0213]

Table 15

[0214]

Table 16

[0215]

Table 17

[0216]

Table 18

[0217]

Table 19

[0218]

Table 20

[0219]

Table 21

[0220] Example 4. Cross-reactivity and specificity of affinity-matured antibodies The 10D10 antibody does not bind to mouse CB1 (muCB1) or human CB2 (huCB2) protein (WO 2014 / 210205 pamphlet). Since all the antibodies isolated in Examples 2 and 3 are variants of 10D10, they are not expected to bind to huCB1 or muCB1. The binding activities of exemplary antibodies against muCB1 and huCB2 confirm that there is no cross-reactivity against these two proteins (Table 14).

[0221]

Table 22

[0222] Example 5. Characterization of Affinity-Matured Variants The top clones isolated from Examples 2 and 3 above were cloned, expressed as IgG1 SEFL2 mAb, and then tested in a cAMP functional assay to measure antagonist or inverse agonist activity.

[0223] Antagonist and Inverse Agonist Activities Antagonist activity. Stably transfected CB1 CHO cells were serum-starved overnight using DMEM (Sigma catalog #D5671) containing 0.5% FBS and 25 mM HEPES before assaying at 37 °C / 5% CO2. On the day of the assay, the CB1 antibody and rimonabant were titrated 4-fold and 1 / 4-fold at a final concentration of 4 using DMEM (Sigma catalog #D5671) containing 0.1% BSA (from the PE kit) and 25 mM HEPES, and the titrated antibodies were transferred to a 384-well assay plate at a volume of 7.5 μL per well. Cells were harvested using Accutase (Sigma) and neutralized with DMEM (Sigma catalog #D5671) containing 0.5% FBS and 25 mM HEPES. These cells were pelleted and resuspended at a density of 1.33×10 6Resuspended in cells / mL. Anti-cAMP antibody from the Lance Ultra cAMP detection kit (Perkin Elmer; TRF0263) was added to the cells at an antibody ratio of 1 to 150, and 3-isobutyl-1-methylxanthine (IBMX; Sigma) was also added to the cells at a final concentration of 1 mM. The cells were transferred to the assay plate at a volume of 15 μL per well or 20,000 cells per well. Forskolin and CP55,940 cocktail were diluted to a 4-fold final concentration using DMEM (Sigma catalog #D5671) containing 0.1% BSA (from the PE kit) and 25 mM HEPES, and transferred to the assay plate at a volume of 7.5 μL per well. After incubating the assay plate at 37 °C / 5% CO2 for 1 / 2 hour, the EU-cAMP tracer (Perkin Elmer; TRF0263) was added to each well, and further incubated at room temperature protected from light for 1 hour. After the final incubation, the assay plate was read using an EnVision plate reader equipped with EU615 and APC665 emission filters. The data were analyzed using the following calculation: TR-FRET (665 nm / 615 nm) × 10,000. Lower values (lower FRET) were interpreted as more potent inhibition of CB1, and the IC50 was calculated using the data. The antagonist activities of the clones obtained from Examples 2 and 3 are shown in Table 15A (n = 1 and n = 2) and Table 15B (n = 3).

[0224]

Table 23

[0225]

Table 24

[0226]

Table 25

[0227] In addition, the antagonist activity of exemplary 10D10 variants and benchmark anti-CB1 antibodies was measured in the presence of the endogenous huCB1 ligands anandamide and 2-arachidonoylglycerol (2). Specifically, CHOK1 human CB1 receptor cells were plated overnight in DMEM containing 0.5% FBS, P / S glutamine, 25 mM HEPES, NEAA, and sodium pyruvate, and 400 μg / mL G418 (Invitrogen) was added to 96-well white flat-bottom plates (Costar, catalog #3917) at a density of 20,000 cells per well and placed in a humidified incubator maintained at 37 °C with 5% CO2. The cells were sequentially treated with the dose response of the antibody, then the CB1 receptor ligand at a concentration of 100 nM WIN55,212, 10 μM anandamide or 500 nM 2-AG (Sigma), and finally 35 μM forskolin (Sigma) in assay buffer of DMEM containing 0.1% BSA. After a 30-minute incubation, d2-cAMP and Eu3+-cryptate-cAMP antibody (Cisbio, catalog number 62AM4PEC) were added, followed by incubation at room temperature for 1 hour. The wavelengths of 665 nm and 620 nm were measured using an EnVision Multilabel Reader, and the 665 / 620 ratio was calculated. The graph was created by plotting the concentration of the CB1 antibody on the x-axis against the mean of two replicate values for the cAMP level (665 / 620 ratio). Next, the data points were fitted using log(agonist) vs. response, variable slope (4-parameter) in GraphPad Prism 7.04 to obtain the IC50 value. The results are shown in Tables 15C and 15D and Figure 5A.

[0228] Inverse agonist activity. The inverse agonist activity of exemplary 10D10 variants was also measured. CHOK1 human CB1 receptor cells were plated overnight in DMEM containing 0.5% FBS, P / S glutamine, 25 mM HEPES, NEAA, and sodium pyruvate, and 400 μg / mL G418 was placed in a 96-well white flat-bottom plate at a density of 20,000 cells per well and placed in a humidified incubator maintained at 37 °C with 5% CO2. Cells were continuously treated with the antibody in a dose response and then with 2 μM forskolin in assay buffer of DMEM containing 0.1% BSA. After a 30-minute incubation, d2-cAMP and Eu3+-cryptate-cAMP antibody were added, followed by a 1-hour incubation at room temperature. The wavelengths of 665 nm and 620 nm were measured using an EnVision Multilabel Reader, and the 665 / 620 ratio was calculated. The graph was created by plotting the concentration of the CB1 antibody on the x-axis against the mean of two replicate values for cAMP levels (665 / 620 ratio). Next, the data points were fitted using log(agonist) vs. response, variable slope (4 parameters) in GraphPad Prism 7.04 to obtain the IC50 value. The results are shown in Table 15C and Figure 5B.

[0229] [Table 26]

[0230] [Table 27]

[0231] The titers of affinity matured antibodies (both homologous and LC-swapped affinity matured antibodies) were analyzed using an in vitro cAMP assay, and the results are summarized in Tables 15A - 15D and Figures 5A and 5B. Most variants from affinity maturation were more potent than the 26970 - 1, 10D10 variant that was obtained in previous attempts and used as a benchmark in the assay (Tables 15A and 15B). 26970 - 1 contained the HC D83K / LC N35Y substitution and was the best available benchmark antibody. Some affinity matured variants showed titers similar to or slightly better than rimonabant in multiple runs of the functional assay. The most potent antibodies were also about 30 - 66-fold and 3.6 - 8-fold more potent than 10D10 and 26970 - 1, respectively (Table 15A). The LIBC528116 - 1 antibody was consistently the best antagonist in the CP55,940 assay (Tables 15A - 15C), and the LIBC523797 - 1 antibody was the most potent in assays conducted in the presence of the endogenous CB1 receptor agonists 2-AG and anandamide (Table 15C). Additionally, the LIBC523797 - 1 antibody was the best inverse agonist (Table 15C), and its titer was equivalent to rimonabant in the 2-AG assay and 3 - 4-fold more potent in the anandamide and inverse agonist assays (Table 15C). Finally, when the antibodies were tested side by side, the most potent 10D10 variant was shown to be about 30-fold more potent than the benchmark anti-CB1 antibody in clinical use (Table 15D). The titer results demonstrated that attempts at affinity maturation achieved the lofty design goal of obtaining antibodies with titers in the single-digit nM range, a goal that had remained elusive for many years.

[0232] Binding affinity Affinity assay using Octet. The affinity of the anti-CB1 antibody was determined using an Octet (registered trademark) HTX (Sartorius, Germany) instrument. Before the experiment, streptavidin (SA) biosensors (ForteBio, #18-5021) were pre-hydrated for 10 minutes in Octet assay buffer at pH 7.4 containing 10 mM Tris (Fisher, #BP152-1), 150 mM NaCl (Fisher, #S271-10), 1 mM CaCl2 (Fisher, #BP510-500), 0.1 mg / mL BSA (BioShop, #ALB001.1), and 0.1% Triton-X100 (Calbiochem, #9410-OP). Biotinylated anti-human Fc antibody (Invitrogen, #A18827) was loaded onto the SA biosensors, followed by one baseline step for 60 seconds in Octet assay buffer. The anti-CB1 antibody was prepared in Octet assay buffer and captured by the anti-human Fc antibody, then immersed in wells containing various concentrations of CB1 nanodisks (#CB1-008, in-house) for 5 minutes and subsequently dissociated for 10 minutes. The CB1 nanodisks were titrated 1:2 from 400 nM to 6.25 nM and prepared in Octet assay buffer. The SA biosensors were used once without regeneration. For data evaluation, ForteBio Data Analysis v11.0 software was used. The rate constants, association rate constant (ka, M-1s-1), dissociation rate constant (kd, s-1), and equilibrium rate constant (KD, M) were determined using a 1:1 Langmuir model.

[0233] Using on-cell ranking with a KinExA® 3200 (Sapidyne, Boise, ID) instrument, the binding of anti-CB1 antibodies to full-length CB1 (DNA 391968-1, in-house) transiently expressed on HEK293T cells was ranked. Prior to the on-cell ranking experiment, the on-cell affinity for the benchmark anti-CB1 antibody, LIBC523661, was determined. Affinity experiments were performed using HEK 293T CB1 cells 1 day after transfection and titrated 1-3 times from 2.5 million cells / mL to 51 cells / mL. HEK293T CB1 cells were equilibrated with LIBC523661 at a final antibody concentration of either 5 pM, 100 pM, or 1 nM (the binding arm concentration is 2-fold the antibody concentration) in cell culture medium containing FreeStyle 293T culture medium (Gibco, #12338-018), 2% ultra-low IgG FBS (Invitrogen, #16250-078), 50 μg / mL of G418 (Sigma, #G8168), and 0.05% w / v of sodium azide (Sigma, #S2002). Cells were separated by centrifugation, free antibody in the equilibrium supernatant was recovered, and measured by KinExA® technology by passing the supernatant through PMMA beads (Sapidyne) pre-coated with goat anti-human Fc (Invitrigen, #31125) and detected with goat anti-human IgG (H+L) AlexFluor649 (Jackson Immunoresearch, #109-605-088). KD was obtained from non-linear regression of the curve using the one-site homogeneous binding model provided by KinExA® Pro software Version 4.2.12. The KD of LIBC523661 was measured to be 64.0 pM, 95% CI was 46.6 - 88.2 pM, and the number of receptors per cell was equal to 1.1 million copies / cell. Using the KD control curve, it was found that approximately 12,000 cells / mL were required to achieve 50% KD of LIBC523661. Therefore, the on-cell ranking of anti-CB1 antibodies was performed using 20 pM of each antibody at 12,000 cells / mL. Anti-CB1 antibodies were prepared in 293 culture media either without cells or containing HEK293T CB1 cells and incubated at room temperature for 24 hours.Similarly, in the affinity experiment, cells were separated by centrifugation, the free antibody in the supernatant was recovered, and measured by the KinExA® technology. The binding of each anti-CB1 antibody was reported as the percent inhibited free fraction (%IFF), which was calculated as the value obtained by subtracting the signal from the blank from the antibody signal obtained in the presence of 12,000 cells / mL, divided by the value obtained by subtracting the signal from the blank from the antibody signal obtained in the absence of cells (the lower the %IFF, the higher the affinity; the higher the %IFF, the lower the affinity). The relative binding of all anti-CB1 antibodies was compared to LIBC523661 by taking the %IFF of LIBC523661 divided by the %IFF of the antibody.

[0234] The Octet and Kinexa binding assays of the affinity matured 10D10 variant confirmed that the increase in titer was accompanied by an improvement in binding affinity to huCB1 (Table 16). Octet showed that faster on-rates and slower off-rates contributed to a 10 - 50-fold higher binding affinity to huCB1-ND for 10D10, while Kinexa confirmed an improvement in binding to wt huCB1 expressed on cells.

[0235]

Table 28

[0236] Example 6. Improvement in the Expression of Affinity Matured Variants Isotypes and LC swapping affinity matured variants were tested for transient and stable expression in HEK293 and CHO cells. Four out of the five most potent isotype 10D10 variants transiently expressed at 100 - 200 mg / L in HEK293-6E cells (Table 17), showing titers >3-fold or >7-fold higher than 10D10 or benchmark variants. Testing of the LC sequences suggests that a decrease in hydrophobicity at key positions (notably L58Q) can be associated with improved expression titers (Table 17). The most potent LC swapping 10D10 HC variant transiently expressed at 15 - 80 mg / L and was well expressed at >670 mg / L in stable production runs from CHO-K1.

[0237]

Table 29

[0238] Example 7. Effect of affinity matured antibodies in animal models The SEFL2 format LIBC528116-1 (37940-4) antibody (F.W. et al., J Biol Chem. 2017 Feb 3;292(5):1865-1875) was scaled up for testing in diet-induced obesity (DIO) transgenic mice with a huCB1 donor DNA knocked in using CRISPR-Cas9 technology, mediating substitution of the mouse Cnr1 coding sequence with the human CNR1 counterpart. Specifically, 6- to 10-week-old huCB1 knock-in (KI) mice were fed a high-fat diet (HFD, Research Diets D12492 60% kcal from fat) for 13 weeks to induce obesity. The LIBC528116-1 antibody and huIgG1 control antibody were administered to the mice at 30 mg per week (QW) / kg for 60 days (n = 7 mice / group), and body weight and food intake were measured every 3 to 4 days. On the 58th day of treatment, total fat mass was measured by MRI, and triglyceride and insulin levels in the animals were analyzed, and the results are shown in Table 18 and Figures 6A-6C. On the 60th day, the affinity-matured 10D10 variant reduced body weight by 15.9%, fat mass by 22%, liver weight by 47%, liver triglyceride by 64%, and plasma insulin by 77% compared to the huIgG1 control (Table 18, Figures 6A, and 6C). In contrast, a previous study with the D83K / N35Y variant (26970-2) of 10D10 had no effect on body weight gain compared to the isotype control. These results demonstrated that the potent peripheral-acting anti-CB1 antibodies described herein are effective treatments for anti-obesity, hyperinsulinemia, and non-alcoholic steatohepatitis (NASH).

[0239]

Table 30

[0240] Fibrosis and inflammation markers in the kidneys were also measured in animals. Specifically, RNA was extracted and isolated from mouse kidney tissue by TRIzol extraction. 50 - 100 mg of tissue and 5 mm stainless steel beads (Qiagen, #69989) were placed in a microcentrifuge tube containing 1 mL of TRIzol (Ambion by life technologies, #15596018). The TissueLyser II device was used at a frequency (1 / S) of 30.0 for 3 minutes and repeated once. The lysate was then incubated for 5 minutes and subsequently quickly spun down. Then, 200 μL of chloroform (Sigma - Aldrich, #372978 - 100 mL) was added to each tube and vortexed for 30 seconds. The tubes were then incubated for 5 minutes and then spun down at 4°C, 12,000 g for 15 minutes. The top aqueous layer was removed and placed into a new deep well plate and mixed with an equal volume of 70% EtOH. Once mixed, the solution was transferred from the RNeasy Plus 96 kit to an RNA plate and RNA isolation was performed according to the kit instructions (Qiagen, #74192), and the Qiagen DNase step was performed according to the kit instructions (Qiagen, #79254). The RNA was diluted in RNase / DNase - free water and the concentration of the RNA was confirmed by NanoDrop 8000. The RNA was then diluted to 50 ng / 8.5 μL of water and then run on a Quantstudio7 Flex real - time PCR system. The TaqMan RNA - to - CT one - step kit (Applied Biosystems, #4392938) was used together with a TaqMan gene primer - probe set. The relative RNA expression levels were calculated using the CT values. Treatment with the anti - CB1 antibody reduced the expression levels of fibrosis and inflammatory markers in the kidneys compared to control IgG (Figure 6D).

[0241] Example 8. Novel XENOMOUSE Antibody Campaign Mouse immunization: Several groups of xenomice were immunized with different combinations of immunogens. The immunogens were: plasmid DNA encoding full-length huCB1 with the E3k epitope for T cell activation. The DNA was coated onto gold particles and delivered intradermally; HEK293T cells were transiently transfected with full-length huCB1 using the E3k epitope. The cells were delivered subcutaneously, and nanoparticles (nanodiscs) containing the huCB1 protein were engineered to enhance stability. The nanoparticles were delivered subcutaneously.

[0242] Immunization took several months; blood samples were collected from the animals and the CB1 antibody titers were measured periodically. Since previous experience and the published CB1 structure suggested that the large N-terminal domain was likely to be irrelevant to drug function, only animals showing titers against both full-length CB1 and truncated CB1 (i.e., lacking the 1-98 aa N-terminus) were selected for recovery in order to improve the chance of finding functional antibodies during screening. Titration was performed by FACS on CHO-S cells transiently transfected with full-length huCB1 or truncated huCB1 or empty vector.

[0243] Screening of plasma B cells: Conventional hybridoma generation relies on the immortalization of B cells by fusion with myeloma cells. Due to the low fusion efficiency, valuable B cell clones, if they are rare, can be difficult to recover by this process. To complement hybridoma generation, direct screening of antibody-secreting plasma B cells was performed. The latter approach focused on memory B cells and was used to increase the chance of finding valuable clones by sampling different cell populations.

[0244] B cells for screening were obtained from the spleens, lymph nodes and bone marrow of slaughtered animals. Plasma B cells were separated from memory B cells according to CD138 cell surface marker expression using a commercially available CD138 enrichment kit (Stemcell Technologies 17877). The CD138+ cells were then combined with HEK293T cells transfected with truncated huCB1 and HEK293T cells transfected with an empty vector. The three types of cells were combined together in B cell medium and then mixed with a warm agarose solution (Sigma A5030, final concentration 1%), placed in fluorinated oil (RAN Biotechnologies 008 - FluoroSurfactant - 2wtH - 50G), and emulsified by vortexing. As a result, an oil-in-medium emulsion containing microdroplets in which the cells were trapped was obtained. After 1 hour of incubation and 10 minutes of cooling, the agarose microgels were extracted from the oil, washed with 1% FBS in PBS, and screened using a fluorescently labeled anti - human antibody as a secondary detection reagent (Jackson 109 - 545 - 098). The microgels were examined under a fluorescence microscope, and those containing a fluorescent signal were manually collected using an Eppendorf micromanipulator. The isolated microgels were placed into individual PCR tubes containing lysis buffer and subjected to an RT - PCR reaction to rescue the antibody sequences from the B cells trapped in the microgels for cloning and expression.

[0245] Molecular rescue and sequencing of CB1 antibodies from B cells: RNA (total or mRNA) was purified from samples containing CB1 antibody-producing single B cells using Agencourt RNA Clean XP magnetic beads. The purified RNA was used as a template in template-switching PCR to generate first-strand cDNA via reverse transcription, followed by cDNA amplification. The cDNA was cleaned up using the Agencourt AMPure XP PCR clean-up kit and used as a template for amplifying the variable regions (V) of the antibody heavy and light chains. Using cDNA as a template and multiplex PCR, the variable region of the gamma heavy chain was amplified to obtain a full human antibody gamma heavy chain. The 5' gamma chain-specific primer annealed to the signal sequence of the antibody heavy chain, while the 3' primer annealed to the region of the gamma constant domain. Using cDNA as a template and multiplex PCR, the variable region of the kappa light chain was amplified to obtain a full human antibody kappa light chain. The 5' kappa light chain-specific primer annealed to the signal sequence of the antibody light chain, while the 3' primer annealed to the region of the kappa constant domain. Using cDNA as a template and multiplex PCR, the variable region of the lambda light chain was amplified to obtain a full human antibody lambda light chain. The 5' lambda light chain-specific primer annealed to the signal sequence of the light chain, while the 3' primer annealed to the region of the lambda constant domain. The primers used in the multiplex PCR reaction also had overhangs that facilitated golden gate cloning into the expression vector. The amplicons from the multiplex PCR reaction were purified using the Agencourt AMPure XP PCR clean-up kit. Next, the clean PCR amplicons were cloned into the expression vector using standard golden gate cloning methods and subsequently transformed into chemically competent Escherichia coli (E. coli) bacterial cells. Bacterial colonies derived from the transformation were cultured and then sequenced directly. The amino acid sequence was deduced by bioinformatics from the corresponding nucleic acid sequence. Next, the obtained amino acid sequence was analyzed to determine the germline sequence origin of the antibody and to identify the differences from the germline sequence.

[0246] Generation and Screening of Hybridomas: Hybridomas were generated by immortalizing B cells with mouse myeloma cells using standard techniques. Hybridomas were first plated at a density of several clones per well and screened for secreted antibodies for binding to both full-length and truncated CB1 transiently expressed on HEK293T cells or CHO-S cells. Polyclonal wells showing CB1 binding were sorted for single cells to obtain individual antibody-secreting clones.

[0247] A total of 1325 Xenomouse clones were screened, and 71 of them bound to both full-length huCB1 and truncated huCB1. Most of the 71 clones were non-functional, and only 9 clones met the titer criteria set for the mouse campaign. Three unique antibodies were isolated from the 9 clones. The sequences of the antibodies (Tables 19 and 20) are similar to the sequence of 10D10. In particular, the Y57H mutation in HCDR2 that emerged after affinity maturation of the antibody is also present in these antibodies (Table 19).

[0248] [Table 31]

[0249] [Table 32]

[0250] Characterization of Antibodies: The antibody clones identified above were tested for binding to full-length human CB1 3 and N-terminal truncated human CB1 to confirm that the antibodies bind to the extracellular loop ECL1 and not to the CB1 N-terminus. Binding to human CB2 was also tested to confirm that there are no side effects due to cross-reactivity with CB2. Finally, binding to mouse CB1 was tested. After the binding assay, a functional screening using the TR-FRET cAMP assay was performed to measure the antibody inhibitory effect on CB1.

[0251] To evaluate target specificity, binding was measured by FACS on HEK293T cells transiently transfected with the protein of interest and compared to non-specific binding to cells transfected with the empty vector. Binding results are presented as the ratio of the mean fluorescence intensity of target-transfected and mock-transfected cells. The results showed that all three antibodies bind to full-length and truncated human CB1, but not to human CB2 or mouse CB1 (Table 21).

[0252] The inhibitory effect of the antibodies on CB1 function was evaluated using a time-resolved Förster resonance energy transfer (TR-FRET) cAMP assay. Antibodies that inhibit CB1 cause an increase in cAMP, which in turn results in a decrease in the FRET signal in the assay. Thus, a lower assay signal means better inhibition of CB1 by the antibody. To improve assay sensitivity, CP55,940 and forskolin were used to activate cellular CB1 and adenylyl cyclase, respectively. Rimonabant was used as a positive control.

[0253] Briefly, stably transfected CB1 CHO cells were cultured overnight at 37°C / 5% CO2 in DMEM (Sigma catalog #D5671) containing 0.5% FBS and 25 mM HEPES. On the day of the assay, the CB1 antibody and rimonabant were titrated 1:4 at 4-fold final concentrations using DMEM (Sigma catalog #D5671) containing 0.1% BSA (from the PE kit) and 25 mM HEPES, and the titrated antibody was transferred to a 384-well assay plate at a volume of 7.5 μL per well. Cells were harvested using Accutase (Sigma) and neutralized with DMEM (Sigma catalog #D5671) containing 0.5% FBS and 25 mM HEPES. These cells were pelleted and resuspended at 1.33x106 cells / mL using DMEM (Sigma catalog #D5671) containing 0.1% BSA (from the PE kit) and 25 mM HEPES. Anti-cAMP antibody from the Lance Ultra cAMP detection kit (Perkin Elmer; TRF0263) was added to the cells at an antibody ratio of 1:150, and 3-isobutyl-1-methylxanthine (IBMX; Sigma) was also added to the cells at a final concentration of 1 mM. The cells were transferred to the assay plate at a volume of 15 μL per well or at a quantity of 20,000 cells per well. Forskolin and CP55,940 cocktail were diluted to 4-fold final concentrations using DMEM (Sigma catalog #D5671) containing 0.1% BSA (from the PE kit) and 25 mM HEPES, and transferred to the assay plate at a volume of 7.5 μL per well. After incubating the assay plate at 37°C / 5% CO2 for 1 / 2 hour, the Eu-cAMP tracer (Perkin Elmer; TRF0263) was added to each well and further incubated for 1 hour at room temperature protected from light. After the final incubation, the assay plate was read using an EnVision plate reader equipped with Eu615 and APC665 emission filters. The data were analyzed using the following calculation: (665 nm / 615 nm) × 10,000. Lower values (lower FRET) indicate stronger inhibition of CB1, and the IC50 was calculated using the data.The results showed that all three antibodies were far superior to 10D10 in terms of titer (Table 22 and Figure 9).

[0254]

Table 33

[0255]

Table 34

[0256] Example 9. Further affinity maturation of anti-CB1 antibodies To further improve the functional titer, another round of affinity maturation was performed. Analysis of the affinity matured antibodies from the previous round (Examples 1-3 above) showed that for the cognate 10D10 variant, there was a good correlation between GFP-tagged huCB1-SMALP binding and IC50, indicating that huCB1-GFP-SMALP binding predicted most of the most potent LC-swapping variants. Therefore, improvement of binding to huCB1-GFP-SMALP was the focus during screening and sorting in this round of affinity maturation to obtain antibodies with sub-nanomolar titers.

[0257] For this affinity maturation, two strategies were used. First, fortunately, the VK1 LCs shared by the top LC-swapped variants belong to a well-studied germline that is solvent-exposed and predicted to be resistant to mutation at LCDR positions with high confidence. Thus, to further optimize those LCs, the inventors attempted to pair the HCs of LIBC528116-1 and other top LC-swapped variants with a VK1 / O2 LCDR library previously utilized in an internal affinity maturation project. The co-crystal structure suggests that mutations at solvent-exposed LC positions are unlikely to result in contact with huCB1, but it is hypothesized that LC mutations may provide more optimal structural support for the HC loop conformation to enhance binding and titer. To increase the selection stringency, a monovalent yeast display Fab library was constructed and screened for improved binding to wtCB1-SMALP while retaining huCB1-ND binding (relative to the LIBC58116-1 Fab).

[0258] Second, the inventors exploited the surprising discovery of more 10D10-like antibodies from a new mouse campaign to utilize a better starting point for affinity maturation (see Example 8). Antibodies from the new mouse campaign have shown that specific positions in HCDR2 and HC FR3 can be further explored to improve binding and titer. Additionally, the novel antibodies cluster into two highly related HCDR3s that differ by only one amino acid in length (e.g., LIBC560657-1 and LIBC529593-1 of Example 8), indicating that the length of HCDR3 can vary. Yeast display libraries for further isologous affinity maturation integrated HC and LC mutations from the most potent isologous 10D10 variants from Example 2 into the LIBC529593-1 sequence context and incorporated both HCDR3 lengths. HC library 2 incorporated the shorter HCDR3 sequence and contained a fixture at HCDR3 position 112 of the predicted DS isomerization site. The design of further isologous affinity maturation libraries is summarized in Tables 23 to 25.

[0259]

Table 35

[0260]

Table 36

[0261]

Table 37

[0262] The Fab-yeast library was constructed by combining the 10D10 HC and LC library designs to select for increased binding stringency. The cognate library for further affinity maturation was sorted / screened for improved binding to wtCB1-SMALP (versus LIBC523797-1 Fab) while retaining huCB1-ND binding.

[0263] The libraries constructed for both further affinity maturation campaigns were sorted successively against lower concentrations of huCB1-GFP-SMALP starting at 25 or 50 nM. We performed clone binding screening at the low huCB1-SMALP concentration of 2 - 5 nM, which represents a 20 - 50-fold lower concentration than that used in previous affinity maturation campaigns (Examples 2 - 4). Next, the best binders showing minimal non-specific binding and predicted sequence liability from the further VK1 LC optimization campaign and the further cognate affinity maturation campaign were selected based on the same principles as previously described (Examples 2 and 3). Eight clones were obtained from VK1 LC optimization and 18 clones were obtained from further cognate affinity maturation. These top clones showed improved huCB1-SMALP binding, similar huCB1-ND binding, and low levels of empty ND binding compared to the LIBC528116-1 and LIBC523797-1 Fab-yeast benchmarks from both LC swapping and the respective campaigns.

[0264] The CDR sequences, germline information, and binding data of the top eight further LC swapping affinity matured variants are summarized in Tables 26 and 27 and Figure 7. All top clones showed improved binding to CB1-SMALP and CB1-ND (Table 28), while binding to irrelevant XCR1-SMALP and empty ND was minimal. For LIBC681737-1, when preparing the IgG1 SEFL2 mAb for functional testing, the LC P12S germline revertant mutation was included.

[0265]

Table 38

[0266]

Table 39

[0267]

Table 40

[0268]

Table 41

[0269]

Table 42

[0270]

Table 43

[0271]

Table 44

[0272] The CDR sequences, germline information, and binding data of the top 18 additional germline affinity matured variants are summarized in Tables 30 and 31 and Figures 8A and 8B. Most of the 18 conjugates utilized shorter HCDR3s and had the HC T79R FR3 mutation in the library design of HC library 2, highlighting the advantage of rationally merging features from all available sequence-function relationships. All 18 germline variants met the stringent binding improvement threshold for huCB1-GFP-SMALP, and binding to irrelevant XCR1-SMALP and empty ND was minimal (Table 32). These variants also showed better binding to CB1 SMALP compared to LIBC523797-1. For LIBC674043-1 and LIBC674200-1, the VH R149S and VL G76D germline revertant mutations were also included in a panel of IgG1 SEFL2 mAbs generated for functional testing.

[0273]

Table 45

[0274]

Table 46

[0275]

Table 47

[0276]

Table 48

[0277]

Table 49

[0278]

Table 50

[0279]

Table 51

[0280]

Table 52

[0281] Example 10. Functional Analysis of Further Affinity-Matured Variants The clones isolated from Example 9 above were cloned and expressed as IgG1 SEFL2 mAbs for functional analysis. The antagonist activity of the antibodies was analyzed using the TR-FRET cAMP assay described above (e.g., Example 8), and the titers of the antibodies are summarized in Table 33. The titers of all the antibodies were in the single-digit nM range, and some had titers of less than 1 nM in all four experiments.

[0282]

Table 53

[0283] Example 11. Effect of Further Affinity-Matured Variants in an Animal Model The further affinity matured antibodies LIBC673952(52777) and LIBC680574(52778) in the YTE format (Dall’Acqua et al., The Journal of Immunology, 169:5171 - 5180(2002)) were scaled up for testing in diet - induced obesity (DIO) transgenic mice knocked in with huCB1. HuCB1 knock - in (KI) mice at 6 - 10 weeks of age were fed a high - fat diet (HFD) for 15 weeks to induce obesity. Antibodies (52777, 52778, and 37940) and a control antibody (huIgG control) were administered to the mice at 30 mg / kg QW for 35 days, and the body weight and food intake of the animals were measured every 3 - 4 days, and the results are shown in FIGS. 10A and 10B. Administration of the further affinity matured antibodies decreased the body weight of the animals (FIG. 10A), while the average daily food intake did not change significantly (FIG. 10B). The body fat mass and liver weight of the animals were also measured, and the results are shown in FIGS. 10C and 10D. These results indicated that the potent peripherally acting anti - CB1 antibodies described herein are effective treatments for anti - obesity, hyperinsulinemia, and NASH.

[0284] Example 12. Effect of anti - CB1 mAb in energy consumption and insulin resistance tests Materials and methods: Tests using mice in accordance with all relevant ethical rules and the approvals for the conducted tests were obtained from Amgen's IACUC Institutional Review Board (Thousand Oaks, CA). The lighting in the animal housing room was on a 12:12 hour light::dark cycle, and the ambient temperature and humidity ranges were 68 - 79°F and 30 - 70% respectively. Animals had free access to irradiated pelleted feed and reverse - osmosis chlorinated (0.3 - 0.5 ppm) water via an automatic watering system or by water bottles as directed. The cages were changed weekly.

[0285] Male huCNR1 TG mice (4 - 7 weeks old) were shipped from Charles River Laboratories (Hollister, CA) to Amgen (Thousand Oaks, CA), and after arrival, they were housed individually and started on a high - fat diet (HFD) (60% kcal fat, Research Diets D12492i) for 12 weeks. After 12 weeks of HFD feeding, the mice were acclimated to the water bottles for 1 week, and then n = 24 mice were transferred to a comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments, Columbus, Ohio - Oxymax Model 2018, 0233 - 004M, Oxymax for Windows v5.53 software, Hardware configuration 190395) and further acclimated for 2 weeks. During that period, the amounts of oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange ratio (RER), and energy consumption (HEAT) were measured. At the start of the treatment, the mice were given HFD for a total of 15 weeks, and n = 23 mice were randomized into two groups based on body weight, age, VO2, VCO2, RER, and Heat during the baseline measurements from - 3 days to - 1 day. The mice were administered huIgG1 control PL - 42466 (n = 11 mice) or CB1 mAb 37940 - 17 (n = 12 mice) by intraperitoneal (IP) injection at 30 mg / kg (2 mL / kg) once a week for a total of 5 injections. The mice were housed in the CLAMS system from day - 14 to day 7, returned to their home cages from day 7 to day 14, returned to the CLAMS system from day 14 to day 28, and returned to their home cages on day 28 throughout the test period. Body weight and food intake were measured weekly. For simplicity, the data are presented in a light - on and light - off cycle (12 hours light: 12 hours dark).

[0286] For the insulin tolerance test (ITT), on the 32nd day of treatment (4 days after the 5th IP injection), mice were fasted for 6 hours starting at 6 am. At 12 pm, blood glucose was measured by retro-orbital bleeding using a blood glucose meter (AlphaTrak), and insulin (0.75 U / kg) was immediately administered IP to the mice. Blood glucose was measured by retro-orbital bleeding 30 minutes, 60 minutes, 90 minutes, and 120 minutes after insulin administration. The area under the curve (AUC) was calculated for each mouse using GraphPad Prism (version 9.5.1).

[0287] Results: Energy consumption measured as heat (kcal / hour / kg) and respiratory exchange ratio (RER) was averaged per week (acclimation, week 1, week 3, and week 4) for the light cycle (6 am to 6 pm) and dark cycle (6 pm to 6 am). During the light cycle when the mice were resting, the energy consumption at week 4 of treatment was significantly higher in CB1 mAb-treated mice (+12% compared to the CB1 baseline, +4% compared to the control baseline, p = 0.0325; two-way repeated measures ANOVA with Sidak's test for multiple comparisons, Figure 11A). Furthermore, RER was significantly lower in the CB1 mAb-treated group at week 3 of treatment (treatment p = 0.0125; two-way repeated measures ANOVA using Sidak's test for multiple comparisons), indicating an increase in lipid oxidation (Figure 11A).

[0288] To evaluate insulin sensitivity, an insulin tolerance test (ITT) was performed. After a 6-hour fast, there was no difference in the baseline blood glucose measurements among the mice; however, after insulin administration, CB1 mAb-treated mice had statistically lower blood glucose at 30 minutes, 60 minutes, and 90 minutes after insulin injection (p < 0.01, two-way repeated measures ANOVA with Sidak's test for multiple comparisons), and a 25% lower AUC (p = 0.0006; Student's unpaired t-test), indicating significantly improved insulin sensitivity after CB1 mAb treatment (Figure 11B).

[0289] Example 13. Brain Uptake Test of Anti-CB1 MAB in Tg32 Mice This study was conducted to characterize the single-dose plasma pharmacokinetics and brain uptake of an exemplary anti-CB1 antagonist mAb following intravenous bolus administration in male huFcRn Tg 32 homozygous mice.

[0290] Materials and Methods: Food and water were provided ad libitum throughout the study period. On the day prior to dosing, the stock test article was removed from the -70°C (±10°C) storage and thawed overnight at 2 - 8°C. On the morning of dosing, the stock test article was removed from the 2 - 8°C storage, mixed by gentle inversion, and placed on wet ice for the dilution procedure. The vehicle was removed from the 2 - 8°C storage, mixed by gentle inversion, and placed on wet ice for the dilution procedure. For all groups, the stock test article was diluted with the appropriate formulation buffer to obtain the final dose solution concentration listed in the experimental design. After preparation of the dosing solution, all remaining vehicle (if applicable) was returned to the 2 - 8°C storage. After dosing, all remaining stock test article (if applicable) and remaining dosing solution (if applicable) were placed at -70°C (±10°C) and transferred to the Bioanalytical Principal Investigator for subsequent analysis and storage. The test animals received an intravenous bolus injection of the appropriate dosing solution via the lateral tail vein.

[0291] Approximately 0.05 mL of whole blood per test system was collected at each successive time point via the submandibular vein using a SARSTEDT Microvette® K3 EDTA plasma separation tube. After placing the whole blood® into a Sarstedt Microvette® plasma tube, the tube was mixed by gentle inversion 8 - 10 times and placed on wet ice. The samples were centrifuged at 2 - 8°C at approximately 14,000 rcf for 5 minutes using a calibrated Eppendorf 5417R centrifuge system (Brinkmann Instruments, Inc., Westbury, NY 11590). All plasma samples were stored at approximately -70°C (±10°C) until analysis.

[0292] Cardiac perfusion was performed at the end of the test. Each test system was deeply anesthetized with isoflurane (5% in O2(g), at a rate of 1.5 L / min) and kept under surgical anesthesia throughout the perfusion procedure. An incision was made in the skin just below the sternum and the tip of the sternum was located. The sternum was grasped and cut along both sides of the rib cage through the diaphragm to expose the heart, causing pneumothorax in the animal. The heart was stabilized and a 20-gauge half-inch needle was placed in the left ventricle. The needle was connected via a tube to a perfusion / syringe pump and 0.9% saline was flowed at a rate of approximately 4 mL / min for approximately 5 minutes. Once the flow was established, the right atrium was incised to allow the fluid to flow out of the animal and prevent overpressure in the vascular system. After 5 minutes of perfusion with saline, 10% neutral buffered formalin was perfused for an additional 5 minutes. When the perfusion was complete, the needle was removed from the left ventricle and appropriate tissue was collected for subsequent analysis.

[0293] At the end point, immediately after blood loss as outlined below, tissue was collected from each test system. At the time of extraction, the tissue was rinsed with 0.9% sterile saline, blotted with dry gauze, immediately placed into cryovials (Eppendorf Protein LoBind, catalog #0030108116), weighed, and immersed in liquid nitrogen to instantaneously freeze the tissue. Once the tissue was instantaneously frozen, they were kept on dry ice during the autopsy procedure and stored at -70°C (±10°C) for subsequent analysis.

[0294] Plasma and tissue specimens for PK test substance concentration determination were analyzed using an enzyme-linked immunosorbent assay (ELISA). The specimens were maintained at -70°C (±10°C) prior to analysis. Non-compartmental analysis was performed on the mean plasma test substance concentration vs. time data from all mice at each sampling time per dose group. The parameters of interest included the tissue-to-plasma percentage ratio on day 10, and the half-life (t 1 / 2 ) is included.

[0295] Results: The PK and brain exposure of the antibody are shown in Table 34 below. The results show that the brain exposure of the anti-CB1 mAb is 0.2% of the plasma concentration of the antibody. Transgenic Tg32 mice expressing the human fetal Fc receptor (hFcRn) are known to predict the human PK of mAb therapeutics (Avery et.al. mAbs 20161). These results indicate that 1 / 0.002 of the mAb plasma concentration may be distributed to the brain. These results are also consistent with previously published literature on immunoglobulin G1 (IgG1) mAbs in other mouse models (e.g., Avery L.B. et al., MAbs. 2016 Aug-Sep;8(6):1064-78.doi:10.1080 / 19420862.2016.1193660.Epub 2016 May 27.PMID:27232760;PMCID:PMC4968115; Shah D.K. et al., MAbs. 2013 Mar-Apr;5(2):297-305.doi:10.4161 / mabs.23684.Epub 2013 Feb 13.PMID:23406896;PMCID:PMC3893240; and Garg A. and Balthasar J.P., AAPS J. 2009 Sep;11(3):553-7.doi:10.1208 / s12248-009-9129-9.Epub 2009 Jul 28.PMID:19636712;PMCID:PMC2758122).

[0296]

Table 54

[0297] Example 14. CRYO-EM Structure of the CB1-Fab Complex The cryo-EM structure of huCB1 complexed with the Fab of 10D10 and four affinity matured (AM) antibodies was segmented at a resolution of approximately 3.1 - 3.7 Å, which is sufficient to determine epitope / paratope interactions. The antibody titers range from 1.77 - 47 nM. The cryo-EM structure confirmed that only the heavy chain of the antibody is involved in binding and that ECL2 of CB1 is the major epitope. In addition, HCDR1 plays a major role in the binding of CB1 ECL2, which is consistent with the fact that the HCDR1 sequences of the AM antibodies are highly conserved. R32 (AHo numbering), adjacent to HCDR1, is also involved in epitope interaction. HCDR2 has a diverse sequence, and specific residues in HCDR2 (e.g., Y59, AHo numbering) are thought to contribute to binding affinity. Y131 and Y132 (AHo numbering) of HCDR3 are conserved among the antibodies tested and bind to CB1.

[0298]

Table 55

[0299]

Table 56

[0300] While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that variations of the preferred compounds and methods may be used and that the invention may be practiced in ways other than those specifically described herein. Accordingly, the invention includes all modifications within the spirit and scope of the invention as defined by the following claims.

Claims

**Claim 1** An isolated antibody that binds to the human cannabinoid receptor 1 (huCB1), said antibody comprising a heavy chain variable region (HV) comprising heavy chain complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region (LV) comprising light chain complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein said heavy and light chain CDRs are as follows: (a) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 135-137, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 138-140; (b) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 426-428, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 423-425; (c) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 266-268, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 263-265; (d) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 129-131, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 132-134; (e) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 159-161, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 162-164; (f) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 207-209, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 210-212; (g) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 1-3, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 4-6; and (h) HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences of SEQ ID NOs: 55-57, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 58-60 the antibody being selected from any of the above. **Claim 2** The antibody according to claim 1, wherein the antibody comprises an HV comprising an amino acid sequence selected from SEQ ID NO: 228, SEQ ID NO: 460, SEQ ID NO: 368, SEQ ID NO: 100, SEQ ID NO: 236, SEQ ID NO: 226, SEQ ID NO: 252, SEQ ID NO: 98, and SEQ ID NO:

116.

3. The antibody according to claim 1 or 2, wherein the antibody comprises an LV comprising an amino acid sequence selected from SEQ ID NO: 227, SEQ ID NO: 459, SEQ ID NO: 367, SEQ ID NO: 99, SEQ ID NO: 235, SEQ ID NO: 225, SEQ ID NO: 251, SEQ ID NO: 97, and SEQ ID NO:

115.

4. The antibody is as follows: i) an HV comprising the amino acid sequence of SEQ ID NO: 228 and an LV comprising the amino acid sequence of SEQ ID NO: 227; ii) an HV comprising the amino acid sequence of SEQ ID NO: 460 and an LV comprising the amino acid sequence of SEQ ID NO: 459; iii) an HV comprising the amino acid sequence of SEQ ID NO: 368 and an LV comprising the amino acid sequence of SEQ ID NO: 367; iv) an HV comprising the amino acid sequence of SEQ ID NO: 100 and an LV comprising the amino acid sequence of SEQ ID NO: 99; v) an HV comprising the amino acid sequence of SEQ ID NO: 236 and an LV comprising the amino acid sequence of SEQ ID NO: 235; vi) an HV comprising the amino acid sequence of SEQ ID NO: 226 and an LV comprising the amino acid sequence of SEQ ID NO: 225; vii) an HV comprising the amino acid sequence of SEQ ID NO: 252 and an LV comprising the amino acid sequence of SEQ ID NO: 251; viii) an HV comprising the amino acid sequence of SEQ ID NO: 98 and an LV comprising the amino acid sequence of SEQ ID NO: 97; and ix) an HV comprising the amino acid sequence of SEQ ID NO: 116 and an LV comprising the amino acid sequence of SEQ ID NO: 115 The antibody according to any one of claims 1 to 3, comprising an HV and an LV selected from any of the above.

5. An isolated antibody that binds to huCB1, wherein the antibody comprises an HV and an LV, a) the HV comprises an HCDR1, an HCDR2, and an HCDR3, wherein HCDR1 comprises the amino acid sequence of RGGDY WX1 (SEQ ID NO: 530) [wherein X1 is A, S, or G]; HCDR2 comprises the amino acid sequence of HX2Y X3X4G X5TX6YN PX7X8X9X10 (SEQ ID NO: 531) [wherein X2 is I or V; X3 is H, Y, or Q; X4 is E, T, or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; HCDR3 contains the amino acid sequence of X11YD X12X13X14G X15SYYYYYGMDV (SEQ ID NO: 532) [wherein, X11 is D, E, or N; X12 is A, I, P, T, or V; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y], and b) the antibody, wherein the LV is of the germline of VK1 / O2 / JK4, VK2 / A19 / JK1, or VK3 / A27 / JK1. **Claim 6** a) HCDR1 contains the amino acid sequence of RGGDY WX1 (SEQ ID NO: 530) [wherein, X1 is A or S], HCDR2 contains the amino acid sequence of HX2Y X3X4G X5TX6YN PX7X8X9X10 (SEQ ID NO: 531) [wherein, X2 is I or V; X3 is Y or Q; X4 is E, T, or S; X5 is S or Q; X6 is A or K; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N], HCDR3 contains the amino acid sequence of X11YD X12X13X14G X15SYYYYYGMDV (SEQ ID NO: 532) [wherein, X11 is D, E, or N; X12 is A, I, P, or V; X13 is L or V; X14 is S or T; X15 is H or Y], b) the antibody according to claim 5, wherein the LV is of the germline of VK2 / A19 / JK1. **Claim 7** The antibody according to claim 6, wherein X4 is T and X15 is Y. **Claim 8** b) the LV contains LCDR1, LCDR2, and LCDR3, where LCDR1 contains the amino acid sequence of RSSQSL LX16SX17G X18NYX19D (SEQ ID NO: 533) [wherein, X16 is H, S, or T; X17 is S, T, or Y; X18 is A, N, I, or Y; X19 is L or V], LCDR2 contains the amino acid sequence of X20GSNRA (SEQ ID NO: 534) [wherein, X20 is L or Q], LCDR3 contains the amino acid sequence of X21QAX22X23X24PRT (SEQ ID NO: 535) [wherein, X21 is M or R; X22 is L, I, R, or V; X23 is Q, E, T, A, or G; X24 is T, I, L, or Q], and the antibody according to any one of claims 5 to 7. **Claim 9** The antibody according to claim 8, wherein X24 is L. **Claim 10** a) HCDR1 comprises the amino acid sequence of RGGDY WX1 (SEQ ID NO: 530) [wherein X1 is A, G, or S]; HCDR2 comprises the amino acid sequence of HX2Y X3X4G X5T X6YNP X7X8X9X10 (SEQ ID NO: 531) [wherein X2 is I or V; X3 is H, Y, or Q; X4 is T or S; X5 is K, S, or Q; X6 is A, K, or N; X7 is N, S, K, or R; X8 is F or L; X9 is E or K; X10 is G, D, S, or N]; HCDR3 comprises the amino acid sequence of X11YDX12X13X14G X15SYYYYYGMDV (SEQ ID NO: 532) [wherein X11 is D or N; X12 is A, I, or T; X13 is F, L, or V; X14 is S or T; X15 is H, N, or Y]; b) The antibody according to claim 5, wherein the LV is of the VK1 / O2 / JK4 or VK3 / A27 / JK1 germline. **Claim 11** a) The antibody according to claim 10, wherein X1 is A or G; X3 is Q or Y, X4 is T, X8 is F, X14 is T; X15 is Y. **Claim 12** b) The antibody according to claim 10 or 11, wherein the LV is of the VK1 / O2 / JK4 germline. **Claim 13** b) The antibody according to claim 10 or 11, wherein the LV is of the VK1 / O2 / JK4 germline and comprises light chain LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the amino acid sequence of RASQSISNYLN (SEQ ID NO: 132), RASQSII SYLN (SEQ ID NO: 150), or RASQSISSYLN (SEQ ID NO: 186); LCDR2 comprises the amino acid sequence of AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151); LCDR3 comprises the amino acid sequence of QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); or b) The antibody according to claim 10 or 11, wherein the LV is of the VK3 / A27 / JK1 germline and comprises light chain LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the amino acid sequence of RASQSVSSYL G (SEQ ID NO: 168); LCDR2 comprises the amino acid sequence of GASSRAT (SEQ ID NO: 169); LCDR3 comprises the amino acid sequence of QQYGSSPR T (SEQ ID NO: 170). **Claim 14** The antibody according to claim 13, wherein LCDR1 comprises the amino acid sequence of RASQSISNYLN (SEQ ID NO: 132), LCDR2 comprises the amino acid sequence of AASSLHHS (SEQ ID NO: 133), and LCDR3 comprises the amino acid sequence of QQYQSYPLT (SEQ ID NO: 134).

15. a) HCDR1 comprises the amino acid sequence of RGGDY WX1 (SEQ ID NO: 530) [wherein X1 is A or G], HCDR2 comprises the amino acid sequence of HX2Y X3X4GX5TX6YN PX7X8X9X10 (SEQ ID NO: 531) [wherein X2 is I or V; X3 is Y or Q; X4 is T; X5 is S; X6 is K or N; X7 is S or R; X8 is F; X9 is K; X10 is G or D], HCDR3 comprises the amino acid sequence of X11YD X12X13X14GX15SYYYYYGMDV (SEQ ID NO: 532) [wherein X11 is N; X12 is T; X13 is L or V; X14 is T; X15 is Y], b) the LV is of the VK1 / O2 / JK4 germline, the antibody according to claim 10.

16. a) HCDR1 comprises the amino acid sequence of RGGDYA (SEQ ID NO: 414) or RGGDYG (SEQ ID NO: 408), HCDR2 comprises the amino acid sequence of HVYYTGSTKYNP SFK D (SEQ ID NO: 427), HVYYTGSTNYNP RFK D (SEQ ID NO: 445), HIYQTGSTNYNP RFK G (SEQ ID NO: 415), or HVYQTGSTKYNP SFK D (SEQ ID NO: 409), HCDR3 comprises the amino acid sequence of NYDTLTGY SYYYYYGMDV (SEQ ID NO: 410) or NYDTVTGY SYYYYYGMDV (SEQ ID NO: 446), the antibody according to claim 15.

17. b) The LV comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of RASQSISSYLN (SEQ ID NO: 405); LCDR2 comprises the sequence of X39ARX40LX41S (SEQ ID NO: 536) [wherein X39 is N, S, K, or G; X40 is R, K, L, or A; X41 is A, G, or S]; and LCDR3 comprises the sequence of QQX42X43X44X45PX46T (SEQ ID NO: 537) [wherein X42 is Y or F; X43 is R, A, S, G, or Y; X44 is S, K, R, or H; X45 is S, L, F, Y, P, or M; X46 is L, I, or V], the antibody according to claim 15 or 16.

18. The antibody according to claim 17, wherein X42 is Y.

19. The antibody according to any one of claims 5 to 18, wherein the amino acid at position 83 of the HV is N and the amino acid at position 85 of the HV is Y.

20. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ b) the LV comprises LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the amino acid sequence of RSSQSLlHRsGYNYLd (SEQ ID NO: 257); LCDR2 comprises the amino acid sequence of LGSNRAS (SEQ ID NO: 258) or QGSNRAS (SEQ ID NO: 264); LCDR3 comprises the amino acid sequence of MQSLQTPRT (SEQ ID NO: 259), RQSVAlPRT (SEQ ID NO: 271), or RQARAIPRT (SEQ ID NO: 265), the antibody according to claim 21 or 22.

24. The antibody according to any one of claims 21 to 23, wherein the amino acid at position 83 of the HV is N and the amino acid at position 85 of the HV is Y.

25. The antibody according to claim 24, wherein the amino acid at position 79 of the HV is R.

26. The antibody according to any one of claims 21 to 25, wherein the amino acid at position 94 of the LV is S, or the amino acid at position 76 of the LV is D, or both.

27. The antibody according to any one of claims 5 to 26, wherein the HCDR2 of the antibody comprises no more than 5 mutations as compared with HVYYTGSTNYNPRFKd (SEQ ID NO: 136).

28. The antibody according to claim 27, wherein the HCDR3 of the antibody comprises no more than 3 mutations as compared with NYDTvTGSYYYYYYGMDV (SEQ ID NO: 137).

29. The antibody according to any one of claims 5 to 26, wherein the HCDR2 of the antibody comprises no more than 5 mutations as compared with HVYYTGSTKYNPnFKg (SEQ ID NO: 8).

30. The antibody according to claim 29, wherein the HCDR3 of the antibody comprises no more than 4 mutations as compared with DYDIltTGSYYYYYYGMDV (SEQ ID NO: 9).

31. An isolated antibody or antigen-binding fragment thereof that binds to huCB1, wherein the antigen-binding molecule comprises an HV comprising HCDR1, HCDR2, and HCDR3, and an LV comprising LCDR1, LCDR2, and LCDR3, HCDR1 comprises the amino acid sequence of RGGDYWS (SEQ ID NO: 474) or RGGDYWN (SEQ ID NO: 480), HCDR2 comprises the amino acid sequence of HIYYSSGSTNYNPSLRS (SEQ ID NO: 475), HIYYSSGSKNYNPSLKS (SEQ ID NO: 481), or HIYYTG TKYYNPSLKS (SEQ ID NO: 487), HCDR3 comprises the amino acid sequence of DYDIYGYSYYYYYGLDV (SEQ ID NO: 476) or GYDS SGYSYYYYYGMDV (SEQ ID NO: 475), LCDR1 comprises the amino acid sequence of RSSQSL LHRSGYNYLD (SEQ ID NO: 471), RSSQSL LYSNGHNF LD (SEQ ID NO: 477), or RSSQSL LYSNGHNYLD (SEQ ID NO: 483), LCDR2 comprises the amino acid sequence of LGSNRAS (SEQ ID NO: 472) or LGSNRAP (SEQ ID NO: 484), and LCDR3 comprises the amino acid sequence of MQSLQTPRT (SEQ ID NO: 473) or MQALQTPRT (SEQ ID NO: 479), an isolated antibody or antigen-binding fragment thereof.

32. The antibody according to claim 31, wherein the HV comprises the amino acid sequence of SEQ ID NO: 489, SEQ ID NO: 491, or SEQ ID NO: 493, and the LV comprises the amino acid sequence of SEQ ID NO: 490, SEQ ID NO: 492, or SEQ ID NO:

494.

33. The antibody according to claim 5 or 21, wherein the amino acid at position 32 according to Aho numbering in the heavy chain is R.

34. The antibody according to any one of claims 1 to 33, wherein the antibody is a monoclonal antibody.

35. The antibody according to claim 34, wherein the monoclonal antibody is a chimeric antibody, a humanized antibody, or a human antibody.

36. The antibody according to claim 34 or 35, wherein the monoclonal antibody is an antagonist antibody of huCB1.

37. The antibody according to claim 34 or 35, wherein the monoclonal antibody is an inverse agonist antibody of huCB1.

38. An antibody according to claim 36 or 37, having at least 3-fold higher binding affinity for huCB1 compared to an antibody (10D10 LC N35Y) comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 256 and a light chain variable region having the amino acid sequence of SEQ ID NO:

255.

39. The antibody according to claim 36, having an IC50 of 10 nM or less when measured using a cell-based cAMP assay.

40. The antibody according to claim 39, having an IC50 of 5 nM or less, or 1 nM or less when measured using a cell-based cAMP assay.

41. The antibody according to any one of claims 34 to 40, wherein the monoclonal antibody is a human IgG1, IgG2, IgG3, or IgG4 antibody.

42. The monoclonal antibody according to claim 41, wherein the monoclonal antibody is a human IgG1 antibody, preferably a human IgG1z antibody.

43. The monoclonal antibody according to claim 41 or 42, wherein the antibody comprises a mutation at amino acid position N297 in the heavy chain according to EU numbering.

44. The monoclonal antibody according to claim 43, wherein the mutation is N297G.

45. The monoclonal antibody according to claim 43 or 44, wherein the antibody further comprises R292C and V302C mutations in the heavy chain according to EU numbering.

46. The monoclonal antibody according to any one of claims 41 to 45, wherein the antibody comprises mutations at amino acid positions M252, S254, and T256 in the heavy chain according to EU numbering.

47. The monoclonal antibody according to claim 46, wherein the mutations are M252Y, S254T, and T256E.

48. The monoclonal antibody according to claim 40, wherein the antibody comprises a heavy chain constant region amino acid sequence selected from SEQ ID NOs: 520 to 529 and a light chain constant region amino acid sequence selected from SEQ ID NOs: 513 to 519.

49. The antibody is: i) a heavy chain constant region amino acid sequence of SEQ ID NO: 525, 526, or 527 and a light chain constant region amino acid sequence of SEQ ID NO: 518 or 519; or ii) a heavy chain constant region amino acid sequence of SEQ ID NO: 525, 526, or 527 and a light chain constant region amino acid sequence of SEQ ID NO: 518 The antibody according to claim 40, comprising.

50. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 49 and a pharmaceutically acceptable excipient.

51. An isolated polynucleotide encoding the antibody according to any one of claims 1 to 50.

52. An expression vector comprising the polynucleotide according to claim 51.

53. A host cell comprising the expression vector according to claim 52.

54. A method for producing an antibody that binds to huCB1, comprising culturing the host cell according to claim 53 under conditions that allow expression of the antibody; and recovering the antibody from the culture medium or the host cell.

55. A method for treating a subject in need of antagonizing or inverse agonizing a CB1 receptor, comprising administering to the subject an effective amount of the antibody according to any one of claims 1 to 49 or the pharmaceutical composition according to claim 50.

56. A method for treating a disease or disorder in a subject responsive to antagonizing or inverse agonizing a CB1 receptor, comprising administering to the subject the antibody according to any one of claims 1 to 49 or the pharmaceutical composition according to claim 50.

57. The method according to claim 55 or 56, wherein the CB1 receptor is a peripheral CB1 receptor.

58. The method according to claim 57, wherein the administration results in one or more of a decrease in body weight, a reduction in appetite, an improvement in metabolic parameters, a reduction in blood glucose level, a reduction in insulin level, a reduction in triglyceride level, a reduction in kidney injury, a reduction in kidney fibrosis, a reduction in kidney inflammation, and an improvement in kidney function.

59. The method according to claim 57, wherein the disease or disorder is selected from obesity, diabetes, dyslipidemia, metabolic diseases, liver diseases, fibrosis, non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis, kidney diseases, kidney fibrosis, chronic kidney diseases, IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular diseases, cancer, inflammatory diseases, and combinations thereof.

60. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an antibody according to any one of claims 1 to 49 or a pharmaceutical composition according to claim 50, wherein the disease or disorder is obesity, diabetes, dyslipidemia, metabolic disease, liver disease, fibrosis, NASH, primary biliary cirrhosis, kidney disease, renal fibrosis, chronic kidney disease, IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular disease, cancer, inflammatory disease, or a combination thereof.

61. The method according to claim 60, wherein the disease or disorder is obesity, diabetes, dyslipidemia, metabolic disease, liver disease, NASH, kidney disease, renal fibrosis, chronic kidney disease, or a combination thereof.

62. The method according to claim 60 or 61, wherein the administration results in one or more of weight loss, reduced appetite, improved metabolic parameters, reduced blood glucose level, reduced insulin level, reduced triglyceride level, reduced kidney injury, reduced renal fibrosis, reduced kidney inflammation, and improved kidney function.

63. The method according to any one of claims 54 to 62, wherein the subject is a human.

64. An antibody according to any one of claims 1 to 49 or a pharmaceutical composition according to claim 50 for use in antagonizing or inverse agonizing the CB1 receptor.

65. An antibody according to any one of claims 1 to 49 or a pharmaceutical composition according to claim 50 for use in the treatment of a disease or disorder selected from obesity, diabetes, dyslipidemia, metabolic disease, fibrosis, liver disease, NASH, primary biliary cirrhosis, kidney disease, renal fibrosis, chronic kidney disease, IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular disease, cancer, inflammatory disease, and combinations thereof.

66. The antibody according to claim 64 or 65, wherein the use results in one or more of weight loss, reduced appetite, improved metabolic parameters, reduced blood glucose level, reduced insulin level, reduced triglyceride level, reduced kidney injury, reduced renal fibrosis, reduced kidney inflammation, and improved kidney function.

67. The method according to claim 59 or 60, or the antibody according to claim 65, wherein the disease or disorder is obesity.

68. The method or antibody according to claim 67, wherein the subject has a BMI of at least 27 kg / m ２ .

69. The subject has a BMI of at least 30 kg / m ２ and the method or antibody according to claim 68.

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