Cannabinoid receptor type 1 binding proteins and uses thereof

Abstract

Described herein are anti-CB1 (e.g., huCB1) antibodies and antigen-binding fragments, compositions comprising the same, and their use for the treatment of various disorders or diseases, such as obesity and complications thereof, chronic kidney disease, liver disease (e.g., NAFLD), and NASH.

Description

Technical Field

[0001] The present disclosure relates to cannabinoid type 1 receptor (CB1) binding proteins and their use for treating diseases or disorders.

[0002] Incorporation by Reference of Electronically Submitted Materials

[0003] Incorporated herein by reference in its entirety is the Amino Acid Sequence Listing filed concurrently herewith and identified as follows: 7027 kilobyte XML document named "10129-WO01-SEC Sequence Listing.xml", created on July 20, 2023.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 392,891, filed on July 28, 2022. Background Art

[0006] Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor (GPCR) that is expressed in the central nervous system (CNS) and peripheral organs such as the liver, skeletal muscle, adipose tissue, and endocrine pancreas. (Cinar R. et al., Pharmacology & Therapeutics 208 (2020) 107477.) CB1 receptors are expressed at lower levels in peripheral organs than in the CNS, and the receptors are involved in regulating metabolic homeostasis in both systems. Due to its role in energy homeostasis, efforts have long been made to develop CB1 receptor antagonists and / or inverse agonists to treat obesity and its comorbidities. (See, e.g., Cinar R. et al., Pharmacology & Therapeutics 208 (2020) 107477; Thomas Murphy and Bernard Le Foll, Biomolecules 2020, 10, 855.)

[0007] In clinical trials, the small molecule CB1 receptor antagonist / inverse agonist rimonabant (also known as SR141716) can reduce the weight of overweight people and significantly improve 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 can also cause serious psychiatric side effects, such as anxiety, depression and suicidal tendencies, which led to the withdrawal of the drug from the market in 2008. Additional efforts have been made in the development of peripherally restricted small molecule CB1 receptor antagonists (e.g., Tam et al., J. Clin. Invest. [Journal of Clinical Research] (2010) 120: 2953-66; US2011 / 0144157), but success in this field remains to be seen.

[0008] Since antibodies do not significantly cross the blood-brain barrier and penetrate the CNS, scientists assume that CB1 antagonist antibodies with rimonabant-like efficacy may have a beneficial effect on metabolism (e.g., treating obesity and its comorbidities) without producing side effects associated with the CNS. However, the discovery and engineering of effective antibody antagonists of CB1 have proven difficult in this field. For example, the efficacy of antagonist CB1 antibodies described in the art is in the double-digit or triple-digit nanomolar range (see, for example, WO 2014 / 210025, WO 2015 / 148948, and WO 2019 / 211665); in contrast, the efficacy of rimonabant is in the single-digit nanomolar range. (See, for example, Congy C. et al., FEBS Letters [European Biochemical Society Express] 350 (1994) 240-244; Bauer M. et al., The Journal of Biological Chemistry [Journal of Biological Chemistry], 287 (44) (2016) 36944–36967). Furthermore, the binding affinity of these antibodies to CB1 (eg, huCB1) is lower than that of rimonabant.

[0009] Several factors are thought to contribute to this difficulty. It is generally difficult to generate antibodies against GPCRs with small extracellular loops (e.g., CB1) by immunization (see, e.g., Jo Migyeong and Jung Sang Take, Experimental & Molecular Medicine (2016) 48, e207). In addition, many CB1 antibodies generated in animal experiments are often non-functional. In addition, the potency and / or affinity of antagonist antibodies isolated from animal experiments are usually not high enough to meet potency and / or affinity requirements (e.g., comparable to or better than rimonabant). Although affinity maturation can be used to improve the potency of antagonist antibodies, it is challenging to use this strategy for CB1 antibodies due to the lack of stable and high-quality preparations of conformationally related CB1 proteins.

[0010] There remains a need in the art to identify CB1 antagonist antibodies with rimonabant-like potency and affinity that can achieve therapeutic efficacy while avoiding CNS-related side effects. Summary of the invention

[0011] The present application relates to antagonist or inverse agonist anti-CB1 (e.g., huCB1) antibodies and antigen-binding fragments with high potency (e.g., comparable or better potency than rimonabant). Compared with previously known anti-CB1 antibodies, the antibodies disclosed herein have much higher potency and are suitable for safe and effective treatment of various diseases, such as obesity and its complications, 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 recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only routine experiments. Such equivalents are intended to be covered by the following embodiment (E).

[0012] E1: An isolated antibody that binds huCB1, the antibody comprising an HV and a LV, wherein a) the HV comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A, S, or G; HCDR2 comprises the amino acid sequence 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; and X10 is G, D, S, or N; and HCDR3 comprises the amino acid sequence X11YDX12X13X14GX15SYYYYYGMDV (SEQ ID NO: 532). 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; and X15 is H, N or Y; and b) the LV belongs to the VK1 / 02 / JK4, VK2 / A19 / JK1 or VK3 / A27 / JK1 germline.

[0013] E2 is an antibody as described in E1, wherein a) HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A or S; HCDR2 comprises the amino acid sequence 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; and X10 is G, D, S or N; and HCDR3 comprises the amino acid sequence X11YDX12X13X14GX15SYYYYYGMDV (SEQ ID NO: 532). IDNO: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; and X15 is H or Y; and b) the LV belongs to the VK2 / A19 / JK1 germline.

[0014] E3 is the antibody as described in E2, wherein X4 is T and X15 is Y.

[0015] E4 is an antibody as described in any one of E1-E3, wherein b) the LV comprises LCDR1, LCDR2 and LCDR3, and wherein 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; and X19 is L or V; LCDR2 comprises the amino acid sequence X20GSNRA (SEQ ID NO:534), wherein X20 is L or Q; and LCDR3 comprises the amino acid sequence 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; and X24 is T, I, L or Q.

[0016] E5 is the antibody as described in E4, wherein X24 is L.

[0017] E6. The antibody as described in E1, wherein a) HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A, G or S; HCDR2 comprises the amino acid sequence 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; and X10 is G, D, S or N; and HCDR3 comprises the amino acid sequence X11YDX12X13X14GX15SYYYYYGMDV (SEQ ID NO: 532). 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; and X15 is H, N or Y; and b) the LV belongs to the VK1 / 02 / JK4 or VK3 / A27 / JK1 germline.

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

[0019] E8. The antibody of E6 or E7, wherein b) the LV belongs to the VK1 / 02 / JK4 lineage.

[0020] E9. The antibody of E6 or E7, wherein b) the LV belongs to the VK1 / 02 / JK4 germline and comprises light chains LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises the amino acid sequence RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO: 150) or RASQSISSYLN (SEQ ID NO: 186); LCDR2 comprises the amino acid sequence AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151); and LCDR3 comprises the amino acid sequence QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); or

[0021] b) the LV belongs to the VK3 / A27 / JK1 germline and comprises light chains LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises the amino acid sequence RASQSVSSYLG (SEQ ID NO: 168); LCDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO: 169); and LCDR3 comprises the amino acid sequence QQYGSSPRT (SEQ ID NO: 170).

[0022] The antibody of E10.E9, wherein LCDR1 comprises the amino acid sequence RASQSISNYLN (SEQ ID NO: 132), LCDR2 comprises the amino acid sequence AASSLHS (SEQ ID NO: 133), and LCDR3 comprises the amino acid sequence QQYQSYPLT (SEQ ID NO: 134).

[0023] E11.E6 antibody, wherein a) HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO:530), wherein X1 is A or G, HCDR2 comprises the amino acid sequence HX2YX3X4GX5TX6YNPX7X8X9X10 (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; and X10 is G or D; and HCDR3 comprises the amino acid sequence X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO:532), wherein X11 is N; X12 is T; X13 is L or V; X14 is T; and X15 is Y, and b) the LV belongs to the VK1 / O2 / JK4 germline.

[0024] E12. The antibody of E11, wherein a) HCDR1 comprises the amino acid sequence RGGDYWA (SEQ ID NO: 414) or RGGDYWG (SEQ ID NO: 408); HCDR2 comprises the amino acid sequence HVYYTGSTKYNPSFKD (SEQ ID NO: 427), HVYYTGSTNYNPRFKD (SEQ ID NO: 445), HIYQTGSTNYNPRFKG (SEQ ID NO: 415) or HVYQTGSTKYNPSFKD (SEQ ID NO: 409); and HCDR3 comprises the amino acid sequence NYDTLTGYSYYYYGMDV (SEQ ID NO: 410) or NYDTVTGYSYYYYGMDV (SEQ ID NO: 446).

[0025] E13. An antibody as described in E11 or E12, wherein b) the LV comprises LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises the sequence RASQSISSYLN (SEQ ID NO:405); LCDR2 comprises the sequence X39ARX40LX41S (SEQ ID NO:536), wherein X39 is N, S, K or G; X40 is R, K, L or A; and X41 is A, G or S; and LCDR3 comprises the sequence 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; and X46 is L, I or V.

[0026] E14. The antibody of E13, wherein X42 is Y.

[0027] E15. An isolated antibody that binds huCB1, the antibody comprising HV and LV, wherein a) the HV comprises CDRH1, CDRH2 and CDRH3, wherein CDRH1 comprises the amino acid sequence RGGDYWS (SEQ ID NO: 1); CDRH2 comprises the amino acid sequence 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; and X33 is G, S or N; and CDRH3 comprises the amino acid sequence 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; and X38 is M or L; and b) the LV belongs to the VK2 / A19 / JK1 germline.

[0028] E16. The antibody of E15, wherein a) X26 is Y, X34 is G, X36 is absent, and X37 is S.

[0029] E17. The antibody as described in E15 or E16, wherein b) the LV comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence RSSQSLLHRSGYNYLD (SEQ ID NO: 257); LCDR2 comprises the amino acid sequence LGSNRAS (SEQ ID NO: 258) or QGSNRAS (SEQ ID NO: 264); and LCDR3 comprises the amino acid sequence MQSLQTPRT (SEQ ID NO: 259), RQSVALPRT (SEQ ID NO: 271) or RQARALPRT (SEQ ID NO: 265).

[0030] E18. The antibody as described in E1-E14, wherein the antibody comprises LIBC523596-1, LIBC523603-1, LIBC523661-1, LIBC523670-1, LIBC523748-1, LIBC523760-1, LIBC523797-1, LIBC523813-1, LIBC523815-1, LIBC52384 4-1, LIBC523857-1, LIBC523862-1, LIBC523868-1, LIBC523969-1, LIBC524049-1, LIBC52 7906-1, LIBC527814-1, LIBC527997-1, LIBC528141-1, LIBC527912-1, LIBC528116-1, LIB HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of C527879-1, 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, or a group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0031] E19. The antibody as described in E15-E17, wherein the antibody comprises LIBC673948-1, LIBC673952-1, LIBC673965-1, LIBC673982-1, LIBC673972-1, LIBC674024-1, LIBC674035-1, LIBC674043-1, LIBC674090-1, LIBC674002-1, LIBC674153 ... HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of BC674200-1, LIBC674214-1, LIBC674216-1, LIBC674229-1, LIBC674235-1, LIBC674257-1, and LIBC674276-1, or a group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0032] E20. The antibody of E18 or E19, wherein the antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of LIBC680574, LIBC528116, LIBC673952, LIBC523797, LIBC527814, LIBC527997, LIBC527919, LIBC523661, and LIBC524049, or a group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0033] E21. The antibody of any one of E1-E20, wherein the amino acid at position 83 of the HV is N and the amino acid at position 85 of the HV is Y.

[0034] E22. The antibody as described in E21, wherein the amino acid at position 94 of the LV is S.

[0035] E23. The antibody as described in E19, wherein the amino acid at position 83 of the HV is N, the amino acid at position 85 of the HV is Y, and the amino acid at position 79 of the HV is R.

[0036] E24. The antibody of E23, wherein the amino acid at position 94 of the LV is S, and the amino acid at position 76 is D.

[0037] E25. The antibody of any one of E1-E24, wherein HCDR2 of the antibody comprises 5 or fewer mutations, or 3 or fewer mutations compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136).

[0038] E26. The antibody of any one of E1-E24, wherein the HCDR2 of the antibody comprises 5 or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8), and optionally, the HCDR3 of the antibody comprises 4 or fewer mutations compared to DYDILTGYSYYYYGMDV (SEQ ID NO: 9).

[0039] E27. The antibody of any one of E1-E26, wherein the antibody comprises LIBC523596-1, LIBC523603-1, LIBC523661-1, LIBC523670-1, LIBC523748-1, LIBC523760-1, LIBC523797-1, LIBC523813-1, LIBC523815-1, LIBC523844-1, LIBC523857-1, LIBC523862-1, LIBC5238 68-1, 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, LIBC52 7827-1, 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, LI HV and / or LV of any one of BC673972-1, 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.

[0040] E28. The antibody as described in E27, wherein the antibody comprises the HV and / or LV of any one of LIBC680574, LIBC528116, LIBC673952, LIBC523797, LIBC527814, LIBC527997, LIBC527919, LIBC523661 and LIBC524049.

[0041] E29. An isolated antibody that binds to huCB1 and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, or a group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of LIBC529593, LIBC560340, or LIBC560657.

[0042] E30. The antibody as described in E29, wherein the antibody comprises the HV and / or LV of LIBC529593, LIBC560340, or LIBC560657.

[0043] E31. The antibody of any one of E1-E30, wherein the antibody is a monoclonal antibody, optionally, the monoclonal antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0044] E32. The monoclonal antibody of E31, wherein the antibody is an antagonist and / or inverse agonist antibody of huCB1.

[0045] E33. The antibody of E31 or E32, wherein the antibody has a binding affinity for huCB1 that is at least 3-fold higher than 10D10N35Y.

[0046] E34. The antibody of 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, as determined using a cell-based cAMP assay.

[0047] E35. An isolated monoclonal antibody that binds huCB1, wherein the antibody is an antagonist and / or inverse agonist antibody to huCB1 and has an IC50 of 10 nM or less, or 8 nM or less, as determined using a cell-based cAMP assay.

[0048] E36. The antibody of E35, wherein the antibody binds to extracellular loop 2 of huCB1 or to a region contained within extracellular loop 2 of huCB1.

[0049] E37. The antibody of any one of E34-E36, wherein the cell-based cAMP assay is a cell-based cAMP assay using CP55,940.

[0050] E38. The antibody of any one of E31-E37, wherein the monoclonal antibody is a human IgG1, IgG2, IgG3 or IgG4 antibody, preferably a human IgG1 antibody.

[0051] E39. The antibody of E38, wherein the antibody comprises in its heavy chain a mutation at amino acid position N297 according to EU numbering, eg, N297G.

[0052] E40. The antibody of E39, wherein the antibody further comprises R292C and V302C mutations according to EU numbering in its heavy chain.

[0053] E41. The antibody of any one of E37-E40, wherein the antibody comprises in its heavy chain mutations at amino acid positions M252, S254 and T256, preferably M252Y, S254T and T256E according to EU numbering.

[0054] E42. A method for treating a subject in need of antagonism or inverse agonism of a CB1 receptor, the method comprising administering to the subject the antibody of any one of E1-E41.

[0055] E43. A method for treating a disease or disorder in a subject responsive to antagonism or inverse agonism of CB1 receptors, the method comprising administering to the subject the antibody of any one of E1-E41.

[0056] E44. A method as described in E42 or E43, wherein the administration results in one or more of weight loss, decreased appetite, improved metabolic parameters, reduced blood glucose levels, reduced insulin levels, reduced triglyceride levels, reduced renal damage, reduced renal fibrosis, reduced renal inflammation, and improved renal function.

[0057] E45. A method as described in 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, renal fibrosis, chronic kidney disease, IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular diseases, cancer and inflammatory diseases.

[0058] E46. An antibody as described in any one of E1-E41, for use in treating a disease or disorder in a subject, wherein the disease or disorder is 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 and inflammatory disease.

[0059] E47. The antibody as described in E46, wherein the use results in one or more of weight loss, decreased appetite, improved metabolic parameters, reduced blood sugar levels, reduced insulin levels, reduced triglyceride levels, reduced kidney damage, reduced kidney fibrosis, reduced kidney inflammation and improved kidney function.

[0060] E48. The method of E45 or the antibody of E46, wherein the disease or disorder is obesity, optionally the subject has a body mass index of at least 27 kg / m 2 or a BMI of at least 27 kg / m 2 BMI.

[0061] E49. The method of any one of E42-45 or the antibody of E47 or E48, wherein the subject is a human.

[0062] E50. An isolated polynucleotide encoding the antibody of any one of E1-E41.

[0063] E51. An expression vector comprising the polynucleotide described in E50.

[0064] E52. A host cell comprising the expression vector of E51. E53. The antibody of E5 or E15, wherein the amino acid at position 32 according to AHo numbering in the heavy chain is R. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1A and 1B The binding profiles of the top affinity matured homologous variants are shown.

[0066] Figure 2A and 2B The binding profiles of the top affinity matured LC exchange variants are shown.

[0067] Figure 3A An alignment of the HV CDR and FR3 sequences of the top cognate affinity matured variants is shown. Figure 3B An alignment of the LV CDR and FR3 sequences of the top cognate affinity matured variants is shown.

[0068] Figure 4A An alignment of the HV CDR and FR3 sequences of the top LC-swap affinity-matured variants is shown. Figure 4B An alignment of the LV CDR sequences of the top LC-swap affinity-matured variants is shown.

[0069] Figure 5A Antagonists of exemplary affinity matured antibodies are shown. Figure 5B Inverse agonist activity of exemplary affinity matured antibodies is shown.

[0070] Fig. 6A and 6B Shown is the reduction of body weight and food intake by exemplary affinity matured antibodies in obese human CB1 knock-in mice. Figure 6CA reduction in fat mass, liver triglycerides, and insulin levels was shown with the antibody. Fig.6D The effects of exemplary affinity matured antibodies on markers of kidney injury, fibrosis and inflammation are shown.

[0071] Figure 7 An alignment of the HV and LV CDR sequences of the top LC-swap affinity-matured variants is shown.

[0072] Fig. 8A An alignment of the HV CDR and FR3 sequences of the top additional cognate affinity matured variants is shown. Figure 8B An alignment of the LV CDR sequences of the top additional cognate affinity matured variants is shown.

[0073] Fig. 9 Antagonist activity of antibodies isolated from mouse antibody experimental activity is shown.

[0074] Fig. 10A Exemplary additional affinity matured antibodies are shown to reduce body weight in obese human CB1 knock-in mice. Fig. 10B The effect of antibodies on food intake is shown. Fig. 10C showed the effect of antibodies on fat mass, Fig. 10D The effect of the antibodies on liver weight is shown.

[0075] Fig.11A Treatment with an exemplary anti-CB1 antibody was shown to increase energy expenditure and decrease respiratory exchange ratio in an animal model (mice). Fig. 11B Treatment with exemplary anti-CB1 antibodies was shown to increase insulin sensitivity in animal models. DETAILED DESCRIPTION

[0076] Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor for endogenous cannabinoids such as 2-arachidonoylglycerol (2-AG), which mediates many cannabinoid-induced effects such as food intake. Activation of the receptor leads to 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. [Journal of Biological Chemistry] 279: 129-134 (1991)). The human CB1 (huCB1) receptor is a 472 amino acid polypeptide (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 is a membrane protein with 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).

[0077]

[0078]

[0079] CB1 receptors are expressed in the CNS and peripheral systems and are involved in regulating energy homeostasis. The receptor has become a target for developing treatments for obesity and its comorbidities. Given the experience with rimonabant, the field has focused on developing peripherally acting CB1 receptor antagonists or inverse agonists to confer metabolically beneficial effects without producing CNS-related side effects.

[0080] Antibodies against CB1 receptors can be used as peripherally acting antagonists or inverse agonists because they do not significantly penetrate the CNS. Efforts have been made to identify antagonist or inverse agonist anti-CB1 antibodies with rimonabant-like potency to achieve therapeutic effects without side effects. The 10D10 antibody is a lead antagonist anti-CB1 antibody isolated from the Xenomouse antibody experimental activities, but its potency is significantly lower than that of rimonabant. (WO 2014 / 210205, the disclosure of which is incorporated herein by reference in its entirety). Multiple rounds of affinity maturation were performed to improve the potency of the antibody. Although several 10D10 affinity matured variants isolated from early affinity maturation experimental activities had improved potency compared to 10D10, none of the variants reached the design goal of rimonabant-like potency. It is believed that the lack of stable, high-quality conformation-related CB1 protein preparations has resulted in the inability to isolate anti-CB1 antibodies with rimonabant-like potency. Furthermore, the 10D10 antibody and its early affinity matured variants had poor manufacturability (eg, low expression levels and instability).

[0081] The technical proficiency of producing high-quality, soluble complex membrane protein preparations continues to develop, allowing huCB1 to be formulated as 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). Compared to detergent-solubilized cell lysates prepared from cells expressing huCB1, both CB1-NDs and -SMALPs are purified reagents that remain stable after multiple freeze / thaw cycles and are suitable for long-term storage. In addition, they can be added to binding experiments at user-defined huCB1 concentrations. The availability of CB1-NDs and -SMALPs enables the isolation of highly potent antagonist or inverse agonist anti-CB1 antibodies disclosed herein.

[0082] The present application relates to antagonist or inverse agonist anti-CB1 antibodies and antigen-binding fragments, in particular anti-huCB1 antibodies and antigen-binding fragments, which are comparable or better than rimonabant in efficacy. Antibodies that inhibit CB1 can lead to an increase in cAMP, which can be measured using an in vitro cAMP assay (e.g., a cell-based cAMP assay). As illustrated in the examples, the efficacy of the antibodies and antigen-binding fragments disclosed herein is in the single-digit nanomolar range, for example, they have an IC50 of less than about 10nM in a cell-based cAMP assay (see Examples 5, 8, and 9). As shown in the examples, the highly potent antibodies and antigen-binding fragments disclosed herein reduce body weight, food intake, and multiple metabolic parameters in obese animal models (e.g., body fat, insulin levels, and liver triglyceride levels are reduced, Example 7), and increase energy consumption, lipid oxidation, and insulin sensitivity in animal models, while having minimal brain exposure, indicating that they can safely and effectively treat various diseases and disorders (e.g., obesity and its comorbidities, chronic kidney disease, NAFLD, NASH). On the contrary, antagonists or inverse agonist antibodies with efficacy values ​​lower than rimonabant are considered insufficient for such therapeutic purposes. Furthermore, the anti-CB1 antibodies and antigen-binding fragments disclosed herein are stable and express well for manufacturing purposes.These highly effective anti-CB1 antibodies and antigen-binding fragments are described and exemplified herein.

[0083] definition

[0084] The present invention provides antagonist or inverse agonist antibodies for human CB1. "Antibody" is a protein comprising an antigen-binding fragment that specifically binds to an antigen and a scaffold or framework portion that allows the antigen-binding fragment to present a conformation that promotes antibody binding to the antigen. As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin comprising 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 comprising a single immunoglobulin light chain variable region (VL or LV) and a single immunoglobulin light chain constant domain (CL or LC) from the amino terminus to the carboxyl terminus. The immunoglobulin light chain constant domain (CL) can be a human kappa (k) or human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino terminus to 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 into mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε) chains, and they define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Antibodies of the IgG and IgA classes are further subdivided into several subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains in IgG, IgA and IgD antibodies have three domains (CH1, CH2 and CH3), while the heavy chains in IgM 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. The antibody chains are linked together 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.

[0085] In human antibodies, CH1 means the region having the amino acid sequence at positions 118 to 215 of the EU index or EU numbering system, which is based on the sequence numbering of the first human IgG1 (i.e., "EU antibody") sequenced (Edelman et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 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 the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region having the amino acid sequence at positions 341 to 446 of the EU index.

[0086] "CL" means the constant region of the light chain. In the case of the κ chain of a human antibody, CL means the region having the amino acid sequence at positions 108 to 214 of the EU index. In the λ chain, CL means the region having the amino acid sequence at positions 108 to 215.

[0087] "Variable domain" refers to the variable region of an antibody light chain (VL or LV) or the variable region of an antibody heavy chain (VH or HV), whether alone or in combination. As is known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FR) connected by three complementary determining regions (CDRs), and contribute to the formation of the antigen binding site of the antibody. From N-terminus to C-terminus, both naturally occurring light and heavy chain variable regions typically follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0088] The "complementarity determining regions" (CDRs) of an antibody can be defined according to the cumulative, AbM, contact, North and / or conformational definitions of Kabat, Chothia, both Kabat and Chothia, or any CDR determination method well known in the art. See, e.g., 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 the AbM antibody modeling software from Oxford University Molecular (Oxfbrd Molecular). The identity of the amino acid residues in a particular antibody that comprise a CDR can be determined using methods well known in the art.

[0089] The term "antigen-binding fragment" refers to a molecule derived from an antibody and retaining the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Examples of antigen-binding fragments include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and (v) a dAb fragment (Ward et al., 1989 Nature 341: 544-546), which consists of a VH domain. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, recombinant methods can be used to connect the two domains through a synthetic linker that enables them to form a single protein chain, in which the VL region and the VH region pair to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al. Science 242:423-426 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0090] "About" or "approximately," when used in conjunction with a measurable numerical variable, refers to the indicated value of the variable and all values ​​of the variable that are within the experimental error range of the indicated value (e.g., within a 95% confidence interval for the mean) or ±10% of the indicated value, whichever is greater. Numerical ranges are inclusive of the numbers defining the range.

[0091] The term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, antibody, or antigen-binding fragment) is a molecule that, depending on its origin or source of derivation, is (1) not associated with naturally associated components that accompany it in its native state; (2) substantially free of other molecules from the same species; (3) expressed by cells of a different species; or (4) not found in nature. Thus, a molecule (which is chemically synthesized or expressed in a cell system different from the cells from which it naturally originates) will be "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by separation using purification techniques well known in the art. Molecular purity or homogeneity may be determined by a variety of methods well known in the art. For example, the purity of a polypeptide sample may be determined using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For some purposes, HPLC or other purification methods well known in the art may be used to provide higher resolution.

[0092] Unless otherwise indicated, throughout the specification and claims, the numbering of amino acid residues in an immunoglobulin heavy or light chain is according to the AHo numbering scheme described in Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001. In some embodiments, when referring to amino acid positions in immunoglobulin constant regions, the EU numbering scheme is used as described in Edelman et al., Proc. Natl. Acad. USA, Vol. 63:78-85 (1969). Both numbering schemes are well known in the art.

[0093] "Antagonist" refers to an agent that binds to a protein (e.g., a receptor) and, in particular, partially or completely inhibits, blocks, reduces, prevents, delays, inactivates, desensitizes, or downregulates the signaling activity of a protein (e.g., a receptor agonist) ligand. An inverse agonist is a drug that causes the opposite effect of an agonist, e.g., it reduces the basal activity of a receptor.

[0094] Anti-CB1 antibodies and antigen-binding fragments

[0095] Disclosed herein are antibodies and antigen-binding fragments directed against huCB1 (SEQ ID NO: 559) and are antagonists and / or inverse agonists of huCB1. The potency of the antibodies and antigen-binding fragments is comparable to or better than that of rimonabant, which enables them to achieve therapeutic effects (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.

[0096] 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 previous antibody experiment campaign (WO 2014 / 210205), 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, in which amino acids at specific HV and LV positions of 10D10 were optimized to improve binding affinity and potency. The 10D10 LC belongs to the VK2 germline, and as disclosed herein, only the HC of the antibody binds to the CB1 receptor (Examples 1 and 14). LC-exchanged affinity-matured antibodies (i.e., 10D10 LC was exchanged with a VK1 or VK3 germline light chain having 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, disclosed herein is 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

[0097] a) the HV comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A, S or G; HCDR2 comprises the amino acid sequence 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; and X10 is G, D, S or N; and HCDR3 comprises the amino acid sequence X11YDX12X13X14GX15SYYYYYGMDV (SEQ ID NO: 532). 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; and X15 is H, N or Y; and

[0098] b) The LV belongs to or is derived from the VK1 / 02 / JK4, VK2 / A19 / JK1 or VK3 / A27 / JK1 germline.

[0099] As used herein, "germline" or "derived from germline" variable region sequences (e.g., variable region or CDR amino acid sequences) refer to sequences with structural features of a specific germline rather than another system. In order for a host organism to produce an antibody that binds to an antigen, the rearranged immunoglobulin germline genes (rearranged immunoglobulin germline genes of the B cells of the host organism) must first encode an initial antibody with a three-dimensional structure and biochemical properties that can interact with the antigen. Each host organism has a defined immunoglobulin germline gene library that can be rearranged to produce initial antibodies. Through the somatic hypermutation process, a host organism can select mutations or mutation sets in the genes encoding antibodies, thereby further enhancing its interaction with the antigen. Therefore, an antibody sequence "of" or "derived from" a specific germline can have a variable region sequence that contains differences compared to the germline. However, the antibody sequence will contain structural features, such as specific amino acids, CDR lengths, and / or conformational features of a specific germline, indicating that a specific germline is the closest germline associated with the sequence. This interaction can be likened to a hand (antigen) putting on a glove (germline antibody). The initial structure defined by the germline genes must be sufficient to allow the glove to fit the hand first, and then can be customized to fit more precisely. Since the specific combination of rearranged immunoglobulin germline genes encoding the antibody must have a specific three-dimensional structure (glove) for antigen (hand) interaction, the germline identity of the antibody will be understood as the structural features that define the paratope that interacts with the antigen. Since the light chain of the CB1 antibody disclosed herein does not make direct contact with the antigen, the light chain germline identity will be understood as the structural features of the light chain that are used to pair with the heavy chain for paratope interaction with the antigen.

[0100] Germline gene repertoires commonly used for producing antibodies, including Homo sapiens and Mus musculus, are sequences and are known and can be obtained, 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). The sequence of an antibody can be aligned to the germline gene repertoire, for example using an algorithm such as the Basic Local Alignment Search Tool (BLAST) under default settings, to determine to which host organism germline gene segment (or segments) the antibody belongs.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A or S; HCDR2 comprising the sequence 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; and X10 is G, D, S or N, and HCDR3 comprising the sequence X11YDX12X13X14GX15SYYYYYGMDV (SEQ ID NO: 532). 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; and X15 is H or Y. In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or S; a 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; and X10 is G, D, S or N, and a 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; and X15 is Y. In various embodiments, wherein HCDR1, HCDR2 and HCDR3 comprise the sequences defined above, the LV belongs to or is derived from the VK1 / 02 / JK4, VK2 / A19 / JK1 or VK3 / A27 / JK1 germline. In some embodiments, the LV belongs to or is derived from the VK2 / A19 / JK1 germline.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or S; a 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; and X10 is G, D, S or N, and a 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; and X15 is H or Y, and b) the LV comprises LCDR1, LCDR2, and LCDR3, wherein 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; and X19 is L or V; LCDR2 comprises the amino acid sequence X20GSNRA (SEQ ID NO:534), wherein X20 is L or Q; and LCDR3 comprises the amino acid sequence 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; and X24 is T, I, L or Q.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or S; 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; and X10 is G, D, S or N, and 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; and X15 is Y, and b) LCDR1 comprising the sequence of SEQ ID NO:533 NO:533, wherein X16 is H, S or T; X17 is S, T or Y; X18 is A, N, I or Y; and X19 is L or V; LCDR2 comprises the sequence SEQ ID NO:534, wherein X20 is L or Q; and LCDR3 comprises the sequence 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; and X24 is T, I, L or Q.

[0104] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or S; a 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; and X10 is G, D, S or N, and a 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; and X15 is H or Y, and b) a LCDR1 comprising the sequence of SEQ ID NO:533 NO:533, wherein X16 is H, S or T; X17 is S, T or Y; X18 is A, N, I or Y; and X19 is L or V; LCDR2 comprises the sequence SEQ ID NO:534, wherein X20 is L or Q; and LCDR3 comprises the sequence 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; and X24 is L.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or S; 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; and X10 is G, D, S or N, and 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; and X15 is Y, and b) LCDR1 comprising the amino acid sequence of SEQ ID NO:534. NO:533, wherein X16 is H, S or T; X17 is S, T or Y; X18 is A, N, I or Y; and X19 is L or V; LCDR2 comprises the amino acid sequence of SEQ ID NO:534, wherein X20 is L or Q; and LCDR3 comprises 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; and X24 is L.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A, G or S; 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; and X10 is G, D, S or N; and 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; and X15 is H, N or Y. In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or G; 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; and X10 is G, D, S or N; and 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; and X15 is Y. In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence SEQ ID NO: 530, wherein X1 is A or G; HCDR2 comprising the sequence 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; and X10 is G or D; and HCDR3 comprising the sequence SEQ ID NO: 532, wherein X11 is N; X12 is T; X13 is L or V; X14 is T; and X15 is Y. In various embodiments, wherein HCDR1, HCDR2 and HCDR3 comprise the sequences defined above, LV belongs to or is derived from the VK1 / 02 / JK4 or VK3 / A27 / JK1 germline. In some embodiments, LV belongs to or is derived from the VK1 / 02 / JK4 germline.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence of SEQ ID NO: 530, wherein X1 is A, G or S; 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; and X10 is G, D, S or N; and HCDR3 comprising the sequence of SEQ ID NO: 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; and X15 is H, N or Y, and b) LV belongs to or is derived from the VK1 / O2 / JK4 or VK3 / A27 / JK1 lineage, preferably, LV belongs to or is derived from the VK1 / O2 / JK4 lineage.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or G; a 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; and X10 is G, D, S or N; and a 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; and X15 is Y, and b) the LV belongs to or is derived from the VK1 / 02 / JK4 germline.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A, G or S; a 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; and X10 is G, D, S or N; and a 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; and X15 is H, N or Y, and b) a LV belongs to or is derived from VK1 / O2 / JK4 and comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO: 150) or RASQSISSYLN (SEQ ID NO: 186); LCDR2 comprises the amino acid sequence AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151); and LCDR3 comprises the amino acid sequence QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); or, the VL belongs to the VK3 / A27 / JK1 germline and comprises light chain LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises the amino acid sequence RASQSVSSYLG (SEQ ID NO: 168); LCDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO: 169); and LCDR3 comprises the amino acid sequence QQYGSSPRT (SEQ ID NO: 170).

[0110] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A, G or S; 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; and X10 is G, D, S or N; and 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; and X15 is H, N or Y, and b) LCDR1 comprising the sequences RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO:150) or RASQSISSYLN (SEQ ID NO:186); LCDR2, which comprises the sequence AASSLHS (SEQ ID NO:133) or AASSLRS (SEQ ID NO:151); and LCDR3, which comprises the sequence QQYQSYPLT (SEQ ID NO:134) or QQYSNYPLT (SEQ ID NO:152); preferably, b) LCDR1, which comprises the sequence RASQSISNYLN (SEQ ID NO:132); LCDR2, which comprises the sequence AASSLHS (SEQ ID NO:133); and LCDR3, which comprises the sequence QQYQSYPLT (SEQ ID NO:134).

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or G; a 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; and X10 is G, D, S or N; and a 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; and X15 is Y, and b) a LV belongs to or is derived from the VK1 / 02 / JK4 germline and comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence RASQSISNYLN (SEQ ID NO:132), RASQSIISYLN (SEQ ID NO:150), or RASQSISSYLN (SEQ ID NO:186); LCDR2 comprises the amino acid sequence AASSLHS (SEQ ID NO:133) or AASSLRS (SEQ ID NO:151); and LCDR3 comprises the amino acid sequence QQYQSYPLT (SEQ ID NO:134) or QQYSNYPLT (SEQ ID NO:152); preferably, b) LCDR1 comprises the sequence RASQSISNYLN (SEQ ID NO:132); LCDR2 comprises the sequence AASSLHS (SEQ ID NO:133); and LCDR3 comprises the sequence QQYQSYPLT (SEQ ID NO:134).

[0112] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or G; 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; and X10 is G or D; and HCDR3 comprising the sequence of SEQ ID NO:532, wherein X11 is N; X12 is T; X13 is L or V; X14 is T; and X15 is Y, and b) LV belongs to or is derived from the VK1 / 02 / JK4 germline. In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the amino acid sequence RGGDYWA (SEQ ID NO:414) or RGGDYWG (SEQ ID NO:408); HCDR2 comprising the amino acid sequence HVYYTGSTKYNPSFKD (SEQ ID NO:427), HVYYTGSTNYNPRFKD (SEQ ID NO:445), HIYQTGSTNYNPRFKG (SEQ ID NO:415), or HVYQTGSTKYNPSFKD (SEQ ID NO:409); and HCDR3 comprising the amino acid sequence NYDTLTGYSYYYYGMDV (SEQ ID NO:410) or NYDTVTGYSYYYYGMDV (SEQ ID NO:446), and b) the LV belongs to or is derived from the VK1 / 02 / JK4 germline.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a HCDR1 comprising the sequence of SEQ ID NO:530, wherein X1 is A or G; a 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; and X10 is G or D; and a HCDR3 comprising the sequence of SEQ ID NO:532, wherein X11 is N; X12 is T; X13 is L or V; X14 is T; and X15 is Y, and b) a LV belongs to or is derived from a VK1 / 02 / JK4 germline comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence RASQSISSYLN (SEQ ID NO:405); LCDR2 comprises the sequence X39ARX40LX41S (SEQ ID NO:536), wherein X39 is N, S, K or G; X40 is R, K, L or A; and X41 is A, G or S; and LCDR3 comprises the sequence 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; and X46 is L, I or V. In some embodiments, X42 is Y and X1-X15, X39-X41 and X43-X44 are as defined above.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the amino acid sequence RGGDYWA (SEQ ID NO:414) or RGGDYWG (SEQ ID NO:408); HCDR2 comprising the amino acid sequence HVYYTGSTKYNPSFKD (SEQ ID NO:427), HVYYTGSTNYNPRFKD (SEQ ID NO:445), HIYQTGSTNYNPRFKG (SEQ ID NO:415), or HVYQTGSTKYNPSFKD (SEQ ID NO:409); and HCDR3 comprising the amino acid sequence NYDTLTGYSYYYYGMDV (SEQ ID NO:410) or NYDTVTGYSYYYYGMDV (SEQ ID NO:446), and b) LCDR1 comprising the sequence RASQSISSYLN (SEQ ID NO:405); LCDR2 comprising the sequence X39ARX40LX41S (SEQ ID NO:427). NO:536), wherein X39 is N, S, K or G; X40 is R, K, L or A; and X41 is A, G or S; and LCDR3 comprises the sequence 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; and X46 is L, I or V. In some embodiments, X42 is Y and X39-X41 and X43-X44 are as defined above.

[0115] In certain embodiments, the anti-CB1 antibodies or antigen-binding fragments disclosed herein comprise a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 from any of the CB1 antibodies described herein. Thus, in some embodiments, described herein are 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, LIBC527970-1, LIBC5238141-1, LIBC52374 ... or an anti-CB1 antibody or an antigen-binding fragment thereof of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of any one of LIBC23857-1, 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 HCDR1, HCDR2 and HCDR3 of any one of these antibodies, and LCDR1, LCDR2 and LCDR3. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 of any one of LIBC523661-1, LIBC523797-1, LIBC524049-1, LIBC527814-1, LIBC527997-1, LIBC528116, LIBC527919-1, and LIBC680574-1, and LCDR1, LCDR2 and LCDR3. The HV and LV CDR sequences of these antibodies are listed in Tables 7, 10 and 26.

[0116] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises 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, LIBC523816-1, LIBC523817-1, LIBC523818-1, LIBC523819-1, LIBC523819-1, LIBC523819-1, LIBC523817-1, LIBC523816-1, LIBC523817-1, LIBC523818-1, LIBC523819 ... 3844-1, 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 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, LIBC523816-1, LIBC523817-1, LIBC523818-1, LIBC523819-1, LIBC523819-1, LIBC523819-1, LIBC523817-1, LIBC523816-1, LIBC523817-1, LIBC523818-1, LIBC523819 ... HV and LV of any one of 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.

[0117] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises HV or LV of any one 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 HV and LV of any one of LIBC523661-1, LIBC523797-1, LIBC524049-1, LIBC527814-1, LIBC527997-1, LIBC528116, LIBC527919-1, and LIBC680574-1.

[0118] Certain mutations in the HC_FR3 de-loop of 10D10 associated with enhanced CB1 binding were included in the affinity maturation library design (Example 1). In addition, all test sequences identified after affinity maturation include the R94S germline reversion of the non-shared mutation of LC FR3 (Examples 2 and 3). In addition, R32 (AHo numbering) adjacent to HCDR1 also participates in epitope interactions. Therefore, in some embodiments of the above-mentioned antibodies and antigen-binding fragments, 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 above-mentioned antibodies and antigen-binding fragments, 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, in the above-mentioned antibodies and antigen-binding fragments, the amino acid at HV position 32 is R.

[0119] In various embodiments, the antibodies and antigen-binding fragments disclosed herein are antagonists and / or inverse agonists of huCB1, for example, the antibodies and binding fragments inhibit signaling of huCB1. The potency of the antibodies and antigen-binding fragments is in the nanomolar or subnanomolar range, for example, 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, as measured in a cell-based cAMP assay. In some embodiments, the potency of the antibodies and antigen-binding fragments disclosed herein is comparable to or better than the potency of rimonabant (e.g., at least two-fold, three-fold, five-fold, eight-fold, or ten-fold better than rimonabant).

[0120] In another aspect, disclosed herein is 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

[0121] a) the VH comprises HCDR1, HCDH2 and HCDR3, wherein CDRH1 comprises the amino acid sequence RGGDYWS (SEQ ID NO: 1); CDRH2 comprises the sequence 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; and X33 is G, S or N; and CDRH3 comprises the sequence X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539); IDNO:539), wherein X34 is D or G; X35 is A, I, T or V; X36 is L or absent; X37 is S or T; and X38 is M or L; and

[0122] b) The VL belongs to or is derived from the VK2 / A19 / JK1 germline.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a CDRH1 comprising the sequence RGGDYWS (SEQ ID NO: 1); a CDRH2 comprising the sequence 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; and X33 is G, S or N; and a CDRH3 comprising the sequence X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539); NO:539), wherein X34 is G; X35 is A, I, T or V; X36 is absent; X37 is S; and X38 is M or L, and b) the VL belongs to or is derived from the VK2 / A19 / JK1 germline.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a CDRH1 comprising the sequence RGGDYWS (SEQ ID NO: 1); a CDRH2 comprising the sequence 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; and X33 is G, S or N; and a CDRH3 comprising the sequence X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539). 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; and X38 is M or L, and b) the VL belongs to or is derived from the VK2 / A19 / JK1 germline and comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence RSSQSLLHRSGYNYLD (SEQ ID NO:257); LCDR2 comprises the sequence 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; and X51 is T or L.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a CDRH1 comprising the sequence RGGDYWS (SEQ ID NO: 1); a CDRH2 comprising the sequence 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; and X33 is G, S or N; and a CDRH3 comprising the sequence X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539); NO:539), wherein X34 is G; X35 is A, I, T or V; X36 is absent; X37 is S; and X38 is M or L, and b) LCDR1 comprising the sequence RSSQSLLHRSGYNYLD (SEQ ID NO:257); LCDR2 comprising the sequence 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; and X51 is T or L.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a CDRH1 comprising the sequence RGGDYWS (SEQ ID NO: 1); a CDRH2 comprising the sequence 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; and X33 is G, S or N; and a CDRH3 comprising the sequence X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539). 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; and X38 is M or L, and b) LCDR1 comprising the amino acid sequence RSSQSLLHRSGYNYLD (SEQ ID NO:257); LCDR2 comprising the amino acid sequence LGSNRAS (SEQ ID NO:258) or QGSNRAS (SEQ ID NO:264); and LCDR3 comprising the amino acid sequence MQSLQTPRT (SEQ ID NO:259), RQSVALPRT (SEQ ID NO:271) or RQARALPRT (SEQ ID NO:265).

[0127] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) a CDRH1 comprising the sequence RGGDYWS (SEQ ID NO: 1); a CDRH2 comprising the sequence 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; and X33 is G, S or N; and a CDRH3 comprising the sequence X34YDX35X36X37GYSYYYYGX38DV (SEQ ID NO: 539); NO:539), wherein X34 is G; X35 is A, I, T or V; X36 is absent; X37 is S; and X38 is M or L, and b) LCDR1 comprises the amino acid sequence RSSQSLLHRSGYNYLD (SEQ ID NO:257); LCDR2 comprises the amino acid sequence LGSNRAS (SEQ ID NO:258) or QGSNRAS (SEQ ID NO:264); and LCDR3 comprises the amino acid sequence MQSLQTPRT (SEQ ID NO:259), RQSVALPRT (SEQ ID NO:271) or RQARALPRT (SEQ ID NO:265).

[0128] In certain embodiments, the anti-CB1 antibodies or antigen-binding fragments disclosed herein comprise a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 from any of the CB1 antibodies described herein. For example, in some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of any one 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 HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of any one 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.

[0129] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises the HV or LV of any one 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 HV and LV of any one 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.

[0130] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of any one of LIBC529593-1, LIBC560340-1 and LIBC560657-1. In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 of any one of LIBC529593-1, LIBC560340-1 and LIBC560657-1 and LCDR1, LCDR2 and LCDR3. The HV and LV CDR sequences of the antibodies are listed in Table 19. For example, the antibody or antigen-binding fragment thereof comprises a) HCDR1 comprising the amino acid sequence RGGDYWS (SEQ ID NO: 474) or RGGDYWN (SEQ ID NO: 480); HCDR2 comprising the amino acid sequence HIYYSGSTNYNPSLRS (SEQ ID NO: 475), HIYYSGSKNYNPSLKS (SEQ ID NO: 481), or HIYYTGTKYYNPSLKS (SEQ ID NO: 487), and HCDR3 comprising the amino acid sequence DYDILYGYSYYYYGLDV (SEQ ID NO: 476), or GYDSSGYSYYYYGMDV (SEQ ID NO: 475), and b) LCDR1 comprising the amino acid sequence RSSQSLLHRSGYNYLD (SEQ ID NO: 471), RSSQSLLYSNGHNFLD (SEQ ID NO: 477), or RSSQSLLYSNGHNYLD (SEQ ID NO: 488). NO:483), LCDR2 comprising the amino acid sequence of LGSNRAS (SEQ ID NO:472) or LGSNRAP (SEQ ID NO:484), and LCDR3 comprising the amino acid sequence of MQSLQTPRT (SEQ ID NO:473) or MQALQTPRT (SEQ ID NO:479).

[0131] In some embodiments, the antibody or its antigen-binding fragment comprises any one of LIBC529593-1, LIBC560340-1 and LIBC560657-1 HV or LV. In some embodiments, the antibody or its antigen-binding fragment comprises any one of LIBC529593-1, LIBC560340-1 and LIBC560657-1 HV and LV. The HV and LV sequences of the antibody are listed in Table 20.

[0132] In some embodiments, the anti-CB1 antibody or its antigen-binding fragment comprises amino acid mutations in the HV CDR compared to the reference antibody. For example, in some embodiments, the antibody or its antigen-binding fragment comprises a) HV comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR2 of the antibody comprises 5 or fewer mutations compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136). In some embodiments, the HCDR2 of the antibody or its antigen-binding fragment comprises 4 or fewer mutations compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136). In some embodiments, the HCDR2 of the antibody or its antigen-binding fragment comprises 5 or fewer mutations compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136), and the HCDR3 of the antibody or its antigen-binding fragment comprises 3 or fewer mutations compared to NYDTVTGYSYYYYGMDV (SEQ ID NO: 137). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises 4 or fewer mutations compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136), and the HCDR3 of the antibody or antigen-binding fragment thereof comprises 3 or fewer mutations compared to NYDTVTGYSYYYYGMDV (SEQ ID NO: 137). In such embodiments, the HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A, S or G, preferably A or S, and the LV of the antibody or antigen-binding fragment thereof belongs to or is derived from VK1 / 02 / JK4, VK2 / A19 / JK1 or VK3 / A27 / JK1 germline. Preferably, the LV belongs to or is derived from VK1 / 02 / JK4 or VK2 / A19 / JK1 germline, more preferably VK1 / 02 / JK4 germline. Exemplary sequences of germline or germline-derived LV sequences are described above and in Tables 8, 11, 27, and 31.

[0133] In some embodiments, the antibody or antigen-binding fragment thereof comprises a) an HV comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR2 of the antibody or antigen-binding fragment thereof comprises 5 or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises 4 or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises 5 or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8), and the HCDR3 of the antibody or antigen-binding fragment thereof comprises 3 or fewer mutations compared to DYDILTGYSYYYYGMDV (SEQ ID NO: 9). In some embodiments, the HCDR2 of the antibody or antigen-binding fragment thereof comprises 4 or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8), and the HCDR3 of the antibody or antigen-binding fragment thereof comprises 3 or fewer mutations compared to DYDILTGYSYYYYGMDV (SEQ ID NO: 9). In such embodiments, the HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A, S or G, preferably A or S, and the LV of the antibody or antigen-binding fragment thereof belongs to or is derived from the VK1 / 02 / 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 in Tables 8, 11, 20, 27 and 31.

[0134] In some embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a sequence having at least 90% identity, or at least 95% identity (e.g., at least 96%, at least 97%, or at least 98% identity) with the sequence of SEQ ID NO: 228. The LV of the antibody or antigen-binding fragment thereof belongs to or is derived from the VK1 / O2 / JK4, VK2 / A19 / JK1, or VK3 / A27 / JK1 germline. Preferably, the LV belongs to or is 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 having at least 90% identity, or at least 95% identity (e.g., at least 96%, at least 97%, or at least 98% identity) with the sequence of SEQ ID NO: 100. The LV of the antibody or antigen-binding fragment thereof belongs to or is derived from the VK1 / 02 / 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 in Tables 8, 11, 20, 27 and 31.

[0135] As used herein, the term "identity" refers to the relationship between sequences as determined by comparing and comparing the sequences of two or more polypeptide molecules or two or more nucleic acid molecules. As used herein, "identity percentage" means the percentage of identical residues between amino acids or nucleotides in the molecules compared, and is calculated based on the size of the smallest molecule compared. For these calculations, the spaces (if any) in the comparison must be resolved 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 those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, 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. [Journal of the Society for Industrial and Applied Mathematics] Applied Math. [Applied Mathematics] 48:1073. For example, sequence identity can be determined by standard methods commonly used to compare the similarity of amino acid positions of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptide or two polynucleotide sequences are aligned to achieve the best match of their respective residues (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences).These programs provide default open and gap penalties, and scoring matrices, such as PAM 250 (Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3, 1978), or BLOSUM62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) can be used in conjunction with a calculator program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100, then divided by the length of the longer sequence within the matching range and the sum of the number of gaps introduced into the longer sequence to align the two sequences. When calculating the percent identity, the compared sequences are aligned in a manner that achieves maximum matching between the sequences.

[0136] The GCG package is a computer program that can be used to determine the percentage of identity, which 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 compare two polypeptides or two polynucleotides whose percentage of sequence identity is to be determined. The sequences are aligned to achieve the best match of their respective amino acids or nucleotides (as determined by the algorithm as the "match range"). A gap open penalty (calculated as 3x diagonal average, where the "diagonal average" is the average of the diagonals of the comparison matrix used; "diagonal" is the score or value assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (which is typically 1 / 10×gap open penalty) and a comparison matrix (such as PAM 250 or BLOSUM 62) are used in conjunction with the algorithm. In certain embodiments, the algorithm also uses a standard comparison matrix (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0137] Recommended parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program include the following:

[0138] Algorithm: Needleman et al. 1970, J. Mol. Biol. 48:443-453;

[0139] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

[0140] Gap Penalty: 12 (but no penalty for end gaps)

[0141] Gap length penalty: 4

[0142] Similarity threshold: 0

[0143] Certain alignment schemes used to align two amino acid sequences can match only shorter regions of the two sequences, and this smaller alignment region can have a very high sequence identity, even if there is no obvious relationship between the two full-length sequences. Therefore, if necessary, the selected alignment method (GAP program) can be adjusted to align over at least 50 consecutive amino acids of the target polypeptide.

[0144] As described above, compared to 10D10, the anti-CB1 antibodies or antigen-binding fragments thereof disclosed herein may contain mutations in the HC_FR3 region. For antibodies or antigen-binding fragments thereof comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 538, and HCDR3 of SEQ ID NO: 539, in addition to mutations at HV positions 83 and / or 85, the amino acid at HV position 79 may 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 antigen-binding fragment thereof is R, the amino acid at HV position 83 of the antibody or antigen-binding fragment thereof is N, and the amino acid at HV position 85 of the antibody or antigen-binding fragment thereof is Y. In some embodiments of the above antibodies, the amino acid at HV position 83 of the antibody or antigen-binding fragment thereof is N, the amino acid at HV position 85 of the antibody or antigen-binding fragment thereof is Y, and the amino acid at LV position 94 of the antibody or antigen-binding fragment thereof is S. In some embodiments, the amino acid at position 79 of the antibody or antigen-binding fragment thereof is R, the amino acid at HV position 83 of the antibody or antigen-binding fragment thereof is N and the amino acid at HV position 85 of the antibody or antigen-binding fragment thereof is Y, and the amino acid at LV position 94 of the antibody or antigen-binding fragment thereof is S.

[0145] In certain preferred embodiments, the anti-CB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of any one 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 a set of heavy chain and light chain CDRs of any one of the following:

[0146] (a) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 135-137, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 138-140, respectively;

[0147] (b) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 426-428, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 423-425, respectively;

[0148] (c) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 266-268, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 263-235, respectively

[0149] (d) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 129-131, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 132-134, respectively;

[0150] (e) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 159-161, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 162-164, respectively;

[0151] (f) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 207-209, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 210-212, respectively;

[0152] (g) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1-3, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4-6, respectively; and

[0153] (h) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 55-57, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 58-60, respectively.

[0154] In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises an HV comprising an amino acid sequence selected from the group consisting of 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 antigen-binding fragment thereof comprises an LV comprising an amino acid sequence selected from the group consisting of 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 comprises an HV and a LV selected from any one 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: NO:97; and ix) HV comprising the amino acid sequence of SEQ ID NO:116, LV comprising the amino acid sequence of SEQ ID NO:115.

[0155] In various embodiments, the antibodies or antigen-binding fragments thereof disclosed herein have a higher binding affinity for CB1 (e.g., huCB1) than 10D10 LC N35Y (i.e., 10D10 containing an N to Y substitution at LC amino acid position 35). In some embodiments, the binding affinity of the antibody or antigen-binding fragment thereof for CB1 is at least three times higher than 10D10 LC N35Y. In some embodiments, the binding affinity of the antibody or antigen-binding fragment thereof for CB1 is at least five times higher (e.g., at least eight times higher, at least ten times higher, at least twelve times higher, at least fifteen times higher, or at least twenty times higher) than 10D10 LC N35Y.

[0156] Affinity is determined using a variety of techniques, one example of which is an affinity ELISA assay. In various embodiments, affinity is determined by surface plasmon resonance assay (e.g., based on Using this method, the association rate constant (k a , with M -1 s -1 ) and the dissociation rate constant (k d , with s -1 The ratio of the kinetic rate constants (k d / k a ) Calculate the equilibrium dissociation constant (K D , represented by M). In some embodiments, affinity is determined by a kinetic method such as the kinetic exclusion assay (KinExA) described in Rathanaswami et al. Analytical Biochemistry, Vol. 373: 52-60, 2008. Using the KinExA assay, the equilibrium dissociation constant (K D , represented by M) and the association rate constant (k a , with M -1 s -1 Dissociation rate constant (k d , with s -1 ) can be calculated from these values ​​(K D xk a In other embodiments, the biofilm interferometry method is used, such as described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278: 165-82, 2015 and used for The affinity was determined by the method of the Pall ForteBio system. The kinetic constant (k a and k d ) and affinity constant (K D) can be calculated in real time using biofilm interferometry. Binding to CB1 (e.g., huCB1) can be measured using these techniques and an antigen source (e.g., cells expressing CB1-ND, CB1-SMALP, or CB1 (e.g., 293T cells)).

[0157] The equilibrium dissociation constant (K) of an antibody or antigen-binding fragment that specifically binds to an antigen D )≤1x 10 -6 M. When K D ≤3x 10 -8 When M, the antibody or antigen-binding fragment specifically binds to the antigen with "high affinity". In some embodiments, the anti-CB1 antibody or antigen-binding fragment binds to the target antigen (e.g., huCB1), K D ≤100 nM (e.g., less than about 90 nM, 70 nM, 50 nM, 30 nM, 20 nM, 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) of less than about 70 nM-10 nM (e.g., about 60 nM-20 nM, or about 40 nM-10 nM). D ) binds to CB1 (e.g., huCB1, such as huCB1-ND), such as using In some embodiments, the antibody or antigen-binding fragment is ≤1x10 -8 M (e.g., ≤ 1x 10 -9 M, ≤5x 10 -10 M, or ≤1x 10 -10 M) KD binding to CB1 (eg, hu-CB1) as determined using the KinExA assay and cells expressing huCB1-SMAP or CB1.

[0158] 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 potency of the antibodies and antigen-binding fragments is in the single-digit nanomolar range or the sub-nanomolar range, for example, they have an IC50 of less than about 10nM, less than about 5nM, less than about 3nM or less than about 1nM, as measured in a cell-based cAMP assay (e.g., a cell-based cAMP assay performed in the presence of CP 55,940). In some embodiments, the anti-CB1 antibodies and antigen-binding fragments disclosed herein have a potency comparable to that of rimonabant. In some embodiments, the potency of the anti-CB1 antibodies and antigen-binding fragments disclosed herein is greater than that of rimonabant (e.g., at least two, three, five, eight or ten times greater than rimonabant). In various embodiments, potency can be measured using a cell-based cMAP assay.

[0159] The 10D10 antibody binds to the extracellular loop 2 region of huCB1 (i.e., amino acid residues 256 to 273 of SEQ ID NO: 559) (WO2014210205). 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. Therefore, 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, as measured in a cell-based cAMP assay (e.g., a cell-based cAMP assay in the presence of CP 55,940).

[0160] The anti-CB1 antibody of the present invention may comprise any immunoglobulin constant region. The term "constant region", which may be used interchangeably with "constant domain", herein refers to all domains in an antibody except the variable region. The constant region is not directly involved in the binding of an antigen, but exhibits a variety of 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) depending on the amino acid sequence of their heavy chain constant region. The light chain constant region may be, for example, a κ-type or λ-type light chain constant region present in all five antibody isotypes, such as a human κ-type or λ-type light chain constant region. Examples of human immunoglobulin light chain constant region sequences are shown in Table 1:

[0161] Table 1. Exemplary human immunoglobulin light chain constant regions

[0162]

[0163] The heavy chain constant region of the anti-CB1 antibody of the present invention can be, for example, an α, Δ, ε, γ or μ type heavy chain constant region, such as a human α, Δ, ε, γ or μ type heavy chain constant region. In some embodiments, the anti-CB1 antibody comprises a heavy chain constant region from an IgG1, IgG2, IgG3 or IgG4 immunoglobulin, such as a human IgG1, IgG2, IgG3 or IgG4 immunoglobulin. In one embodiment, the anti-CB1 antibody comprises a heavy chain constant region from a human IgG1 immunoglobulin. In such an embodiment, the human IgG1 immunoglobulin constant region may comprise one or more mutations that prevent glycosylation of the antibody and / or extend the half-life of the antibody, as described in more detail herein. In another embodiment, the anti-CB1 antibody comprises a heavy chain constant region from a human IgG2 immunoglobulin. In yet another embodiment, the anti-CB1 antibody comprises a heavy chain constant region from a human IgG4 immunoglobulin. Examples of human IgG1, IgG2 and IgG4 heavy chain constant region sequences are shown in Table 2 below.

[0164] Table 2. Exemplary human immunoglobulin heavy chain constant regions

[0165]

[0166]

[0167]

[0168] Each of the light chain variable regions and each of the heavy chain variable regions disclosed herein (Tables 8, 11, 20, 27 and 31) can be attached to the above light chain constant region (Table 1) and heavy chain constant region (Table 2) to form complete antibody light and heavy chains, respectively. In some embodiments, each light chain variable region and each heavy chain variable region disclosed in Tables 8, 11, 20, 27 and 31 are attached to SEQ ID NO: 518 or 519 and SEQ ID NO: 525, 526 or 527 to form complete antibody light and heavy chains, respectively. In some embodiments, each light chain variable region and each heavy chain variable region disclosed in Tables 8, 11, 20, 27 and 31 are attached to SEQ ID NO: 518 and SEQ ID NO: 525, 526 or 527 to form complete antibody light and heavy chains, respectively. In some embodiments, each light chain variable region and each heavy chain variable region disclosed in Tables 8, 11, 20, 27 and 31 are attached to SEQ ID NO: 518 and SEQ ID NO: 525 to form complete antibody light and heavy chains, respectively. In some embodiments, the monoclonal antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 562 and a light chain having an amino acid sequence of SEQ ID NO: 563. In some embodiments, the monoclonal antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 562 and a C-terminal amino acid residue K is deleted and a light chain having an amino acid sequence of SEQ ID NO: 563. In addition, the heavy and light chain sequences thus generated can each be combined to form a complete antibody structure. It should be understood that the heavy and light chain variable regions provided herein can also be attached to other constant domains having sequences different from the exemplary sequences listed above.

[0169] 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 with 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. Therefore, in some embodiments, the anti-CB1 antibody can have a human IgG1, IgG2, IgG3 or IgG4 constant domain. 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.

[0170] As used herein, the term "monoclonal antibody" (or "mAb") refers to an antibody obtained from a substantially homologous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations present in trace amounts. Relative to polyclonal antibody preparations that typically include different antibodies for different epitopes, monoclonal antibodies are highly specific for individual antigenic sites or epitopes. Monoclonal antibodies can be produced using any technique known in the art, such as by immortalizing spleen cells collected from an animal after completing an immunization regimen. Any technique known in the art can be used, such as by immortalizing spleen cells by fusing spleen cells with myeloma cells to produce hybridomas. See, for example, Antibodies; Harlow and Lane, Cold Spring Harbor Laboratory Press, 1st edition, e.g., from the 1988 edition, or the 2nd edition, e.g., from the 2014 edition. Myeloma cells in the fusion procedure for producing hybridomas preferably do not produce antibodies, have high fusion efficiency, and have enzyme defects that prevent them from growing in certain selective culture media that only support the growth of the desired fusion cells (hybridomas). Examples of cell lines suitable for fusion 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 fusion with rat cells include, but are not limited to, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines that can be used for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6. Other exemplary methods for separating monoclonal antibodies include screening plasma B cells from animals after the immunization program is completed. (See, e.g., 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 (e.g., to improve binding) using in vitro affinity maturation techniques such as yeast display-based affinity maturation known in the art. (See, e.g., Cherf, GM and Cochran, JR, Yeast Surface Display: Methods, Protocols, and Applications. Yeast Surface Display: Methods, Protocols, and Applications 2015, 155-175).

[0171] In some embodiments, the anti-CB1 antibody or antigen-binding fragment of the present invention is a chimeric or humanized antibody or antigen-binding fragment 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 covalently joined to produce a functional immunoglobulin light chain or heavy chain or binding fragment. In general, a portion of the heavy chain and / or light chain is consistent or homologous to a 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 consistent or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. For methods involving chimeric antibodies, see, for example, U.S. Pat. No. 4,816,567; and Morrison et al., 1985, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81: 6851-6855, all of which are hereby incorporated by reference in their entirety.

[0172] In general, the purpose of preparing chimeric antibodies is to produce chimeras in which the number of amino acids from a predetermined species is maximized. An example is a "CDR-grafted" antibody, in which the antibody comprises one or more CDRs from a particular species or belonging to a particular antibody class or subclass, and the remainder of the antibody chain (etc.) is consistent or homologous with the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. CDR grafting is described in, for example, U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089 and 5,530,101. For use in humans, variable regions or selected CDRs from rodent or rabbit antibodies are usually transplanted into human antibodies, replacing the naturally occurring variable regions or CDRs of human antibodies.

[0173] A useful type of chimeric antibody is a "humanized" antibody. In general, humanized antibodies are prepared from monoclonal antibodies originally produced in non-human animals, such as rodents or rabbits. Certain amino acid residues in the monoclonal antibody, typically amino acid residues from the non-antigen recognition portion of the antibody, are modified to be homologous to corresponding residues in human antibodies of the corresponding isotype. Humanization can be performed, for example, using various methods by replacing the corresponding region of a human antibody with at least a portion of a rodent or rabbit variable region (see, for example, U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., 1986, Nature [Nature] 321: 522-525; Riechmann et al., 1988, Nature [Nature] 332: 323-27; Verhoeyen et al., 1988, Science [Science] 239: 1534-1536).

[0174] In one aspect, the CDRs of the light chain and heavy chain variable regions (see Tables 7, 10, 19, 26 and 30) of the antibodies provided herein are transplanted to the framework regions (FRs) of antibodies from the same or different phylogenetic species. For example, the CDRs of the heavy chain and light chain variable regions listed in Tables 7, 10, 19, 26 and 30 can be transplanted to the common human FRs. In order to produce a common human FR, the FRs from several human heavy chain or light chain amino acid sequences can be compared to identify the common amino acid sequence. Alternatively, the variable regions transplanted from a heavy chain or light chain can be used together with the constant regions of the constant regions of the specific heavy chain or light chain disclosed herein.

[0175] In certain embodiments, the anti-CB1 antibody or antigen-binding fragment of the present invention is a fully human antibody or antigen-binding fragment thereof. A "fully human antibody" is an antibody comprising a variable region and a constant region derived from or indicative of a human germline immunoglobulin sequence. A specific approach provided for producing fully human antibodies is the "humanization" of the mouse humoral immune system. Introducing human immunoglobulin (Ig) loci into mice with inactivated endogenous Ig genes is a way to produce fully human monoclonal antibodies (mAbs) in mice, which are animals that can be immunized with any desired antigen. The use of fully human antibodies can minimize immunogenicity and allergic responses, which are sometimes caused by administering mice or mouse-derived mAbs to humans as therapeutic agents.

[0176] The transgenic mice described above are called "HuMab" mice, which contain human immunoglobulin gene mini-locus encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences and targeted mutations that inactivate endogenous μ and κ chain loci (Lonberg et al., 1994, Nature 368: 856-859). As a result, the mice exhibit lower mouse IgM and κ expression and respond to immunization with the introduced human heavy and light chain transgenes undergoing class switching and somatic mutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764: 536-546). The preparation of HuMab mice is described in detail in the following: 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 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. NY Acad. Sci. 764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851; the above references are hereby incorporated by reference in their entirety. A particular transgenic mouse line suitable for producing fully human anti-CB1 antibodies is Transgenic mice are described in U.S. Pat. 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, all of which are hereby incorporated by reference in their entirety. Antibodies of human origin can also be produced using phage display technology. Phage display is described, for example, in Dower et al., WO 91 / 17271; McCafferty et al., WO 92 / 01047; and Caton and Koprowski, 1990, Proc. Natl. Acad. Sci. USA, 87:6450-6454, each of which is incorporated herein by reference in its entirety.

[0177] In certain embodiments, the anti-CB1 antibodies and antigen-binding fragments of the present invention may comprise one or more mutations or modifications of the constant region. For example, the heavy chain constant region or Fc region of the anti-CB1 antibody may comprise 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 the heavy chain of an immunoglobulin, which can be produced by papain digestion of a complete antibody. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. In certain embodiments, the Fc region is an Fc region from an IgG1, IgG2, IgG3 or IgG4 immunoglobulin. In certain embodiments, the Fc region comprises CH2 and CH3 domains from human IgG1 or human IgG2 immunoglobulins. The Fc region can 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 function and / or improve half-life, as described in more detail below.

[0178] In some embodiments, the anti-CB1 antibodies of the present invention comprise one or more amino acid substitutions in the Fc region that reduce effector function. Amino acid substitutions in an amino acid sequence are typically designated herein by a single-letter abbreviation for the amino acid residue in the specific position, followed by the amino acid position number relative to the original sequence of interest, followed by a single-letter abbreviation for the substituted amino acid residue. For example, "C220S" symbolically represents a cysteine ​​residue at amino acid position 220 relative to the original sequence of interest replaced by a serine residue. Exemplary amino acid substitutions (according to the EU numbering scheme) that may 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 a combination of any of the foregoing.

[0179] Glycosylation can promote the effector function of antibodies, particularly IgG1 antibodies. Therefore, in some embodiments, the anti-CB1 antibodies of the present invention may contain one or more amino acid substitutions that affect the level or type of antibody glycosylation. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the connection of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatically connecting a carbohydrate moiety to an asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the connection of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0180] In some embodiments, the glycosylation of the anti-CB1 antibodies described herein is reduced or eliminated by, for example, removing one or more glycosylation sites from the Fc region of the antibody. In some embodiments, the anti-CB1 antibody is a glycosylated human monoclonal antibody, such as a glycosylated human IgG1 monoclonal antibody. Amino acid substitutions that eliminate or change 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 position N297 (according to the EU numbering scheme), such as N297Q, N297A, or N297G. In some embodiments, the anti-CB1 antibodies of the present invention include Fc regions with mutations at position N297 from human IgG1 antibodies. In a specific embodiment, the anti-CB1 antibodies of the present invention include Fc regions with N297G mutations from human IgG1 antibodies. For example, in some embodiments, the anti-CB1 antibodies of the present invention include heavy chain constant regions containing sequence SEQ ID NO:524.

[0181] In order to improve the stability of the molecule comprising 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 the formation of disulfide bonds in the dimer state. The residues corresponding to V259, A287, R292, V302, L306, V323 or I332 (according to the EU numbering scheme) in the IgG1 Fc region can therefore be substituted with cysteine. Preferably, a specific residue pair is substituted with cysteine ​​so that they preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond competition. 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 with R292C and V302C mutations from a human IgG1 antibody. In such embodiments, the Fc region may further comprise an N297 mutation, such as an N297G mutation. In some embodiments, the anti-CB1 antibody of the present invention comprises a heavy chain constant region comprising the sequence of SEQ ID NO:525.

[0182] It may also be necessary to modify the anti-CB1 antibodies of the present invention to increase serum half-life (e.g., in vivo half-life). One method of achieving increased serum half-life is by amino acid mutations at certain positions in the constant domain, such as those described in WO 2002060919. Such mutations increase the affinity of the antibody to FcRn and its serum half-life. In some embodiments, the anti-CB1 antibodies described herein comprise an Fc region 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 antibodies of the present invention comprise a heavy chain constant region comprising the sequence SEQ ID NO: 527.

[0183] In some embodiments, the Fc region of the anti-CB1 antibodies described herein may include modifications that change the glycosylation, stability, and serum half-life of the antibody. For example, in some embodiments, the anti-CB1 antibodies described herein include Fc regions with mutations R292C and V302C, N297 mutations (e.g., N297G mutations) (according to the EU numbering scheme) from human IgG1 antibodies, and may also include one or more mutations at positions 252, 254, and 256, such as M252Y, S254T, and T256E (according to the EU numbering scheme). In some embodiments, the anti-CB1 antibodies of the present invention include a heavy chain constant region containing the sequence SEQ ID NO: 526.

[0184] It is expected that the anti-CB1 antibodies and antigen-binding fragments described herein do not cross-react with (i.e., do not substantially recognize or bind to) human cannabinoid receptor type 2 (CB2). Human CB2 is a G protein-coupled receptor from the cannabinoid receptor family, encoded by the CNR2 gene. Human CB2 contains 360 amino acids (UniProtKB-P34972, SEQ ID NO: 560) and has about 44% sequence similarity to huCB1. The anti-CB1 antibodies and antigen-binding fragments described herein also do not cross-react with mouse CB1 protein (UniProtKB-P47746, SEQ ID NO: 561).

[0185] The present invention includes one or more polynucleotides or nucleic acids encoding the anti-CB1 antibodies or antigen-binding fragments described herein. In addition, the present invention encompasses vectors comprising these nucleic acids, host cells or cell lines comprising these nucleic acids, and methods for preparing the anti-CB1 antibodies and antigen-binding fragments of the present invention. These nucleic acids include, for example, polynucleotides encoding all or part of an antibody or antigen-binding fragment, such as one or two chains of an antibody of the present invention, or fragments, derivatives or variants thereof; PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides; antisense oligonucleotides for inhibiting the expression of polynucleotides; and the aforementioned complementary sequences. Where appropriate, these nucleic acids can be of any length for the desired use or function, and can include one or more additional sequences, such as regulatory sequences, and / or as part of a longer nucleic acid, such as a vector. The nucleic acid molecules of the present invention include DNA and RNA in single-stranded and double-stranded forms, and corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the present invention include full-length genes or cDNA molecules, and combinations of fragments thereof. The nucleic acids of the present invention can be derived from human sources as well as non-human species.

[0186] The relevant amino acid sequence or polypeptide of interest from an immunoglobulin or its region (e.g., variable region, Fc region, etc.) can be determined by direct protein sequencing, and suitable encoding nucleotide sequences can be designed according to a universal codon table. Alternatively, the genome or cDNA encoding the monoclonal antibody of the invention or its binding fragment can be isolated and sequenced from cells producing such antibodies using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody).

[0187] The present invention also includes vectors containing one or more nucleic acids encoding one or more components (e.g., variable regions, light chains, and heavy chains) of the antibodies or antigen-binding fragments of the present invention. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein coding information to a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors (e.g., 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 a suitable nucleic acid control sequence necessary for expressing an operably connected coding sequence in a specific host cell. An expression vector may include, but is not limited to, sequences that affect or control transcription, translation, and (if introns are present) RNA splicing of a coding region operably connected thereto. Nucleic acid sequences necessary for expression in prokaryotes include promoters, optionally operator sequences, ribosome binding sites, and possible other sequences. It is known that eukaryotic cells utilize promoters, enhancers, and termination and polyadenylation signals.

[0188] A secretory signal peptide sequence may also be optionally encoded by an expression vector operably linked to the target coding sequence, so that the expressed polypeptide can be secreted by the recombinant host cell (if desired) to more easily separate the target polypeptide from the cell. For example, in some embodiments, the signal peptide sequence can be attached / 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 MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 541) is fused to the amino terminus of any of the variable region polypeptide sequences in Tables 8, 11, 20, 27, and 31. In other embodiments, a signal peptide having the amino acid sequence MAWALLLLTLLTQGTGSWA (SEQ ID NO: 542) is fused to the amino terminus of any of the variable region polypeptide sequences in Tables 8, 11, 20, 27, and 31. In yet other embodiments, a signal peptide having the amino acid sequence MTCSPLLLTLLIHCTGSWA (SEQ ID NO: 543) is fused to the amino terminus of any of the variable region polypeptide sequences in Tables 8, 11, 20, 27, and 31. Other suitable signal peptide sequences that can be fused to the amino terminus of any 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), 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:556) NO:558).Other signal or secretory peptides are known to those of ordinary skill in the art and may 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.

[0189] Typically, expression vectors for producing anti-CB1 antibodies and antigen-binding fragments of the present invention in host cells will contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences encoding components of antibodies and antigen-binding fragments. In certain embodiments, such sequences (collectively referred to as "flanking sequences") typically will include one or more of the following nucleotide sequences: a promoter (e.g., a promoter suitable for the host cell), one or more enhancer sequences (e.g., a viral enhancer sequence), an origin of replication (e.g., a plasmid origin of bacteria or a viral origin of a mammalian system), 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 a polypeptide to be expressed, and a selective marker element (e.g., a sequence encoding a protein that confers antibiotic resistance or complements a host cell auxotrophic defect). Flanking sequences and vectors are well known in the art. For example, expression vectors are commercially available. In addition, commercially available expression vectors can be modified as desired or constructed using sequences known in the art. Nucleic acids encoding different components of anti-CB1 antibodies or binding fragments (eg, HV and / or LV) can be inserted into the same vector or different vectors at appropriate sites for expression in host cells.

[0190] "Host cell" refers to a cell that has been transformed or is capable of being transformed with a nucleic acid and thereby expresses a gene of interest. The term includes progeny of a parent cell, whether or not the progeny is identical to the original parent cell in morphology or genetic makeup, as long as the gene of interest is present. A host cell comprising an isolated nucleic acid of the invention, preferably 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 vectors into host cells are well known in the art.

[0191] Suitable host cells include prokaryotic cells 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, e.g., murine, rat, macaque, or human.

[0192] Prokaryotic host cells include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, and Bacillus, e.g., B. subtilis and B. licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are in common use and can be used herein, such as Pichia, e.g., P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi, such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0193] Host cells for expressing glycosylated antibodies and antigen-binding fragments can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from the following hosts, such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. Multiple virus strains for transfecting such cells are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

[0194] Vertebrate host cells are also suitable hosts, and recombinant production of antibodies and antigen-binding fragments from such cells has become routine. Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Germplasm Collection Center (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 77:4216, 1980); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 or subcloned 293 cells grown in suspension culture (Graham et al., J. Gen Virol. [Journal of General Virology] 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. [Reproduction Biology] 23:243-251, 1980); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells, and many other cell lines. In some embodiments, CHO cells are preferred host cells for expressing the anti-CB1 antibodies and antigen-binding fragments of the present invention.

[0195] The host cells are transformed or transfected with the above nucleic acids or vectors to produce anti-CB1 antibodies or antigen-binding fragments, and cultured in conventional nutrient media appropriately modified for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Therefore, the present invention also provides a method for producing the anti-CB1 antibodies or antigen-binding fragments described herein, the method comprising culturing a host cell containing one or more expression vectors described herein in a culture medium under conditions that allow expression of the antibody or antigen-binding fragment encoded by the one or more expression vectors described herein; and recovering the antibody or antigen-binding fragment from the culture medium or host cells.

[0196] After culturing the host cell, the antibody or antigen binding fragment can be produced in the intracellular, periplasmic space, or directly secreted into the culture medium. If the antibody or antigen binding fragment is produced intracellularly, as a first step, the granular fragments of the host cell or dissolved fragments are removed, for example, by centrifugation or ultrafiltration. The antibody or antigen binding fragment can be purified using methods known in the art, such as hydroxyapatite chromatography, cation or anion exchange chromatography, or preferably affinity chromatography, using one or more target antigens or protein A or protein G as affinity ligands. Protein A can be used to purify proteins including polypeptides based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. [Journal of Immunology Methods] 62: 1-13, 1983). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. [Journal of the European Molecular Biology Association] 5: 1567-1575, 1986). The matrix to which the affinity ligand is connected is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene can increase flow rates and reduce processing times compared to agarose. In the case of proteins containing CH3 domains, Bakerbond ABX TM Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification such as ethanol precipitation, reverse phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also possible, depending on the specific antibody or antigen-binding fragment to be recovered.

[0197] Composition

[0198] Compositions comprising anti-CB1 antibodies or antigen-binding fragments are also disclosed herein. Suitable compositions include those comprising the anti-CB1 antibodies or antigen-binding fragments 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 employed and / or do not produce allergic or adverse reactions when administered to humans. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0199] In some embodiments, the pharmaceutical composition may include materials for changing, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, buffers (e.g., 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 (e.g., serum albumin, gelatin, or immunoglobulins); amino acids (e.g., glycine, glutamic acid, asparagine, histidine, arginine, or lysine), colorants and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEGs, sorbitan esters, polysorbates (e.g., polysorbate 20, polysorbate 80), triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutically acceptable adjuvants. Methods and suitable materials for formulating molecules for therapeutic use are known in the pharmaceutical art and are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., (AR Genrmo ed.), 1990, Mack Publishing Company.

[0200] In some embodiments, the compositions disclosed herein are liquid compositions, such as aqueous solutions. In some embodiments, the compositions comprise a buffer, a tonicity agent or a bulking agent, a surfactant, and have a pH in the range of about 4.5 to about 7.5. Optionally, the composition further comprises a salt and / or a preservative.

[0201] In some embodiments, the pH of the composition is in the range of about 4.5 to about 7.0, e.g., about 4.5 to about 6.5, or about 4.8 to about 5.5, or optionally about 5.0. Examples of buffers suitable for pH values ​​within this range include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconates, histidine, citrates, and other organic acid buffers. Depending on, for example, the buffer and the desired isotonicity of the composition, the buffer concentration may be about 1 mM to about 200 mM, or about 10 mM to about 60 mM.

[0202] A tonicity agent may be included in the composition, which may also stabilize the antibody or antigen binding fragment. Exemplary tonicity agents include polyols, such as sucrose, mannitol, or trehalose. Preferably, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may also be suitable. Exemplary concentrations of the polyol in the formulation may range from about 1% to about 15% w / v.

[0203] Surfactants can also be added to the formulation to reduce the aggregation of the antibody or Fab prepared and / or minimize the formation of particles in the formulation and / or reduce adsorption. Exemplary surfactants include nonionic surfactants, such as polysorbates (e.g., polysorbate 20 or polysorbate 80) or poloxamer (poloxamer) (e.g., poloxamer 188). Exemplary concentrations of surfactants can be in the range of about 0.001% to about 0.5%, or about 0.005% to about 0.2%, or alternatively about 0.004% to about 0.01% w / v.

[0204] In one embodiment, the composition contains the above identified medicament (i.e., antibody or Fab, buffer, polyol and surfactant) and is substantially free of one or more preservatives, such as benzyl alcohol, phenol, metacresol, chlorobutanol and benzethonium chloride. In another embodiment, the formulation may include, for example, a concentration of about 0.1% to about 2%, or alternatively about 0.5% to about 1% preservative. In one embodiment, the composition may further include salt such as sodium chloride or one or more other pharmaceutically acceptable excipients. The formulation may include one or more pharmaceutically acceptable carriers, excipients or stabilizers, such as REMINGTON'S PHARMACEUTICAL SCIENCES [Remington's Pharmaceutical Science], the 18th edition, (AR Genrmo edited), 1990, Mack Publishing Company [Mark Publishing Company] described in those, as long as it does not adversely affect the desired formulation characteristics.

[0205] The formulation to be 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 easily achieved by filtering through a sterile filtration membrane. The resulting solution can be packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation will be combined with a sterile solution before administration.

[0206] In some embodiments, the 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. Lyophilization is generally used to stabilize the polypeptide for a longer storage period, particularly when the polypeptide is relatively unstable in a liquid composition. The lyophilization cycle generally consists of three steps: freezing, primary drying, and secondary drying (see Williams and Polli, Journal of Parenteral Science and Technology, Vol. 38, No. 2, pp. 48-59, 1984). In the freezing step, the solution is cooled until it is fully frozen. At this stage, the overall water in the solution forms ice. The ice sublimates in the primary drying stage, which is performed by using a vacuum to reduce the chamber pressure to less than the vapor pressure of ice. Finally, in the secondary drying stage, the absorbed or bound water is removed at the reduced chamber pressure and elevated shelf temperature. The process produces a substance called a lyophilized cake. Afterwards, the cake can be reconstituted before use. Standard reconstitution practice for lyophilized materials is to add back a volume of purified water (typically equal to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used when preparing drugs for parenteral administration (see Chen, Drug Development and Industrial Pharmacy, Vol. 18: 1311-1354, 1992).

[0207] It has been noted that in some cases, excipients act as stabilizers for freeze-dried products (see Carpenter et al., Vol. 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). In addition, polyols and sugars are also commonly used to protect polypeptides from damage induced by freezing and drying and to enhance stability during storage in a dried state. In general, sugars, especially disaccharides, are effective during the freeze-drying process and during storage. It has also been reported that other classes of molecules, including monosaccharides and disaccharides and polymers such as PVP can act as stabilizers for freeze-dried products.

[0208] As described herein, the formulation of the present invention can be designed to be short-acting, quick-release, long-acting or sustained-release. Therefore, the pharmaceutical formulation can also be formulated for controlled release or slow release. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies or antigen-binding fragments, and these matrices are formed articles, such as film or microcapsule forms. Examples of sustained-release matrices include polyesters, hydrogels (such as poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Patent number 3,773,919), copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot TM (Injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Although polymers (such as ethylene-vinyl acetate and lactic acid-glycolic acid) can release molecules for more than 100 days, some hydrogels release proteins for a shorter period of time. When the encapsulated polypeptide is kept in the body for a long time, it may be denatured or aggregated due to exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be designed to achieve stability based on the mechanisms involved. For example, if it is found that the aggregation mechanism is to form intermolecular SS bonds through sulfo-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives and developing specific polymer matrix compositions. Excipients and methods for preparing sustained release compositions are described in, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, (AR Genrmo, editor), 1990.

[0209] The composition comprising an anti-CB1 antibody or antigen-binding fragment and one or more of the above-mentioned 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 dose is administered by injection, most preferably subcutaneous, intramuscular or intravenous injection. Other methods of administration are contemplated, including topical, particularly transdermal, transmucosal, rectal, oral or local administration, for example via a catheter placed near the desired site.

[0210] use

[0211] Disclosed herein also include the use of anti-CB1 antibodies or antigen-binding fragments or compositions comprising the same for inhibiting, reducing or neutralizing the amount, activity or signal transduction of CB1 receptors (e.g., huCB1). The antibodies and antigen-binding fragments of the present invention can be used to treat, prevent and / or improve any disease or disorder associated with or mediated by CB1 expression or activity, or by blocking the interaction between CB1 and CB1 ligands (e.g., cannabinoids) or otherwise inhibiting CB1 activity and / or signal transduction and / or promoting receptor internalization and / or reducing the number of cell surface receptors. Any disease or disorder that can be treated. In various embodiments, CB1 is peripheral CB1.

[0212] Exemplary disorders, diseases and conditions that can be treated by the anti-CB1 antibodies or antigen-binding fragments described herein include obesity and comorbidities, such as syndromic obesity, including Prader-Willi syndrome, Alstrom syndrome, Bardet-Biedel syndrome, syndrome, BBS), Albright hereditary osteodystrophy (AHO), and SIM1 deletion syndrome; diabetes and related complications (e.g., abnormal plasma glucose, insulin, and / or resistin levels); dyslipidemia (e.g., abnormal HDL, plasma cholesterol, and / or triglyceride levels); liver disease, such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and primary biliary cirrhosis; fibrosis, such as renal fibrosis; chronic kidney disease (CKD), such as CKD caused by metabolic disorders; IgA nephropathy; renal disease; metabolic diseases, osteoporosis, atherosclerosis, inflammatory diseases, cardiovascular diseases, cancer, pain, systemic sclerosis, multiple sclerosis spasticity, glaucoma, and nicotine addiction.

[0213] Obesity refers to abnormal or excessive fat accumulation that may impair an individual's health and can be measured or categorized by body mass index (BMI). BMI is defined as a person's weight (e.g., kilograms) divided by the square of their height (in meters) (e.g., kg / m 2 ).

[0214] Thus, in one aspect, disclosed herein are methods for treating a disease or disorder in a subject that is responsive to antagonizing or inversely agonizing a CB1 receptor (e.g., huCB1), or a method for treating a disease or disorder in a subject that requires antagonizing or inversely agonizing a CB1 receptor, the method comprising administering the above-described antibodies or antigen-binding fragments. In some embodiments, the antibodies or antigen-binding fragments are effective antagonists and / or inverse agonists of the CB1 receptor, for example, they have a potency (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 (e.g., a cell-based cAMP assay in the presence of CP 55,940). In some embodiments, the antibodies or antigen-binding fragments comprise heavy and / or light chain CDRs as described above. The 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 a heavy chain and / or light chain variable region as described above. In other embodiments, the antibody comprises a heavy chain and / or light chain as described above. The 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 an obstacle or disease (e.g., to cause a potential benefit or therapeutic effect). In various embodiments, the CB1 receptor is a peripheral CB1 receptor.

[0215] The term "subject in need" or "subject in need of treatment" includes subjects with a disorder or disease, as well as subjects that do not yet clinically manifest a disorder or disease. "Subject" includes humans and other mammalian subjects receiving prophylactic or therapeutic treatment. In a preferred embodiment, the subject is a human.

[0216] 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, renal fibrosis, chronic kidney disease (e.g., chronic kidney disease caused by metabolic disorders), IgA nephropathy, osteoporosis, atherosclerosis, cardiovascular disease, cancer or inflammatory disease. In some embodiments, the disorder or disease is obesity. In some embodiments, the treated subject suffers from chronic kidney disease, such as chronic kidney disease driven by metabolic disorders or renal injury. In some embodiments, the treated subject suffers from a BMI of 27 kg / m 2 In some embodiments, the subject being treated has a BMI of 30 kg / m2 or higher obesity. In other embodiments, the disorder or disease is a liver disease (eg, NASH).

[0217] In some embodiments, administration or treatment results in one or more of the following in the subject: weight loss, decreased 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 renal damage, fibrosis, inflammation, improved renal function.

[0218] Reagent test kit

[0219] In some embodiments, kits comprising compositions are disclosed herein, which comprise anti-CB1 antibodies or antigen-binding fragments and one or more of the above-mentioned pharmaceutically acceptable excipients. For example, such a kit includes a composition comprising an anti-CB1 antibody or antigen-binding fragment as described herein, such as the above-mentioned aqueous composition or lyophilized composition, which is packaged in a container such as a sealed bottle, a vessel, a single-use or multiple-use vial, a prefilled syringe or a prefilled injection device. A suitable medium or carrier, such as water for injection or a physiological saline solution, can be provided for the lyophilized composition. Other materials commonly found in parenteral administration preparations may also be included. In one aspect, the composition is packaged as a unit dosage form. The kit may further include a device suitable for administering the preparation according to a specific route of administration. Preferably, the kit further contains a label describing the use of the antibody or antigen-binding fragment or a composition comprising it.

[0220] All references cited herein (including patents, patent applications, literature publications, etc.) are hereby incorporated by reference in their entirety.

[0221] The following examples are given merely to illustrate the present invention and are not intended to limit its scope in any way.

[0222] Examples

[0223] Example 1. Design of deep mutational scanning and affinity maturation libraries

[0224] Materials and methods: CB1-ND preparation. 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% CHS and 10uM ligand-1981202#5. POPC lipids were solubilized with the help of 100mM sodium cholate and mixed with MSP1D1 scaffold protein and CB1 at a molar ratio of 480:8:1. Nanodisc formation was achieved by incubation with SM2 BioBeads at 4°C for 1 hour.

[0225] Binding to CB1-ND and empty-ND. Luna V. et al. (Luna V. et al., European Polymer Journal 109 (2018) 483–488) describe the proliferation and induction of yeast cells displaying antibodies for binding experiments. Take out at least 2x10 5 Yeast cells displaying IgG were added and washed with 1x phosphate buffered saline (Mg2+-free, Ca2+-free) supplemented with 5 mg / mL bovine serum albumin (PBS-B). huCB1-ND or empty-ND with His tag were 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 anti-His antibody conjugated with allophycocyanin (APC) to detect binding and anti-huFc antibody conjugated with phycoerythrin (PE) to detect IgG surface display. Cells were washed with PBS-B before flow cytometric analysis using FACS-Canto (BD Biosciences). The median fluorescence intensity (MFI) of APC and PE was recorded.

[0226] FACS library sorting. As described above, at least 10 7 After fluorescent antibody staining, cells were sorted using a FACS Aria (BD Biosciences) using appropriate allophycocyanin and phycoerythrin detection settings.

[0227] The anti-CB1 antibody 10D10 was previously produced by immunizing Xenomouse with HEK293 cells expressing human CB1. (See WO 2014210205). Although affinity maturation is a common strategy for improving the potency of antagonist antibodies, previous efforts to affinity mature 10D10 failed to produce antibodies with sufficient potency (e.g., potency in the single-digit nM range in a cell-based cAMP assay) to produce the desired therapeutic effect (e.g., weight loss) without CNS-related side effects. The lack of high-quality, soluble preparations of complex membrane proteins (e.g., CB1) is considered to be the main reason for the previous failure. The development of CB1 nanodiscs (NDs) and CB1 styrene maleic acid lipid particles (SMALPs) has greatly improved the quality of CB1 soluble preparations. (Luna V. et al., European Polymer Journal [European Polymer Journal] 109 (2018) 483–488). Both CB1-ND and -SMALP are purified reagents that remain stable after multiple freeze / thaw cycles and are suitable for long-term storage. Furthermore, these reagents can be added to binding experiments at user-defined huCB1 concentrations. The availability of these reagents enables additional affinity maturation work.

[0228] Deep mutational scanning of antibodies provides information for the design of affinity maturation libraries. Deep mutational scanning requires the generation of comprehensive point mutation libraries, selection for binding under different conditions, and evaluation of changes in mutation frequencies after selection by high-throughput sequencing. (See, e.g., Araya, CL and Fowler, DM, Trends Biotechnol. 2011 Sep;29(9):435–442). Identification of a subset of mutations associated with improved and specific huCB1 binding enables focused combinatorial library design for affinity maturation without the aid of high-resolution antibody-antigen co-crystal structures.

[0229] Fifty-three and thirty-seven positions 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 generated, in which the comprehensive set of 10D10 point mutations in the heavy chain and light chain CDRs and the heavy chain (HC) and light chain FR3 de-loops was divided into four libraries (2 for each heavy chain and light chain). Each library was sorted for surface expression using FACS, and non-specific binding was evaluated for huCB1-ND under two different stringencies and for empty-ND, respectively. The lower stringency huCB1-ND binding gate collected all cells that showed at least 10D10-like binding (10-20% of the population), while the higher stringency gate only collected the top binders (1.5-6% of the population). After FACS, plasmids were harvested from the sorted pool using the Zymoprep II kit (Zymo Research), and amplicons were generated by minimal PCR amplification of the recovered plasmids.

[0230] Illumina MiSeq was used to rank 1710 point mutations by comprehensively evaluating the change in mutation frequency within the sorting gate compared to the starting frequency (freq) within the expression sorted reference pool. The binding enrichment (EF) factor (EF = freq) was calculated for each mutation based on NGS analysis and using the expression sorted pool as the reference sample. 结合 / freq 参考(表达) ). The enrichment factor was then used to generate a sequence-binding fitness graph for each mutation in the CDR and FR3 de loop. Mutations that resulted in better, comparable, and worse binding to CB1 compared to the 10D10 antibody were called beneficial, neutral, and harmful mutations, respectively. According to the fitness graph, many changes within HCDR1 and HCDR3 are harmful, indicating that these regions appear to have been well optimized and may have less optimization requirements and / or potential. Many mutations in HCDR2 and HC_FR3 de loop appear to promote improved binding. Data from libraries containing LCDR1 and LCDR2 mutations indicate a lack of beneficial mutations in these regions. Overall, the binding fitness graph indicates that these HCDRs are regions that are more promising for diversification for affinity maturation to improve binding.

[0231] Based on experience, ideal positions for diversification for affinity maturation generally contain variations in both deleterious and beneficial mutations. To identify the most promising mutations at a single position for inclusion in combinatorial library design, each neutral or beneficial mutation for huCB1-ND binding was also evaluated for binding to empty-ND. The most promising mutations were enriched for huCB1-ND binding, while lacking empty-ND binding (relative to the 10D10 parent). Mutations that exhibited positive enrichment factors for both huCB1-ND and empty-ND were discarded from consideration for library design.

[0232] Two NGS-guided HC yeast libraries were designed and constructed for affinity maturation. Each library was designed to explore a separate CDR loop region and maintain combinatorial theoretical diversity that could be managed through conventional yeast library transformations. Tables 3-4 below summarize the diversification strategy for the HC libraries. By introducing multiple degenerate codons during library gene synthesis, combinations of desired mutations were fully explored within discrete regions of the 10D10 HC to construct an initial set of two yeast-displayed IgG HC variant libraries paired with the 10D10 parental LC. Each degenerate codon at a specific position was selected to maximize the representation of the desired mutation within the encoded theoretical diversity.

[0233] Table 3. Design of 10D10 H1H3 Library 1*

[0234]

[0235]

[0236] *Theoretical combination diversity: 6.9E3 (paired with 10D10 LC)

[0237] Table 4. Design of 10D10 H2 / de loop library 2*

[0238] 10D10 residue Location Diversification strategy Y57 HCDR2 Y, H I58 HCDR2 I.V Y60 HCDR2 Y, H, Q, Terminate S65 HCDR2 S, E, T, A, K, Terminate S67 HCDR2 S, Q N69 HCDR2 N, A, K S73 HCDR2 S, K, N, R L74 HCDR2 L.F. K75 HCDR2 K, E S76 HCDR2 S, G, N, D D83 de ring (HC_FR3) D, K, E, N D85 de ring (HC_FR3) S.Y

[0239] *Theoretical combination diversity: 3.0E5 (paired with 10D10 LC)

[0240] A NGS-guided yeast library was designed and constructed for LC optimization to improve the manufacturability of 10D10. It is believed that long HCDR3 and LCDR1 may lead to poor expression and aggregation tendency of early 10D10 mutants, and the design of the LC library incorporates strategies to improve these properties (e.g., reducing the hydrophobicity of LC residues exposed to key solvents in LCDR1). Since the fitness graph of LC mutations shows a lack of beneficial mutations, the LC library design includes mutations that show 10D10-like binding enrichment to CB1-ND and lack of empty ND. Table 5 below summarizes the diversification strategy of the LC optimization library. By introducing multiple degenerate codons during library gene synthesis, the combination of desired mutations is fully explored in discrete regions of 10D10 LC to construct a yeast display IgG LC variant library paired with the parent 10D10 HC. Each degenerate codon at a specific position is selected to maximize the representation of the desired mutation group in the theoretical diversity of the encoding. The library is used for the following homologous affinity maturation (Example 2).

[0241] Table 5. Design of 10D10 LC optimized library**

[0242] 10D10 residue Location Diversification strategy H33 LCDR1 H, S, T N35 LCDR1 N, S*, T*, Y# Y38 LCDR1 Y, A*, E*, I*, N*, F L41 LCDR1 L, V L58 LCDR2 L, Q* R94 LC FR3 R.S. M107 LCDR3 M.R L110 LCDR3 L, F, I, R, V, C, G, S Q111 LCDR3 Q, E, K, R, T, A, G, P T135 LCDR3 T, I, L, Q, K, P

[0243] *Amino acids with reduced hydrophobicity compared to the corresponding 10D10 residues

[0244] **Theoretical combination diversity: 5.0E5 (paired with 10D10 HC)

[0245] 10D10 LC belongs to the VK2 germline. It is known in the art that LCs of germlines such as VK1 or VK3 confer better biophysical properties and better manufacturability (see, e.g., Ewert S. et al., J. Mol. Biol. [Journal of Molecular Biology], (2003) 325 (3): 531-53). In a second strategy for LC optimization, the 10D10 LC was exchanged with LCs of different germlines (e.g., VK1 or VK3) that were expected to have more favorable biophysical properties. Both low-resolution X-ray and CryoEM structures of mAb / antigen complexes showed that only the HC loop of 10D10 interacted with the CB1 receptor. These results suggest that the 10D10 VK2 LC can indeed be exchanged for another LC belonging to a germline (e.g., VK1 or VK3) that is expected to have superior biophysical properties to improve manufacturability. To this end, the 10D10 HC was paired with a diverse human LC library, and the binders that retained equivalent binding to 10D10 were screened and sorted. Next, those clones with LCs related to the 10D10 LC optimization design described above were removed from the positive clones. This process yielded yeast clones that exhibited CB1 binding equivalent to 10D10 but contained LCs from both 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 expanded to a set of 22 LCs to repair germline derivation within the FR region of four clones.

[0246] Table 6. Six new LCs can replace 10D10 LC without affecting CB1 binding

[0247]

[0248] Example 2. Homologous affinity maturation

[0249] Materials and Methods: GFP-fused huCB1-SMALP was prepared as described in Luna V. et al., European Polymer Journal 109 (2018) 483–488.

[0250] Preparation of yeast cells for binding experiments and measurement of binding of huCB1-SMALP fused to GFP by flow cytometry were performed as described in Luna V. et al., European Polymer Journal [European Polymer Journal] 109 (2018) 483–488, except that AlexaFluor647-conjugated anti-huFab antibodies were used to detect IgG surface display. The median fluorescence intensity (MFI) of GFP (binding) and AlexaFluor647 (display) was recorded.

[0251] For the nanodisc binding experiments, the binding / display ratio of each tested yeast clone was calculated by dividing the APC MFI by the PE MFI. The binding / display ratio of each clone was further normalized to that of the 10D10 LC N35Y control to calculate a "relative binding fold change" ratio, enabling comparison of yeast clones. Specific binding to huCB1-ND relative to empty-ND was assessed by dividing the huCB1-ND binding / display ratio by the empty-ND binding / display ratio. For the SMALP binding experiments, 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.

[0252] The two HC libraries and one LC optimized library designed and constructed in Example 1 were used for homologous affinity maturation of 10D10. According to established methods, improved binders in the two HC libraries were enriched by FACS respectively relative to 10D10 (see, e.g., Boder, ET and KD Wittrup, Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol, 2000. 328: 430-44; and Chao, G., et al., Isolating and engineering human antibodies using yeast surface display. Nat Protoc, 2006. 1 (2): 755-68). The LC optimized library was enriched for cells that exhibited 10D10-like binding signals. HC and LC mutations that persisted after enrichment were recovered from the yeast pool by zymoprep and PCR, and the pools of HC and LC were mixed as designed during yeast transformation to construct a new set of shuffled HC / LC libraries (mutH1H3 / mutLC, mutH2 / mutLC, and mutH1H2H3 / mutLC). Chain shuffled libraries were subjected to more stringent FACS enrichment using successively lower concentrations of huCB1-ND. Clones from the enriched shuffled libraries were screened in two rounds to select the top clones. In the first round, approximately 600 clones were screened from the three libraries for clones that bound to huCB1-ND better than the 10D10 LC N35Y control (10D10 with N35Y in the LC).

[0253] In the second round of screening, the 200 best binding clones from the first round were retested for more stringent binding to 200pM huCB1-ND, 100nM GFP-tagged huCB1-SMALP, and nonspecific binding to empty ND. After ranking the positive clones by CB1-specific binding, clones with N-linked glycosylation and unpaired Cys Tier 1 hotspots were removed, and clones with predicted Tier 1.5 deamidation and isomerization sites other than DT were deprioritized. In addition, positive clones with R94S repair for LC FR3 consensus sequence disruption were prioritized. Positive clones with HC D83K FR3 mutation (thought to be associated with enhanced anti-CB1 function) will also be prioritized if they meet the minimum threshold for improved binding. In addition, clones with reduced hydrophobicity of key residues in LCDR1 and LCDR2 were prioritized. Tables 7 and 8 below summarize the sequence information of the top 15 homology affinity matured 10D10 variants. Figure 1A and 1B Flow cytometry binding profiles of 15 variants are shown; 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). In addition, Y57H within HCDR2 and D83N and S85Y within the HC FR3 de loop were observed in all top clones (Table 9). Figure 3A ). None of the top 15 binders contained predicted hotspots that pose the highest risk for chemical modifications during therapeutic antibody manufacturing and storage.

[0254] Table 7. CDR sequences of the first 15 affinity matured homologous 10D10 variants

[0255]

[0256]

[0257]

[0258] Table 8. Variable region sequences of the top 15 homology affinity matured 10D10 variants

[0259]

[0260]

[0261]

[0262]

[0263] Table 9. Top 15 affinity matured homologous 10D10 variants: binding information

[0264]

[0265] Example 3. Light chain exchange affinity maturation

[0266] In a parallel group of affinity maturation, 22 VK1 and VK3 LCs obtained in Example 1 were transformed with 10D10 mutant HC genes or gene fragments extracted from yeast cells, and these yeast cells obtained variants showing improved binding after FACS enrichment of H1H3 and H2 / de-loop libraries (Example 2). Using more stringent FACS enrichment, three LC exchange libraries (mutH1H3 / VK LC, mutH2 / VK LC and mutH1H2H3 / VK LC) for affinity maturation were enriched to obtain the most improved binders. The clones isolated from the LC exchange library were screened in two rounds to select the top clones. The screening and selection criteria for the LC exchanged 10D10 affinity maturation variants were the same as those described in Example 2 for the homologous affinity maturation, except that the priority of the sequences with reduced hydrophobicity in LCDR1 and LCDR2 was not used. A total of 600 positive clones entered the first round of screening and 200 positive clones entered the second round of screening. The 200 positive clones were then ranked for CB1 specific binding to obtain the final top 15 LC exchange binders. The CDR and variable region sequences of the top 15 LC exchange affinity matured 10D10 variants are shown in Tables 10 and 11 below. Table 12 below summarizes the binding data of the top 15 LC exchange affinity matured 10D10 variants. Figure 2A and 2B Flow cytometry binding profiles of these variants are shown. Similar to the homologous affinity matured variants, all LC-swapped top clones exhibited significantly improved binding to huCB1-ND and huCB1-SMALP compared to 10D10 LC N35Y, while maintaining minimal binding to empty-ND (Table 12). In addition, Y57H within HCDR2 and D83N and S85Y mutations within the HC FR3 de loop were observed in the top 15 LC-swapped 10D10 affinity matured variants ( Figure 4A ). In addition, VK1 LCs accounted for a large proportion of the top LC-swapped 10D10 variants, with only one variant having a VK3 LC (Table 13). If the R94S germline reversion was not present in the original sequence, the R94S germline reversion was imposed on all VK LC sequences of the top variants.

[0267] Table 10. CDR sequences of the first 15 affinity matured LC-swapped 10D10 variants

[0268]

[0269]

[0270]

[0271]

[0272] Table 11 Variable region sequences of the first 15 LC-swapped 10D10 variants

[0273]

[0274]

[0275]

[0276]

[0277] Table 12. Top 15 LC-swapped 10D10 variants: binding information

[0278]

[0279]

[0280] Table 13. Germline information of the top 15 LC-swapped 10D10 variants

[0281] name VH germline VL line 10D10 VH4|4-31 / D3|3-9|RF2 / JH6 VK2|A19 / JK1 10D10N35Y VH4|4-31 / D3|3-9|RF2 / JH6 VK2|A19 / JK1 LIBC527906-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC527814-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC527997-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC528141-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC527912-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK4 LIBC528116-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC527879-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK4 LIBC527919-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK4 LIBC527984-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC527968-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC528169-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC528131-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK3|A27 / JK1 LIBC527827-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC527869-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1 LIBC528148-1 VH4|4-31 / D3|3-9|RF2 / JH6 VK1|O2 / JK1

[0282] Example 4 Cross-reactivity and specificity of affinity matured antibodies

[0283] The 10D10 antibody does not bind to mouse CB1 (muCB1) or human CB2 (huCB2) proteins (WO 2014 / 210205). Since all antibodies isolated in Examples 2 and 3 are variants of 10D10, they are not expected to bind to huCB1 or muCB1. The binding activity of the exemplary antibodies against muCB1 and huCB2 demonstrated the absence of cross-reactivity against these two proteins (Table 14).

[0284] Table 14. No cross-reactivity against muCB1 and huCB2

[0285]

[0286] Example 5 Characterization of affinity matured variants

[0287] The top clones isolated from Examples 2 and 3 above were cloned and expressed as IgG1SEFL2 mAbs and then tested in a cAMP functional assay to measure antagonist or inverse agonist activity.

[0288] Antagonist and inverse agonist activity

[0289] Antagonist activity. Stably transfected CB1 CHO cells were serum starved overnight at 37°C / 5% CO2 using DMEM (Sigma catalog number D5671) containing 0.5% FBS, 25mM HEPES. On the day of the assay, CB1 antibodies and rimonabant were titrated at 1:4 at 4 times the final concentration using DMEM (Sigma catalog number D5671) containing 0.1% BSA (from PE kit) and 25mM HEPES, and the titrated antibodies were transferred to a 384-well assay plate with a volume of 7.5 μL / well. Cells were harvested using Accutase (Sigma) and neutralized with DMEM (Sigma catalog number D5671) containing 0.5% FBS, 25mM HEPES. The cells were pelleted and resuspended to 1.33 x 10 cells / mL using DMEM (Sigma Catalog No. D5671) containing 0.1% BSA (from the PE kit), 25 mM HEPES. 6 cells / mL. Anti-cAMP antibody from the Lance Ultra cAMP Assay Kit (Perkin Elmer; TRF0263) was added to the cells at a ratio of 1 to 150 antibody to cell volume, 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. The forskolin and CP55,940 mixture was diluted to 4 times the final concentration using DMEM (Sigma Catalog No. D5671) containing 0.1% BSA (from the PE kit), 25 mM HEPES, and transferred to the assay plate at a volume of 7.5 μL per well. The assay plate was incubated at 37°C / 5% CO2 1 / 2 hours, then EU-cAMP tracer (PerkinElmer; TRF0263) was added to each well and further incubated for 1 hour at room temperature in the dark. After the final incubation, the assay plate was read using an Envision plate reader with EU615 and APC 665 emission filters. The data were analyzed using the following calculation: TR-FRET (665nm / 615nm) x 10000. Lower values ​​(lower FRET) were interpreted as stronger inhibition of CB1 and the data were used to calculate IC50. The antagonistic activity of the clones obtained from Examples 2 and 3 is shown in Table 15A (n=1 and n=2) and Table 15B (n=3).

[0290] Table 15A. IC50 values ​​of homologous and LC-swap affinity matured variants

[0291]

[0292]

[0293]

[0294] Table 15B. IC50 values ​​of exemplary 10D10 variants

[0295]

[0296]

[0297] In addition, the antagonist activity of exemplary 10D10 variants and benchmark anti-CB1 antibodies was measured in the presence of endogenous huCB1 ligands anandamide and 2-arachidonoylglycerol (2-AG). Specifically, CHOK1 human CB1 receptor cells were seeded overnight in DMEM containing 0.5% FBS, P / S glutamine, 25 mM HEPES, NEAA, sodium pyruvate and 400 μg / ml G418 (Invitrogen) at a density of 20,000 cells per well in 96-well white flat-bottom plates (Costar, catalog number 3917) and placed in a humidified incubator maintained at 37° C., 5% CO 2 . Cells were treated sequentially with the corresponding dose of antibody, then with 100 nM concentration of CB1 receptor ligand WIN 55,212, 10 μM cannabinoids or 500 nM 2-AG (Sigma), and finally with 35 μM forskolin (Sigma) in assay buffer containing 0.1% BSA in DMEM. After 30 minutes of incubation, d2-cAMP and Eu3+-cryptate-cAMP antibodies (Cisbio, catalog number 62AM4PEC) were added, followed by incubation at room temperature for 1 hour. Wavelengths at 665 nm and 620 nm were measured on an EnVision multi-label reader, and the 665 / 620 ratio was calculated. The concentration curve of the CB1 antibody was plotted on the x-axis by the average of two replicates for cAMP levels (665 / 620 ratio) to generate a graph. The data points were then fitted using log(agonist) vs. response, variable slope (4 parameters) in GraphPad Prism 7.04 to obtain IC50 values. The results are shown in Tables 15C and 15D and Figure 5A middle.

[0298] Inverse agonist activity. The inverse agonist activity of the exemplary 10D10 variants was also measured. CHOK1 human CB1 receptor cells were inoculated overnight in DMEM containing 0.5% FBS, P / S glutamine, 25mM HEPES, NEAA, sodium pyruvate and 400ug / ml G418 (Invitrogen) in 96-well white flat-bottom plates at a density of 20,000 cells per well and placed in a humidified incubator maintained at 37°C, 5% CO2. The cells were treated with the response dose of the antibody in turn and then treated with 2μM forskolin in DMEM assay buffer containing 0.1% BSA. After incubation for 30 minutes, d2-cAMP and Eu3+-cryptate-cAMP antibodies were added and then incubated at room temperature for 1 hour. The wavelengths at 665nm and 620nm were measured on an EnVision multi-label reader, and the 665 / 620 ratio was calculated. The concentration curve of CB1 antibody was plotted on the x-axis by the average of two replicates against cAMP levels (665 / 620 ratio) to generate a graph. The data points were then fitted using log (agonist) vs. response, variable slope (4 parameters) in GraphPad Prism 7.04 to obtain IC50 values. The results are shown in Table 15C and Figure 5B middle.

[0299] Table 15C. IC50 values ​​of exemplary variants in antagonist and inverse agonist assays

[0300]

[0301] Table 15D. IC50 of exemplary variants and benchmark antibody antagonist assays

[0302] name IC50(nM) LIBC527814-1 3.8 LIBC528116-1 3.4 10D10 143.8 Benchmark mAb* 95.8

[0303] *Clinical benchmark mAb

[0304] The potency of affinity matured antibodies (homologous and LC-swap affinity matured antibodies) was analyzed using an in vitro cAMP assay and the results are summarized in Tables 15A-15D and Figure 5A and 5BIn. Most variants from affinity maturation are more effective than 26970-1, which is a 10D10 variant obtained in previous work and used as a benchmark in the assay (Tables 15A and 15B). 26970-1 contains HC D83K / LC N35Y substitutions and is the most available benchmark antibody. In multiple functional assays, some affinity maturation variants showed similar or slightly better efficacy than rimonabant. The most effective antibodies were about 30-66x and 3.6-8x more effective than 10D10 and 26970-1, respectively (Table 15A). The LIBC528116-1 antibody is always the best antagonist in the CP 55,940 assay (Tables 15A-15C), while the LIBC523797-1 antibody is the most effective in the assay run in the presence of endogenous CB1 receptor agonist 2-AG and cannabinoids (Table 15C). In addition, the LIBC523797-1 antibody was the most superior inverse agonist (Table 15C), with potency comparable to rimonabant in the 2-AG assay and 3-4x more potent in the cannabinoid and inverse agonist assays (Table 15C). Finally, when the antibodies were tested side-by-side, the most potent 10D10 variant showed approximately 30x greater potency than the clinical benchmark anti-CB1 antibody (Table 15D). The potency results indicate that the affinity maturation work achieved the lofty design goal of obtaining antibodies with potency in the single-digit nM range, a goal that has been elusive for many years.

[0305] Binding affinity

[0306] Affinity determination was performed using Octet. HTX (Sartorius, Germany) instrument was used to determine the affinity of anti-CB1 antibodies. Prior to the experiment, streptavidin (SA) biosensors (ForteBio, #18-5021) were prehydrated for 10 min in Octet assay buffer (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-X 100 (Calbiochem, #9410-OP). The SA biosensor was loaded with biotinylated anti-human Fc antibody (Invitrogen, #A18827) followed by a 60-second baseline step in Octet assay buffer. Anti-CB1 antibodies were prepared in Octet assay buffer and captured by anti-human Fc antibodies, then immersed in wells containing different concentrations of CB1-nanodisks (#CB1-008, in-house) for 5 minutes and then dissociated for 10 minutes. CB1-nanodisks were titrated from 400nM to 6.25nM in a 1:2 ratio and prepared in Octet assay buffer. The SA biosensor was used once without regeneration. For data evaluation, Fortebio Data Analysis v11.0 software was used. The 1:1 Langmuir model was used to determine the kinetic rate constants, association rate constants (ka, M-1s-1), dissociation rate constants (kd, s-1), and equilibrium rate constants (KD, M).

[0307] Cell sorting using KinExA The 3200 (Sapidyne, Boise, ID) instrument was used to sort the binding of anti-CB1 antibodies to full-length CB1 transiently expressed on HEK 293T cells (DNA391968-1, in-house). Prior to the cell sorting experiments, the cell affinity of the benchmark anti-CB1 antibody LIBC523661 was determined. Affinity experiments were performed using HEK 293T CB1 cells one day after transfection and titrated 1 to 3 from 2.5 million cells / mL to approximately 51 cells / mL. HEK 293T CB1 cells were equilibrated at room temperature for 24 hours with LIBC523661 at a final antibody concentration (the concentration of the binding group was twice the antibody concentration) of 5 pM, 100 pM or 1 nM in cell culture medium containing FreeStyle 293T medium (Gibco, #12338-018), 2% ultra-low IgG FBS (Invitrogen, #16250-078), 50 μg / mL G418 (Sigma, #G8168) and 0.05% w / v / sodium azide (Sigma, #S2002). The cells were separated by centrifugation, and the free antibody in the equilibration supernatant was collected and stained by The KD was measured using the ELISA technique by passing the supernatant over PMMA beads (Sapidyne) pre-coated with goat anti-human Fc (Invitrogen, #31125) and probed with goat anti-human IgG (H+L) AlexFluor649 (Jackson Immunoresearch, #109-605-088). The one-site homogeneous binding model provided in Pro software version 4.2.12 was obtained from nonlinear regression of the curves. The KD for LIBC523661 was determined to be 64.0 pM, 95% CI 46.6-88.2 pM, and the number of receptors per cell equal to 1.1 million copies / cell. Using the KD control curve, it was determined that LIBC523661 required approximately 12,000 cells / mL to achieve 50% KD. Therefore, cell sorting for anti-CB1 antibodies was performed using 20 pM of each antibody and 12,000 cells / mL. Anti-CB1 antibodies were prepared in 293 medium without cells or containing HEK 293T CB1 cells and incubated at room temperature for 24 hours. Similar to the affinity experiments, cells were separated by centrifugation, and the free antibodies in the supernatant were collected and purified by The binding of each anti-CB1 antibody was measured using the ELISA technique. The binding of each anti-CB1 antibody was reported as the percentage of inhibition of the free fraction (% IFF), calculated as the antibody signal minus the blank signal obtained in the presence of 12,000 cells / mL divided by the antibody signal minus the blank signal obtained in the absence of cells (the lower the % IFF, the higher the affinity, and the higher the % IFF, the lower the affinity). The relative binding of all anti-CB1 antibodies was compared to LIBC523661 by dividing the % IFF of LIBC523661 by the % IFF of the antibody.

[0308] Octet and Kinexa binding assays of affinity matured 10D10 variants demonstrated that the potency enhancement was accompanied by improved binding affinity to huCB1 (Table 16). Octet revealed that faster on-rates and slower off-rates resulted in huCB1-ND binding affinities 10-50x higher than 10D10, while Kinexa demonstrated improved binding to wt huCB1 expressed on cells.

[0309] Table 16. Octet and Kinexa of exemplary variants

[0310]

[0311] *Calculated based on the fraction of free mAb compared to LIBC523661 mAb

[0312] #Clinical Benchmark mAb

[0313] Example 6. Improved expression of affinity matured variants

[0314] Homologous and LC exchange affinity maturation variants were tested for transient and stable expression in HEK293 and CHO cells. Four of the five most effective homologous 10D10 variants were transiently expressed in HEK 293-6E cells at 100-200 mg / L (Table 17), representing titers higher than 10D10 or benchmark variants by >3x or >7x. Inspection of the LC sequence showed that the reduction in hydrophobicity at key positions (especially L58Q) may be associated with improved expression titers (Table 17). The most effective LC exchanged 10D10 HC variants were transiently expressed at 15-80 mg / L and well expressed at >670 mg / L in stable production runs of CHO-K1.

[0315] Table 17. Improved expression of exemplary homology affinity matured variants

[0316] Example 7. Effects of affinity matured antibodies in animal models

[0317] The SEFL2 version of the LIBC528116-1 (37940-4) antibody (see Jacobsen FW et al., J Biol Chem. 2017 Feb 3;292(5):1865–1875) was scaled up for testing in diet-induced obese (DIO) transgenic mice in which huCB1 donor DNA was knocked in using CRISPR-Cas9 technology to mediate replacement 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 Diet D12492 60% kcal from fat) for 13 weeks to induce obesity. The LIBC528116-1 antibody and huIgG1 control antibody were administered to mice at a dose of 30 mg / kg (QW) per week for 60 days (n=7 mice / group), and body weight and food intake were measured every 3-4 days. Total fat mass was measured by MRI on treatment day 58, and the animals were analyzed for triglyceride and insulin levels. The results are shown in Tables 18 and Figures 6A-6C At day 60, the affinity matured 10D10 variant reduced body weight by 15.9%, fat mass by 22%, liver weight by 47%, liver triglycerides by 64%, and plasma insulin by 77% compared to the huIgG1 control (Table 18, Fig. 6A and 6C ). In contrast, an earlier study with the D83K / N35Y variant of 10D10 (26970-2) had no effect on weight gain compared to isotype control. These results suggest that the potent peripherally acting anti-CB1 antibodies described herein are effective treatments against obesity, hyperinsulinemia, and nonalcoholic steatohepatitis (NASH).

[0318] Table 18. Effects of affinity matured antibodies on body weight, fat mass and liver weight of huCB1 KI mice

[0319]

[0320] Fibrosis and inflammatory markers in animal kidneys were also measured. In particular, RNA was extracted and isolated from mouse kidney tissue by TRIzol extraction. 50-100mg tissue and 5mm stainless steel beads (Qiagen, #69989) were placed in a microcentrifuge tube equipped with 1mL TRIzol (Ambion, #15596018, Life Technologies). The TissueLyser II machine was used for 3 minutes and repeated at a frequency (1 / S) 30.0. The lysate was then incubated for 5 minutes and then centrifuged quickly. Then 200 μL of chloroform (Sigma-Aldrich, #372978-100mL) was added to each tube and vortexed for 30 seconds. Then the tube was incubated for 5 minutes, and then the tube was rotated at 4°C, 12,000g for 15 minutes. The top water layer was removed and placed in a new deep well plate and mixed with 70% EtOH of equal parts. After mixing, the solution was transferred from the RNeasy Plus 96 kit to the RNA plate and RNA isolation was performed according to the kit instructions (Qiagen, #74192). The Qiagen DNase step (Qiagen, #79254) was performed according to the kit instructions. RNA was diluted in RNase / DNase-free water and the concentration of RNA was confirmed by NanoDrop 8000. The RNA was then diluted to 50ng / 8.5uL water and run on the Quantstudio 7Flex Real-Time PCR System. The TaqMan RNA-to Ct One-Step Kit with TaqMan Gene Primer Probe Set (Applied Biosystems, #4392938) was used according to the instructions. CT values ​​were used to calculate relative RNA expression levels. Anti-CB1 antibody treatment reduced the expression levels of fibrosis and inflammatory markers in the kidney compared to control IgG ( Fig.6D ).

[0321] Example 8. New xenogeneic mouse antibody experimental activities

[0322] Mouse immunization: Several xenogeneic mouse groups 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 on gold particles and delivered intradermally; HEK293T cells were transiently transfected with full-length huCB1 with the E3k epitope. The cells were delivered subcutaneously; and nanoparticles (nanodiscs) containing huCB1 protein engineered for enhanced stability. The nanoparticles were delivered subcutaneously.

[0323] Immunizations take several months; blood samples are collected from animals and CB1 antibody titers are measured periodically. Because previous experience and published CB1 structures suggest that the large N-terminal domain of CB1 may not be relevant to drug function, only animals that show titers to both full-length and truncated CB1 (i.e., lacking 1-98 N-terminal amino acids) are selected for harvest to improve the chances of finding functional antibodies during screening. Titers are measured by FACS on CHO-S cells transiently transfected with full-length or truncated huCB1 or empty vector.

[0324] Plasma B cell screening: Traditional hybridoma generation relies on immortalization of B cells by fusion with myeloma cells. Due to low fusion efficiency, valuable B cell clones are difficult to recover through this process if there are few of them. To complement hybridoma generation, direct screening of antibody-secreting plasma B cells has been performed. The latter approach focuses on memory B cells and is used to increase the chances of discovering valuable clones by sampling different cell populations.

[0325] The B cells for screening are taken from the spleen, lymph nodes and bone marrow of the animals put to death. Plasma B cells are separated from memory B cells according to CD138 cell surface marker expression using commercial CD138 enrichment kit (Stemcell Technologies 17877). Then CD138+ cells are combined with HEK293T cells transfected with truncated huCB1 and HEK293T cells transfected with empty vectors. Three types of cells are mixed together in B cell culture medium, then mixed with warm agarose solution (Sigma A5030, final concentration 1%), put into fluorinated oil (RAN Biotechnologies 008- fluorinated surfactant-2wtH-50G) and emulsified by vortex. The result is a kind of oil-in-oil medium emulsion, wherein containing droplets, and cells are captured in the droplets. 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 fluorescent signals were manually picked using an Eppendorf micromanipulator. The isolated microgels were placed in separate PCR tubes containing lysis buffer and RT-PCR reactions were performed to rescue antibody sequences from B cells captured in the microgels for cloning and expression.

[0326] Molecular rescue and sequencing of CB1 antibodies in B cells: RNA (total or mRNA) was purified from samples containing single B cells producing CB1 antibodies using Agencourt RNA Clean XP magnetic beads. The purified RNA was used as a template in template switching PCR to generate first-strand cDNA by reverse transcription, followed by cDNA amplification. The cDNA was cleaned up using the Agencourt AMPure XP PCR Cleanup Kit and used as a template to amplify antibody heavy and light chain variable region (V) genes. Fully human antibody γ heavy chains were obtained by using cDNA as a template and amplifying the variable region of the γ heavy chain using multiplex PCR. The 5'γ chain-specific primer was annealed to the signal sequence of the antibody heavy chain, while the 3' primer was annealed to the region of the γ constant domain. Fully human antibody κ light chains were obtained by using cDNA as a template and amplifying the variable region of the κ light chain using multiplex PCR. The 5'κ light chain-specific primer was attached to the signal sequence of the antibody light chain, while the 3' primer was attached to the region of the κ constant domain. Fully human antibody λ light chains were obtained by using cDNA as a template and amplifying the variable region of the λ light chain using multiplex PCR. The 5'λ light chain specific primer is annealed to the signal sequence of the light chain, while the 3' primer is annealed to the region of the λ constant domain. The primers used in the multiplex PCR reaction also have overhangs, which facilitate Golden Gate cloning into an expression vector. The amplicon from the multiplex PCR reaction is purified using the Agencourt AMPure XP PCR cleanup kit. The clean PCR amplicon is then cloned into an expression vector using the standard Golden Gate cloning method and then transformed into chemically competent Escherichia coli bacterial cells. The bacterial colonies from the transformation are cultured and then sequenced directly. The amino acid sequence is derived from the corresponding nucleic acid sequence by bioinformatics methods. The derived amino acid sequence is then analyzed to determine the germline sequence origin of the antibody and to identify deviations from the germline sequence.

[0327] Hybridoma Generation and Screening: 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 secreted antibodies were screened for binding to full-length and truncated CB1 transiently expressed on HEK293T or CHO-S cells. Polyclonal wells showing CB1 binding were single cell sorted to obtain clones secreting individual antibodies.

[0328] A total of 1325 Xenomouse clones were screened, of which 71 bound to full-length and truncated huCB1. Most of the 71 clones were non-functional, and only 9 clones met the potency criteria set by the mouse experimental activities. Three unique antibodies were isolated from the nine clones. The sequences of the antibodies (Tables 19 and 20) were similar to that of 10D10. Notably, the Y57H mutation in HCDR2 that appeared after affinity maturation of the antibodies was also present in these antibodies (Table 19).

[0329] Table 19. CDR sequences of antibodies in mouse experimental activities

[0330]

[0331]

[0332] Table 20 Variable region sequences of antibodies from mouse experimental activities

[0333]

[0334]

[0335] Antibody Characterization: The antibody clones identified above were tested for binding to full-length human CB1 and N-terminally truncated human CB1 to confirm that the antibodies bind to the extracellular loops ECL1-3 and not to the CB1 N-terminus. Binding to human CB2 was also tested to ensure that no side effects would occur due to cross-reactivity with CB2. Finally, binding to mouse CB1 was tested. Following binding studies, functional screening was performed using the TR-FRET cAMP assay to measure the inhibitory effect of the antibodies on CB1.

[0336] To assess target specificity, binding on HEK293T cells transiently transfected with the target protein was measured by FACS and compared with nonspecific binding of cells transfected with an empty vector. Binding results were expressed as the ratio of the mean fluorescence intensity of target-transfected cells to mock-transfected cells. Results showed that all three antibodies bound to full-length and truncated human CB1, but not to human CB2 or mouse CB1 (Table 21).

[0337] The inhibitory effect of antibodies on CB1 function was evaluated using a time-resolved Forster resonance energy transfer (TR-FRET) cAMP assay. Antibodies that inhibit CB1 will result in an increase in cAMP, which in turn leads to a decrease in the FRET signal in the assay. Therefore, a lower assay signal means better inhibition of CB1 by the antibody. To increase assay sensitivity, CP55,940 and forskolin were used to activate cellular CB1 and adenylate cyclase, respectively. Rimonabant was used as a positive control.

[0338] Briefly, stably transfected CB1 CHO cells were cultured overnight in DMEM (Sigma Catalog No. D5671) containing 0.5% FBS, 25mM HEPES at 37°C / 5% CO2. On the day of the assay, CB1 antibody and rimonabant were titrated 1:4 at 4 times the final concentration using DMEM (Sigma Catalog No. D5671) containing 0.1% BSA (from PE kit), 25mM HEPES, and the titrated antibody was transferred to a 384-well assay plate at a volume of 7.5 μL / well. Cells were harvested using Accutase (Sigma) and neutralized with DMEM (Sigma Catalog No. D5671) containing 0.5% FBS, 25mM HEPES. These cells are precipitated and resuspended to 1.33x106 cells / mL using DMEM (Sigma catalog number D5671) containing 0.1% BSA (from PE kit), 25mM HEPES. Anti-cAMP antibodies from Lance Ultra cAMP detection kit (Perkin Elmer; TRF0263) are added to cells at a ratio of 1 to 150 of antibody to cell volume, and 3-isobutyl-1-methylxanthine (IBMX; Sigma) is added to cells at a final concentration of 1mM. Cells are transferred to assay plates with a volume of 15 μL per well or 20,000 cells per well. Forskolin and CP55,940 mixtures are diluted to 4 times the final concentration using DMEM (Sigma catalog number D5671) containing 0.1% BSA (from PE kit), 25mM HEPES, and transferred to assay plates with a volume of 7.5 μL per well. The assay plates were incubated at 37°C / 5% CO2 for 1 / 2 hour, then EU-cAMP tracer (PerkinElmer; TRF0263) was added to each well and further incubated for 1 hour at room temperature in the dark. After the final incubation, the assay plates were read using an Envision plate reader with EU615 and APC 665 emission filters. The data were analyzed using the following calculation: (665nm / 615nm) x 10,000. Lower values ​​(lower FRET) indicate stronger inhibition of CB1 and the data were used to calculate IC50. The results show that the potency of all three antibodies is much better than 10D10 (Table 22 and Fig. 9 ).

[0339] Table 21. Antibody binding data

[0340]

[0341] Table 22. Antibody efficacy data

[0342]

[0343] Example 9. Further affinity maturation of anti-CB1 antibodies

[0344] Another round of affinity maturation was performed to further improve functional potency. Analysis of the previous round of affinity matured antibodies (Examples 1-3 above) showed that there was a good correlation between GFP-tagged huCB1-SMALP binding and the IC50 of the homologous 10D10 variants, and that huCB1-GFP-SMALP binding predicted the majority of the most potent LC-exchanged variants. Therefore, improving binding to huCB1-GFP-SMALP was the focus of the screening and sorting process in this round of affinity maturation to obtain antibodies with subnanomolar potency.

[0345] Two strategies were used in this affinity maturation. First, the VK1 LC shared by the top LC-exchange variants unexpectedly belonged to a well-studied germline with high confidence LCDR positions predicted to be solvent-exposed and tolerant to mutation. Therefore, to further optimize their LCs, we sought to pair the HCs of LIBC528116-1 and other top LC-exchange variants with a VK1 / O2 LCDR library previously used in an in-house affinity maturation program. While co-crystal structures suggested that mutations at solvent-exposed LC positions were unlikely to make contacts with huCB1, we speculated that LC mutations might provide more optimal structural support for HC loop conformations to enhance binding and potency. To increase selection stringency, a monovalent yeast-displayed Fab library was constructed and sorted / screened for improved binding to wtCB1-SMALP while retaining huCB1-ND binding (compared to LIBC58116-1 Fab).

[0346] Secondly, we utilize the unexpected discovery (referring to Example 8) of more 10D10-like antibodies from new mouse experimental activities to utilize better affinity maturation starting points.Antibodies from new mouse experimental activities show that certain positions in HCDR2 and HC FR3 can be further explored to improve binding and effectiveness.In addition, new antibodies are clustered into two highly correlated HCDR3s, whose lengths differ by one amino acid (such as LIBC560657-1 and LIBC529593-1 of Example 8), indicating that the length of HCDR3 can be changed.The yeast display library for further homology affinity maturation merges the HC and LC mutations of the most effective homology 10D10 variants from Example 2 into the LIBC529593-1 sequence background and incorporates two HCDR3 lengths.HC library 2 incorporates shorter HCDR3 sequences, and is also included in the repair of the DS isomerization site for prediction at HCDR3 position 112.The designs of other homology affinity maturation libraries are summarized in Tables 23-25.

[0347] Table 23. Design of 10D10-like HC Library 1 for further homologous AM*

[0348] 10D10 residue Location Diversification strategy Y57 HCDR2 <![CDATA[H # ]]> I58 HCDR2 I.V Y60 HCDR2 Y, Q S65 HCDR2 E, A, K, T S67 HCDR2 S, Q N69 HCDR2 E, A, K, T S73 HCDR2 S, N L74 HCDR2 L.F. K75 HCDR2 <![CDATA[K、R # ]]> S76 HCDR2 S, G, N, D A78 HFR3 <![CDATA[V # ]]> T79 HFR3 <![CDATA[T、R # ]]> D83 de ring (HFR3) N S85 de ring_(HFR3) Y M136 HCDR3 <![CDATA[M、L # ]]>

[0349] #: Mutations found in LIBC529593-1

[0350] *Theoretical portfolio diversity: 1.6E4

[0351] Table 24. Design of 10D10-like HC Library 2 for further homologous AM*

[0352]

[0353]

[0354] #: Mutations found in LIBC529593-1

[0355] *Theoretical portfolio diversity: 6.5E4

[0356] Table 25. Design of 10D10-like LC library for further homologous AM*

[0357] 10D10 residue Location Diversification strategy S34 LCDR1 <![CDATA[R # ]]> N35 LCDR1 <![CDATA[S # ]]> L58 LCDR2 L, Q R94 LC FR3 <![CDATA[S # ]]> M107 LCDR3 R A109 LCDR3 <![CDATA[A、S # ]]> L110 LCDR3 V.R Q111 LCDR3 A.G. T135 LCDR3 I.L

[0358] #: Mutations found in LIBC529593-1

[0359] * Theoretical combination diversity: 33 (32+1)

[0360] 10D10 HC and LC library designs were combinatorially paired to construct a Fab yeast library to select for increased binding stringency. The homologous library for further affinity maturation was sorted / screened to improve binding to wtCB1-SMALP while retaining huCB1-ND binding (compared to LIBC523797-1 Fab).

[0361] Libraries constructed for further affinity maturation experiments were sorted against successively lower concentrations of huCB1-GFP-SMALP (starting from 25 or 50 nM). We performed clone binding screening at concentrations of huCB1-SMALP as low as 2-5 nM (which represents a concentration 20-50x lower than that used in previous affinity maturation experiments (Examples 2-4). The most improved binders showing minimal non-specific binding and predicted sequence propensity were then selected from further VK1 LC optimization experiments and further homologous affinity maturation experiments based on similar principles as described previously (Examples 2 and 3). Eight clones were obtained from VK1 LC optimization and 18 clones were obtained from further homologous affinity maturation. Compared with the LIBC528116-1 and LIBC523797-1 Fab-yeast benchmarks from LC exchange and homologous experiments, respectively, these top clones showed improved huCB1-SMALP binding, similar huCB1-ND binding and low levels of empty ND binding.

[0362] Tables 26 and 27 and Figure 7 The CDR sequences, germline information and binding data of the first 8 further LC exchange affinity matured variants are summarized. All top clones showed improved binding to CB1-SMALP and CB1-ND (Table 28), while showing minimal binding to the unrelated XCR1-SMALP and empty-ND. For LIBC681737-1, the LC P12S germline reversion mutation was included when preparing the IgG1SEFL2 mAb for functional testing.

[0363] Table 26. CDR sequences of the first 8 further VK1 LC optimized variants

[0364]

[0365]

[0366] Table 27. Variable region sequences of the first 8 further VK1 LC optimized variants

[0367]

[0368]

[0369] Table 28. Germline information of the first 8 further VK1 LC optimized variants

[0370]

[0371]

[0372] Table 29. Binding data for the first 8 further VK1 LC optimized variants

[0373]

[0374] *Relative binding fold change compared to LIBC528116 (Fab), calculated after background subtraction

[0375] Tables 30 and 31 and Fig. 8A and 8B Summarize the CDR sequence, germline information and binding data of the first 18 further homologous affinity maturation variants.Most of 18 binders utilize shorter HCDR3, and possess HC T79R FR3 mutation in the library design of HC library 2, which highlights the benefit of rationally merging the features from all available sequence-function relationships.All 18 homologous variants meet the strict combination improvement threshold for huCB1-GFP-SMALP, and have minimum combination (table 32) with unrelated XCR1-SMALP and empty-ND.Relative to LIBC523797-1, these variants also show better combination with CB1 SMALP.For LIBC674043-1 and LIBC674200-1, VH R149S and VL G76D germline reversion mutations are also included in the IgG1 SEFL2 mAb group produced for functional testing.

[0376] Table 30. CDR sequences of the first 18 further homologous variants

[0377]

[0378]

[0379]

[0380] Table 31. Variable region sequences of the first 18 further homologous variants

[0381]

[0382]

[0383]

[0384]

[0385]

[0386] Table 32. Binding data for the first 18 further homologous variants

[0387]

[0388]

[0389] *Relative binding fold change compared to LIBC523797-1 (Fab), calculated after background subtraction

[0390] Example 10. Functional analysis of further affinity matured variants

[0391] The clones isolated from Example 9 above were cloned and expressed as IgG1 SEFL2 mAbs for functional analysis. The antibodies were analyzed for antagonist activity using the TR-FRET cAMP assay as described above (e.g., Example 8), and the potency of the antibodies is summarized in Table 33. The potency of all antibodies in all four experiments was in the single digit nM, with some antibodies having a potency of less than 1 nM.

[0392] Table 33. IC50 values ​​of further affinity matured exemplary antibodies

[0393]

[0394] Example 11. Effects of further affinity matured variants in animal models

[0395] Further affinity matured antibodies LIBC673952 (52777) and LIBC680574 (52778) in YTE format (Dall'Acqua et al., The Journal of Immunology, 169:5171-5180 (2002)) were scaled up for testing in diet-induced obese (DIO) transgenic mice into which huCB1 was knocked in. 6-10 week old huCB1 knock-in (KI) mice were fed a high fat diet (HFD) for 15 weeks to induce obesity. Antibodies (52777, 52778 and 37940) and control antibody (huIgG control) were administered to 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 Table 1. Fig. 10A and 10B Administration of further affinity matured antibodies reduced the body weight of the animals ( Fig. 10A ), while average daily food intake did not change significantly ( Fig. 10B ). The animals’ fat mass and liver weight were also measured and the results showed Fig. 10C and 10D These results suggest that the potent peripherally acting anti-CB1 antibodies described herein are effective treatments against obesity, hyperinsulinemia and NASH.

[0396] Example 12. Effects of anti-CB1 mAbs on energy expenditure and insulin tolerance testing

[0397] Materials and Methods: All studies using mice complied with all relevant ethical regulations, and the studies performed were approved by the IACUC Institutional Review Board of Amgen (Thousand Oaks, CA). The lighting in the animal housing room was maintained at a 12:12 h light:dark cycle, and the ambient temperature and humidity ranged from 68-79°F and 30-70%, respectively. Animals had ad libitum access to irradiated pelleted diet and reverse osmosis chlorinated water (0.3 to 0.5 ppm) via an automatic watering system or via water bottles as directed. Cages were changed weekly.

[0398] Male huCNR1 TG mice (4-7 weeks old) were shipped from Charles River Laboratories (Hollister, CA) to Amgen (Thousand Oaks, CA), housed individually upon arrival, and started on a high-fat diet (HFD) (60% kcal fat, research diet D12492i) for 12 weeks. After 12 weeks of HFD feeding, mice were adapted to water bottles for one week, and then n = 24 mice were transferred to a comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments, Columbus, OH - Oxymax Model 2018, 0233-004M, Oxymax Windows v5.53 software, hardware configuration 190395) for an additional two weeks of adaptation. Oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange ratio (RER), and energy expenditure (HEAT) were measured during this period. At the start of treatment, mice had been fed a 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 calories during baseline measurements from day -3 to day -1. Mice were given huIgG1 control PL-42466 (n = 11 mice) or CB1 mAb 37940-17 (n = 12 mice) IP at 30 mg / kg (2 mL / kg) per week for a total of 5 injections. Mice were housed in the CLAMS system from day -14 to day 7, returned to the cage on days 7-14, returned to the CLAMS system from day 14 to day 28, and returned to the cage on day 28 for study. Body weight and food intake were measured weekly. For simplicity, data are shown as lights on and lights off cycles (12 hours on: 12 hours off).

[0399] For insulin tolerance test (ITT), mice were fasted for 6 hours at 6 am on treatment day 32 (4 days after the 5th IP injection). At 12 noon, blood glucose was measured by retro-orbital blood sampling using a glucometer (AlphaTrak), and insulin (0.75 U / kg) was given to mice IP immediately. Blood glucose was measured by retro-orbital blood sampling at 30, 60, 90, and 120 minutes after insulin administration. The area under the curve (AUC) for each mouse was calculated using GraphPad Prism (version 9.5.1).

[0400] Results: Energy expenditure measured as calories (kcal / hr / kg) and respiratory exchange ratio (RER) were averaged weekly (acclimation, week 1, week 3, and week 4) for both the light cycle (6am-6pm) and dark cycle (6pm-6am). Energy expenditure was significantly higher in CB1 mAb treated mice during week 4 of treatment when mice were resting during the light cycle (+12% increase from baseline for CB1 vs. +4% increase from baseline for controls, p=0.0325; two-way repeated measures ANOVA with Sidak's multiple comparison test, Fig.11A In addition, during the third week of treatment, the RER in the CB1 mAb-treated group was significantly decreased (treatment p = 0.0125; two-way repeated measures ANOVA with Sidak's multiple comparison test), indicating increased lipid oxidation ( Fig.11A ).

[0401] To assess insulin sensitivity, an insulin tolerance test (ITT) was performed. After fasting for 6 hours, there was no difference in baseline blood glucose measurements of mice; however, after administration of insulin, 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 multiple comparison test) and a 25% lower AUC (p=0.0006; Student's unpaired t-test), indicating that insulin sensitivity was significantly improved after CB1 mAb treatment ( Fig. 11B ).

[0402] Example 13. Brain uptake study of ANT-CB1 MAB in Tg32 mice

[0403] This study was conducted to characterize the single-dose plasma pharmacokinetics and brain uptake of exemplary anti-CB1 antagonist mAbs following intravenous bolus administration in male huFcRn Tg 32 homozygous mice.

[0404] Materials and methods: Food and water were provided ad libitum throughout the study. One day before dosing, the reserve test article was removed from -70°C (±10°C) storage and placed at 2-8°C to thaw overnight. On the morning of dose administration, the reserve test article was removed from 2-8°C storage, mixed by gentle inversion, and placed on wet ice for dilution procedures. The vehicle was removed from 2-8°C storage and mixed by gentle inversion and placed on wet ice for dilution procedures. For all groups, the reserve test article was diluted in the appropriate formulation buffer to obtain the final dose solution concentration listed in the experimental design. After the dose solution was prepared, all remaining vehicles (if applicable) were returned to 2-8°C for storage. After dose administration, all remaining reserve test articles (if applicable) and remaining dosing solutions (if applicable) were placed at -70°C (±10°C) and transferred to the bioanalysis principal investigator for subsequent analysis and storage. Test animals received intravenous bolus doses of appropriate dosing solutions through the lateral tail vein.

[0405] SARSTEDT K3 EDTA plasma separator tubes were used to collect approximately 0.05 mL of whole blood from each test system at each serial time point via submandibular venipuncture. After the plasma tubes were placed, the tubes were mixed by gently inverting 8-10 times and placed on wet ice. The samples were centrifuged at about 14,000 rcf for 5 minutes at 2-8°C using a calibrated Eppendorf 5417R centrifuge system (Brinkmann Instruments, Inc., Westbury, New York 11590). All plasma samples were stored at about -70°C (± 10°C) before analysis.

[0406] Transcardial perfusion was performed at the end of the study. Each test system was deeply anesthetized with isoflurane (5% O2(g) at a rate of 1.5 L / min) and maintained at a surgical plane of anesthesia throughout the perfusion. 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 through the diaphragm and along both sides of the rib cage to expose the heart, allowing the animal to develop a pneumothorax. The heart was stabilized and a 20-gauge, 1 A 1 / 2" needle is placed into the left ventricle. The needle is connected by tubing to a perfusion / syringe pump, and 0.9% saline is allowed to run at a rate of approximately 4 mL / minute for approximately 5 minutes. Once flow is established, the right atrium is cut open to allow fluid to flow out of the animal and prevent over-pressurization of the vasculature. After 5 minutes of perfusion with saline, 10% neutral buffered formalin is perfused for an additional 5 minutes. Once perfusion is complete, the needle is removed from the left ventricle and one or more appropriate tissues are harvested for subsequent analysis.

[0407] Tissues were harvested from each test system immediately after bleeding at the final time point, as described below. After extraction, tissues were rinsed with 0.9% sterile saline, blotted with dry gauze, immediately placed in cryovials (Eppendorf Protein LoBind, catalog # 0030108116), weighed, and immersed in liquid nitrogen to snap-freeze the tissues. Once the tissues were snap-frozen, they were stored on dry ice during necropsy and stored at -70°C (±10°C) for subsequent analysis.

[0408] Plasma and tissue samples for PK test article concentration determination were analyzed using enzyme-linked immunosorbent assay (ELISA). Samples were kept at -70°C (±10°C) prior to analysis. Non-compartmental analysis of mean plasma test article concentrations for all mice in each dose group at each sampling time versus time data was performed. Parameters of interest included tissue-to-plasma percentage ratios and half-life (t 1 / 2 ).

[0409] Results: The PK and brain exposure of the antibodies 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 human neonatal Fc receptor (hFcRn) are known to predict the human PK of mAb therapeutics (Avery et al. mAbs [monoclonal antibodies] 20161). These results suggest that 0.002 fractions of mAb plasma concentrations can 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 LB 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 DK 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 et al., 2014 Mar-Apr;5(2):297-305. doi:10.4161 / mabs.23684. Epub 2013 Feb 13. PMID:23406896; PMCID:PMC3893240; and Garg A. and Balthasar et al., 2014 Mar-Apr;5(2):297-305. doi:10.4161 / mabs.23684. Epub 2013 Feb 13. PMID:23406896; PMCID:PMC3893240; and JP, AAPS J. 2009 Sep;11(3):553-7. doi:10.1208 / s12248-009-9129-9. Published July 28, 2009. PMID:19636712; PMCID:PMC2758122).

[0410] Table 34 PK and brain exposure of anti-CB1 mAbs

[0411]

[0412] Example 14. CRYO-EM structure of CB1-Fab complex

[0413] The cryo-EM structure of huCB1 in complex with 10D10 Fab and four affinity matured (AM) antibodies was 3.1 to 1. The resolution of the antibody was resolved, which is sufficient to determine the epitope / paratope interaction. The potency of the antibody ranged from 1.77 to 47nM. The cryo-EM structure confirmed that only the heavy chain of the antibody was involved in binding and that ECL2 of CB1 was 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 sequence of the AM antibody is highly conserved. R32 (AHo numbering), adjacent to HCDR1, is also involved in epitope interactions. HCDR2 is diverse in sequence, and certain residues in HCDR2 (e.g., Y59, AHo numbering) appear to contribute to binding affinity. Y131 and Y132 (AHo numbering) of HCDR3 are conserved among the antibodies tested and bind to CB1.

[0414] Table 35. Consensus sequences of affinity matured variants

[0415]

[0416]

[0417]

[0418] Although the present invention has been described with emphasis on preferred aspects, it will be apparent to those skilled in the art that variations of the preferred compounds and methods may be used, and that the present invention is intended to be practiced in a manner other than as specifically described herein. Therefore, the present invention includes all modifications encompassed within the spirit and scope of the present invention as defined by the following claims.

Claims

1. An isolated antibody that binds to human cannabinoid receptor 1 (huCB1), wherein the antibody comprises a heavy chain variable region (HV) comprising heavy chain complementary regions (CDRs) HCDR1, HCDR2 and HCDR3, and a light chain variable region (LV) comprising light chain complementary regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein the heavy chain and light chain CDRs are selected from any of the following: (a) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 135-137, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 138-140, respectively; (b) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 426-428, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 423-425, respectively; (c) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequence of SEQ ID NOs: 266-268, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence of SEQ ID NOs: 263-235, respectively; (d) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 129-131, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 132-134, respectively; (e) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 159-161, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 162-164, respectively; (f) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 207-209, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 210-212, respectively; (g) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1-3, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4-6, respectively; and (h) HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 55-57, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 58-60, respectively.

2. The antibody of claim 1, wherein the antibody comprises an HV comprising an amino acid sequence selected from the group consisting of 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 of claim 1 or 2, wherein the antibody comprises a LV comprising an amino acid sequence selected from the group consisting of 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 of any one of claims 1-3, wherein the antibody comprises an HV and a LV selected from any one of the following: i) HV comprising the amino acid sequence of SEQ ID NO: 228, LV comprising the amino acid sequence of SEQ ID NO: 227; ii) HV comprising the amino acid sequence of SEQ ID NO: 460, LV comprising the amino acid sequence of SEQ ID NO: 459; iii) HV comprising the amino acid sequence of SEQ ID NO: 368, LV comprising the amino acid sequence of SEQ ID NO: 367; iv) HV comprising the amino acid sequence of SEQ ID NO: 100, LV comprising the amino acid sequence of SEQ ID NO: 99; v) HV comprising the amino acid sequence of SEQ ID NO: 236, LV comprising the amino acid sequence of SEQ ID NO: 235; vi) HV comprising the amino acid sequence of SEQ ID NO: 226, LV comprising the amino acid sequence of SEQ ID NO: 225; vii) HV comprising the amino acid sequence of SEQ ID NO: 252, LV comprising the amino acid sequence of SEQ ID NO: 251; viii) HV comprising the amino acid sequence of SEQ ID NO: 98, LV comprising the amino acid sequence of SEQ ID NO: 97; and ix) HV comprising the amino acid sequence of SEQ ID NO:116, and LV comprising the amino acid sequence of SEQ ID NO:

115.

5. An isolated antibody that binds to huCB1, wherein the antibody comprises HV and LV, wherein a) the HV comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A, S or G; HCDR2 comprises the amino acid sequence 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; and X10 is G, D, S or N; and HCDR3 comprises the amino acid sequence 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; and X15 is H, N or Y; and b) The LV belongs to the VK1 / O2 / JK4, VK2 / A19 / JK1 or VK3 / A27 / JK1 lineage.

6. The antibody according to claim 5, wherein a) HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A or S; HCDR2 comprises the amino acid sequence 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; and X10 is G, D, S or N; and HCDR3 comprises the amino acid sequence 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; and X15 is H or Y; and b) The LV belongs to the VK2 / A19 / JK1 lineage.

7. The antibody of claim 6, wherein X4 is T and X15 is Y.

8. The antibody of any one of claims 5-7, wherein b) The LV includes 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; and X19 is L or V; LCDR2 comprises the amino acid sequence X20GSNRA (SEQ ID NO:534), wherein X20 is L or Q; and LCDR3 comprises the amino acid sequence 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; and X24 is T, I, L or Q.

9. The antibody of claim 8, wherein X24 is L.

10. The antibody of claim 5, wherein a) HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A, G or S; HCDR2 comprises the amino acid sequence 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; and X10 is G, D, S or N; and HCDR3 comprises the amino acid sequence 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; and X15 is H, N or Y; and b) The LV belongs to the VK1 / O2 / JK4 or VK3 / A27 / JK1 lineage.

11. The antibody of claim 10, wherein a) X1 is A or G; X3 is Q or Y, X4 is T, X8 is F, X14 is T; and X15 is Y.

12. The antibody of claim 10 or 11, wherein b) the LV belongs to the VK1 / 02 / JK4 germline.

13. The antibody according to claim 10 or 11, wherein b) the LV belongs to the VK1 / O2 / JK4 germline and comprises light chains LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises the amino acid sequence RASQSISNYLN (SEQ ID NO: 132), RASQSIISYLN (SEQ ID NO: 150), or RASQSISSYLN (SEQ ID NO: 186); LCDR2 comprises the amino acid sequence AASSLHS (SEQ ID NO: 133) or AASSLRS (SEQ ID NO: 151); and LCDR3 comprises the amino acid sequence QQYQSYPLT (SEQ ID NO: 134) or QQYSNYPLT (SEQ ID NO: 152); or b) the LV belongs to the VK3 / A27 / JK1 germline and comprises light chains LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises the amino acid sequence RASQSVSSYLG (SEQ ID NO: 168); LCDR2 comprises the amino acid sequence GASSRAT (SEQ ID NO: 169); and LCDR3 comprises the amino acid sequence QQYGSSPRT (SEQ ID NO: 170).

14. The antibody of claim 13, wherein LCDR1 comprises the amino acid sequence RASQSISNYLN (SEQ ID NO: 132), LCDR2 comprises the amino acid sequence AASSLHS (SEQ ID NO: 133), and LCDR3 comprises the amino acid sequence QQYQSYPLT (SEQ ID NO: 134).

15. The antibody of claim 10, wherein a) HCDR1 comprises the amino acid sequence RGGDYWX1 (SEQ ID NO: 530), wherein X1 is A or G, HCDR2 comprises the amino acid sequence HX2YX3X4GX5TX6YNPX7X8X9X10 (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; and X10 is G or D; and HCDR3 comprises the amino acid sequence X11YDX12X13X14GX15SYYYYGMDV (SEQ ID NO: 532), wherein X11 is N; X12 is T; X13 is L or V; X14 is T; and X15 is Y, and b) The LV belongs to the VK1 / O2 / JK4 lineage.

16. The antibody of claim 15, wherein a) HCDR1 comprises the amino acid sequence RGGDYWA (SEQ ID NO: 414) or RGGDYWG (SEQ ID NO: 408); HCDR2 comprises the amino acid sequence HVYYTGSTKYNPSFKD (SEQ ID NO:427), HVYYTGSTNYNPRFKD (SEQ ID NO:445), HIYQTGSTNYNPRFKG (SEQ ID NO:415), or HVYQTGSTKYNPSFKD (SEQ ID NO:409); and HCDR3 comprises the amino acid sequence NYDTLTGYSYYYYGMDV (SEQ ID NO: 410) or NYDTVTGYSYYYYGMDV (SEQ ID NO: 446).

17. The antibody of claim 15 or 16, wherein b) the LV comprises LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises the sequence RASQSISSYLN (SEQ ID NO:405); LCDR2 comprises the sequence X39ARX40LX41S (SEQ ID NO:536), wherein X39 is N, S, K or G; X40 is R, K, L or A; and X41 is A, G or S; and LCDR3 comprises the sequence 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; and X46 is L, I or V.

18. The antibody of claim 17, wherein X42 is Y.

19. The antibody of any one of claims 5-18, 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. The antibody of claim 19, wherein the amino acid at position 94 of the LV is S.

21. An isolated antibody that binds to huCB1, the antibody comprising HV and LV, wherein a) The HV comprises CDRH1, CDRH2 and CDRH3, wherein CDRH1 comprises the amino acid sequence RGGDYWS (SEQ ID NO: 1); CDRH2 comprises the amino acid sequence 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; and X33 is G, S or N; and CDRH3 comprises the amino acid sequence 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; and X38 is M or L; and b) The LV belongs to the VK2 / A19 / JK1 lineage.

22. The antibody of claim 21, wherein a) X26 is Y, X34 is G, X36 is absent, and X37 is S.

23. The antibody of claim 21 or 22, wherein b) the LV comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence RSSQSLLHRSGYNYLD (SEQ ID NO: 257); LCDR2 comprises the amino acid sequence LGSNRAS (SEQ ID NO: 258) or QGSNRAS (SEQ ID NO: 264); and LCDR3 comprises the amino acid sequence MQSLQTPRT (SEQ ID NO: 259), RQSVALPRT (SEQ ID NO: 271) or RQARALPRT (SEQ ID NO: 265).

24. The antibody of any one of claims 21-23, 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 of claim 24, wherein the amino acid at position 79 of the HV is R.

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

27. The antibody of any one of claims 5-26, wherein the HCDR2 of the antibody comprises 5 or fewer mutations compared to HVYYTGSTNYNPRFKD (SEQ ID NO: 136).

28. The antibody of claim 27, wherein HCDR3 of the antibody comprises 3 or fewer mutations compared to NYDTVTGYSYYYYGMDV (SEQ ID NO: 137).

29. The antibody of any one of claims 5-26, wherein HCDR2 of the antibody comprises 5 or fewer mutations compared to HVYYTGSTKYNPNFKG (SEQ ID NO: 8).

30. The antibody of claim 29, wherein HCDR3 of the antibody comprises 4 or fewer mutations compared to DYDILTGYSYYYYGMDV (SEQ ID NO: 9).

31. An isolated antibody or antigen-binding fragment thereof that binds huCB1, wherein the antigen-binding molecule comprises an HV comprising HCDR1, HCDR2 and HCDR3 and a LV comprising LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence RGGDYWS (SEQ ID NO: 474) or RGGDYWN (SEQ ID NO: 480), HCDR2 comprises the amino acid sequence HIYYSGSTNYNPSLRS (SEQ ID NO: 475), HIYYSGSKNYNPSLKS (SEQ ID NO: 481) or HIYYTGTKYYNPSLKS (SEQ ID NO: 487), and HCDR3 comprises the amino acid sequence DYDILYGYSYYYYGLDV (SEQ ID NO: 476) or GYDSSGYSYYYYGMDV (SEQ ID NO: 475), and wherein LCDR1 comprises the amino acid sequence RSSQSLLHRSGYNYLD (SEQ ID NO: 488). NO:471), RSSQSLLYSNGHNFLD (SEQ ID NO:477) or RSSQSLLYSNGHNYLD (SEQ ID NO:483), LCDR2 comprises the amino acid sequence LGSNRAS (SEQ ID NO:472) or LGSNRAP (SEQ ID NO:484), and LCDR3 comprises the amino acid sequence MQSLQTPRT (SEQ ID NO:473) or MQALQTPRT (SEQ ID NO:479).

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

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

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

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

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

38. The antibody of claim 36 or 37, wherein the antibody has a binding affinity for huCB1 that is at least 3-fold higher than that of an antibody 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 (10D10 LC N35Y).

39. The antibody of claim 36, wherein the antibody has an IC50 of 10 nM or less as determined using a cell-based cAMP assay.

40. The antibody of claim 39, wherein the antibody has an IC50 of 5 nM or less, or 1 nM or less, as determined using a cell-based cAMP assay.

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

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

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

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

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

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

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

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

49. The antibody of claim 40, wherein the antibody comprises: i) the heavy chain constant region amino acid sequence of SEQ ID NO: 525, 526 or 527 and the light chain constant region amino acid sequence of SEQ ID NO: 518 or 519; or ii) the heavy chain constant region amino acid sequence of SEQ ID NO:525, 526 or 527 and the light chain constant region amino acid sequence of SEQ ID NO:

518.

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

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

52. An expression vector comprising the polynucleotide of claim 51.

53. A host cell comprising the expression vector of claim 52.

54. A method of producing an antibody that binds to huCB1, the method comprising culturing the host cell of 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 of treating a subject in need of antagonism or inverse agonism of a CB1 receptor, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-49 or the pharmaceutical composition of claim 50.

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

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

58. The method of claim 57, wherein the administration results in one or more of weight loss, decreased appetite, improved metabolic parameters, decreased blood glucose levels, decreased insulin levels, decreased triglyceride levels, decreased renal injury, decreased renal fibrosis, decreased renal inflammation, and improved renal function.

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

60. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an antibody as described in any one of claims 1-49 or a pharmaceutical composition as described in 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 of 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 of claim 60 or 61, wherein the administration results in one or more of weight loss, decreased appetite, improved metabolic parameters, decreased blood glucose levels, decreased insulin levels, decreased triglyceride levels, decreased renal injury, decreased renal fibrosis, decreased renal inflammation, and improved renal function.

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

64. The antibody of any one of claims 1-49 or the pharmaceutical composition of claim 50, for use in antagonizing or inverse agonizing peripheral CB1 receptors.

65. An antibody as described in any one of claims 1-49 or a pharmaceutical composition as described in claim 50, for use in treating a disease or disorder selected from the group consisting of obesity, diabetes, dyslipidemia, metabolic diseases, 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 of claim 64 or 65, wherein the use results in one or more of weight loss, decreased appetite, improved metabolic parameters, reduced blood glucose levels, reduced insulin levels, reduced triglyceride levels, reduced renal injury, reduced renal fibrosis, reduced renal inflammation, and improved renal function.

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

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

69. The method or antibody of claim 68, wherein the subject has a blood pressure of at least 30 kg / m 2 BMI.

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