FIELD OF THE INVENTION

[0001] The present invention relates to antibodies and antigen-binding fragments thereof that bind to cannabinoid receptor 1 (CBl) receptor, and methods of using such antibodies and antigen-binding fragments.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: 15-343-PRO_Seq_List_2015-09-30_ST25, date recorded: September 30, 2015, file size 803 KB).

BACKGROUND

[0003] Cannabinoid 1 (CBl) receptor is a member of the G protein-coupled receptor (GPCR) superfamily. The CBl receptor is expressed in the central nervous system (CNS), lungs, liver, adipose tissue and kidneys, and has been implicated in many human diseases including obesity, diabetes, fibrosis, liver diseases, cardiovascular disease, cancer, pain, MS spasticity, and glaucoma, among others. More specifically, CBl receptor has been shown to exhibit detrimental activity in, for example, obesity, diabetes, fibrosis, liver diseases, cardiovascular disease and cancer; and has been shown to exhibit beneficial activity in pain, MS spasticity and glaucoma, among others.

[0004] There is a need in the art for new CBl receptor antagonists and agonists for therapeutic purposes as well as selective binders for diagnostic/imaging purposes. In particular, a CB 1 receptor-targeting compound that lacks the capacity for CNS penetration would be desirable to reduce potential CNS-mediated side effects of CBl receptor modulation, highlighted by the psychiatric adverse events associated with the CBl inverse agonist rimonabant. SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof that bind to cannabinoid 1 receptor (also referred to herein as "CBl receptor" or "CBl"). In some embodiments, the CBl receptor is a human CBl receptor. In some embodiments, the antibody or fragment thereof recognizes one or more extracellular epitopes on the CBl receptor. In some embodiments, the CBl receptor binding antibodies and fragments thereof provided herein are functional antibodies or antigen binding fragments thereof. In some embodiments, the CBl receptor binding antibodies or fragments thereof inhibit or increase CBl receptor signaling activity. In some embodiments, the CBl receptor binding antibodies or fragments thereof are antagonistic antibodies, in that they inhibit CBl receptor signaling activity. In some embodiments, the CBl receptor binding antibodies or fragments thereof are agonistic antibodies, in that they enhance CBl receptor signaling activity. In some embodiments, the CBl receptor binding antibodies or fragments thereof are modulators of CBl receptor signaling activity or are allosteric modulators of CBl receptor signaling activity. In some embodiments the CBl receptor binding antibodies or fragments thereof are selective binders without agonist or antagonist activity. In some embodiments, the CBl receptor binding antibodies or fragments thereof are selective binders without agonist or antagonist activity that are useful for diagnostic and/or imaging purposes.

[0006] The isolated antibodies or antigen binding fragments thereof, in some embodiments, are at least as potent as small molecule CBl receptor modulators such as, for example, AM6545, AM251, or rimonabant. In some embodiments, the antibodies or fragments thereof have CBl receptor inhibiting or activating activity that is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, or at least 20 fold more potent relative to the small molecules AM6545, AM251, or rimonabant. In some embodiments, the isolated antibodies or antigen binding fragments thereof inhibit CBl receptor agonist-mediated signal transduction. In some embodiments, the inhibition of CBl receptor agonist-mediated signal transduction is measured by determining intracellular cAMP levels and/or downstream ERK phosphorylation. [0007] In some embodiments, the isolated antibodies and antigen-binding fragments thereof have the advantage of reduced or absent brain penetration. In some embodiments, the brain penetration of the isolated antibodies and antigen-binding fragments thereof exhibit reduced brain penetration relative to small molecule CB1 receptor agonists or antagonists (e.g., AM6545, AM251 , or rimonabant). In some embodiments, the CB1 receptor binding antibodies and fragments thereof provided herein provide a therapeutic benefit with reduced central nervous system side effects relative to a small molecule CB1 receptor agonist or antagonist. CNS side effects associated with small molecule CB1 receptor antagonist rimonabant include anxiety, depression, agitation, eating disorders, irritability, aggression, and insomnia (Moreira, 2009, Rev Bras Psiquiatr., 31(2): 145-53).

[0008] In some embodiments, the isolated antibodies and antigen-binding fragments thereof provided herein are generated from hybridoma cell lines. In other embodiments, the isolated antibodies and antigen-binding fragments thereof provided herein are generated from phage display libraries.

[0009] In some embodiments, the isolated antibodies and antigen-binding fragments thereof provided herein have an affinity for native human CB 1 receptor that is at least nM range. For example, in some embodiments, the affinity for CB1 receptor is about 1 μΜ or less, or about 750 nM or less, or about 500 nM or less, or about 250nM or less, or about 100 nM or less, or about 75 nM or less, or about 50 nM or less, or about 25 nM or less, or about 10 nM or less, or about 1 nM or less. In other embodiments, the isolated antibodies and antigen-binding fragments disclosed herein have an affinity for CB1 receptor of about 15 nM or less. In some embodiments, the isolated antibodies and antigen-binding fragments thereof have an affinity for human CB1 receptor that is from about 0.01 nM to about 500 nM, about 0.02 nM to about 250 nM, about 0.02 to about 200 nM, about 0.05 to about 100 nM, about 0.05 to about 50 nM.

[0010] The isolated antibodies and antigen binding fragments thereof of the present invention may be derived from any species including, but not limited to, mouse, rat, rabbit, hamster, guinea pig, primate, llama or human. In some embodiments, the isolated antibodies and antigen binding fragments thereof are murine antibodies. In other embodiments, the isolated antibodies and antigen binding fragments thereof are chimeric antibodies. In still other embodiments, the isolated antibodies and antigen binding fragments thereof are humanized antibodies. In some embodiments, the isolated antibodies and antigen binding fragments thereof are fully human antibodies.

[0011] In one embodiment, the isolated antibodies and antigen binding fragments thereof are humanized or chimeric P1C4 antibodies, as described herein. In one embodiment, the humanized P1C4 antibodies are selected from the group consisting of P1C4-H0, P1C4-H2, and P1C4-H4, as described herein. In one embodiment, the isolated antibodies and antigen binding fragments thereof comprise Fc modifications that result in reduced, impaired, or eliminated antibody effector function. In a further embodiment, the isolated antibodies and antigen binding fragments thereof are selected from the group consisting of PlC4-H0-IgG2-4 Hybrid, P1C4-H0- IgG2A330S/P331S, PlC4-H0-IgG4S228P, PlC4-H2-IgG2-4 Hybrid, P1C4-H2- IgG2A330S/P331S, PlC4-H2-IgG4S228P, PlC4-H4-IgG2-4 Hybrid, P1C4-H4- IgG2A330S/P331S, PlC4-H4-IgG4S228P.

[0012] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1, 9, 17, 25, 33, 41, 49, and 57. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 10, 18, 26, 34, 42, 50, and 58. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain constant region comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 3, 11, 19, 27, 35, 43, 51, and 59. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 4, 12, 20, 28, 36, 44, 52, and 60. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain variable region comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5, 13, 21, 29, 37, 45, 53, and 61. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 6, 14, 22, 30, 38, 46, 54, and 62. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 7, 15, 23, 31, 39, 47, 55, and 63. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 8, 16, 24, 32, 40, 48, 56, and 64.

[0013] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 1; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 3; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 5; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 7.

[0014] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 2; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 4; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 6; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 8.

[0015] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 9; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 11; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 13; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 15.

[0016] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 10; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 12; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 14; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 16. [0017] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 17; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 19; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 21; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 23.

[0018] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 18; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 20; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 22; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 24.

[0019] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 25; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 27; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 29; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 31.

[0020] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 26; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 28; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 30; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 32.

[0021] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 33; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 35; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 37; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 39.

[0022] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 34; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 36; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 38; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 40.

[0023] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 41; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 43; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 45; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 47.

[0024] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 42; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 44; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 46; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 48.

[0025] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 49; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 51; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 53; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 55.

[0026] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 50; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 52; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 54; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 56.

[0027] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 57; a heavy chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 59; a light chain variable region comprising a nucleic acid sequence according to SEQ ID NO: 61; and a light chain constant region comprising a nucleic acid sequence according to SEQ ID NO: 63.

[0028] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 58; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 60; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 62; and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 64.

[0029] In one embodiment, the invention provides an isolated antibody or fragment thereof that comprises a nucleic acid sequence or an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-351.

[0030] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises heavy chain complementary determining regions (CDRs) independently selected from the CDRs present in the heavy chain variable regions according to SEQ ID NOs: 2, 10, 18, and 26. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises light chain CDRs independently selected from the CDRs present in the light chain variable regions according to SEQ ID NOs: 6, 14, 22, and 30.

[0031] In one embodiment, the isolated antibody or antigen binding fragment thereof is a humanized antibody comprising heavy chain complementary determining regions (CDRs) independently selected from the CDRs present in the heavy chain variable regions according to SEQ ID NOs: 2, 10, 18, and 26. In another embodiment, the isolated antibody or antigen binding fragment thereof is a humanized antibody comprising light chain complementary determining regions (CDRs) independently selected from the CDRs present in the light chain variable regions according to SEQ ID NOs: 6, 14, 22, and 30.

[0032] In one embodiment, the heavy chain variable region comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 339-341. In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%o or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 339-341. In a further embodiment, the heavy chain variable region comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 339-341.

[0033] In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain amino acid sequence that is least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 343-351. In a further embodiment, the heavy chain comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:343-351.

[0034] In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain variable region amino acid sequence that is least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence according to SEQ ID NO: 337. In a further embodiment, the heavy chain variable region comprises, consists essentially of, or consists of an amino acid sequence according to SEQ ID NO: 337. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain amino acid sequence that is least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence according to SEQ ID NO: 338. In a further embodiment, the light chain comprises, consists essentially of, or consists of an amino acid sequence according to SEQ ID NO: 338. [0035] In one embodiment, the invention provides a humanized isolated antibody or antigen binding fragment thereof that binds CB1. In a further embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain variable region according to SEQ ID NO: 337 and a heavy chain variable region according to SEQ ID NO: 339. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain variable region according to SEQ ID NO: 337 and a heavy chain variable region according to SEQ ID NO: 340. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain variable region according to SEQ ID NO: 337 and a heavy chain variable region according to SEQ ID NO: 341. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a full light chain according to SEQ ID NO: 338 and a full heavy chain according to a sequence selected from the group consisting of SEQ ID NOs: 343-351.

[0036] In other embodiments, the isolated antibody or antigen binding fragment thereof comprises a light chain sequence according to SEQ ID NO: 829, 830, 831, or 832. In some embodiments, the isolated antibody or antigen binding fragments comprise a light chain sequence according to SEQ ID NO: 829, 830, 831, or 832, and a heavy chain sequence according to SEQ ID NO: 437.

[0037] In one embodiment, the isolated antibody or fragment thereof comprises a heavy chain CDR1 sequence having at least 80%, at least 85%, at least 90%>, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 352 (YYWMN). In another embodiment, the isolated antibody or fragment thereof comprises a heavy chain CDR2 sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 96%), at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 353 (QIYPGDGETKY). In another embodiment, the isolated antibody or fragment thereof comprises a heavy chain CDR3 sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 354 (SHGNYLPY). In another embodiment, the isolated antibody or fragment thereof comprises a light chain CDR1 sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 355 (SSYLH). In another embodiment, the isolated antibody or fragment thereof comprises a light chain CDR2 sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 356 (STSNLAS). In another embodiment, the isolated antibody or fragment thereof comprises a light chain CDR3 sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 357 (HQ YHRSPPTF) .

[0038] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain CDR1, CDR2, and CDR3 comprising amino acid sequences according to SEQ ID NOs: 352, 353, and 354, respectively. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain CDR1, CDR2, and CDR3 comprising amino acid sequences according to SEQ ID NOs: 355, 356, and 357, respectively. In a further embodiment, the isolated antibody or antigen binding fragment thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising amino acid sequences according to SEQ ID NOs: 352, 353, 354, 355, 356, and 357, respectively. In a still further embodiment, the isolated antibody or fragment thereof is chimeric or humanized.

[0039] In still another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain CDR1 comprising amino acid sequence according to SEQ ID NO: 833. In further embodiments, the isolated antibody or antigen binding fragment thereof comprises a light chain CDR2 selected from a group comprising amino acid sequence according to SEQ ID NOs: 694, 834 and 835. In still further embodiments, the isolated antibody or antigen binding fragment thereof comprises a light chain CDR3 selected from a group comprising amino acid sequence according to SEQ ID NOs: 779 and 836.

[0040] In an embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain CDR1 comprising amino acid sequence according to SEQ ID NO: 833, a light chain CDR2 comprising amino acid sequence according to SEQ ID NO: 694, and a light chain CDR3 comprising amino acid sequence according to SEQ ID NO: 836. [0041] In another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain CDRl comprising amino acid sequence according to SEQ ID NO: 833, a light chain CDR2 comprising amino acid sequence according to SEQ ID NO: 835, and a light chain CDR3 comprising amino acid sequence according to SEQ ID NO: 836.

[0042] I yet another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain CDRl comprising amino acid sequence according to SEQ ID NO: 833, a light chain CDR2 comprising amino acid sequence according to SEQ ID NO: 694, and a light chain CDR3 comprising amino acid sequence according to SEQ ID NO: 357.

[0043] In still another embodiment, the isolated antibody or antigen binding fragment thereof comprises a light chain CDRl comprising amino acid sequence according to SEQ ID NO: 833, a light chain CDR2 comprising amino acid sequence according to SEQ ID NO: 834, and a light chain CDR3 comprising amino acid sequence according to SEQ ID NO: 779.

[0044] In still another embodiment, disclosed herein is an isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CB1), wherein the antibody or antigen binding fragment thereof comprises a light chain CDRl comprising an amino acid sequence according to SEQ ID NOs: 355 or 833; a light chain CDR2 comprising an amino acid sequence according to SEQ ID NOs: 356, 694, 834, or 835; and a light chain CDR3 comprising an amino acid sequence according to SEQ ID NOs: 357, 779, or 836.

[0045] The person of skill in the art will understand that the heavy and light chain CDRs of the antibodies provided herein may be independently selected, or mixed and matched, to form an antibody or binding fragment thereof comprising any light chain CDRl, CDR2, and CDR3; and any heavy chain CDRl, CDR2, and CDR3 from the antibodies provided herein. The skilled person will further understand that the heavy and light chain variable regions of the antibodies provided herein may be independently selected, or mixed and matched, to form an antibody or binding fragment comprising any heavy and light chain from the antibodies provided herein.

[0046] In one embodiment, the antibody or antigen binding fragment thereof provided herein is a chimeric antibody or fragment containing heavy and light chain CDRs selected from the CDRs provided herein, or conservative variants of the CDRs provided herein. In another embodiment, the antibody or antigen binding fragment thereof provided herein is a humanized antibody or fragment containing heavy and light chain CDRs selected from the CDRs provided herein, or conservative variants of the CDRs provided herein. In one embodiment, the antibody or antigen binding fragment thereof provided herein comprises a light chain and/or a heavy chain comprising a sequence provided herein, or a conservative variant thereof. In one embodiment, the conservative variants have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%o, at least 97%, at least 98%> or at least 99% homology to the reference sequence provided herein. In one embodiment, the conservative variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, insertions, or deletions.

[0047] In some embodiments, the isolated antibody or antigen binding fragment thereof binds CB1 and exhibits reduced effector function. In one embodiment, the isolated antibody or antigen binding fragment thereof binds CB1 and comprises one or more Fc region modifications. In a further embodiment, the antibody or antigen binding fragment thereof binds CB1 and comprises an amino acid sequence comprising one or more mutations in the Fc region. In a further embodiment, the isolated antibody or antigen binding fragment thereof has a mutation at position 228 and/or 330 and/or 331. In another embodiment, the isolated antibody or antigen binding fragment thereof has a mutation at position 228 of the Fc region, wherein the Fc region is of the IgG4 isotype. In a further embodiment, the mutation is S228P. In another embodiment, the isolated antibody or antigen binding fragment thereof has a mutation at position 330 and/or position 331. In a further embodiment, the isolated antibody or antigen binding fragment thereof has a mutation at position 330 and/or 331, wherein the Fc region is of the IgG2 isotype. In a further embodiment, the isolated antibody or antigen binding fragment thereof has the following mutations in the Fc region: A330S and P331S. In another embodiment, the isolated antibody or antigen binding fragment thereof comprises an Fc region that is a hybrid Fc region. For example, in one embodiment, the Fc region is a hybrid IgG2/IgG4 Fc region, wherein the CHI and hinge regions are derived from IgG2, and the CH2 and CH3 regions are derived from IgG4.

[0048] Thus, in one embodiment, the antibody or antigen binding fragment thereof provided herein is a chimeric or humanized antibody or fragment containing heavy and light chain CDRs selected from the CDRs provided herein, or conservative variants of the CDRs provided herein, wherein the isolated antibody or fragment thereof comprises an Fc region comprising modifications that alter antibody effector functions. For example, in one embodiment, the isolated antibody or antigen binding fragment thereof comprises light and heavy chain CDRs according to SEQ ID NOs: 352-357, or conservative variants thereof, and further comprises an IgG2-IgG4 hybrid Fc region, an IgG2 Fc region comprising amino acid mutations at positions 330 and 331 (e.g., A330S and P331 S), or an IgG4 Fc region comprising an amino acid mutation at position 228 (e.g., S228P).

[0049] In one embodiment, the present invention provides an isolated antibody or antigen binding fragment thereof that binds to CB l , wherein the antibody or fragment has a binding affinity Kd for CB l receptor of about 70 nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 25nM or less, about 20nM or less, about 15nM or less, about ΙΟηΜ or less, about 8nM or less, about 6nM or less, about 5 nM or less, about 4nM or less, about 3nM or less, about 2nM or less, or about InM or less. In one embodiment, present invention provides an isolated antibody or fragment thereof that binds to CBl , wherein the antibody or fragment has a binding affinity Kd for CBl receptor in the range of about InM to about ΙΟΟηΜ, about 2nM to about 75nM, about 3nM to about 50nM, about 4nM to about ΙΟηΜ, or has a binding affinity Kd for CB2 receptor that is about 50nM, or about 40nM, or about 30nM, or about 20nM, or about ΙΟηΜ, or about 5nM, or about 4nM, or about 3nM or about 2nM, or about InM.

[0050] In one embodiment, the present invention provides an isolated antibody or antigen binding fragment thereof that is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 1 1 fold, at least 12 fold, at least 13 fold, at least 14 fold, or at least 15 fold more potent than the small molecule rimonabant, wherein the potency of the antibody or fragment or rimonabant is measured by inhibition of CBl receptor antagonist-mediated signal transduction in a cAMP assay. In a further embodiment, the isolated antibody or antigen binding fragment thereof is humanized.

[0051] In one embodiment, the present invention provides an isolated humanized antibody or antigen binding fragment thereof that binds to CB l , wherein the antibody or fragment exhibits greater binding affinity and/or greater potency than a corresponding non-humanized or chimeric antibody, wherein the humanized antibody or fragment and the corresponding non-humanized or chimeric antibody comprise the same heavy and light chain CDRs. For example, in one embodiment, the present invention provides a humanized antibody or fragment thereof comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 352, 353, 354, 355, 356, and 357, respectively, wherein the humanized antibody exhibits greater binding affinity for CBl receptor and/or greater potency with respect to inhibition of CBl receptor agonist. In one embodiment, the humanized antibodies and fragments provided herein exhibit at least 50% greater, at least 100% greater, at least 2 fold greater, at least 3 fold greater, at least 4 fold greater, at least 5 fold greater, or at least 10 fold greater potency relative to the corresponding non-humanized or chimeric antibody. In a further embodiment, the potency is measured by inhibition of CBl - cAMP production.

[0052] Potency of CB 1 receptor antibodies provided herein may be measured by any method known in the art. For example, in one embodiment, potency of the antibodies and fragments provided herein is measured by intracellular cAMP levels or ER phosphorylation. For example, potency may be measured by the level of inhibition cAMP production in a cAMP functional assay (Cisbio) or inhibition of WIN55,212-induced ERK phosphorylation in a Western blot.

[0053] In some embodiments, the present invention provides an antibody or antigen binding fragment thereof that is capable of competing with the antibody or antigen binding fragments thereof disclosed herein for binding to CBl receptor. In some other embodiments, the present invention provides an antibody or antigen binding fragment thereof that is capable of specifically binding to essentially the same epitope on CBl receptor as the antibodies or antigen binding fragments disclosed herein. Such antibodies can be identified using routine competition binding assays. In certain embodiments, competition is measured by ELISA, flow cytometry, or surface plasmon resonance (SPR) assay.

[0054] In some embodiments, the antibodies and fragments thereof are conjugated to one or more agents selected from the group including an additional therapeutic agent, a cytotoxic agent, an immunoadhesion molecule, and an imaging agent. In some embodiments, the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. [0055] In one aspect, methods are provided for modulating the signaling activity of CBl receptor comprising contacting a cell expressing CBl receptor with the antibody or fragment thereof disclosed herein. In some embodiments, the methods provided result in inhibition of the activity of CBl receptor signaling. In some embodiments, the methods provided result in increased activity of CBl receptor signaling. In some embodiments, the modulation of CBl receptor signaling activity is indirect, such as though an allosteric modulator. In some embodiments, the modulation of CBl receptor signaling activity is biased for Galpha i/o mediated signaling versus beta arrestin mediated signaling.

[0056] In one aspect, methods for treating a disease or disorder responsive to antagonism or agonism of CBl receptor in a subject in need thereof are provided. In some embodiments, the methods comprise administering to the subject an anti-CBl receptor antibody or antigen binding fragment thereof as disclosed herein. In one embodiment, the subject is a mammal. In a further embodiment, the subject is a human. In some embodiments, the disease or disorder is obesity, diabetes, dyslipidemia, metabolic diseases, fibrosis, non-alcoholic steatohepatitis (NASH), liver disease, primary biliary cirrhosis, renal disease, kidney fibrosis, chronic kidney disease, osteoporosis, atherosclerosis, cardiovascular disease, cancer, an inflammatory disease, pain, MS spasticity, and ocular diseases, including glaucoma. In some embodiments, the disease or disorder is, for example, obesity, diabetes, fibrosis, liver disease, cardiovascular disease, or cancer, and the method provided results in inhibition of the activity of CBl receptor. In some embodiments, the disease or disorder is, for example, pain or glaucoma, and the method provided results in activation or increase of CB 1 receptor activity.

[0057] In one aspect, a method for detecting CBl receptor in a cell, tissue, or subject is provided, the method comprising contacting a cell with a CBl receptor binding antibody or antigen binding fragment provided herein. In one embodiment, the cell is present in a subject. In another embodiment, the cell is present in a human subject. In another embodiment, the CBl receptor expression level on cells is correlated with a disease state. Thus, in one aspect, the present invention provides methods of using antibodies and fragments thereof that specifically bind to CBl receptor as tools for the diagnosis and/or prognosis of human diseases. In one embodiment, the present invention provides methods for imaging CBl receptor comprising the use of the CBl receptor antibodies and fragments disclosed herein. In one embodiment, the method for detecting CBl receptor is achieved with a CBl receptor antibody or fragment thereof disclosed herein that selectively binds CBl receptor. In a further embodiment, the selective CBl receptor antibody or fragment thereof does not exhibit agonistic or antagonistic activity. In a further embodiment, the selective CBl receptor antibody or fragment thereof does not internalize. In one embodiment, the present invention provides diagnostic and imaging methods comprising the use of a CBl receptor antibody or fragment that is conjugated to an imaging agent such as, for example, a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.

[0058] In one embodiment, the invention provides a host cell expressing an isolated antibody or fragment thereof that specifically binds to CBl receptor. In another embodiment, a method for making an antibody or fragment thereof that specifically binds to CB 1 receptor is provided, the method comprising immunizing mammals with purified CBl receptor or an antigenic fragment thereof, CBl/lipid complexes, CBl receptor iCAPS, and/or CBl receptor DNA. In a further embodiment, the immunized mammals are mice. In another embodiment, the mammals are immunized one, two, three, four, five, or more times with purified CBl or an antigenic fragment thereof, CBl/lipid complex, CBl receptor iCAPS, and/or CBl receptor DNA prior to harvesting cells from the immunized mammals. In a further embodiment, the antibody or fragment thereof that specifically binds to CBl receptor is generated from a hybridoma cell line comprising cells derived from the immunized mammals. In another embodiment, the antibody or fragment thereof that specifically binds to CBl receptor is generated from a phage display library. In a further embodiment, the phage display library is derived from cells isolated from the immunized mammals. In a further embodiment, the phage display library is derived from naive human immunoglobulin sequences.

BRIEF DESCRIPTION OF THE FIGURES

[0059] Figure 1A is a set of histograms showing binding of P2A12 Bril mAb (left column), PA2LR3-P2D3 (middle column), or PA2R3-P1A7 (right column) at 300 nM (top row) or 30 nM (bottom row) to Trex-CHO Native human CBl cell line (native CBl receptor expressing; dark gray lines), Trex-CHO CBl T21 OA/fusion partner (overexpressed CBl; medium gray lines), or Trex-CHO parental cell line (no CBl receptor expression; light gray lines). Figure IB is a set of histograms showing binding of PA2R3-P1F1 (left column), PA2LR3-P2E5 (middle column), or PA2LR3-P3B10 (right column) at 300nM (top row) or 30nM (bottom row) to Trex-CHO Native CBl cell line (native human CBl receptor expressing; dark gray lines), Trex-CHO CBl T21 OA/fusion partner (overexpressed CBl; medium gray lines), or Trex-CHO parental cell line (no CBl receptor expression; light gray lines). Figure 1C is a set of histograms showing binding of PA2LR3-P3B8 (left column) or PA2LR3-P1H4 (right column) at 300 nM (top row) or 30 nM (bottom row) to Trex-CHO Native human CBl cell line (native CBl receptor expressing; dark gray lines), Trex-CHO CBl T21 OA/fusion partner (overexpressed CBl; medium gray lines), or Trex-CHO parental cell line (no CBl receptor expression; light gray lines). Figure ID is a set of histograms showing binding of PA2LR3-P4B1 (left column), PA2LR3-P4B5 (middle column), or PA2LR3-P4C6 (right column) at 300nM (top row) or 30nM (bottom row) to Trex- CHO Native human CB 1 cell line (native CB 1 receptor expressing; dark gray lines), Trex-CHO CBl T21 OA/fusion partner (overexpressed CBl; medium gray lines), or Trex-CHO parental cell line (no CBl receptor expression; light gray lines). Figure IE is a set of histograms showing binding of PA2LR3-P4G10 (left column) or PA2LR3-P6D7 (right column) at 300nM (top row) or 30nM (bottom row) to Trex-CHO Native human CBl cell line (native CBl receptor expressing; dark gray lines), Trex-CHO CBl T21 OA/fusion partner (overexpressed CBl; medium gray lines), or Trex-CHO parental cell line (no CBl receptor expression; light gray lines). Figure IF is a set of histograms showing binding of PA2LR3-P1G6 (left column), PA2LR3-P1H4 (middle column), or PA2LR3-P2B8 (right column) to Trex-CHO parental cell line (no CBl receptor expression; top row), Trex-CHO CBl T21 OA/fusion partner cell line (overexpressed CBl; middle row), or Trex-CHO A156 cell line (native human CBl receptor expressing; bottom row).

[0060] Figure 2 shows the selectivity of two of the CBl receptor antibodies, PA13R3-P1C4 and 36E12B6C2, for binding to cells expressing CBl . Figure 2A (showing PA13R3-P1C4 binding) and Figure 2B (showing 36E12B6C2 binding) show that both antibodies bound to A156 (native human CBl receptor expressing) and, to an even greater extent, A56 (over- expresses CB1 receptor modified by T210A mutation and ICL3 replacement with fusion partner fusion partner) but did not exhibit binding to non-CBl receptor expressing CHO cells, CB2 expression cell line, or 5HT2b expression cell line. Expression of CB2 (Figure 2C) and 5HT2b (Figure 2D) was confirmed in CB2 expression and 5HT2b expression cell lines, respectively.

[0061] Figure 3 shows the results of a competition assay to determine if 36E12B6C2 and P1C4 bind to similar epitopes. Trex CHO A156 native human CB1 cells were incubated with competitor IgGs (PA13R3-P1C4 IgG or Fab and 36E12B6C2) followed by different concentrations of staining IgGs (300nM or 75nM of P1C4, Figure 3 A; 80nM or 25nM of 36E12B6C2, Figure 3B).

[0062] Figure 4 shows the results of the cAMP functional antagonist assay. Antibodies 36E12B2H8 (Figure 4A) and PA13R3-P1C4 (Figure 4B) exhibited antagonistic activity that was equipotent (36E12B2H8) or more potent (PA13R3-P1C4) relative to positive control small molecule CB1 receptor inhibitors AM251 (Figure 4C), SR141716A (rimonabant) (Figure 4D), and AM6545 (Figure 4E). P2A12 and hybridoma IgG isotype were used as negative controls (Figures 4F and 4G).

[0063] Figure 5A is a set of Western blots showing phosphorylated ER (pER ) and total ER in Trex-CHO native human CB1 receptor cells following CB1 receptor expression and treatment with control IgG, positive control small molecule AM6545, or phage derived mAb PA13R3-P1C4 or PA13R3-P1E4, followed by treatment with lOOnm of CB1 receptor agonist WIN55,212. The Western blots shown are from 10 minutes following WIN55,212 activation (left panels) or 15 minutes following WIN55,212 activation (right panels). Figure 5B is a set of Western blots showing pERK and total ERK in Trex-CHO native human CB1 receptor cells following CB1 receptor expression and treatment with control IgGl, control IgG2, WIN55,212, AM6545, or hybridoma-derived mAb 36E12B2E5, 36E12B6C2, or 36E12B2F2, followed by WIN55,212 activation.

[0064] Figure 6A shows the results of the cAMP functional assay performed in the absence of CP55940 to assess potential agonist activity of PA2LR3-P2D3, PA2LR3-P4B1, PA2LR3- P6B12, and PA2LR3-P6G6, relative to controls depicted in Figure 6B: CP55940 (positive control), P2A12 (negative control), or PA2R3-P1A7 (negative control). Figure 6C shows the results of the cAMP functional assay performed in the presence of CP55940 to assess potential allosteric modulator activity of PA2LR3-P2D3, PA2LR3-P4B1, PA2LR3-P6B12, and PA2LR3- P6G6, relative to controls depicted in Figure 6D: CP55940 alone (positive control), P2A12 (negative control), or PA2R3-P1A7 (negative control).

[0065] Figure 7 shows the results of a cAMP assay conducted to assess the inverse agonist or neutral antagonist activity of PA13R3-P1C4 and 36E12B6C2. AM6545 and SR141716A were used as positive controls for neutral antagonist and inverse agonist respectively.

[0066] Figure 8A shows an iCAPS ELISA binding assay assessing 36E12B6C2 Fab (top left panel) or IgG (top right panel), or P1F7 Fab (bottom left panel) or Fab (bottom right panel) to rBril-0918, empty iCAPS, iCAPS that do not express CBl receptor (hl3h iCAPS), or iCAPS that express human CBl receptor (A138 iCAPS and A139 iCAPS). Figure 8B shows an iCAPS ELISA binding assay assessing PA13R3-P1C4 Fab (top left panel) or IgG (top right panel), or P1F7 Fab (bottom left panel) or Fab (bottom right panel) to rBril-0918, empty iCAPS, iCAPS that do not express CBl receptor (hl3h iCAPS), or iCAPS that express human CBl receptor (A138 iCAPS and A139 iCAPS).

[0067] Figures 9A and 9B show CBl receptor internalization following various treatments. Figure 9 A shows that CBl antibodies do not block WIN55,212 induced receptor internalization. The top row of histograms in Figure 9A shows surface expression of CBl following treatment with WIN55,212 or control, or pre-treatment with CBl specific neutral antagonist AM6545 followed by WIN55,212. The middle and bottom rows of histograms in Figure 9A show surface expression of CBl following pre-treatment with CBl antibodies PA2LR3-P3A8, PA2LR3-P3F8, PA2LR3-P5B11, PA2LR3-P5E7, PA2LR3-P6B12, PA2LR3-P6G7, PA3R3-P4D5, PA2LR3- P4B1, PA2LR3-P4B5, PA2LR3-P4C6, and PA2LR3-P4G10, or negative control P2A12 followed by treatment with WIN55,212. Figure 9B shows that CBl antibodies alone do not induce CBl receptor internalization. The top row of histograms in Figure 9B show surface expression of CBl following treatment with WIN55,212 or control, or pre-treatment with CBl specific neutral antagonist AM6545 followed by WIN55,212. The middle and bottom rows of histograms in Figure 9B show surface expression of CBl following treatment with CBl antibodies PA2LR3-P3A8, PA2LR3-P3F8, PA2LR3-P5B11, PA2LR3-P5E7, PA2LR3-P6B12, PA2LR3-P6G7, PA3R3-P4D5, PA2LR3-P4B1, PA2LR3-P4B5, PA2LR3-P4C6, and PA2LR3- P4G10, or negative control P2A12

[0068] Figure 10 shows the results of the cAMP functional antagonist assay for humanized versus chimeric P1C4 antibodies. Humanized antibody P1C4-H0 exhibited antagonistic activity that was similar to the chimeric P1C4 antibody. Humanized antibodies P1C4-H4 and PIC4-H2 exhibited antagonistic activity that was more potent relative to the chimeric P1C4 antibody or to positive control small molecule CB 1 receptor inhibitor rimonabant.

[0069] Figure 11 shows the binding affinity, cross-reactivity, and specificity of humanized PI C4 antibodies as measured by flow cytometry. Humanized P1C4 antibodies P1C4-H2 and PIC4-H4 exhibited superior binding affinity to both native human CB1 cells (Figure 11 A, top panel) and to overexpressed CB1 cells (Figure 11A, bottom panel) relative to the P1C4 chimeric antibody. None of the chimeric or humanized antibodies bound to mouse cells expression mouse CB1, TRex-CHO parental cells, or TRex-CHO cells expressing human CB2 (Figure 1 IB).

[0070] Figure 12 shows the affinity of unlabeled P4B5 antibody versus Vivotag 680 XL- labeled P4B5 antibody to CB1 on cells.

[0071] Figure 13A shows the detection of labeled P4B5 antibody in the heart, lungs, liver, kidneys, stomach, intestines, and bladder at time points Oh, lh, 5h, 24h, 48h, 72h, 96h, and 144h (13A.1); and the detection of labeled P4B5 antibody in the brain at timepoints Oh, lh, 5h, 24h, 48h, 72h, 96h, and 144h (13A.2). Figure 13B shows the detection of labeled antibody in the brain including both tissue and blood (left panel) versus detection of labeled antibody in the brain with the blood signal subtracted (i.e., brain tissue only; right panel).

[0072] Figure 14 shows the SEC profiles and SDS-PAGE analyses for PA13R3-P1C4 humanized variants expressed in 293 and CHO-K1 cells. Figure 14A shows the SEC profile (top) and SDS-PAGE (bottom) analyses for one of the 293 FreeStyle batches. Figure 14B shows the SEC profile (top) and SDS-PAGE (bottom) analyses for one of the CHO-K1 batches.

[0073] Figure 15 shows cAMP functional assays for PA13R3-P1C4 humanized variants compared with parental chimeric PA13R3-P1C4 and P2A12 mAb, a non-GPCR targeting mAb negative control antibody of IgGl isotype. [0074] Figure 16 shows a comparison of the activity of humanized variants PlC4-h2-IgG2 and PlC4-h2-IgG4 with rimonabant, AM6545 and the P2A12-IgGl negative control antibody in 1.5 μΜ forskolin stimulated TRex-CHO CB1 cells (Figure 16 A) as well as 5 μΜ forskolin stimulated TRex-CHO CB1 cells (Figure 16B).

[0075] Figure 17 shows the effect of increasing PlC4-h2-IgG4 concentrations on CP55,940 (Figure 17A and WIN55,212 (Figure 17C). Schild plots for each treatment are also shown (Figures 17B and 17D).

[0076] Figures 18A and 18B show Western blot ER activation assays measuring the ability of PA13R3-P1C4 humanized variants to block WIN55,212 mediated ERK activation.

[0077] Figure 19A shows a flow-cytometry based CB1 receptor internalization study under various induction conditions, in the absence of inhibitor (Panel A), or the presence of rimonabant (Panel B) or PA13R3-P1C4 humanized variants (Panels C-G). Figure 19B shows the same CB1 receptor internalization assay investigating the effect of PlC4-h2 cloned into the different human Fc frameworks IgG2 and IgG4.

[0078] Figure 20, A-D show flow cytometry data measuring binding of humanized PA13R3- P1C4 antibody variants to TRex-CHO cells stably transfected with tetracycline inducible human CB1. Figure 20, E-M show binding selectivity and cross-reactivity of humanized PA13R3- P1C4 variants to human CB1 versus human CB2 and mouse CB1.

[0079] Figure 21 shows flow cytometry data measuring the binding of antibodies (indicated at top) to cells expressing various CB1 constructs (indicated at left).

[0080] Figure 22 shows antibody mediated cytotoxicity and complement dependent cytotoxicity of humanized P1C4 variants PlC4-h2-IgG2 and PlC4-h2-IgG4 in Daudi cells. Figures 22A-C show the effect of P14C variants in antibody mediated toxicity assays. Figure 22D shows the effect of P14C variants in complement dependent cytotoxicity assays.

[0081] Figure 23 shows Western Blot analysis assessing recognition of denatured CB1 protein by the indicated P1C4 primary antibodies or control antibodies, and anti-human (Figure 23A) or anti-mouse (Figure 23B) secondary antibodies. Purified human IgG with human secondary (Figure 23A, Lane 1), and mouse primary with anti-rabbit secondary (Figure 23B, Lane 11) are presented as negative controls. [0082] Figure 24 shows the results of flow cytometry binding experiments (Figure 24A), and inhibition of cAMP production (Figure 24B), by chimeric and humanized P1C4 Fab antibody fragments incubated with cells expressing CBl receptor.

[0083] Figure 25 shows positive CBl-specifc staining in macrophage, hepatocytes, and hepatic myofibroblasts in early NASH (left panel), NASH fibrosis (middle panel) and late fibrosis (right panel) samples.

[0084] Figure 26 shows that no staining was observed with isotype controlled irrelevant antibodies in cells derived from either normal (middle panel) or NASH fibrosis (right panel) cells.

[0085] Figure 27 shows no CBl specific staining in normal tissues.

[0086] Figure 28 shows RT-PCR expression data measuring Pro-collagen A1(I), in primary hepatic stellate cells treated with PBS, non-functional control antibody, and PlC4-h2 antibodies.

[0087] Figure 29 shows RT-PCR expression data measuring TGFP expression levels in primary hepatic stellate cells treated with the indicated antibodies, concentrations, and controls.

[0088] Figure 30 shows RT-PCR expression data measuring TIMP1 expression levels in primary hepatic stellate cells treated with the indicated antibodies, concentrations, and controls.

[0089] Figure 31 shows RT-PCR expression data measuring a-SMA expression levels in primary hepatic stellate cells treated with the indicated antibodies, concentrations, and controls.

DETAILED DESCRIPTION

[0090] In one aspect, the present invention provides antigen binding proteins such as antibodies and antigen-binding fragments thereof that bind selectively to human cannabinoid 1 (CBl) receptor. The antibodies and fragments thereof are functional antibodies that agonize or antagonize CBl receptor, or are selectively recognizing CBl without agonist or antagonist activity.

[0091] As used herein, the term "antibody" refers to binding proteins having at least one antigen-binding domain and includes monoclonal antibodies fragments and/or variants thereof including recombinant polypeptides, fusion proteins, and immunoconjugates. Thus, the terms "antibody," "antibody fragment," and "antibody variant" are used interchangeably herein. Examples of antibody fragments of the invention include, but are not limited to, the Fab fragment, consisting of VL, VH, CL and CHI domains; the Fc fragment, consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH; the dAb fragment consisting of a VH domain; isolated CDR regions; F(ab') ₂ a bivalent fragment comprising two linked Fab fragments; and single chain Fv molecules (scFv). The CBl receptor binding antibodies provided herein may be generated from any species including, but not limited to, mouse, rat, rabbit, primate, llama and human. The CBl receptor binding antibodies may be chimeric, humanized, or fully human antibodies.

[0092] As used herein, the term "derived" when used to refer to a molecule or polypeptide relative to a reference antibody or other binding protein, means a molecule or polypeptide that is capable of binding with specificity to the same epitope as the reference antibody or other binding protein.

[0093] The use of the singular includes the plural unless specifically stated otherwise. The word "a" or "an" means "at least one" unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more." Furthermore, the use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components comprising more than one unit unless specifically stated otherwise.

[0094] The antibodies and antigen-binding fragments thereof disclosed herein are specific for cannabinoid 1 (CBl) receptor. By "specific for" is meant that the antibodies and fragments thereof bind CBl receptor with greater affinity (i.e., a lower binding affinity Kd value) than any other target. Thus, antibodies and fragments thereof that are selective for CBl receptor bind CBl receptor with greater affinity (i.e., a lower binding affinity Kd value)than any other cannabinoid receptor or any other GPCR or any other target. The antibodies and fragments or variants thereof may have a binding affinity Kd value for CBl receptor in the range of about 0.01 nM to about 500 nM, about 0.02 nM to about 250 nM, about 0.02 to about 200 nM, about 0.05 to about 100 nM, about 0.05 to about 50 nM. The antibodies and fragments thereof may have a binding affinity Kd value for CBl receptor of about 500 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 1 nM, about 500 M, about 250pM, about Ι ΟΟρΜ, about 50pM, or about ΙΟρΜ. The antibodies and fragments thereof may have a binding affinity Kd value for CB 1 receptor of about ΙΟΟηΜ or less, about 75nM or less, about 50nM or less, about Ι ΟηΜ or less, about InM or less, about 500pM or less, or about ΙΟΟρΜ or less.

[0095] As used herein, the term "agonist" refers to a compound that enhances the signaling activity of another compound or receptor site.

[0096] As used herein, the term "antagonist" refers to a compound that inhibits, diminishes or prevents the signaling activity of another compound at a receptor site and more generally refer to a compound that diminishes or prevents the activation and/or the signaling activity of a receptor.

[0097] An "allosteric modulator" is a compound that indirectly modulates the agonistic effects of another compound. For example, an allosteric modulator may indirectly modulate the agonistic effect of a receptor agonist by inducing a conformational change within the protein structure. Allosteric modulators may be positive (amplify the agonistic effect of the agonist compound) or negative (diminish the effect of the agonist compound) modulators.

[0098] As used herein, the terms "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventive measures. A subject in need of treatment is a subject that already has the disease or disorder as well as those that may develop the disease or disorder and in whom the object is to prevent, delay, or diminish the disease or disorder. The methods of "treatment" disclosed herein employ administration to a subject, an antibody or antigen binding fragment disclosed herein, for example, a subject having a CB1 -associated disease or disorder (e.g., a fibrotic disease) or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the invention is a human. [0099] A "therapeutically effective amount," as used herein, refers to the amount of a compound or composition that is necessary to provide a therapeutic and/or preventative benefit to the subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 1 ug to about 5,000 mg, about 1 mg to about 1,000 mg, about 10 mg to about 100 mg, of an antibody or antigen binding fragment thereof, disclosed herein. Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of an antibody or antigen binding fragment thereof are minimized or outweighed by the beneficial effects.

I. Modified Anti-CBl Antibodies

[0100] In certain embodiments, anti-CBl receptor antibodies disclosed herein may comprise one or more modifications. Modified forms of anti-CBl receptor antibodies disclosed herein can be made using any techniques known in the art. Non-exhaustive examples of modified anti-CBl receptor antibodies are disclosed in U.S. Application No. 14/774,582, filed September 10, 2015, International Application No. PCT/US 15/23108, filed March 27, 2015, International Application No. PCT/CN2014/074199, filed March 27, 2014, and International Application No. PCT/CN2014/081797, filed July 8, 2014, the disclosures of each of which are incorporated herein by reference in their entireties.

[0101] In some embodiments, the anti-CBl receptor antibodies and fragments thereof are conjugates further comprising an agent selected from the group including an additional therapeutic agent, a cytotoxic agent, an immunoadhesion molecule, and an imaging agent. In some embodiments, the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In some embodiments, the imaging agent is a radiolabel selected from the group consisting of: ³ H ¹⁴ C ³⁵ S, ⁶⁴ Cu, ⁸⁹ Zr, ⁹⁰ Y, ⁹⁹ Tc, ^m In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm. In some embodiments, the therapeutic agent or cytotoxic agent is selected from the group including an immunosuppressive agent, an immuno-stimulatory agent, an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a toxin, and an apoptotic agent.

[0102] In one embodiment, the isolated anti-CBl receptor antibody or antigen binding fragment disclosed herein is conjugated to a CB 1 antagonist. Non- limiting examples of known CB1 antagonists include rimonabant, taranabant, VD60, Isis-414930 Antisense CB1, JD5037, AM6545, and TM38837. In one embodiment, the isolated anti-CBl receptor antibody or antigen binding fragment disclosed herein is conjugated to rimonabant. In one embodiment, the isolated antibody or antigen binding fragment thereof that is conjugated to the cytotoxic agent is a CB1 receptor agonist. In another embodiment, the isolated antibody or antigen binding fragment that is conjugated to the cytotoxic agent is a CB1 receptor neutral binder that allows receptor internalization to occur.

[0103] In another aspect, the isolated anti-CBl receptor antibody or antigen binding fragment disclosed herein is conjugated to a chemotherapeutic agent. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',22"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in the definition are proteasome inhibitors such as bortezomib (Velcade), BCL-2 inhibitors, IAP antagonists (e.g. Smac mimics/xIAP and cIAP inhibitors such as certain peptides, pyridine compounds such as (S)-N- {6-benzo[ 1 ,3]dioxol-5-yl- 1 -[5-(4-fluoro-benzoyl)-pyridin-3-ylmethyl]-2-oxo-l ,2-dihydro- pyridin-3-yl}-2-methylamino-propionamide, xIAP antisense), HDAC inhibitors (HDACI) and kinase inhibitors (Sorafenib). In one embodiment, the isolated antibody or antigen binding fragment that is conjugated to the cytotoxic agent is a CB 1 receptor agonist.

[0104] In some embodiments, the binding protein is conjugated directly to the agent. In other embodiments, the binding protein is conjugated to the agent via a linker. Suitable linkers include, but are not limited to, amino acid and polypeptide linkers disclosed herein. Linkers may be cleavable or non-cleavable.

[0105] In certain embodiments, the antibodies and fragments thereof are bispecific or bi- functional antibodies. The term "bispecific antibodies" refers to molecules which combine the antigen-binding sites of two antibodies within a single molecule. Thus, a bispecific antibody is able to bind two different antigens simultaneously. A bispecific antibody typically is an artificial hybrid antibody having two different heavy chain/light chain pairs and two different binding sites or epitopes. Bispecific antibodies can be monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.

[0106] In one embodiment, the bispecific antibody and/or fragment thereof has binding specificities directed towards CBl and a second target antigen. In certain embodiments, the bispecific antibody and/or fragment thereof has binding specificities directed toward CBl and TGF-β, 2-AG, PDGF-β, IL-6, anandamide (AEA), or LOXL-2.

[0107] The antibodies and fragments disclosed herein may bind to one or more target antigens selected from the group consisting of carbonic anhydrase IX, alpha-fetoprotein, a- actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CDla, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDK 2A, CXCR4, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM1, CEACAM6, c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gplOO, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-

1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, la, IGF-1R, IFN-γ, IFN-a, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor- 1 (IGF-1), KC4-antigen, KS-1 -antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-

2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PIGF, IGF, IGF-1R, IL-6, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-a, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-lA-antigen, complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET, an oncogene marker and an oncogene product (see, e.g., Sensi et al, Clin Cancer Res 2006, 12:5023-32; Parmiani et al, J Immunol 2007, 178: 1975-79; Novellino et al. Cancer Immunol Immunother 2005, 54: 187- 207).

[0108] Methods for making bispecific antibodies are well known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, the hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J 10:3655 (1991). Other methods for making bispecific antibodies are provided in, for example, Kufer et al, Trends Biotech 22:238-244, 2004.

[0109] Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, C _H2 , and C _H3 regions. It may have the first heavy chain constant region (C _HI ) containing the site necessary for light chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al, Meth Enzym 121 :210 (1986). A variety of recombinant methods have been developed for efficient production of bispecific antibodies, both as antibody fragments (Carter et al. (1995), J. Hematotherapy 4: 463-470; Pluckthun et al. (1997) Immunotechology 3: 83-105; Todorovska et al. (2001) J. Immunol. Methods 248: 47-66) and full length IgG formats (Carter (2001) J. Immunol. Methods 248: 7-15).

[0110] Unless otherwise stated, the practice of the present invention employs conventional molecular biology, cell biology, biochemistry, and immunology techniques that are well known in the art and described, for example, in Methods in Molecular Biology, Humana Press; Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989), Current Protocols in Immunology (J. E. Coligan et al, eds., 1991); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Phage display: a laboratory manual (C. Barbas III et al, Cold Spring Harbor Laboratory Press, 2001); and Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999).

[0111] In one aspect, the invention provides methods for producing the antibodies and fragments or variants described herein comprising immunizing mice with a CB1 receptor immunogen such as, for example, CB1 receptor DNA, CB1 receptor protein, or CBl/lipid complex. In some embodiments, mice are immunized 1, 2, 3, 4, 5, or more times. In some embodiments, mice are immunized with CBl receptor DNA and the CBl receptor response is boosted by further immunization with CBl receptor DNA and/or purified CBl receptor protein, membranes comprising CBl receptor protein, or CBl receptor iCAPS. In some embodiments, cells from immunized mice are used to generate hybridoma and phage libraries.

[0112] In some embodiments, CBl receptor antibodies are generated by recovering B cells from immunized mice and generating CBl receptor antibody-producing hybridoma cells. The generation of hybridomas is a technique well known in the art and involves fusion of antibody- producing cells with myeloma or other immortalized cells to generate the immortalized antibody- producing hybridoma cell line (see, for example, Kohler and Milstein, 1975, Nature, 256:495). Monoclonal antibodies produced by the hybridoma cells can be isolated from the supernatant by conventional means of immunoglobulin purification such as precipitation, chromatography, ultrafiltration, centrifugation, gel electrophoresis, and/or any other method known in the art. Supernatants or isolated monoclonal antibodies may be tested for binding to CBl receptor by assessing binding to CBl receptor membranes relative to naive membranes. For example, supernatants or isolated monoclonal antibodies may be tested for binding to CBl receptor in an ELISA.

[0113] Another aspect of the invention provides methods of producing the antibodies and fragments or variants described herein comprising the use of a phage library. Methods for recombinantly generating antibodies via phage display technology are known in the art (see, for example, Winter et al, Annu. Rev. Immunol. 12:433-455 (1994), McCafferty et al, Nature 348: 552-553 (1990), and Clackson et al Nature 352:624 (1991)). In some embodiments, spleens from immunized mice are used to isolate an array of anti-CBl receptor antibodies and form a random combinatorial library of variable genes derived from those antibodies. In some embodiments, rather than utilizing the cells from immunized mice to generate the phage display library, the library is generated from variable heavy and light chain genes of human primary blood lymphocytes.

[0114] In some embodiments, the phage library is panned for CBl receptor binding phage in at least 3 rounds of panning, and phage binders are subsequently screened for specific binding to CB1 receptor by ELISA. Specific binders may then be selected and converted into full antibodies.

[0115] In some embodiments, the antibodies and fragments provided herein are chimeric antibodies or humanized antibodies. Methods for generating chimeric and humanized antibodies are well known in the art and summarized, for example, in Lo, Benny, K. C, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004.

[0116] A "chimeric antibody" is an antibody having at least a portion of the heavy chain variable region and at least a portion of the light chain variable region derived from one species; and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody may comprise murine variable regions and a human constant region. A "humanized antibody" is an antibody containing complementarity determining regions (CDRs) that are derived from a non-human antibody; and framework regions as well as constant regions that are derived from a human antibody.

[0117] As used herein, the term "CDR" or "complementarity determining region" means the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The Kabat definition is based on sequence variability. The IMGT unique numbering for all IG and TR V-regions of all species relies on the high conservation of the structure of the variable region (Lefranc, Mp et al., Dev comp. Immunol. 27:55-77, 2003). IMGT numbering, set up after aligning more than 5,000 sequences takes into account and combines the definition of the framework and CDRs. The Clothia definition is based on the location of the structural loop regions. The Contact definition (MacCallum et al) is based on an analysis of the complex crystal structures and antibody-antigen interactions. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison. In one embodiment disclosed herein, the term "CDR" is a CDR as defined by the Kabat definition. In another embodiment disclosed herein, the CDR is a CDR as defined by IMGT.

[0118] The CDRs generally are of importance for epitope recognition and antibody binding. However, changes may be made to residues that comprise the CDRs without interfering with the ability of the antibody to recognize and to bind the cognate epitope. For example, changes that do not impact epitope recognition, yet increase the binding affinity of the antibody for the epitope, may be made. Several studies have surveyed the effects of introducing one or more amino acid changes at various positions in the sequence of an antibody, based on the knowledge of the primary antibody sequence, on the properties thereof, such as binding and level of expression (Yang et al, 1995, J Mol Biol 254:392-403; Rader et al, 1998, Proc Natl Acad Sci USA 95:8910-8915; and Vaughan et al, 1998, Nature Biotechnology 16, 535-539).

[0119] Thus, equivalents of an antibody of interest can be generated by changing the sequences of the heavy and light chain genes in the CDR1, CDR2 or CDR3, or framework regions, using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling or mutator-strains of E. coli (Vaughan et al., 1998, Nat Biotech 16:535-539; and Adey et al, 1996, Chap. 16, pp. 277-291, in Phage Display of Peptides and Proteins, eds. Kay et al, Academic Press). The methods of changing the nucleic acid sequence of the primary antibody can result in antibodies with improved affinity (Gram et al, 1992, Proc Natl Acad Sci USA 89:3576-3580; Boder et al, 2000, Proc Natl Acad Sci USA 97: 10701-10705; Davies & Riechmann, 1996, Immunotech 2: 169-179; Thompson et al, 1996, J Mol Biol 256:77-88; Short et al, 2002, J Biol Chem 277: 16365-16370; and Furukawa et al, 2001, J Biol Chem 276:27622-27628).

[0120] For example, the CB1 antibodies provided herein may comprise CDRs derived from one or more murine antibodies and human framework and constant regions. Thus, in one embodiment, the humanized antibody provided herein binds to the same epitope on CB1 as the murine antibody from which the antibody's CDRs are derived. Exemplary humanized antibodies are provided herein. Additional humanized CB1 antibodies comprising the heavy and light chain CDRs provided herein, or variants thereof, may be generated using any human framework sequence, and are also encompassed in the present invention. In one embodiment, framework sequences suitable for use in the present invention include those framework sequences that are structurally similar to the framework sequences provided herein. In some embodiments, human frameworks were selected based on homology between the parent antibody and the human germline VH and VK genes. Selected frameworks, in some embodiments, had the highest homology with the parent antibody VH and VK genes and also were predicted, based on computer modeling or other means, to support the CDR structure predicted to be presented by the parent antibody.

[0121] Further modifications in the framework regions may be made to improve the properties of the antibodies provided herein. Such further framework modifications may include chemical modifications; point mutations to reduce immunogenicity or remove T cell epitopes; or back mutation to the residue in the original germline sequence. In one embodiment of the present invention, the humanized antibodies and fragments thereof comprise a human framework and grafted CDRs provided herein, without further modifications to the variable region. Humanized antibodies that do not comprise a human framework backmutation are herein termed HO (e.g., P1C4-H0). In another embodiment of the present invention, the humanized antibodies and fragments thereof comprise a human framework and grafted CDRs provided herein, wherein the amino acid at position 27 and/or and 28 of the heavy chain framework region 1 is backmutated. In a further embodiment, the amino acid at position 27 is backmutated from Gly (G) to Tyr (Y); and the amino acid at position 28 is backmutated from Thr (T) to Glu (E). Humanized antibodies having such mutations at positions 27 and 28 are herein described as "H2" or "H2 (YE)" (e.g., P1C4-H2 or P1C4-H2 (YE)). In another embodiment of the present invention, the humanized antibodies and fragments thereof comprise a human framework and grafted CDRs provided herein, wherein the amino acid at position 27 and/or and 28 of the heavy chain framework region 1 and the amino acid at position 60 and/or 61 of the heavy chain framework region 3 is backmutated. In a further embodiment, the amino acid at position 27 is backmutated from Gly (G) to Tyr (Y); the amino acid at position 28 is backmutated from Thr (T) to Glu (E); the amino acid at position 60 is backmutated from Ala (A) to Asn (N); and the amino acid at position 61 is backmutated from Gin (Q) to Gly (G). Humanized antibodies having such mutations at positions 27, 28, 60, and 61 are herein described as "H4" or "H4 (YENG)" (e.g., P1C4-H4 or P1C4-H4 (YENG)). In one embodiment of the present invention, the antibodies and antigen binding fragments thereof comprise framework modifications such as backmutations in the light chain. For example, in one embodiment, the antibodies comprise a mutation at position 45 and/or 47 of the light chain framework region 2. In a further embodiment, the amino acid at position 45 is mutated from Arg (R) to Lys (K) and the amino acid at position 47 is mutated from Leu (L) to Trp (W). The present invention also encompasses humanized antibodies that bind to CB l and comprise framework modifications corresponding to the exemplary modifications described herein with respect to any suitable framework sequence, as well as other framework modifications that otherwise improve the properties of the antibodies. The CB l antibodies and fragments thereof disclosed herein may be of an IgGl , IgG2, IgG3, or IgG4 isotype, or any combination thereof. The term "isotype" refers to the antibody class encoded by the heavy chain constant region genes. In addition, the heavy chain constant region may be derived from any species including, but not limited to, mouse, rat, rabbit, hamster, guinea pig, primate, llama or human. For example, in one embodiment, the CBl antibodies and fragments thereof of the present invention comprise a human IgGl Fc constant region. In another embodiment, the CBl antibodies and fragments thereof comprise a human IgG2, human IgG4, or hybrid IgG2-IgG4 Fc constant region.

II. Effector Functions and Fc Modifications

[0122] In some embodiments, present invention provides CB l antibodies comprising variant Fc regions. The Fc region of an antibody is the portion of the antibody that binds to Fey receptors (FcyRs) and the complement molecule C lq. The Fc region plays a role in mediating antibody effector functions. "Effector functions," as used herein in connection with antibody Fc, refers to antibody functions such as, for example, Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; opsonization; transcytosis; and down-regulation of cell surface receptors (e.g. B cell receptor). Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using various assays known in the art for evaluating such antibody effector functions. Variant Fc regions are Fc regions that comprise modifications that alter effector functions. In some embodiments, the CBl antibodies provided herein comprise Fc region modifications that reduce, impair, or eliminate one or more effector functions. For example, in one embodiment, the antibodies and fragments thereof disclosed herein bind CBl and exhibit reduced, impaired, or absent Clq binding and/or CDC and/or ADCC. Fc modifications may be amino acid insertions, deletions, or substitutions, or may be chemical modifications. For example, Fc region modifications may be made to increase or decrease complement binding; to increase or decrease antibody-dependent cellular cytoxicity; or to modify glycosylation. Various Fc modifications are known in the art and have been described, for example, in Labrijin et al, Nature Biotech 27(8):767-71 (2009); Idusogie, et al. J Immunol 2000; Greenwood et al Eur J Immunol 23: 1098-104 (1993); Mueller et al . Mol Immunol 1997; 34:441-52; and Rother et al Nature Biotechnol 2007; 25: 1256-64. Any of the Fc modifications known in the art may be applied to the exemplary CBl antibodies disclosed herein to alter effector function. Moreover, various therapeutic antibodies have been engineered to have Fc region modifications to alter effector function. Such therapeutic antibodies are known in the art and include, for example, alemtuzumab, benralizumab, bevacizumab, bimekizumab, cantuzumab, codrituzumab, dalotuzumab, efalizumab, elotuzumab, enavatuzumab, enokizumab, etrolizumab, farletuzumab, ficlatuzumab, imgatuzumab, itolizumab, lifastuzumab, ligelizumab, lodelcizumab, lorvotuzumab, mogamulizumab, motavizumab, obinutuzumab, ocaratuzumab, omalizumab, parsatuzumab, pateclizumab, perakizumab, pertuzumab, pidilizumab, quilizumab, rontalizumab, sofituzumab, solanezumab, suvizumab, teplizumab, tildrakizumab, tocilizumab, trastuzumab, trastuzumab emtansine, tregalizumab, vedolizumab, vorsetuzumab, vorsetuzumab mafodotin, yttrium (90 Y) clivatuzumab tetraxetan, anrukinzumab, dacetuzumab, daclizumab, etaracizumab, milatuzumab, ozanezumab, pinatuzumab vedotin, polatuzumab vedotin, tigatuzumab, veltuzumab, abituzumab, bococizumab, demcizumab, gevokizumab, ponezumab, ralpancizumab, romosozumab, tanezumab, blosozumab, concizumab, crenezumab, ibalizumab, ixekizumab, lebrikizumab, olokizumab, pembrolizumab, simtuzumab, ulocuplumab, vatelizumab, and samalizumab (see, e.g., SEQ ID NOs: 358-432). Any of the Fc modifications known in the art may be applied to the CBl receptor antibodies provided herein to alter effector function, antibody half life, or other antibody properties. [0123] In one embodiment, the CBl antibody exhibits reduced effector function. In a further embodiment, the CBl antibody comprises an IgG4 Fc region having a mutation at position 228. In a further embodiment, the amino acid at position 228 is mutated from serine (S) to proline (P) (i.e., S228P). In another embodiment, the CBl antibody exhibits reduced effector function and comprises an IgG2 Fc region having a mutation at position 330 and/or 331. In a further embodiment, the amino acid at position 330 is mutated from alanine (A) to serine (S), and/or the amino acid at position 331 is mutated from proline (P) to serine (S). In a further embodiment, the CBl antibody comprises an IgG2 Fc domain having both A330S and P331S mutations. In another embodiment, the CBl antibody comprises an IgG2/IgG4 hybrid Fc region. For example, in one embodiment, the CBl antibody comprises a CHI and hinge region derived from IgG2, and a CH2 and CH3 region derived from IgG4.

[0124] Conformation Antigen Presenting System (iCAPS). iCAPS enable the purified, isolated, conformationally correct presentation of functional GPCRs. Purified GPCRs are stabilized in lipid bilayers surrounded by a belt protein.

[0125] In one embodiment, the invention provides an isolated nucleic acid encoding any one of the antibodies and antigen binding fragments or variants thereof disclosed herein. In some embodiments, a vector comprising the isolated nucleic acid is provided. In some embodiments, a host cell transformed with the vector is provided. In some embodiments, the host cell is a prokaryotic cell. In further embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is a eukaryotic cell. In further embodiments, the eukaryotic cell is selected from the group consisting of protist cell, animal cell, plant cell and fungal cell. In some embodiments, the host cell is a mammalian cell including, but not limited to, 293, COS, NS0, and CHO and; or a fungal cell such as Saccharomyces cerevisiae; or an insect cell such as Sf9. One embodiment of the invention provides methods of producing the antibodies and fragments or variants described herein comprising culturing any one of the host cells also herein in a culture medium under conditions sufficient to produce the binding protein.

[0126] Exemplary CBl receptor binding antibodies of the invention are provided below in Table 1. Additional exemplary CBl receptor binding antibodies of the invention are defined by SEQ ID NO. and provided in the sequence listing as shown in Table 2. Sequences for the exemplary humanized CB 1 receptor binding antibodies of the invention are provided in Table 3 and Table 35.

Table 1. Nucleic acid and amino acid sequences of heavy chain variable regions and light chain variable regions of exemplary CBl receptor binding antibodies

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCC

TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG

GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

CTCCGGGTAAATGA

HC constant 4 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT amino acid QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE sequence LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain 5 GATATTGTTCTCACCCAGTCTCCAGCAATCATGTCTG

(LC) CATCTCTAGGGGAACGGGTCACCATGACCTGCACTG variable CCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTA region CCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGAT

TTATAGCACATCCAACCTGGCTTCTGGAGTCCCAGCT

nucleic acid

CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC

sequence

TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA

CTTATTACTGCCACCAGTATCATCGTTCCCCACCCAC

GTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

LC variable 6 DIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQ region QKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS amino acid MEAEDAATYYCHQYHRSPPTFGAGTKLELK sequence

LC constant 7 CGAACTGTGGCTGCACCATCTGTCTTCATCTTCC region CGCCATCTGATGAGCAGTTGAAATCTGGAACTG nucleic acid CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC sequence CAGAGAGGCCAAAGTACAGTGGAAGGTGGATA

ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG

TCACAGAGCAGGACAGCAAGGACAGCACCTACA

GCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG

ACTACGAGAAACACAAAGTCTACGCCTGCGAAG

TCACCCATCAGGGCCTGAGCTTGCCCGTCACAA

AGAGCTTCAACAGGGGAGAGTGTTAA

LC constant 8 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE region AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS amino acid TLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNPv sequence GEC 36E12B6 Heavy chain 9 CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGTG C2 (HC) AGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTT

(chimeric) variable CTGGCTATGAATTCAGTTACTACTGGATGAACTGGGT region GAAGCAGAGGCCTGGACAGGGTCTTCAGTGGATTGG

ACAGATTTATCCTGGAGATGGTGATACTAACTACAAT

nucleic acid

GGAAAGTTCAAGGGTAAAGCCACACTGACTGCAGAC

sequence

AAATCCTCCAGCACAGCCTACATGCACCTCACCAGC

CTAACGTCTGAGGACTCTGCGGTCTATTTCTGTGCAA

GATCGGGGGGTAACCCCTTTGCTTTCTGGGGCCAAG

GGACTCTGGTCACTGTCTCTGCA

HC variable 10 QVQLQQSGAELVRPGSSVKISCKASGYEFSYYWMNWV region KQRPGQGLQWIGQIYPGDGDTNYNGKFKGKATLTADK amino acid SSSTAYMHLTSLTSEDSAVYFCARSGGNPFAFWGQGTL sequence VTVSA

HC constant 1 1 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC region CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC nucleic acid TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT sequence GACGGTGTCATGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA

CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA

ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA

GTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC

TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT

GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC

CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA

CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC

CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA

GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCC

TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG

GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

CTCCGGGTAAATGA

HC constant 12 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT amino acid QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE sequence LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain 13 GATATCCAGATGACACAGACTTCATCCTCCCTGTCTG

(LC) CCTCTCTGGGAGACAGAGTCACCTTCAGTTGCAGGG variable CAAGTCAGGACATTAGCAATTATTTAAACTGGTATCA region GCAGAAACCAGATGGAACTGTTAAACTCCTGATCTA

CTACACATCAAGATTACACTCAGGAGTCACATCAAG

nucleic acid

GTTCCGTGGCAGTGGGTCTGGAACAGATTATTCTCTC

sequence

ACCATTAGCAACCTGGAGCAAGAAGACGTTGCCACT

TACTTTTGCCAACAGGGTCATACGCTTCCGTGGTCGT

TCGGTGGAGGCACCAAGCTGGAAATCAAA

LC variable 14 DIQMTQTSSSLSASLGDRVTFSCRASQDISNYLNWYQQ region KPDGTVKLLIYYTSRLHSGVTSRFRGSGSGTDYSLTISN amino acid LEQEDVATYFCQQGHTLPWSFGGGTKLEIK sequence

LC constant 15 CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGC region CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT nucleic acid TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC sequence AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG

GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC

AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG

CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC

GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC

GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

LC constant 16 RTVAAPSVFIFPPSDEQLKSGTASVVCLLN FYPREAKV region QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA amino acid DYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence

36E12B6 Heavy chain 17 CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGTG C2 (HC) AGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTT

(mouse) variable CTGGCTATGAATTCAGTTACTACTGGATGAACTGGGT region GAAGCAGAGGCCTGGACAGGGTCTTCAGTGGATTGG

ACAGATTTATCCTGGAGATGGTGATACTAACTACAAT

nucleic acid

GGAAAGTTCAAGGGTAAAGCCACACTGACTGCAGAC

sequence

AAATCCTCCAGCACAGCCTACATGCACCTCACCAGC

CTAACGTCTGAGGACTCTGCGGTCTATTTCTGTGCAA

GATCGGGGGGTAACCCCTTTGCTTTCTGGGGCCAAG

GGACTCTGGTCACTGTCTCTGCA

HC variable 18 QVQLQQSGAELVRPGSSVKISCKASGYEFSYYWMNWV region KQRPGQGLQWIGQIYPGDGDTNYNGKFKGKATLTADK amino acid SSSTAYMHLTSLTSEDSAVYFCARSGGNPFAFWGQGTL sequence VTVSA HC constant 19 GCTAAAACAACAGCCCCATCGGTCTATCCACTGGCC region CCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTC nucleic acid TAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGT sequence GACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGT

GTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCT

ACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCA

CCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCA

CCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGA

GCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATG

CAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCC

GTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCA

TGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGT

GGATGTGAGCGAGGATGACCCAGATGTCCAGATCAG

CTGGTTTGTGAACAACGTGGAAGTACACACAGCTCA

GACACAAACCCATAGAGAGGATTACAACAGTACTCT

CCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGA

CTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAA

CAACAAAGACCTCCCAGCGCCCATCGAGAGAACCAT

CTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGT

ATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAA

GAAACAGGTCACTCTGACCTGCATGGTCACAGACTT

CATGCCTGAAGACATTTACGTGGAGTGGACCAACAA

CGGGAAAACAGAGCTAAACTACAAGAACACTGAACC

AGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGC

AAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAG

AAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTG

CACAATCACCACACGACTAAGAGCTTCTCCCGGACT

CCGGGTAAATGA

HC constant 20 AKTTAP S VYPLAP VCGDTTG S S VTLGCLVKGYFPEPVT region LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS amino acid QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNL sequence LGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQ

ISWFVN VEVHTAQTQTHREDYNSTLRVVSALPIQHQD

WMSGKEFKCKVN KDLPAPIERTISKPKGSVPvAPQVYV

LPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTN GKT

ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS

CSVVHEGLHNHHTTKSFSRTPGK

Light chain 21 GATATCCAGATGACACAGACTTCATCCTCCCTGTCTG

(LC) CCTCTCTGGGAGACAGAGTCACCTTCAGTTGCAGGG variable CAAGTCAGGACATTAGCAATTATTTAAACTGGTATCA region GCAGAAACCAGATGGAACTGTTAAACTCCTGATCTA nucleic acid CTACACATCAAGATTACACTCAGGAGTCACATCAAG

GTTCCGTGGCAGTGGGTCTGGAACAGATTATTCTCTC

sequence

ACCATTAGCAACCTGGAGCAAGAAGACGTTGCCACT

TACTTTTGCCAACAGGGTCATACGCTTCCGTGGTCGT

TCGGTGGAGGCACCAAGCTGGAAATCAAA

LC variable 22 DIQMTQTSSSLSASLGDRVTFSCRASQDISNYLNWYQQ region KPDGTVKLLIYYTSRLHSGVTSRFRGSGSGTDYSLTISN amino acid LEQEDVATYFCQQGHTLPWSFGGGTKLEIK sequence

LC constant 23 CGGGCAGATGCTGCACCAACTGTATCCATCTTCCCAC region CATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAG nucleic acid TCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACAT sequence CAATGTCAAGTGGAAGATTGATGGCAGTGAACGACA

AAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

CAAAGACAGCACCTACAGCATGAGCAGCACCCTCAC

GTTGACCAAGGACGAGTATGAACGACATAACAGCTA

TACCTGTGAGGCCACTCACAAGACATCAACTTCACCC

ATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

LC constant 24 RADAAPTVSIFPPSSEQLTSGGASVVCFLN FYPKDINV region KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK amino acid DEYERHNSYTCEATHKTSTSPIVKSFNRNEC sequence

PA2LR3- Heavy chain 25 GAGGTTCAGCTGCAGCAGTCTGGGGCTGAACTGGTG

P4B5 (HC) AGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTT

(chimeric) variable CTGGCTATGCATTCAGTTATTACTGGATGAACTGGGT region GAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGG

ACAGATTTATCCTGGAGATGGTGATACTAACTACAGT

nucleic acid

GGAAGGTTCAAGGGTAAAGCCACACTGACTGCAGAC

sequence

AAATCCTCCAGCACAGCCTACATTCAGCTCAGCAGC

CTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAA

GATCGCACGGTAACTATTTTCCTTACTGGGGCCAAGG

GACTCTGGTCACTGTCTCTGCA

HC variable 26 EVQLQQSGAELVRPGSSVKISCKASGYAFSYYWMNWV region KQRPGQGLEWIGQIYPGDGDTNYSGRFKGKATLTADK amino acid SSSTAYIQLSSLTSEDSAVYFCARSHGNYFPYWGQGTL sequence VTVSA

HC constant 27 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC region CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC nucleic acid TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT sequence GACGGTGTCATGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA

CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA

ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA

GTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC

TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT

GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC

CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC

CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA

GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCC

TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG

GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

CTCCGGGTAAATGA

HC constant 28 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT amino acid QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE sequence LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain 29 GACATTGTTCTCAACCAGTCTCCAGCAATCATGTCTG

(LC) CATCTCTAGGGGAACGGGTCACCATGACCTGCACTG variable CCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTA region CCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGAT

TTATAGCACATCCAACCTGGCTTCTGGAGTCCCAGCT

nucleic acid

CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC

sequence

TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA

CTTATTACTGCCACCAGTATCATCGTTCCCCGCTCAC

GTTCGGTGCTGGGACCAAACTGGAAATAAAA

LC variable 30 DIVLNQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQ region QKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS amino acid MEAEDAATYYCHQYHRSPLTFGAGTKLEIK

sequence

LC constant 31 CGAACTGTGGCTGCACCATCTGTCTTCATCTTCC region CGCCATCTGATGAGCAGTTGAAATCTGGAACTG nucleic acid CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC sequence CAGAGAGGCCAAAGTACAGTGGAAGGTGGATA

ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG

TCACAGAGCAGGACAGCAAGGACAGCACCTACA

GCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG

ACTACGAGAAACACAAAGTCTACGCCTGCGAAG

TCACCCATCAGGGCCTGAGCTTGCCCGTCACAA

AGAGCTTCAACAGGGGAGAGTGTTAA LC constant 32 RTVAAPSVFIFPPSDEQLKSGTASVVCLL NFYPRE region AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS amino acid TLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNR sequence GEC

PA2LR3- Heavy chain 33 GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGTG

P2D3 (HC) AGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTT

(chimeric) variable CTGGCTATGCATTCAGTTACTACTGGATGAACTGGGT region GAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGG

ACAGATTTATCCTGGAGATGGTGATACTAACTACAAT

nucleic acid

GGAAAGTTCAAGGGTAAAGCCACACTGACTGCAGAC

sequence

AAATCCTCCAGTACAGCCTACATGCAGCTCAGCAGC

CTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAA

GATCGCACGGTAGCTATTTTGCTTACTGGGGCCAAGG

GACTCTGGTCACTGTCTCTGCA

HC variable 34 EVQLQQSGAELVRPGSSVKISCKASGYAFSYYWMNWV region KQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADK amino acid SSSTAYMQLSSLTSEDSAVYFCARSHGSYFAYWGQGTL sequence VTVSA

HC constant 35 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC region CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC nucleic acid TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT sequence GACGGTGTCATGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA

CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA

ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA

GTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC

TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT

GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC

CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA

CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC

CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA

GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCC

TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG

GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

CTCCGGGTAAATGA

HC constant 36 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT amino acid QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE sequence LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain 37 GATATTGAGCTGGCCCAATCTCCAGCTTCTTTGGCTG

(LC) TGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAG variable CCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTAT region GCACTGGTACCAGCAGAAACCAGGACAGCCACCCAA nucleic acid ACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGG

GTCCCTGCCAGGTTCAGCGGCAGTGGGTCTAGGGCA

sequence

GACTTCACCCTCACCATTGATCCTGTGGAGGCTGATG

ATGCTGCAACCTATTACTGTCTACAATATGCTAGTTC

TCCTCCTACGTTCGGTGCTGGGACCAAACTGGAAATA

AAA

LC variable 38 DIELAQSPASLAVSLGQRATISCRASESVDSYGNSFMH region WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRADFT amino acid LTIDPVEADDAATYYCLQYASSPPTFGAGTKLEIK sequence

LC constant 39 CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC region CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT nucleic acid TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC sequence AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG

GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC

AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG

CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC

GCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCC

GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA

LC constant 40 RTVAAPSVFIFPPSDEQLKSGTASVVCLLN FYPREAKV region QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA amino acid DYEKHKVYACEVTHQGLSLPVTKSFNRGEC sequence

PA2LR3- Heavy chain 41 GAGGTCCAGCTTCAGCAATCTGGGGCTGAGCTGGTG

P4B1 (HC) AGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTT

(chimeric) variable CTGGCTTTGCATTCAGTAACTACTGGATGAACTGGGT region GAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGG

ACAGATTTATCCTGGAGATGGTGATACTAACTTCAAT

nucleic acid

GGAAAGTTCAAGGGTAGAGCCATACTGACTGCAGAC

sequence

ATATCCTCCAACACAGCCTACATGCAGCTCAGCAGC CTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAA GATCGCACGGTAACTATTTTCCTTACTGGGGCCAAGG GACTCTGGTCACTGTCTCTGCA

HC variable 42 EVQLQQSGAELVRPGSSVKISCKASGFAFSNYWMNWV region KQRPGQGLEWIGQIYPGDGDTNFNGKFKGRAILTADISS amino acid NTAYMQLSSLTSEDSAVYFCARSHGNYFPYWGQGTLV sequence TVSA

HC constant 43 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC region CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC nucleic acid TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT sequence GACGGTGTCATGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA

CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA

ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA

GTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC

TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT

GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC

CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA

CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC

CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA

GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCC

TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG

GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

CTCCGGGTAAATGA

HC constant 44 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT amino acid QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE sequence LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain 45 CAAATTGTGTTGACACAGTCTCCAGCAATCATGTCTG (LC) CATCTCTAGGGGAACGGGTCACCATGACCTGCACTG variable CCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTA region CCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGAT nucleic acid TTATAGCACATCCAACCTGGCTTCTGGAGTCCCAGCT sequence CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC

TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA

CTTATTACTGCCACCAGTATCATCGTTCCCCGCTCAC

GTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

LC variable 46 QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQ region QKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS amino acid MEAEDAATYYCHQYHRSPLTFGAGTKLELK sequence

LC constant 47 CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC region CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT nucleic acid TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC sequence AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG

GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC

AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG

CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC

GCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCC

GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA

LC constant 48 RTVAAPSVFIFPPSDEQLKSGTASVVCLLN FYPREAKV region QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA amino acid DYEKHKVYACEVTHQGLSLPVTKSFNRGEC sequence

PA2LR3- Heavy chain 49 GAGGTCCAGCTTCAGCAATCTGGGGCTGAGCTGGTG

P6B12 (HC) AGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTT

(chimeric) variable CTGGCTATGCATTCAGTTACTACTGGATGAACTGGGT region GAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGG

ACAGATTTATCCTGGAGATGGTGATACTAACTACAAT

nucleic acid

GGAAAGTTCAAGGGTAAAGCCACACTGACTGCAGAC

sequence

AAATCCTCCAGTACAGCCTACATGCAGCTCAGCAGC

CTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAA

GATCGCACGGTAACTATTTTGCTTACTGGGGCCAAGG

GACTCTGGTCACTGTCTCTGCA

HC variable 50 EVQLQQSGAELVRPGSSVKISCKASGYAFSYYWMNWV region KQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADK amino acid SSSTAYMQLSSLTSEDSAVYFCARSHGNYFAYWGQGT sequence LVTVSA

HC constant 51 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC region CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC nucleic acid TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT sequence GACGGTGTCATGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA

CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA

GTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC

TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT

GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC

CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA

CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC

CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA

GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCC

TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG

GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

CTCCGGGTAAATGA

HC constant 52 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT amino acid QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE sequence LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain 53 CAAATTGTACTCACCCAGTCTCCAGCAATCATGTCTG

(LC) CATCTCTAGGGGAACGGGTCACCATGACCTGCACTG variable CCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTA region CCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGAT

TTATAGCACATCCAACCTGGCTTCTGGAGTCCCAGCT

nucleic acid

CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC

sequence

TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA

CTTATTACTGCCACCAGTATCATCGTTCCCCCCTCGC

GTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

LC variable 54 QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQ region QKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS amino acid MEAEDAATYYCHQYHRSPLAFGAGTKLELK sequence

LC constant 55 CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC region CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT nucleic acid TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC sequence AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG

GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC

AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG

CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC

GCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCC

GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA

LC constant 56 RTVAAPSVFIFPPSDEQLKSGTASVVCLLN FYPREAKV region QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA amino acid DYEKHKVYACEVTHQGLSLPVTKSFNRGEC sequence

PA2LR3- Heavy chain 57 GAGGTTCAGCTTCAGCAATCTGGGGCTGAGCTGGTG

P6G7 (HC) AGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTT

(chimeric) variable CTGGCTTTGCATTCAGTAACTACTGGATGAACTGGGT region GAAGCAGAGGCCCGGACAGGGTCTTGAGTGGATTGG

ACAGATTTATCCTGGAGATGGTGATACTAACTTCAAT

nucleic acid

GGAAAGTTCAAGGGTAGAGCCATACTGACTGCAGAC

sequence

ATATCCTCCAACACAGCCTACATGCAGCTCAGCAGC

CTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAA

GATCGCACGGTAACTATTTTCCTTACTGGGGCCAAGG

GACTCTGGTCACTGTCTCTGCA

HC variable 58 EVQLQQSGAELVRPGSSVKISCKASGFAFSNYWMNWV region KQRPGQGLEWIGQIYPGDGDTNFNGKFKGRAILTADISS amino acid NTAYMQLSSLTSEDSAVYFCARSHGNYFPYWGQGTLV sequence TVSA

HC constant 59 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC region CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC nucleic acid TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT sequence GACGGTGTCATGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA

CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA

ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA

GTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC

TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT

GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC

CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA

CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC

CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA

GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC

CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACGCC

TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG

GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

CTCCGGGTAAATGA

HC constant 60 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT amino acid QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE sequence LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain 61 GATATTGTGCTAACTCAGTCTCCAGCAATCATGTCCG

(LC) CATCTCTAGGGGAACGGGTCACCATGACCTGCACTG variable CCAGCTCAAGTGTAAGTTCCAGTTACTTACACTGGTA region CCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGAT

TTATAGCACCTCCAACCTGGCTTCTGGAGTCCCAGCT

nucleic acid

CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC

sequence

TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCA

CTTATTACTGCCACCAGCATCATCGTTCCCCACCCAC

GTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

LC variable 62 DIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQ region QKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS amino acid MEAEDAATYYCHQHHRSPPTFGAGTKLELK sequence

LC constant 63 CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC region CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT nucleic acid TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC sequence AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG

GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC

AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG

CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC

GCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCC

GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA

LC constant 64 RTVAAPSVFIFPPSDEQLKSGTASVVCLLN FYPREAKV region QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA amino acid DYEKHKVYACEVTHQGLSLPVTKSFNRGEC sequence

Table 2. SEQ ID NOs for additional CBl receptor binding antibodies Name Sequence description SEQ ID

NO

PA2LR3-P1G6 (chimeric) Heavy chain (HC) variable nucleic acid 65

HC variable region amino acid 66

HC constant region nucleic acid 67

HC constant region amino acid 68

Light chain (LC) variable region nucleic acid 69

LC variable region amino acid 70

LC constant region nucleic acid 71

LC constant region amino acid 72

PA2LR3-P1H4 (chimeric) Heavy chain (HC) variable nucleic acid 73

HC variable region amino acid 74

HC constant region nucleic acid 75

HC constant region amino acid 76

Light chain (LC) variable region nucleic acid 77

LC variable region amino acid 78

LC constant region nucleic acid 79

LC constant region amino acid 80

PA2LR3-P2B8 (chimeric) Heavy chain (HC) variable nucleic acid 81

HC variable region amino acid 82

HC constant region nucleic acid 83

HC constant region amino acid 84

Light chain (LC) variable region nucleic acid 85

LC variable region amino acid 86

LC constant region nucleic acid 87

LC constant region amino acid 88

PA2LR3-P2E5 (chimeric) Heavy chain (HC) variable nucleic acid 89

HC variable region amino acid 90

HC constant region nucleic acid 91

HC constant region amino acid 92

Light chain (LC) variable region nucleic acid 93

LC variable region amino acid 94

LC constant region nucleic acid 95

LC constant region amino acid 96

PA2LR3-P3A8 (chimeric) Heavy chain (HC) variable nucleic acid 97

HC variable region amino acid 98

HC constant region nucleic acid 99

HC constant region amino acid 100

Light chain (LC) variable region nucleic acid 101

LC variable region amino acid 102

LC constant region nucleic acid 103

LC constant region amino acid 104 PA2LR3-P3B10 (chimeric) Heavy chain (HC) variable nucleic acid 105

HC variable region amino acid 106

HC constant region nucleic acid 107

HC constant region amino acid 108

Light chain (LC) variable region nucleic acid 109

LC variable region amino acid 110

LC constant region nucleic acid 111

LC constant region amino acid 112

PA2LR3-P3B8 (chimeric) Heavy chain (HC) variable nucleic acid 113

HC variable region amino acid 114

HC constant region nucleic acid 115

HC constant region amino acid 116

Light chain (LC) variable region nucleic acid 117

LC variable region amino acid 118

LC constant region nucleic acid 119

LC constant region amino acid 120

PA2LR3-P3F8 (chimeric) Heavy chain (HC) variable nucleic acid 121

HC variable region amino acid 122

HC constant region nucleic acid 123

HC constant region amino acid 124

Light chain (LC) variable region nucleic acid 125

LC variable region amino acid 126

LC constant region nucleic acid 127

LC constant region amino acid 128

PA2LR3-P4C6 (chimeric) Heavy chain (HC) variable nucleic acid 129

HC variable region amino acid 130

HC constant region nucleic acid 131

HC constant region amino acid 132

Light chain (LC) variable region nucleic acid 133

LC variable region amino acid 134

LC constant region nucleic acid 135

LC constant region amino acid 136

PA2LR3-P4G10 (chimeric) Heavy chain (HC) variable nucleic acid 137

HC variable region amino acid 138

HC constant region nucleic acid 139

HC constant region amino acid 140

Light chain (LC) variable region nucleic acid 141

LC variable region amino acid 142

LC constant region nucleic acid 143

LC constant region amino acid 144

PA2LR3-P5E7 (chimeric) Heavy chain (HC) variable nucleic acid 145

HC variable region amino acid 146 HC constant region nucleic acid 147

HC constant region amino acid 148

Light chain (LC) variable region nucleic acid 149

LC variable region amino acid 150

LC constant region nucleic acid 151

LC constant region amino acid 152

PA2LR3-P6D7 (chimeric) Heavy chain (HC) variable nucleic acid 153

HC variable region amino acid 154

HC constant region nucleic acid 155

HC constant region amino acid 156

Light chain (LC) variable region nucleic acid 157

LC variable region amino acid 158

LC constant region nucleic acid 159

LC constant region amino acid 160

PA2R3-P1A7 (chimeric) Heavy chain (HC) variable nucleic acid 161

HC variable region amino acid 162

HC constant region nucleic acid 163

HC constant region amino acid 164

Light chain (LC) variable region nucleic acid 165

LC variable region amino acid 166

LC constant region nucleic acid 167

LC constant region amino acid 168

PA2R3-P1F1 (chimeric) Heavy chain (HC) variable nucleic acid 169

HC variable region amino acid 170

HC constant region nucleic acid 171

HC constant region amino acid 172

Light chain (LC) variable region nucleic acid 173

LC variable region amino acid 174

LC constant region nucleic acid 175

LC constant region amino acid 176

PA13R3-P3A7 (chimeric) Heavy chain (HC) variable nucleic acid 177

HC variable region amino acid 178

HC constant region nucleic acid 179

HC constant region amino acid 180

Light chain (LC) variable region nucleic acid 181

LC variable region amino acid 182

LC constant region nucleic acid 183

LC constant region amino acid 184

PA13R3-P3C3 (chimeric) Heavy chain (HC) variable nucleic acid 185

HC variable region amino acid 186

HC constant region nucleic acid 187

HC constant region amino acid 188 Light chain (LC) variable region nucleic acid 189

LC variable region amino acid 190

LC constant region nucleic acid 191

LC constant region amino acid 192

PA13R3-P3D10 (chimeric) Heavy chain (HC) variable nucleic acid 193

HC variable region amino acid 194

HC constant region nucleic acid 195

HC constant region amino acid 196

Light chain (LC) variable region nucleic acid 197

LC variable region amino acid 198

LC constant region nucleic acid 199

LC constant region amino acid 200

PA13R3-P3D11 (chimeric) Heavy chain (HC) variable nucleic acid 201

HC variable region amino acid 202

HC constant region nucleic acid 203

HC constant region amino acid 204

Light chain (LC) variable region nucleic acid 205

LC variable region amino acid 206

LC constant region nucleic acid 207

LC constant region amino acid 208

PA13R3-P3F6 (chimeric) Heavy chain (HC) variable nucleic acid 209

HC variable region amino acid 210

HC constant region nucleic acid 211

HC constant region amino acid 212

Light chain (LC) variable region nucleic acid 213

LC variable region amino acid 214

LC constant region nucleic acid 215

LC constant region amino acid 216

PA13R3-P4C4 (chimeric) Heavy chain (HC) variable nucleic acid 217

HC variable region amino acid 218

HC constant region nucleic acid 219

HC constant region amino acid 220

Light chain (LC) variable region nucleic acid 221

LC variable region amino acid 222

LC constant region nucleic acid 223

LC constant region amino acid 224

PA13R3-P4F8 (chimeric) Heavy chain (HC) variable nucleic acid 225

HC variable region amino acid 226

HC constant region nucleic acid 227

HC constant region amino acid 228

Light chain (LC) variable region nucleic acid 229

LC variable region amino acid 230 LC constant region nucleic acid 231

LC constant region amino acid 232

PA13R3-P4G11 (chimeric) Heavy chain (HC) variable nucleic acid 233

HC variable region amino acid 234

HC constant region nucleic acid 235

HC constant region amino acid 236

Light chain (LC) variable region nucleic acid 237

LC variable region amino acid 238

LC constant region nucleic acid 239

LC constant region amino acid 240

PA13R3-P4H10 (chimeric) Heavy chain (HC) variable nucleic acid 241

HC variable region amino acid 242

HC constant region nucleic acid 243

HC constant region amino acid 244

Light chain (LC) variable region nucleic acid 245

LC variable region amino acid 246

LC constant region nucleic acid 247

LC constant region amino acid 248

PA15R3-P3A6 (chimeric) Heavy chain (HC) variable nucleic acid 249

HC variable region amino acid 250

HC constant region nucleic acid 251

HC constant region amino acid 252

Light chain (LC) variable region nucleic acid 253

LC variable region amino acid 254

LC constant region nucleic acid 255

LC constant region amino acid 256

PA15R3-P3A7(chimeric) Heavy chain (HC) variable nucleic acid 257

HC variable region amino acid 258

HC constant region nucleic acid 259

HC constant region amino acid 260

Light chain (LC) variable region nucleic acid 261

LC variable region amino acid 262

LC constant region nucleic acid 263

LC constant region amino acid 264

PA15R3-P3C9 (chimeric) Heavy chain (HC) variable nucleic acid 265

HC variable region amino acid 266

HC constant region nucleic acid 267

HC constant region amino acid 268

Light chain (LC) variable region nucleic acid 269

LC variable region amino acid 270

LC constant region nucleic acid 271

LC constant region amino acid 272 PA16R3-P2G6 (chimeric) Heavy chain (HC) variable nucleic acid 273

HC variable region amino acid 274

HC constant region nucleic acid 275

HC constant region amino acid 276

Light chain (LC) variable region nucleic acid 277

LC variable region amino acid 278

LC constant region nucleic acid 279

LC constant region amino acid 280

PA16R3-P1A6 (chimeric) Heavy chain (HC) variable nucleic acid 281

HC variable region amino acid 282

HC constant region nucleic acid 283

HC constant region amino acid 284

Light chain (LC) variable region nucleic acid 285

LC variable region amino acid 286

LC constant region nucleic acid 287

LC constant region amino acid 288

PA16R3-P1B5 (chimeric) Heavy chain (HC) variable nucleic acid 289

HC variable region amino acid 290

HC constant region nucleic acid 291

HC constant region amino acid 292

Light chain (LC) variable region nucleic acid 293

LC variable region amino acid 294

LC constant region nucleic acid 295

LC constant region amino acid 296

PA16R3-P1E5 (chimeric) Heavy chain (HC) variable nucleic acid 297

HC variable region amino acid 298

HC constant region nucleic acid 299

HC constant region amino acid 300

Light chain (LC) variable region nucleic acid 301

LC variable region amino acid 302

LC constant region nucleic acid 303

LC constant region amino acid 304

PA16R3-P1H5 (chimeric) Heavy chain (HC) variable nucleic acid 305

HC variable region amino acid 306

HC constant region nucleic acid 307

HC constant region amino acid 308

Light chain (LC) variable region nucleic acid 309

LC variable region amino acid 310

LC constant region nucleic acid 311

LC constant region amino acid 312

PA18R3-P1D8 (chimeric) Heavy chain (HC) variable nucleic acid 313

HC variable region amino acid 314 HC constant region nucleic acid 315

HC constant region amino acid 316

Light chain (LC) variable region nucleic acid 317

LC variable region amino acid 318

LC constant region nucleic acid 319

LC constant region amino acid 320

PA18R3-P1E5 (chimeric) Heavy chain (HC) variable nucleic acid 321

HC variable region amino acid 322

HC constant region nucleic acid 323

HC constant region amino acid 324

Light chain (LC) variable region nucleic acid 325

LC variable region amino acid 326

LC constant region nucleic acid 327

LC constant region amino acid 328

PA18R3-P1H5 (chimeric) Heavy chain (HC) variable nucleic acid 329

HC variable region amino acid 330

HC constant region nucleic acid 331

HC constant region amino acid 332

Light chain (LC) variable region nucleic acid 333

LC variable region amino acid 334

LC constant region nucleic acid 335

LC constant region amino acid 336

Table 3. Se uences of exem lar humanized antibodies

region

Humanized 340 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4-H2 MGQIYPGDGETKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC heavy chain ARSHGNYLPYWGQGTLVTVSS

variable

region

Humanized 341 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4-H4 MGQIYPGDGETKYNGKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC heavy chain ARSHGNYLPYWGQGTLVTVSS

variable

region

Humanized 342 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV P1C4 heavy HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK chain SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE constant DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG region KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Humanized 343 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYWMNWVRQAPGQGLEW P1C4 HO MGQIYPGDGETKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG2-4 ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV Hybrid KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ

TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPEFLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Humanized 344 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYWMNWVRQAPGQGLEW P1C4 HO MGQIYPGDGETKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG2A330S/ ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV P331S KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ

TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST

FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Humanized 345 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYWMNWVRQAPGQGLEW P1C4 HO MGQIYPGDGETKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG4S228P ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT

LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDS

DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Humanized 346 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4 H2 MGQIYPGDGETKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG2-4 ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV Hybrid KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ

TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPEFLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVL

DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Humanized 347 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4 H2 MGQIYPGDGETKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG2A330S/ ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV P331S KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ

TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST

FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPMLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Humanized 348 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4 H2 MGQIYPGDGETKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG4S228P ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT

YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT

LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDS

DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Humanized 349 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4 H4 MGQIYPGDGETKYNGKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG2-4 ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV Hybrid KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ

TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPEFLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVL

DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Humanized 350 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4 H4 MGQIYPGDGETKYNGKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG2A330S/ ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV P331S KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ

TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST

FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPMLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Humanized 351 QVQLVQSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVRQAPGQGLEW P1C4 H4 MGQIYPGDGETKYNGKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC IgG4S228P ARSHGNYLPYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT

YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT

LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDS

DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0127] In some embodiments, the anti-CBl receptor antibodies provided herein comprise the sequences provided herein or conservative variants thereof. "Conservative variants," as used herein, include conservative amino acid substitutions, insertions, or deletions. The person of skill in the art will recognize that a conservative amino acid substitution is a substitution of one amino acid with another amino acid that has a similar structural or chemical properties, such as, for example, a similar side chain; and a conservative amino acid substitution, insertion or deletion results in a sequence that retains the biological activity of the reference sequence. Exemplary conservative substitutions are described in the art, for example, in Watson et ah, Molecular Biology of the Gene, The Bengamin/Cummings Publication Company, 4 ^th Ed. (1987).

III. Methods of Treating CBl-Associated Disorders

[0128] The antibodies and antigen binding fragments thereof disclosed herein can be administered to a human subject for therapeutic purposes. In some embodiments, methods of treatment comprising administering the antibodies and binding fragments or variants thereof disclosed herein to a subject. [0129] In certain embodiments, methods are provided for treatment of diseases wherein the peripheral CBl receptors are preferentially targeted. "Peripheral CBl receptors", as defined herein, are those CBl receptors that are not localized to the brain or central nervous system (e.g. peripherally restricted CBl receptors). In contrast, the term "global CBl receptors" refers to CBl receptors anywhere in the body, including the brain and CNS.

[0130] In one embodiment, the isolated antibodies and antigen binding fragments thereof are useful in the treatment of various diseases or disorders such as, for example, obesity, diabetes, dyslipidemia, fibrosis, non-alcoholic steatohepatitis (NASH), liver diseases, primary biliary cirrhosis, cardiovascular disease, cancer, pain, multiple sclerosis (MS) spasticity, glaucoma, inflammatory diseases, nephropathies, osteoporosis, metabolic disorders, psychiatric disorders, neurological disorders, neurodegenerative disorders, reproductive disorders, renal disease, kidney fibrosis, chronic kidney disease, atherosclerosis, cancer, and skin disorders, among others.

[0131] CBl receptor signaling has been shown to exhibit detrimental activity in, for example, obesity, diabetes, fibrosis, liver diseases, cardiovascular disease, and cancer. (Kunos et al., 2009, Trends Pharmacol Sci 30: 1-7.) In one aspect, the anti-CBl antibodies, or fragments thereof, disclosed herein are useful for antagonizing CBl activity. Accordingly, in another aspect, the invention provides methods for treating CBl -associated diseases or disorders by administering to a subject in need of thereof a pharmaceutical composition comprising one or more anti-CBl antibodies, or antigen binding fragments thereof disclosed herein. In some embodiments, the antagonistic CBl receptor antibodies and fragments thereof provided herein provide a beneficial effect when used as a treatment for, or for prevention of, obesity, diabetes, fibrosis, liver diseases, cardiovascular diseases, addictions such as nicotine addiction, or cancers.

[0132] Nonalcoholic steatohepatitis (NASH), also known as nonalcoholic fatty liver disease (NAFLD), refers to the accumulation of hepatic steatosis not due to excess alcohol consumption. NASH is a liver disease characterized by inflammation of the liver with concurrent fat accumulation. NASH is also frequently found in people with diabetes and obesity and is related to metabolic syndrome. NASH is the progressive form of the relatively benign non-alcoholic fatty liver disease, for it can slowly worsen causing fibrosis accumulation in the liver, which leads to cirrhosis (reviewed in Smith, et al., 2011, Crit Rev Clin Lab Sci., 48(3):97-l 13). Currently, no specific therapies for NASH exist.

[0133] In one aspect, the anti-CBl antibodies or fragments thereof disclosed herein are used in the treatment, prevention, detection, or study of fibrosis. Several studies in mouse models have confirmed the role of CBl receptor in fibrosis, including liver fibrosis. (See, e.g., Wei et al, 2014, Exp. Biol. Med. 239(2): 183-192; Tarn et al, 2010, J. Clin. Invest. 120(8):2953-66; Wan et al, 2014, Cell Metabolism, 19(6):900-1; Takano et al, 2014, Synapse, 68:89-97). Peripheral CBl has been implicated in several mechanisms contributing to NASH and liver fibrosis, including steatosis (fatty liver), inflammation, and liver injury (reviewed by Mallat et al., 2013, J Hepatology, 59(4):891-896). CBl has been demonstrated to be up-regulated in activated human hepatic stellate cells (HSC), which mediate fibrosis by transitioning into myofibroblasts. (Teixeira-Clerc et al., 2006, Nature Med., 12(6):671-76). CBl has also been implicated in diabetic nephropathy. Lin et al, 2014 J. Mol. Med. 92(7):779-92.)

[0134] Studies in hepatocyte-specific and global CBl -knockout mice have implicated a major role of CBl in peripheral cell type (hepatocytes) relevant to several metabolic diseases and disorders. In a mouse model of diet-induced obesity, both global CBl knockout (CBl-/-) and hepatocyte-specific CBl knockout (LCBl-/-) demonstrated reduced steatosis (fatty liver) and increased liver function, thus demonstrating a role of CBl in peripheral cell type (hepatocytes) relevant to non-alcoholic steatohepatitis (NASH), diabetes, and metabolic syndrome disease pathologies. (Osei-Hyiaman et al, 2008, J. Clin. Invest., 118(9):3160-3169; Liu et al, 2012, Gastroenterology, 142: 1218-1228). Selective knockdown of CBl using a macrophage-specific CBl knockdown siRNA (CBlR-GeRPs) prevents progressive hyperglycemia and decline in plasma insulin and C-peptide in Zucker diabetic fatty (ZDF) rats, which are a common model for T2D insulin resistance, hyperglycemia and beta cell failure. (Jourdan et al., 2013, Nature Med. 19(9): 1132-1140) In a mouse model of alcohol-induced liver steatosis, both global CBl knockout (CBl-/-) and hepatocyte-specific CBl knockout (LCBl-/-) have reduced steatosis and increased liver function, thus demonstrating a role of CBl in peripheral cell type (hepatocytes) relevant to steatosis disease pathology. (Jeong, et al, 2008, Cell Metabolism, 7:227-235). Lipid accumulation was shown to be reduced in epididymal white adipose cell lines generated from CBl knockout mice relative to wild-type control (Wagner et al., 2011, Nutrition and Diabetes, l :el6).

[0135] Studies in different models of disease in mouse have shown that peripherally- restricted CBl receptor small molecule antagonists can effectively inhibit liver fibrosis progression. (See, e.g., Wei et al, 2014, Exp. Biol. Med. 239(2): 183-192; Tarn et al, 2010, J. Clin. Invest. 120(8):2953-66; Wan et al, 2014, Cell Metabolism, 19(6):900-1; Takano et al, 2014, Synapse, 68:89-97.) Non-limiting examples of known CBl antagonists include rimonabant, taranabant , VD60, Isis-414930 Antisense CBl, JD5037, AM6545, and TM38837. CBl antagonists such as rimonabant have been shown to inhibit cell proliferation and down- regulate pro-fibrotic gene expression in primary human hepatic stellate cells (HSC), which mediate fibrosis by transitioning into myofibroblasts (Patsenker et al., 2011, Mol Med., 17(11- 12):1285-1294). In the CCl ₄ -induced liver fibrosis mouse model, CBl antagonist VD60 (3,4,22- 3 -demethoxycarbonyl-3 -hydroxylmethyl-4-deacetyl-vindoline 3 ,4-thionocarbonate) was demonstrated to inhibit production of pro-fibrotic gene expression (alpha collagen) and proliferation in activated hepatic stellate cells (HSC line LX-2), while selective CBl agonist ACEA (N-(2-chloroethyl)-5Z,8Z,l lZ,14Z-eicosatetraenamide) prevented this effect (Wei Y. et al, 2014, Exp. Biol. Med. 239(2): 183-192). CBl antagonist JD5037 has been shown to reverse endocannabinoid-induced inhibition of insulin signaling. (Cinar et al, 2014, Hepatology, 59(1)143-153). CBl blockade using rimonabant reverses inflammation-induced impairment of glucose uptake in adipocytes isolated from high- fat diet rats (Miranville et al, 2010, Obesity 18: 2247-2254).

[0136] Human studies also link peripheral CBl receptors to disease etiology and progression. For example, up-regulation of CBl in liver of NASH and HCV patients correlated with severity of liver steatosis and fibrosis. (Auguet et al, 2014, BioMed Res. Intl. Vol. 2014, Article ID 502542). In addition, chronic CBl agonism (via cannabis use) correlated with increased severity of liver steatosis and fibrosis in HCV patients. (Van der Poorten et al, 2010, PlosOne 5, el2841) Furthermore, it has been shown that CBl blockade in obese patients improves liver steatosis. (Despres et al., 2009, Arterioscler Thromb Vase Biol. 29:416-423). [0137] It will also be recognized that the effect of CBl antagonism differs depending on the location of the receptor. For instance, it is known that the effects of CB 1 antagonism are tissue specific, as beneficial cardiometabolic effects of rimonabant observed in patients are independent of weight loss. (Pi-Sunyeret al, 2006, J Am Coll Cardio. 147: 362A). Furthermore, it is known that rimonabant improves glycemic control in type 2 diabetes patients, (see, e.g., Hollander et al, 2010, Diabetes Care. 33(3):605-7) but that this effect is accompanied by significant psychiatric side effects imparted by CBl receptors located in the CNS. (Kunos et al, 2009, Trends Pharmacol Sci 30: 1-7; Moreira et al, 2009, Rev Bras Psiquiatr. 31(2): 145-53; Pacher et al, 2013, FEBS J. 280(9): 1918-1943.) The CBl receptor antagonist rimonabant was shown to improve the profile of several metabolic risk factors (including adiponectin levels) in overweignt patients according to the Rimonabant in Obesity-Lipids (RIO-Lipids) study. (See, e.g., Despres et al, 2005, N Engl J Med, 353:2121-2134).

[0138] CBl receptor signaling has been shown to exhibit beneficial activity in and pain, MS spasticity, and glaucoma, among others. (Pacher et al, 2013, FEBS J. 280(9): 1918—1943). In some embodiments, the agonistic CBl receptor antibodies and fragments thereof provided herein provide a beneficial effect when used as a treatment for, or for prevention of, pain, MS spasticity, or glaucoma. CBl agonists have been demonstrated to activate liver fatty acid synthesis, gluconeogenesis, and other metabolic pathways. (See, e.g., Osei-Hyiaman et al, 2005, J. Clin. Invest., 115(5): 1298-1305; Chanda et al, 2012, JBC, 287(45):38041-38049).

[0139] Multiple Sclerosis (MS) spasticity refers to feelings of stiffness and a wide range of involuntary muscle spasms (sustained muscle contractions or sudden movements). Spasticity is one of the more common symptoms of MS, and can vary in degree from mild tightness to painful, uncontrollable spasms of extremities. Left untreated, spasticity can lead to serious complications, including contractures (frozen or immobilized joints) and pressure sores. Current treatment options for MS spasticity include baclofen, tizanidine, diazepam, dantrolene, phenol, among others. CBl receptors have been shown to mediate control of spasticity in a mouse model of MS. (Pryce et al, 2007, Br J Pharmacol. 150(4): 519-525).

[0140] Activation of CBl receptors produces analgesic effects in several experimental pain models, including visceral pain arising from the gastrointestinal tract. CBl agonists such as WIN55,212-2 and SAB-378 have also been shown to inhibit pain-related responses to repetitive noxious stimuli (Brusberg et al, 2009, J. Neuroscience, 29(5): 1554-1564; Talwar et al, 2011, CNS Neurol Disord Drug Targets. 10(5):536-44.)

[0141] One skilled in the art would be able, by routine experimentation, to determine what an effective, non-toxic amount of antibody (or additional therapeutic agent) would be for the purpose of treating a CB1 -associated disease or disorder. For example, a therapeutically active amount of a polypeptide may vary according to factors such as the disease stage (e.g., stage I versus stage IV), age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the antibody to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Generally, however, an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day and in an embodiment from about 0.5 to 10, milligrams per kilogram body weight per day.

[0142] In some embodiments, the anti-CBl antibodies and fragments disclosed herein are used in methods utilizing a combination therapy wherein human antibodies are administered to a subject with another therapeutic agent, such as one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Because signaling pathway redundancies can result in lack of response to a single antibody, diverse strategies to use combination therapy with antibodies that bind to different epitopes or different antigens on the same target cell have been proposed. Combinations such as anti-CD20 and anti- CD22 (Stein et al, Clin Cancer Res 2004, 10:2868-2878), anti-CD20 and anti-HLA-DR (Tobin et al, Leuk Lymphoma 2007, 48:944-956), anti-CD20 and anti-TRAIL-Rl (Maddipatla et al, Clin Cancer Res 2007, 13:4556-4564), anti-IGF-lR and anti-EGFR (Goetsche et al, Int J Cancer 2005, 113:316-328), anti-IGF-lR and anti-VEGF (Shang et al, Mol Cancer Ther 2008, 7:2599- 2608), or trastuzumab and pertuzumab that target different regions of human EGFR2 (Nahta et al., Cancer Res 2004, 64:2343-2346) have been evaluated preclinically, showing enhanced or synergistic antitumor activity in vitro and in vivo. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

[0143] The antibodies and fragments disclosed herein can be administered in combination with any desired therapeutic agent. In certain embodiments, the antibodies and fragments disclosed herein are administered in combination with, for example, a LOXL2 antibody, TGFP antibody, nintedanib, tyrosine kinase inhibitor, PPAR agonist, Farnesoid X receptor (FXR) agonist, glucagon-like peptide 1 receptor agonist, or caspase inhibitor.

IV. Pharmaceutical Compositions

[0144] In another aspect, the invention provides pharmaceutical compositions comprising an anti-CBl antibody, or fragment thereof.

[0145] Methods of preparing and administering antibodies, or fragments thereof, disclosed herein to a subject are well known to or are readily determined by those skilled in the art. The route of administration of the antibodies, or fragments thereof, disclosed herein may be oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral administration can be used in certain embodiments. While all these forms of administration are clearly contemplated as being within the scope disclosed herein, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachings herein, the polypeptides can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0146] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M (e.g. 0.05M) phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will in an embodiment be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In certain embodiments, isotonic agents are included, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0147] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., an antibody by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation can be vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which is incorporated herein by reference. Such articles of manufacture will in an embodiment have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders.

[0148] Effective doses of the stabilized antibodies, or fragments thereof, disclosed herein, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0149] For passive immunization with an antibody disclosed herein, the dosage may range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, or in particular embodiments at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope disclosed herein.

[0150] Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimens entail administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered may fall within the ranges indicated.

[0151] Antibodies or fragments thereof, disclosed herein can be administered on multiple occasions. Intervals between single dosages can be, e.g., daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of polypeptide or target molecule in the patient. In some methods, dosage is adjusted to achieve a certain plasma antibody or toxin concentration, e.g., 1-1000 ug/ml or 25-300 ug/ml. Alternatively, antibodies, or fragments thereof, can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies show the longest half-life, followed by chimeric antibodies and nonhuman antibodies. In one embodiment, the antibodies, or fragments thereof, disclosed herein can be administered in unconjugated form. In another embodiment, the antibodies disclosed herein can be administered multiple times in conjugated form. In still another embodiment, the antibodies, or fragments thereof, disclosed herein can be administered in unconjugated form, then in conjugated form, or vice versa.

[0152] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose." In this use, the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose. A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.

[0153] In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and, in particular embodiments, until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0154] In one embodiment, a subject can be treated with a nucleic acid molecule encoding a polypeptide disclosed herein (e.g., in a vector). Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ug to 10 mg, or 30-300 ug DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.

[0155] Therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic or therapeutic treatment. Intramuscular injection or intravenous infusion can be used for administration of an antibody disclosed herein . In some methods, therapeutic antibodies, or fragments thereof, are injected directly into the cranium. In some methods, antibodies, or fragments thereof, are administered as a sustained release composition or device, such as a Medipad™ device.

[0156] Agents disclosed herein can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic). Additional agents are those which are art recognized and are routinely administered for a particular disorder.

[0157] While a great deal of clinical experience has been gained with 1311 and 90Y, other radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging. For example, additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 1231, 1251, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 1321, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac, 211A 213BL In this respect alpha, gamma and beta emitters are all compatible with in the instant invention. Further, in view of the instant disclosure it is submitted that one skilled in the art could readily determine which radionuclides are compatible with a selected course of treatment without undue experimentation. To this end, additional radionuclides which have already been used in clinical diagnosis include 1251, 1231, 99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as l l lln. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. Immunol. Cell Biol. 65: 111, 1987). These radionuclides include 188Re and 186Re as well as 199Au and 67Cu to a lesser extent. U.S. Patent No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.

[0158] As previously discussed, the antibodies, or fragments thereof, disclosed herein can be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be appreciated that the disclosed antibodies, or fragments thereof, will be formulated so as to facilitate administration and promote stability of the active agent. In certain embodiments, pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of an antibody disclosed herein, conjugated or unconjugated to a therapeutic agent, shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell. In the case of tumor cells, the polypeptide will in certain embodiments be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and provide for an increase in the death of those cells. Of course, the pharmaceutical compositions disclosed herein may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.

[0159] In keeping with the scope of the present disclosure, the antibodies disclosed herein may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The polypeptides disclosed herein can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody disclosed herein with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of polypeptides according to the present invention may prove to be particularly effective. [0160] Also disclosed herein is a method of treating a condition caused by increased expression of CBl or increased sensitivity to CBl comprising administering to a patient or other subject orally, parenterally by a solution for injection, by inhalation, or topically a pharmaceutically effective amount of a CBl antibody.

[0161] Also disclosed herein is the use of a pharmaceutically effective amount of a CBl antibody for the manufacture of a medicament for treating a condition caused by increased expression of CBl or increased sensitivity to CBl comprising administering to a patient or other subject orally, parenterally by a solution for injection, by inhalation, or topically.

[0162] In some embodiments, the disclosed isolated antibodies and antigen binding fragments thereof have the advantage of minimal brain penetration. In some embodiments, the isolated antibodies and fragments thereof exhibit high selectivity for CBl receptor and do not penetrate the blood brain barrier, or exhibit reduced penetration of the blood brain barrier relative to small molecule CBl receptor compounds, so that CNS side effects are minimized. In further embodiments, the isolated antibodies and fragments thereof do not penetrate the blood brain barrier, or exhibit reduced penetration of the blood brain barrier relative to small molecule CBl receptor compounds such as rimonabant, following intravenous injection.

[0163] In one embodiment, the antibodies and binding fragments or variants thereof disclosed herein may be administered to the subject by at least one route selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intratympanic, intrauterine, intravesical, intravitreal, bolus, subconjunctival, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

[0164] The present invention provides isolated antibodies and antigen binding fragments thereof, and nucleic acids encoding such antibodies and fragments, as well as compositions comprising such isolated antibodies, fragments, and nucleic acids. The present invention further provides pharmaceutical compositions comprising the isolated antibodies or fragments thereof, or nucleic acids encoding such antibodies or fragments, and further comprising one or more pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.

DESCRIPTION OF PARTICULAR ASPECTS AND EMBODIMENTS

[0165] The different aspects disclosed herein and their embodiments can be combined with each other. In addition, any of the aspects and their embodiments described above can be combined with any of the particular aspects and embodiments described herein below.

[0166] Some particular aspects and embodiments that further serve to illustrate the present invention are provided in the following:

[0167] 1. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid 1 (CBl) receptor, wherein the antibody or fragment has a binding affinity Kd for CBl receptor of about ΙμΜ or less.

[0168] 2. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment has a binding affinity Kd for CBl receptor of about lOOnM or less.

[0169] 3. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment has a binding affinity Kd for CBl receptor of about lOnM or less.

[0170] 4. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment has a binding affinity Kd for CBl receptor of about InM or less.

[0171] 5. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment binds to an extracellular epitope on CBl receptor.

[0172] 6. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment binds to human CB 1 receptor.

[0173] 7. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment inhibits CB 1 receptor signaling activity.

[0174] 8. The isolated antibody or antigen binding fragment of embodiment 7, wherein the antibody or fragment has CBl receptor signaling inhibiting activity that is at least equivalent in potency relative to small molecule rimonabant, wherein the potency is measured by inhibition of CB 1 receptor agonist-mediated signal transduction in a cAMP assay.

[0175] 9. The isolated antibody or antigen binding fragment of embodiment 7, wherein the antibody or fragment has CB1 receptor signaling inhibiting activity that is at least 3 fold more potent relative to small molecule rimonabant, wherein the potency is measured by inhibition of CB 1 receptor agonist-mediated signal transduction in a cAMP assay.

[0176] 10. The isolated antibody or antigen binding fragment of embodiment 7, wherein the antibody or fragment has CB1 receptor signaling inhibiting activity that is at least equivalent in potency relative to small molecule rimonabant, wherein the potency is measured by the inhibition of CB 1 receptor agonist-mediated ER phosphorylation.

[0177] 11. The isolated antibody or antigen binding fragment of embodiment 7, wherein the antibody or fragment has CB1 receptor signaling inhibiting activity that is at least 3 fold more potent relative to small molecule rimonabant, wherein the potency is measured by the inhibition of CB1 receptor agonist-mediated ERK phosphorylation.

[0178] 12. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment activates or enhances CB1 receptor activity.

[0179] 13. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment is an allosteric modulator of CB1 receptor.

[0180] 14. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment is an inverse agonist of CB1 receptor.

[0181] 15. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment is murine.

[0182] 16. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment is chimeric.

[0183] 17. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment is humanized.

[0184] 18. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment selectively binds CB 1. [0185] 19. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody is conjugated to an agent, for example, an additional therapeutic agent, a cytotoxic agent, an immunoadhesion molecule, or an imaging agent.

[0186] 20. The isolated antibody or antigen binding fragment of embodiment 19, wherein the agent is an additional therapeutic agent, a cytotoxic agent, an immunoadhesion molecule, or an imaging agent.

[0187] 21. The isolated antibody or antigen binding fragment of embodiment 20, wherein the therapeutic agent is rimonabant.

[0188] 22. The isolated antibody or antigen binding fragment of embodiment 20, wherein the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

[0189] 23. The isolated antibody or antigen binding fragment of embodiment 18, wherein the antibody does not have agonistic or antagonistic activity.

[0190] 24. An antibody or antigen binding fragment thereof that is capable of competing for binding to CB 1 receptor with the antibody or antigen binding fragment according to embodiment 1.

[0191] 25. An antibody or antigen binding fragment thereof that is capable of specifically binding to essentially the same epitope on CBl receptor as the antibody or antigen binding fragment according to embodiment 1.

[0192] 26. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or antigen binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, and 26; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, and 30.

[0193] 27. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, and 28, and a light chain constant region selected from the group consisting of SEQ ID NOs: 8, 16, 24, and 32. [0194] 28. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 2; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 4; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 6, and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 8.

[0195] 29. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 10; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 12; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 14, and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 16.

[0196] 30. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 18; a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 20; a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 22, and a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 24.

[0197] 31. The isolated antibody or antigen binding fragment of embodiment 1, wherein the antibody or fragment thereof comprises a heavy chain variable region comprises an amino acid sequence according to SEQ ID NO: 26; a heavy chain constant region according to SEQ ID NO: 28; a light chain variable region according to SEQ ID NO: 30, and a light chain constant region according to SEQ ID NO: 32.

[0198] 32. A method of antagonizing CB1, the method comprising contacting a cell expressing CB1 receptor with an antibody or binding fragment according to embodiment 1.

[0199] 33. A method of agonizing CB1, the method comprising contacting a cell expressing CB 1 receptor with an antibody or binding fragment according to embodiment 1. [0200] 34. A method of treating a disease or disorder responsive to antagonism or agonism of CB1 receptor in a subject in need thereof, the method comprising administering to the subject an antibody or antigen binding fragment according to embodiment 1.

[0201] 35. The method of embodiment 34, wherein the subject is a human.

[0202] 36. A method for detecting CB1, comprising contacting a cell with an antibody or antigen binding fragment according to embodiment 1.

[0203] 37. The method of embodiment 34, wherein the disease or disorder is selected from the group consisting of obesity, diabetes, dyslipidemia, metabolic diseases, fibrosis, nonalcoholic steatohepatitis (NASH), liver disease, primary biliary cirrhosis, renal disease, kidney fibrosis, chronic kidney disease, osteoporosis, atherosclerosis, cardiovascular disease, cancer, and inflammatory disease.

[0204] 38. The method of embodiment 34, wherein the disease or disorder is selected from the group consisting of pain, multiple sclerosis spasticity and glaucoma.

[0205] 39. The method of embodiment 37, wherein the disease or disorder is fibrosis.

[0206] 40. The method of embodiment 37, wherein the antibody or antigen binding fragment antagonizes CB1.

[0207] 41. The method of embodiment 38, wherein the antibody or antigen binding fragment agonizes CB1.

[0208] 42. A method for diagnosing a disease or disorder associated with CB1, the method comprising contacting a cell with an antibody or antigen binding fragment according to embodiment 1.

[0209] 43. A method for determining the prognosis for a subject diagnosed with a disease or disorder associated with CB1, the method comprising measuring CB1 expression by contacting a cell with an antibody or fragment thereof according to embodiment 1.

[0210] 44. The method of embodiment 42-43, wherein the disease or disorder is selected from the group consisting of obesity, diabetes, dyslipidemia, metabolic diseases, fibrosis, NASH, liver disease, primary biliary cirrhosis, renal disease, kidney fibrosis, chronic kidney disease, osteoporosis, atherosclerosis, cardiovascular disease, cancer, an inflammatory disease, pain, MS spasticity, and ocular diseases, including glaucoma. [0211] 45. The method of embodiment 44, wherein the disease or disorder is fibrosis.

[0212] 46. The method of embodiment 36, wherein the isolated antibody or antigen binding fragment thereof is conjugated to an imaging agent.

[0213] 47. The method of embodiment 46, wherein the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

[0214] 48. The method of embodiment 42-43, wherein the cell is present in a subject.

[0215] 49. The method of embodiment 48, wherein the subject is a human.

[0216] 50. A host cell expressing the isolated antibody or fragment according to embodiment

1.

[0217] 51. A method of making an antibody or fragment thereof that specifically binds to CBl, the method comprising immunizing mammals with purified CBl receptor or an antigenic fragment thereof, CBl/lipid complexes, and/or CBl receptor DNA.

[0218] 52. The method of embodiment 51, wherein the antibody or fragment thereof is generated from a hybridoma cell line comprising cells derived from the immunized mammals.

[0219] 53. The method of embodiment 51, wherein the antibody or fragment thereof is generated from a phage library.

[0220] 54. A method of making an antibody or fragment thereof that specifically binds to CBl, the method comprising generating a phage library comprising variable heavy and light chain regions from human primary blood lymphocytes and panning the phage library for CBl receptor binding.

[0221] 55. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CBl), wherein the antibody or antigen binding fragment thereof comprises a heavy chain CDRl, CDR2, and CDR3 according to SEQ ID NOs: 352, 353, and 354, respectively.

[0222] 56. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CBl), wherein the antibody or antigen binding fragment thereof comprises a light chain CDRl, CDR2, and CDR3 according to SEQ ID NOs: 355, 356, and 357, respectively. [0223] 57. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CB1), wherein the antibody or antigen binding fragment thereof comprises a heavy chain CDRl, CDR2, and CDR3 sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NOs: 352, 353, and 354, respectively.

[0224] 58. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CB1), wherein the antibody or antigen binding fragment thereof comprises a light chain CDRl, CDR2, and CDR3 sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence of SEQ ID NOs: 355, 356, and 357, respectively.

[0225] 60. An antibody or antigen binding fragment thereof that specifically binds to the same epitope as the antibody or antigen binding fragment according to any one of embodiments listed or disclosed herein.

[0226] 61. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CB1), wherein the antibody or antigen binding fragment thereof comprises: a heavy chain CDRl comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 443-463; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 464-577, and a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 578- 625.

[0227] 61. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CB1), wherein the antibody or antigen binding fragment thereof comprises: a light chain CDRl comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 626-661; a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 662-742, and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 742-824.

[0228] 62. The isolated antibody or fragment thereof according to embodiment 1, wherein the antibody or antigen binding fragment thereof is a humanized antibody. [0229] 63. The isolated antibody or antigen binding fragment thereof as disclosed herein, wherein the antibody or antigen binding fragment thereof comprises a human IgGl Fc region.

[0230] 64. The isolated antibody or antigen binding fragment thereof of any of the preceding embodiments, wherein the antibody or antigen binding fragment thereof comprises a modified Fc region.

[0231] 65. The isolated antibody or antigen binding fragment of embodiment 64, wherein the antibody or antigen binding fragment thereof comprises an Fc region selected from the group consisting of an IgG2/IgG4 hybrid, an IgG2 comprising A330S and P331S mutations, and an IgG4 comprising an S228P mutation.

[0232] 66. An isolated antibody or antigen binding fragment thereof that binds to cannabinoid receptor 1 (CB1), wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 339-341, and, optionally, a light chain variable region according to SEQ ID NO: 337.

[0233] 67. The isolated antibody or antigen binding fragment of embodiment 66, wherein the antibody or antigen binding fragment thereof comprises a heavy chain constant region according to SEQ ID NO: 342.

[0234] 68. The isolated antibody or antigen binding fragment of embodiment 66, wherein the antibody or antigen binding fragment thereof comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 343-351, and a light chain amino acid sequence according to SEQ ID NO: 338.

[0235] 69. An isolated antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 341 and a heavy chain constant region comprising SEQ ID NO: 433, SEQ ID NO: 434, or SEQ ID NO: 435.

[0236] 70. An isolated antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 340 and a heavy chain constant region comprising SEQ ID NO: 433, SEQ ID NO: 434, or SEQ ID NO: 435.

[0237] 71. An isolated antibody or fragment thereof that comprises a nucleic acid sequence or an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-351 and SEQ ID NOs. 436-824.

[0238] 72. An isolated humanized antibody or antigen binding fragment thereof that binds to CBl, wherein the antibody or fragment has a binding affinity Kd for CBl receptor of about 100 nM or less.

[0239] 73. The isolated humanized antibody or antigen binding fragment of embodiment 72, wherein the antibody or fragment has a binding affinity Kd for CBl receptor of about 5 nM or less.

[0240] 74. An isolated humanized antibody or antigen binding fragment thereof that binds to CBl, wherein the antibody or fragment has CBl receptor inhibiting activity that is at least 10 fold more potent relative to small molecule rimonabant, wherein the potency is measured by the inhibition of CB 1 receptor agonist-mediated signal transduction in a cAMP assay.

[0241] 75. The isolated humanized antibody or antigen binding fragment of embodiment 74, wherein the antibody or fragment has CBl receptor inhibiting activity that is at least 5 fold more potent relative to small molecule rimonabant, wherein the potency is measured by the inhibition of CBl receptor agonist-mediated signal transduction in a cAMP assay.

[0242] 76. The isolated humanized antibody or antigen binding fragment of embodiment 74, wherein the antibody or fragment has CBl receptor inhibiting activity that is at least equivalent in potency relative to small molecule rimonabant , wherein the potency is measured by the inhibition of CB 1 receptor agonist-mediated signal transduction in a cAMP assay.

[0243] 77. An isolated humanized antibody or antigen binding fragment thereof that binds to CBl, wherein the antibody or fragment exhibits greater potency than a corresponding non- humanized or chimeric antibody, wherein the humanized antibody or fragment and the corresponding non-humanized or chimeric antibody comprise the same heavy and light chain CDRs, and wherein the potency is measured by the inhibition of CBl receptor agonist-mediated signal transduction in a cAMP assay.

[0244] 78. The isolated humanized antibody or antigen binding fragment of embodiment 77, wherein the humanized antibody or fragment has CBl receptor inhibiting activity that is at least 2 fold more potent relative to the corresponding non-humanized or chimeric antibody or fragment.

[0245] 79. The isolated humanized antibody or antigen binding fragment of embodiment 78, wherein the humanized antibody or fragment has CBl receptor inhibiting activity that is at least

3 fold more potent relative to the corresponding non-humanized or chimeric antibody or fragment.

[0246] 80. The isolated humanized antibody or antigen binding fragment of embodiment 79, wherein the humanized antibody or fragment has CBl receptor inhibiting activity that is at least 5 fold more potent relative to the corresponding non-humanized or chimeric antibody or fragment.

[0247] 81. The isolated humanized antibody or antigen binding fragment disclosed herein, wherein the antibody or fragment exhibits reduced or absent brain penetration.

[0248] 82. The isolated humanized antibody or antigen binding fragment of embodiment 81, wherein the brain penetration of the antibody or fragment exhibits reduced brain penetration relative to a small molecule CB 1 receptor agonist or antagonist.

[0249] 83. The isolated humanized antibody or antigen binding fragment of embodiment 81, wherein the antibody or fragment exhibits reduced central nervous system (CNS) side effects relative to a small molecule CB 1 receptor agonist or antagonist.

[0250] 84. The isolated humanized antibody or antigen binding fragment of embodiment 83, wherein the small molecule CBl receptor agonist or antagonist is AM6545, AM251, taranabant, or rimonabant.

[0251] 85. A method of treating a disease or disorder responsive to antagonism or agonism of CBl receptor in a subject in need thereof, the method comprising administering to the subject an antibody or antigen binding fragment thereof according to any one of embodiments 55-84.

[0252] 86. The method of embodiment 85, wherein the subject is a human.

[0253] 87. The method of embodiment 85, wherein the disease or disorder is selected from the group consisting of obesity, diabetes, dyslipidemia, metabolic diseases, fibrosis, NASH, liver disease, primary biliary cirrhosis, renal disease, kidney fibrosis, chronic kidney disease, osteoporosis, atherosclerosis, cardiovascular disease, cancer, an inflammatory disease, pain, MS spasticity, and ocular diseases, including glaucoma.

[0254] 88. The method of embodiment 85, wherein the antibody or antigen binding fragment exhibits reduced or absent brain penetration.

[0255] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES

Example 1. Mouse immunization for generation of CBl receptor antibodies

[0256] The cDNA sequence and primers for cloning of human CB 1 receptor were based on pubmed NCBI reference sequence NM_001160258. CBl receptor was expressed by transient expression with lipofectamine in 293 cells or by generation of stable cell lines. For stable cell line generation, pcDNA4/TO native full length human CBl receptor construct was transfected into tetracycline inducible system Trex-CHO and Trex-293 cells. Cells were cultured under the antibiotics zeocin and blasticidine. Clones that expressed CBl upon tetracycline induction were identified by FACS staining with anti-CBl receptor antibody from R&D (Clone 368302). Membranes were prepared and either used for immunization directly, or following solubilization of membrane proteins in detergent followed by talon purification. Purified CBl receptor was stabilized in a lipid bilayer. The CBl/lipid complex was designated CBl receptor iCAPS.

[0257] Balb/c mice were immunized for 2 rounds with CBl receptor DNA followed by boosts with CBl receptor membranes or CBl receptor iCAPS. Blood samples were taken pre- and post-immunizations and serum was tested for binding to CBl receptor expressing membranes versus na ^' ive membranes in ELISAs. Once mouse sera showed a positive signal on CB1 receptor membranes versus naive membranes, mice were sacrificed and spleens were removed for hybridoma and phage library generation.

Example 2. Recovery of lymphocytes, B cell isolation, fusion, and selection of CB1 receptor binding hybridomas

[0258] Spleens from immunized mice were removed and single cell suspensions generated. To generate single cell suspensions, spleens were transferred to a screen placed on top of a 50 mL conical centrifuge tube, and the plunger of a 3 mL syringe was used to grind the cells out of the spleen. Red blood cells were lysed with ice cold ACK buffer for lOmins, 5ml DMEM was added and the cells were centrifuged. This step was repeated once, and cells were resuspended to a concentration of 2xl0 ⁷ /ml in DMEM medium.

[0259] Myeloma cell recovery and preparation: SP2/0 cells were seeded at a density of approximately 5 x 10 ⁴ cells/mL and passed every 2 days. Before the cell fusion, parental myeloma cells were harvested by centrifuging in a 50 mL conical centrifuge tube at room temperature (RT) at 500 x g for 10 minutes. Cells were washed 3 times by adding 30 mL of serum free medium, and repeated the centrifugation. Supernatant was removed by pipetting and the cell pellet was resuspended in 25 mL of Medium and adjusted to a concentration of 2 x 10 ⁷ /ml viable cells.

[0260] Cell Fusion: Myeloma cells and spleen cells were mixed at a ratio of 1 :5. The cell mixture was spun down at 1000 rpm for 5 mins, and then the supernatant discarded to obtain the cell pellet. Cell mixture was washed twice with fusion working buffer and centrifuged to obtain the cell pellet. The cell pellet was gently resuspended with fusion buffer to a final cell concentration of 8xl0 ⁷ /ml. Cell mixtures were added into the electrode bath and electro fusion performed. The cells were allowed to rest for 5 mins after cell fusion, washed lx with DMEM medium, and pre -heated HAT medium was added into the fusion cells to a final concentration of 0.5x l0 ⁶ B cells /ml. Then ΙΟΟμΙ cell suspension (5xl0 ⁴ B cells) was added into each well of a 96 well plate. The average fusion efficiency is 1 hybridoma/lxlO ⁴ B cells, so the protocol aims for 5 hybridomas in each well.

[0261] Hybridoma cell culture. Fused hybridoma were cultured in cell culture incubator at 5% C02, 37°C. The hybridoma growth condition was checked daily. The colonies become visible after 5 days normally. Medium was changed with fresh DMEM medium at day 7 before positive screening.

[0262] Positive screening: after 7 - 9 days of cell fusion, when the colony became bigger, 100 μΐ _^ of supernatant from each hybridoma well was transferred to a separate well on a new 96- well plate and analyzed using ELISA with CB1 protein.

Example 3. Generation and screening of phage libraries

[0263] Total RNA extraction: Spleen tissue was harvested in RNAlater. A small piece of frozen tissue (~30mg) was homogenized in a mortar cooled with liquid N2 and ground into a fine powder. TRIzol® Reagent was added with vigorous shaking by hand for 30 seconds. lmL solution was transferred to 1.5 microfuge tubes at room temperature 2-3min and let stand in room temperature for a few minutes. To the mixture, 0.2 ml chloroform per lmL solution was added and shaken vigorously by hand for 30 seconds. The mixture was incubated at room temperature for 5 min and then centrifuged at 12,000 xg for 20 min at 4°C. The aqueous phase was removed and transferred to a new tube. An equal volume of isopropyl alcohol was added to the tube and mixed for 30 seconds. After incubating at room temperature for 5 min, the mixture was centrifuged at 12,000 g for 15min at 4 °C. The pellet was washed by adding 500 μΐ 75% ethanol and centrifugation at 12,000 g for 15min at 4 °C. The resulting total RNA pellet was air dried and dissolved with 50 μΐ RNase-free water per ^g of RNA expected. OD was measured at 260 nm and 280 nm of a 1 : 10 dilution of the RNA sample.

[0264] cDNA prep: First-Strand cDNA Synthesis was done with a commercial kit (Invitrogen, Cat. No: 18080-051), briefly, 20 μg total RNA was mixed with 5 μΜ oligo(dT)20 and 1 mM dNTP in DEPC-treated water in 40μ1 and incubated at 65 °C for 5 min, then 80 μΐ of RT buffer with 5 mM MgC12, 10 μΜ DTT, 16 unit of RNaseOUT and 80 unit of Superscript III reverse transcriptase was added. The resulting mix was incubated at 50 °C for 50min, and heat inactivated, before 4 μΐ of RNase H was added to remove residue RNA. The cDNA was used for subsequent library construction.

[0265] Chimeric Fab Library was constructed as follows: The variable regions of heavy chain or light chain were amplified by heavy chain or light chain-specific primers representing multiple germline families described in Barbas et al, using the mouse cDNA template prepared above. The human heavy chain and light chain constant region, Chi and CL1, respectively, were amplified from an existing clone pCOM3xTT. The heavy chain variable region and constant region were connected together by overlapping PCR. The resulting heavy chain and light chain were connected by overlapping PCR again to obtain the chimeric Fab DNA fragment, which was cloned into a modified pCOM3x vector as Sfil fragment insert by ligation. The ligated library DNA was cleaned and transformed into SS320 high efficiency competent cell. The number of total unique transformants obtained was at least 5xl0 ⁷ .

[0266] For panning of phage libraries, a phage library from immunized mouse was subtracted twice in Maxisorp immunotubes (Thermo Scientific) coated with empty iCAPS, then subtracted twice with Dynabeads MyOne Streptavidin Tl (Life Technologies) coated with biotinylated Bril protein. All subtraction steps lasted 30 minutes. Meanwhile, biotinylated CB1 receptor iCAPS were coated on Dynabeads MyOne Strepavidin Tl (Life Technologies). The subtracted phage pool and CB1 iCAPS on beads were then mixed, along with un-biotinylated Bril protein and empty iCAPS for competition, and incubated for one hour at room temperature with rotation. Beads were separated from the binding mixture with magnet and washed multiple times to eliminate unbound phage (5 times in Round 1, 10 times each in Round 2 and 3). Bound phage was eluted twice with Glycine buffer pH2.2 for 10 minutes each. The eluates were combined, neutralized with Tris-HCl pH8.0, and used to infect TGI cells to produce phage for the next round of panning. After 3 rounds of panning, single colonies were picked and screened by monoclonal phage ELISA.

[0267] For screening of phage binders, single colonies of TGI infected with panning output were picked into 96-well plates containing 2YT/carbenicillin/glucose medium and shaken overnight at 30C. The next day, a small volume of saturated culture was transferred to fresh 2YT/carbenicillin/glucose medium and shaken at 37C until OD600nm reached 0.6-0.7. Then the culture was infected with K07 helper phage. The infected TGI cells were spun down, re- suspended in 2YT/carbenicillin/kanamycin medium and shaken at 30C overnight. Meanwhile, Maxisorp 96-well plates (Thermo Scientific) were coated with streptavidin (Wako) at 4C overnight. The third day, biotinylated antigens were captured on streptavidin coated plates, which were then blocked with 3% non-fat milk in PBST. TGI cells were spun down again. Phage containing supernatants were blocked in 3% non-fat milk and then loaded to the ELISA plates and incubated for one hour at room temperature. After three rounds of washing by PBST, HRP mouse anti-M13 antibody (GE Healthcare) diluted 1 :2000 in 3% non-fat milk in PBST was added to the plates and incubated for another hour at room temperature. The plates were washed three times with PBST, then developed with TMB (Biopanda). HC1 was added to the plates to stop the reaction. Absorbance at 450nm was read on Emax precision microplate reader (Molecular Devices). Clones that bound to iCAPS specifically were picked for further characterization and sequencing.

[0268] E. coli colonies harboring plasmids that produce Fab displayed on phage were recovered. Plasmid DNA was extracted and sequenced to obtain the Fab DNA information. Specific primers were designed to amplify the V region of heavy chain, the PCR product was cloned into pTT5-HCV3, a modified version of pTT5 expression vector, previously treated with Apal and Sacl restriction enzymes, in front of the human heavy constant region by seamless cloning. Specific primers were also designed to amplify the entire light chain region of Fab fragment. The resulting PCR fragment was cloned into pTT5-IL2-LCC, a modified pTT5 vector, treated with EcoRI and Notl by seamless cloning. The resulting 2 plasmids were sequence- verified.

Example 4. Hybridoma sequencing

[0269] Hybridoma cells (lxlO ⁷ ) were harvested and total RNA was extracted using Tri Reagent as described above for spleen tissue. cDNA was prepared using Superscript III kit according to the manufacturer's instruction, described above. The resulting cDNA product was used as template for PCR with primers VhRevU and VhForU, the resulting 300 bp PCR product was cleaned up using a PCR clean-up kit and sequenced with the same primer. PCR reaction was also performed with light chain V-region specific primer VkRev7 and VkFor (for variable region only) or KappaFor primers (for entire kappa light chain). Sequencing reactions were performed on cleaned PCR product to obtain DNA sequence. Example 5. Expression and analysis of IgG

[0270] IgG expression: Two pTT5-based plasmids, one containing the Heavy chain and the other containing the light chain DNA, were co-transfected into HEK293F cells for IgG expression. 24 hours prior to transfection, 293F cells were diluted to the density of 8X10 ⁵ cells/ml. On the day of transfection, cells were maintained at 1.1-1.3X10 ⁶ cells/ml. One μg of plasmid DNA was used for transfection of 1ml cell suspension culture. 8(^g of DNA were diluted into 4ml of fresh 293F freestyle medium. 24(^g of the transfection reagent Polyethylenimine (PEI) were diluted into a final volume of 4ml 293F freestyle medium. After 3 minutes incubation, 4ml DNA were mixed thoroughly with 4ml PEI. The 8ml of DNA and PEI mixture were incubated for 15 minutes at room temperature and slowly added into 80ml of 293F cells suspension culture. Cells were incubated at an orbital shaking platform at a speed of 130rpm at 37°C with 5% C02 and harvested in 4 days.

[0271] IgG purification: 0.4 ml bed volume of Protein A were placed into a 1 mL column and washed with 10 mL of dH20 and 10ml of pH 8.0 PBS. Transfected 293F cells suspensions were spun down at 4000 rpm for 45 minutes at 4°C. The pellets were discarded and the supernatant was adjusted to pH 8.0 on ice and loaded into the prepared Protein A column. When the supernatant loading was finished, the column was washed with 5ml of pH 8.0 PBS and eluted with 4ml of 0.1 M Na Citrate-HCl pH3.5. The elution containing IgGs was neutralized with 200 μΐ pH 8.8 1.5M Tris-HCl buffer and concentrated with a 30kD 4ml concentrator. 4.5ml of PBS were filled up the concentrator and spun down. Finally, IgGs were exchanged and stored into PBS. The IgGs were detected by OD280 and the purity was determined by SDS-PAGE gel and SEC.

[0272] The concentrations, volumes, and yields of PA13R3-P1C4 achieved in four different experiments are shown in Table 4. The concentrations, volumes, and amounts of various clones are shown below in Table 5.

Table 4. PA13R3-P1C4 expression PA13R3-P1C4 1.01 500 505 3.2

PA13R3-P1C4 0.49 500 245 1.5

PA13R3-P1C4 1.25 1250 1562.5 3.9

Table 5. Expression of clones

Example 6. Binding of IgG to CB1 iCAPS by ELISA

[0273] Purified IgG was tested for binding to purified CB1 receptor (CB 1 Conformation Antigen Presenting System (iCAPS)) by ELISA. Non-biotinylated antigens (e.g. empty iCAPS) were coated directly on Maxisorp plates. The primary antibodies were purified IgGs with 1 :3 serial dilutions, incubated on the plate for 1 hour. After 3 rounds of washing with PBST, secondary antibodies HRP goat anti-mouse IgG (Abmart) or HRP goat anti-human IgG (Sigma), depending on the species of the IgGs were added and incubated for another hour. The plates were washed three times with PBST, then developed with TMB (Biopanda). HCl was added to the plates to stop the reaction. Absorbance at 450 nm was read on Emax precision microplate reader (Molecular Devices). Binding data is summarized in Table 6.

Table 6. Binding to CB1 iCAPS

Example 7. Binding of IgG by FACS

[0274] TRex CHO parental cells, TRex CHO A56 over-expressed CB 1 (CB 1 T21 OA/fusion partner), and Native human CB 1 TRex CHO A156 were harvested from flasks. 100 μΐ of lxlO ⁶ cells/ml of cells were incubated with primary antibody IgGs. Secondary Antibody PE conjugated anti-human and anti-mouse was diluted in 1 :200 folds. Anti-Human Fab FITC was diluted in 1 :32 folds. Cells were washed with 200 μΐ of FACS buffer twice and transferred to BD 5ml Falcon tube and analyzed by flow cytometry. Binding of purified IgG was initially tested at concentrations of 30 nM and 300 nM. A number of binders were identified as shown in Figure 1A-1F.

[0275] To further assess TRex CHO parental cells, TRex CHO A56 overexpressed CBl, Native human CBl TRex CHO A156, 5HT2B, Mouse CBl and human CB2 were used to examine the specificity of IgG binding. ΙΟΟμΙ of lxl0 ⁶ cells/ml of cells were incubated with primary antibody IgGs in 3 -fold serial dilutions starting from 1 μΜ to 0.5 nM for 30 minutes on ice. After being washed with 200μ1 of FACS buffer twice, cells were incubated with secondary antibody for 30 minutes on ice. Cells were washed with 200 μΐ of FACS buffer twice and transferred to BD Falcon 5ml tube and analyzed by FACS.

[0276] Santa Cruz anti-CBl rabbit polyclonal antibody and secondary antibody FITC conjugated anti-rabbit were used to detect the expression of mouse CBl . R&D mouse monoclonal anti-CB2 and human IgG P2C2 were used to confirm the expression of CB2 and 5HT2B respectively. Both anti-mouse and anti-human secondary antibodies were PE-conjugated.

[0277] For selected binders, full binding curves were generated on CB 1 receptor by testing a range of concentrations. Three-fold serial dilutions from 1 μΜ to 0.1 μΜ were prepared. Selectivity was determined by measuring binding to cells expressing 5HT2B or CB2, relative to binding to A156 (native human CBl receptor expressing) or A56 (overexpressed - CBl receptor with the T210A modification and ICL3 replacement with fusion partner) by flow cytometry. As shown in Figure 2C and Figure 2D, the expression of CB2 and 5HT2B was confirmed using mouse monoclonal anti-CB2 and P2C2 human IgG to confirm CB2 and 5HT2B expression, respectively. PE-conjugated anti-mouse (for detection of anti-CB2) and PE conjugated anti- human antibodies were used to detect CB2 and 5HT2B, respectively. Antibodies PA13R3-P1C4 and 36E12B6C2 bound selectively to CBl, as shown in Figure 2A and Figure 2B. In addition, Table 7 shows the concentrations and disassociation constants (Kd) for each of several batches of PA13R3-P1C4 and 36E12B6C2. [0278] The results of the study showed that PA13R3-P1C4 IgG and Fab and 36E12B6C2 bind to both A56 and A156 but not parental TRex CHO and they do not have cross-activity with 5ht2b, human CB2 or Mouse CB1.

Table 7. Flow cytometry results for various batches of antibodies

Example 8. Competition Assay

[0279] TRex CHO A156 Native human CB1 cells were used to test whether 36E12B6C2 and P1C4 bind to similar epitopes. Concentrations at EC80 and EC50 of P1C4 and 36E12B6C2 were used for staining. Excess of PA13R3-P1C4 IgG, Fab and 36E12B6C2 were used for competition. ΙΟΟμΙ of lxlO ⁶ cells/ml of A156 cells were incubated with competitor IgGs for 30 minutes on ice and then staining IgGs were added into the mixture with 30 minutes incubation on ice. After being washed with 200μ1 of FACS buffer twice, cells were incubated with secondary antibody for 30 minutes on ice. PE conjugated anti-human and anti-mouse was diluted in 1 :200 folds. Cells were washed with 200μ1 of FACS buffer twice and transferred to BD Falcon 5ml tube and analyzed by FACS.

[0280] The results of the study showed that PA13R3-P1C4 Fab and IgG competed with 36E12B6C2 for CB1 binding, suggesting PA13R3-P1C4 and 36E12B6C2 bind to overlapping epitopes (Figure 3 A and B). The competitor 36E12B6C2 brought up from ΙΟΟηΜ to 500nM could also compete with PA13R3-P1C4 for binding to CB1.

Example 9. cAMP functional assay

[0281] A cAMP functional assay was performed to measure the antagonism of the antibodies. The cAMP functional assay (Cisbio) was performed on white 384-well low volume plate (Greiner). 8000 cells/well of stably expressed CB1 TRex CHO cells were seeded to the plate followed by incubating antagonist at various concentrations (ranging from 10 μΜ to 0 μΜ) at room temperature for 10 min. 5 μΜ of forskolin (Sigma Aldrich) and 9 μΜ of the cannabinoid CP55940 (Sigma Aldrich) were added to the cell stimulation mixture to and incubated for 30 min at room temperature to activate CB1. After 30 min incubation, 5 μΐ _^ of cAMP-d2 (1 :39 dilution with conjugate and lysis buffer provided by Cisbio) and 5 μΐ, of anti-cAMP cryptate (1 :9 dilution with conjugate and lysis buffer provided by Cisbio) were added to the cell stimulation and incubated for an hour. FRET signal was detected with Envision multilabel plate reader (Perkin Elmer) at anti-cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism.

[0282] As shown in Figure 4, two antibodies, 36E12B2H8 (hybridoma) and PA13R3-P1C4 (phage derived) exhibited antagonistic activity equipotent (36E12B2H8) or more potent (PA13R3-PIC4) than the small molecule positive controls (inverse CB1 receptor agonists SR141716A (rimonabant) and AM251, and neutral antagonist AM6545) with IC50 values of 350 ± 28 nM and 90 + 13 nM, respectively.

Example 10: ERK activation assay [0283] To further confirm antagonist activity of mAbs, ERK activation as part of the CBl receptor signaling pathway was assessed. Two days before the experiment, Trex-CHO CBl receptor cells were seeded at 500,000 cells/well into 6-well plates. 1 μg/mL tetracycline was used to induce CBl receptor expression after 24 hours. Cells were serum starved for at least two hours before the experiment. Purified IgGs at 300 nM were added to the culture media, after 30 minutes, cells were stimulated with CBl receptor agonist WIN55,212 (100 nM) for 10 and 15 minutes. Cell lysates were harvested and the level of ERK activation was determined by western blot. Anti-ERK and Anti-phospho-specific ERK antibodies were obtained from Cell Signaling Inc.

[0284] Treatment with CBl receptor agonist WIN55,212 induced ERK activation as demonstrated by the increase in phosphorylated ERK signal. Total ERK was used as western blot loading control to show equal loading of the samples. As shown in Figure 5 A, phage-derived antibody PA13R3-P1C4 (300 nM) but not control IgG (irrelevant binder) or PA13R3-P1E4, blocked WIN55,212 (100 nM) induced ERK phosphorylation. As shown in Figure 5B, hybridoma-derived antibodies 36E12B2E5, 36E12B6C2, and 36E12B6F2, but not control IgG, blocked WIN55,212 induced ERK phosphorylation. AM6545 (neutral antagonist) was used as positive control as shown in both Figure 5A and 5B.

Example 11. cAMP functional assays

[0285] The cAMP agonist functional assay (Cisbio) was performed on white 384-well low volume plate (Greiner). 8000 cells/well of stably expressed CBl TRex CHO cells were seeded to the plate followed by incubating agonist at various concentrations (ranging from 1.5 μΜ to 0 μΜ) at room temperature for 10 min. To test for agonist activities (Figure 6A and 6B), 5 μΜ of forskolin (Sigma Aldrich) was added to the cell stimulation mixture and incubated for 30 min at room temperature. To assess for positive allosteric modulator activity (Figure 6C and 6D), 5 μΜ of forskolin (Sigma Aldrich) and 1 μΜ of CP55940 were added to the cell stimulation mixture and incubated for 30 min at room temperature.

[0286] After 30 min incubation, 5 μΐ _^ of cAMP-d2 (1 :39 dilution with conjugate and lysis buffer provided by Cisbio) and 5 μΐ, of anti-cAMP cryptate (1 :9 dilution with conjugate and lysis buffer provided by Cisbio) were added to the cell stimulation and incubate for an hour. FRET signal was detected with Envision multilabel plate reader (Perkin Elmer) when anti-cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism software.

[0287] Results from the cAMP agonist screening identified four potential agonist IgGs including PA2LR3-P2D3, PA2LR3-P4B1, PA2LR3-P6G7 and PA2LR3-P6B12. In particular, PA2LR3-P2D3, PA2LR3-P4B1, and PA2LR3-P6G7 exhibited EC50 > 300 nM, as shown in Figure 6A and 6B. Further, PA2LR3-P6B12 is a potential allosteric modulator, with an EC50 of about ΙΟΟΟηΜ in the presence of CP55940, as shown in Figure 6C. Positive and negative controls are shown in Figure 6B and 6D.

[0288] A cAMP assay was also performed to further characterize PA13R3-P1C4 and 36E12B6C2. The cAMP functional assay (Cisbio) was performed on white 384-well low volume plates (Greiner). 8000 cells/well of stably expressing CB1 TRex-CHO cells were seeded to the plate followed by incubating antagonists, including AM6545, SR141716A, PA12R3-P1C4 and 36E12B6C2, at concentrations ranging from 3 μΜ to 0 μΜ for 10 minutes at room temperature. After 10 minutes incubation, 5 μΜ of forskolin (Sigma Aldrich) were added to the cells stimulation mixture and incubated for 30 min at room temperature. To quantify for the cAMP production, 5 μΐ, of cAMP-d2 and 5 μΐ, of anti-cAMP cryptate were added to the cells stimulation and incubated for an hour. FRET signal was detected with EnVision multilabel plate reader (Perkin Elmer) when anti-cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism software. The results of the study are shown in Figure 7. Previously, AM6545 and SR141716A have been characterized as neutral antagonist and inverse agonist, respectively. The results from the cAMP functional assays indicated that PA12R3-P1C4 and 36E12B6C2 have similar inhibition patterns as SR141716A, suggesting that PA12R3-P1C4 and 36E12B6C2 are inverse agonists.

Example 12. iCAPS ELISA binding assay

[0289] An ELISA binding assay was carried out to assess binding of CB1 receptor IgG or Fab antibodies to iCAPS expressing CB1 (A138 containing ICL3 native sequence replacement with protein 1 sequence and A139 containing ICL3 native sequence replacement with BRIL), iCAPS expressing 5HT2B (hl3h), empty iCAPS, or rBril-0918. IgG and Fab molecules tested were 36E12B6C2 IgG, 36E12B6C2 Fab, PA13R3-P1C4 IgG, and PA13R3-P1C4 Fab, compared to negative control BRIL binder P1F7 IgG and P1F7 Fab. For IgG antibodies, the secondary antibody used to detect binding was anti-mouse IgG-HRP; for Fabs, the secondary antibody used to detect binding was anti-human IgG-HRP. The results of the study are shown in Figures 8 A and 8B and below in Table 8. For both A138 iCAPS and A139 iCAPS binding, 36E12B6C2 Fab yielded higher EC50 values relative to 36E12B6C2 IgG. In contrast, PA13R3-P1C4 IgG and Fab yielded approximately equivalent EC50 values for binding to both A139 and A138. None of the CB1 antibodies or Fabs exhibited binding to rBril-0918, empty iCAPs, or iCAPS expressing 5HT2B. The control mAb P1F7 recognizes BRIL, and hence shows binding to A139 containing BRIL fusion in ICL3, but not to Al 38 lacking BRIL.

Table 8. EC50 values in 36E12B6C2 and PA13R3-P1C4 IgG and Fab ELISA

Example 13. CB1 receptor internalization study

[0290] The impact of CB1 antibody on WIN55,212 (CB1 specific agonist) induced CB1 internalization was examined by flow cytometry. 5 x 10 ⁵ cells/well stably expressing CB1 TRex- CHO cells were seeded in a 6-well plate. Tetracycline (1 μg/ml) was added to culture medium for 24 hours to induce CB1 expression. On the day of the experiment, cells were serum starved for 2 hours. Cells were then pre-incubated with CB1 antibody (300 nM), AM6545 (CB1 neutral antagonist) and negative control (BRIL binder) for half an hour. CB1 agonist (1 μΜ WIN55,212) was then added to the culture media for 1 hour to induce receptor internalization. Surface expression of CB1 was stained with anti-CBl N-terminus mouse monoclonal antibody from R&D and the mean fluorescence intensity (MFI) was determined using flow cytometry. The results of the study are shown in Figure 9 A. Treatment with CBl agonist WIN55,212 showed a reduction in MFI compared to control suggesting internalization of CBl (Figure 9A, top row of histograms). Pre-treatment with CBl specific neutral antagonist AM6545 blocked WIN55,212 induced CBl receptor internalization (Figure 9 A, top row of histograms). Pre-treatment of CBl antibodies (300 nM) did not affect WIN55,212 induced CBl receptor internalization (Figure 9 A, middle and bottom rows of histograms).

[0291] The impact of CBl antibody on receptor internalization was also investigated. After 2 hours serum starvation, 300 nM CBl antibodies, P2A12 negative control (BRIL binder) and CBl agonist WIN55,212 were added to the culture media for 1 hour. Cells were harvested and stained with anti-CBl N-terminus mouse monoclonal antibody (R&D). The results of the study are shown in Figure 9B. Again, WIN55212 induced CBl receptor internalization and blocked by pre-treatment with CBl neutral antagonist AM6545 (Figure 9B, top row of histograms). Surface expression of CBl was not affected by CBl antibodies suggesting CBl antibodies did not induce receptor internalization (Figure 9B, middle and bottom rows of histograms).

Example 14. Potency of humanized CBl antibodies

[0292] Humanized P1C4 antibodies were generated and tested for potency, specificity, and affinity. To generate the humanized P1C4 antibodies, human frameworks were selected based on homology between P1C4 and human germline VH and VK genes. The selected frameworks had the highest homology with the PIC4 VH and VK regions and were selected based on computer modeling as being able to support the CDR structure predicted to be presented by PIC4.

The following humanized antibodies were generated: (1) P1C4-H0 - IgG; (2) P1C4-H2 (YE) - IgG (comprising G27Y and T28E mutations in the heavy chain variable region); and (3) P1C4- H4 (YENG)- IgG (comprising G27Y, T28E, A60N, an Q61G mutations in the heavy chain variable region)

[0293] A cAMP assay was performed in order to determine the potency of chimeric PA13R3-P1C4 and humanized PA13R3-P1C4 antibodies. The cAMP functional assay (Cisbio) was performed on white 384-well low volume plates (Greiner). 8,000 cells/well of stably expressing native human CBl TRex-CHO cells were seeded to the plate, followed by incubating Rimonabant (SR141716A), PA12R3-P1C4 chimeric, PA12R3-P1C4 HO (no back mutation), PA12R3-P1C4 H2 (YE), PA12R3-P1C4 H4 (YENG) and P2A12 (negative control), at concentrations ranging from 1 μΜ to 0 μΜ for 10 minutes at room temperature. After 10 minutes, 5 μΜ of forskolin (Sigma Aldrich) was added to the stimulation mixture and incubated for 30 min at room temperature. To quantify cAMP production, 5 μΐ _^ of cAMP-d2 and 5 μΐ _^ of anti-cAMP cryptate were added and incubated for one hour. A FRET signal was detected with En Vision multilabel plate reader (Perkin Elmer) with anti-cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism software.

[0294] The results of the study are provided in Figure 10 and below in Table 9. The cAMP functional assay indicated that humanized P1C4-H2 and P1C4-H4 have an IC50 of 21 nM and 17 nM, respectively. Thus, of the antibodies tested, the humanized antibodies H1C4-H2 and PIC4-H4 exhibited potency even greater than the corresponding chimeric antibody, as measured by inhibition of cAMP.

Table 9. IC50 for chimeric and humanized CB1 antibodies

[0295] The binding affinity, cross-reactivity and specificity of humanized P1C4 antibodies were determined by flow cytometry with TRex CHO parental cells, TRex CHO A56 overexpressed CB1 cells (T21 OA/fusion partner), native human CB1 TRex CHO A156 cells, Trex parental (no CB1) cells, mouse CB1 cells, and human CB2 stable cells. 100 μΐ of lxl0 ⁶ cells/ml of cells were incubated with PA13R3-P1C4 chimeric, humanized P1C4-H0 (no mutation), P1C4-H2 (YE), P1C4-H4 (YENG) or P2A12 (control) IgGs in 3-fold serial dilutions starting from 300 nM to 0.5nM for 30 minutes on ice. After being washed with 200 μΐ of FACS buffer twice, cells were incubated with PE conjugated anti-human secondary antibody (Southern Biotech) for 30 minutes on ice. Cells were washed with 200 μΐ of FACS buffer twice and transferred to BD Falcon 5ml tube and analyzed by flow cytometry (BD FACScalibur). Table 10. Binding affinity and cross-reactivity of humanized P1C4 antibodies

[0296] The results of the study are shown in Figure 11 and above in Table 10. The humanized CBl antibodies bound to A56 cells and A156 cells. Humanized antibodies P1C4-H2 and PIC4-H4 both bound to TRex CHO A56 overexpressed CBl cells as well as to native human CBl A156 cells with higher affinity relative to the chimeric P1C4 antibody (Figure 11A). In addition, none of the antibodies tested exhibited cross-reactivity with mouse CBl or with human CB2 (Figure 11B).

Example 15. Reduced Effector Function CBl Antibodies

[0297] CBl antagonist antibodies designed to exhibit reduced effector function are constructed and tested. CBl antagonist antibodies having one or more of the following Fc modifications are generated: (1) IgG4 constant region with a serine to proline mutation at position 228 (S228P); (2) IgG2 constant region with an alanine to serine mutation at position 330 (A330S) and a proline to serine mutation at position 331 (P331S); and (3) IgG2/IgG4 hybrid constant region. [0298] The resulting humanized CB1 antibodies having 0, 2, or 4 backmutations in the framework regions are provided as SEQ ID NOs: 343-351. The antibodies are tested for the extent of binding to Fey receptors and complement Clq by ELISA. The resulting CB1 antibodies are also tested for their abilities to activate primary human immune cells in vitro. Specifically, the CB1 antibodies having one or more Fc modification are tested for activation of immune cells, for example, by assessing their crosslinking capacity or the ability of the antibodies to induce expression of activation markers. The results of the study show that the CB1 antagonist antibodies have reduced FcyR binding and/or reduced Clq binding and/or reduced immune cell activation relative to the corresponding CB1 antagonist antibody that does not contain the constant region modification.

Example 16. Biodistribution Study

[0299] A study was conducted to determine the biodistribution of CB1 antibody P4B5 in vivo in mice. Antibody P4B5 was labeled with Vivotag 680 XL (Perkin Elmer), and hairless mice (n=4 per group) were injected IV with 5 mg/kg or 25 mg/kg of labeled antibody. Whole body imaging was conducted using fluorescence mediated tomography (FMT) at the following timepoints: 0 hours (Oh), lh, 5h, 24h, 48h, 72h, 96h, and 144h to measure fluorescence in various tissues. Labeled P4B5 exhibited similar binding affinity to CB1 cells relative to unlabeled P4B5, as shown in Figure 12. The EC50 for unlabeled P4B5 was 60.5 nM, and the EC50 for P4B5 antibody labeled with Vivotag 680 XL was 57.8 nM.

[0300] The results of the study are shown in Figures 13A and 13B. Labeled antibody was detected throughout the timecourse, as shown in Figure 13A.1 and A.2, which provides the data from a representative mouse that received the higher antibody dose (25 mg/kg). However, when the background signal from blood was subtracted, anti-CBl antibody was not detectable in the brain (Figure 13B), indicating that the antibody did not penetrate the blood-brain barrier after IV injection.

Example 17. Expression and analysis of IgG for PA13R3-P1C4 humanized variants [0301] Among the different human IgG subclasses, the Fc regions of IgG2 and IgG4 subclasses bind poorly to effector molecules, such as activating FcyRs or complement lq (Clq), resulting in lower effector function activity. In order to minimize the activation of immune effector function humanized lead series antibodies P1C4-H2 and P1C4-H4 were cloned into 3 human Fc framework variants, IgG2, IgG4, and a hybrid between IgG2 and IgG4, for further characterization.

[0302] The variable region of the heavy chain of humanized P1C4-H2 (SEQ ID NO: 340),the variable region of the heavy chain of humanized P1C4-H4 (SEQ ID NO: 341), and the light chain of humanized P1C4 (SEQ ID NO: 338 ) are shown below in Table 11. Bold residues are back mutations and the underlined residues denote CDR regions. To make the Fc variants in different IgG families, three heavy chain constant region sequences were used. The sequence of the IgG2 heavy chain constant region (SEQ ID NO: 433), IgG4 heavy chain constant region (SEQ ID NO: 434), hybrid IgG2/4 heavy chain constant region (SEQ ID NO: 435) are shown below in Table 11.

Table 11. Design of Fc variants

IgG2 heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS 433 chain GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD constant KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV region DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH

QDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPMLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

IgG4 heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS 434 chain GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD constant KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV region DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH

QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDSDGSF

FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Hybrid ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS 435 IgG2/4 GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD heavy chain KTVERKCCVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV constant DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH region QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDSDGSF

FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

P1C4-H2- OVOLVOSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVROAPGOGL 436 IgG2 EWMGOIYPGDGETKYAOKFOGRVTITADKSTSTAYMELSSLRSEDTA

VYYCARSHGNYLPYWGOGTLVTVSSASTKGPSVFPLAPCSRSTSEST

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVP S SNFGTQTYTCNVDHKP SNTKVDKTVERKCC VECPPCPAPPVAG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPS

SIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV

EWESNGQPEN YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGK

P1C4-H2- OVOLVOSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVROAPGOGL 437 IgG4 EWMGOIYPGDGETKYAOKFOGRVTITADKSTSTAYMELSSLRSEDTA

VYYCARSHGNYLPYWGOGTLVTVSSASTKGPSVFPLAPCSRSTSEST

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS

SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV

EWESNGQPEN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK P1C4-H2- OVOLVOSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVROAPGOGL 438 IgG2/4 EWMGOIYPGDGETKYAOKFOGRVTITADKSTSTAYMELSSLRSEDTA

VYYCARSHGNYLPYWGOGTLVTVSSASTKGPSVFPLAPCSRSTSEST

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVP S SNFGTQTYTCNVDHKP SNTKVDKTVERKCC VECPPCPAPEFLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE

VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP

SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPEN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC

SVMHEALHNHYTQKSLSLSLGK

P1C4-H4- OVOLVOSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVROAPGOGL 439 IgG2 EWMGOIYPGDGETKYNGKFOGRVTITADKSTSTAYMELSSLRSEDTA

VYYCARSHGNYLPYWGOGTLVTVSSASTKGPSVFPLAPCSRSTSEST

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVP S SNFGTQTYTCNVDHKP SNTKVDKTVERKCC VECPPCPAPPV AG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPS

SIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGK

P1C4-H4- OVOLVOSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVROAPGOGL 440 IgG4 EWMGOIYPGDGETKYNGKFOGRVTITADKSTSTAYMELSSLRSEDTA

VYYCARSHGNYLPYWGOGTLVTVSSASTKGPSVFPLAPCSRSTSEST

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS

SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK

P1C4-H4- OVOLVOSGAEVKKPGSSVKVSCKASGYEFSYYWMNWVROAPGOGL 441 IgG2/4 EWMGOIYPGDGETKYNGKFOGRVTITADKSTSTAYMELSSLRSEDTA

VYYCARSHGNYLPYWGOGTLVTVSSASTKGPSVFPLAPCSRSTSEST

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVP S SNFGTQTYTCNVDHKP SNTKVDKTVERKCC VECPPCPAPEFLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE

VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP

SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC

SVMHEALHNHYTQKSLSLSLGK

PlC4-Lc EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLHWYQQKPGQAPRLLI 442 humanized YSTSNLASGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCHQYHRSPPT

FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLS SPVTKSFNRGEC [0303] The antibody variants were expressed and purified in 293FreeStyle, CHO-S, and CHO-K1 cells in different batches on different dates.

[0304] For 293 FreeStyle expression separate pTT5 plasmids encoding heavy and light chain sequences were co-transfected into FreeStyle HEK293F cells for expression of full IgG antibodies. Cells were cultured at 37 °C with 5% C0 ₂ in FreeStyle 293 Expression Medium. Twenty four hours prior to transfection, cells were diluted to a density of 8 x 10 ⁵ cells/mL. To prepare the transfection solution, 80 μg DNA (40 μg light chain + 40 μg heavy chain) and 240 μg polyethylenimine (PEI) were diluted into 8 mL Freestyle 293F medium, mixed thoroughly and filtered through a 0.2 μιη syringe-top filter into a 50 mL conical tube, then incubated for 15 minutes at 22 °C. Eight mL of transfection solution were slowly added to 80 mL 293F cell culture (diluted in FreeStyle 293 Expression Medium to a density of 1.1 - 1.3 x 10 ⁶ cells/mL), which were then incubated at 37 °C with 5% C0 ₂ with rotation at 130 rpm for 4 days. Supernatants from transfected cell cultures were harvested by centrifugation at 4000 rpm for 45 minutes at 4 °C, adjusted to pH 8.0 with 0.1 M NaOH and held on ice until protein purification.

[0305] For CHO-S expression separate pTT5 plasmids encoding heavy and light chain sequences were co-transfected into CHO-S cells for expression of full IgG antibodies. Cells were cultured in CD-CHO medium at 37 °C with 5% C0 ₂ . Twenty four hours prior to transfection, cells were diluted to a density of 0.6-0.7 x 10 ⁶ cells/mL in CD-CHO medium. On the day of transfection, cells were diluted to a density of 1.1-1.3 x 10 ⁶ cells/mL in CD-CHO medium in 250 mL shake flasks. To each 250 mL shake flask, 80 mL of cells were added. Eighty μg of plasmid DNA (40 μg light chain + 40 μg heavy chain) was diluted into a final volume of 4 mL CD-CHO medium and filtered through a 0.2 μιη syringe-top filter into a 50 mL conical tube. In a separate 50 mL conical tube, 80 μΐ _^ of FreeStyle Max reagent were diluted into a final volume of 4 mL CD-CHO medium. These 2 mixtures were incubated at 22 °C for 3 minutes before they were combined, mixed and incubated for an additional 15 minutes at 22 °C. The 8 mL DNA/transfection reagent mixture was slowly added to the 80 mL cell culture in the 250 mL flask. This brought the final density of cells to ~1.0 x 10 ⁶ cells/mL. The culture flask was then incubated on an orbital shaking platform at 37 °C with 5% C0 ₂ at a speed of 133 rpm for 6 days. The culture supernatants were then harvested by centrifugation (Allegra X-15R, Beckman) at 4000xg for 40 minutes at 4 °C, adjusted to pH 8.0 with 0.1 M NaOH and held on ice until protein purification.

[0306] IgG purification from 293 FreeStyle and CHO-S was performed as follows: supernatants were loaded onto Protein A columns (0.4 mL bed volume) pre-equilibrated with PBS pH 7.4 and allowed to flow through by gravity. Columns were washed with 5 mL of PBS pH 7.4 and protein was eluted with 4 mL of 0.1 M Na Citrate -HCl pH 3.5. The eluent was neutralized with 200 1.5 M Tris-HCl buffer pH 8.8, concentrated and buffer-exchanged with PBS pH 7.4 using an Amicon 30 kDa 4 mL concentrator (Millipore), according to the manufacturer's instructions, to a final volume of approximately 0.5 - 1 mL. Protein concentrations were determined by absorbance at 280 _nm and the purity was determined by SDS- PAGE and SEC.

[0307] Protein expression and purification in CHO-K1 was done with proprietary methods at a contract research organization (CRO). Briefly, CHO-K1 cells were used for transfection. IgG was purified with MabSelect™ SuRe™ beads and the wash steps used Dulbecco's PBS (Lonza BE17-512Q). IgG was eluted with 0.1M Glycine pH 3.5

[0308] For protein QC, 3μg of antibody was used for each test, SDS-PAGE and SEC analysis. The QC passage criteria were purity > 90% in SDS PAGE and monomeric peak > 90% in SEC. For purified IgG protein which passed the QC tests, protein was aliquoted into screw caps tubes at 100 μΕ/tube, with concentration of ~5 mg/mL. The aliquots were flash frozen in liquid nitrogen and stored at -80 °C.

The protein yields in 293FreeStyle ranged from low mg/L to 53mg/L. The yields in CHO-S cells were low at about 1 mg/L. The yields in CHO-K1 cells ranged from 198 mg/L to 350 mg/L. SDS-PAGE showed intact protein running at about 150 kDa under non-reducing conditions and 2 bands representing heavy chain and light chain with no visible degradation or aggregation. The SEC profiles and SDS-PAGE analyses for one of the 293 FreeStyle batches are shown in Figure 14A and one of the CHO-K1 batches in Figure 14B, as examples. Protein purification data for 293FreeStyle and CHO-S batches are summarized in Table 12.

Table 12. PA13R3-P1C4 mAb Protein Purification Summary

Example 18. cAMP functional assays for PA13R3-P1C4 humanized variants

[0309] A commercially available kit (Cisbio) based on a competitive immunoassay format using cryptate-labeled anti-cAMP antibody and d2-labeled cAMP was used to characterize PA13R3-P1C4 humanized variant antibodies by measuring changes in intracellular cAMP levels in TRex-CHO cells stably expressing CBl . Cell numbers, forskolin concentration and CP55,940 concentration were optimized according to the manufacturer's instructions. The cAMP antagonist functional assay was performed in white 384-well low volume plates (Greiner). TRex- CHO cells expressing human CBl were seeded at a density of eight thousand cells/well in serum free Ham's F12 media followed by incubating mAb or control compound at various concentrations at 22 °C for 10 minutes. Five μΜ forskolin (at EC20, Sigma Aldrich) and 9 nM CP55,940 (at EC80, Sigma Aldrich) were subsequently added to the cells and incubated for 30 minutes at 22 °C to enhance adenylyl cyclase activity and activate CBl signaling, respectively. Five cAMP-d2 (1 :39 dilution with conjugate and lysis buffer provided by Cisbio) and 5 anti-cAMP cryptate (1 :9 dilution with conjugate and lysis buffer provided by Cisbio) were then added to the cells and incubated for 1 hour at 22 °C. FRET signal was detected with Envision multilabel plate reader (Perkin Elmer) at anti-cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism.

[0310] Activities of humanized PA13R3-P1C4 antibodies in this assay were compared with parental chimeric PA13R3-P1C4, rimonabant, a small molecule inverse agonist of CB l , and P2A12 mAb, a non-GPCR targeting mAb negative control antibody of IgGl isotype. PA13R3- P1C4 mAb and its humanized variants dose-dependently inhibited CP55,940-induced reduction in intracellular cAMP levels while negative control P2A12 mAb did not have any effects (Figure 15). The mean IC50s ± SD of PA13R3-P1C4 humanized variants are listed in Table 13.

Table 13. Summary of IC ₅ o of humanized P1C4 variants determined by cAMP antagonist assay

Rimonabant 417 ± 82 [3]* N/A N/A N/A p < 0.05, **p < 0.005; and compared to rimonabant, p < 0.05, ^m p < 0.02.

[0311] Humanized PlC4-h2 and h4 variants were more potent (1.6 - 3.3 fold) than chimeric PA13R3-P1C4 mAb in a cAMP antagonist assay (p<0.005). PlC4-h2 and PlC4-h4 humanized variants had comparable potency, while among the different isotypes of humanized PA13R3- P1C4 variants, a trend of higher potency of the IgGl and IgG4 variants versus the IgG2 and IgG2/4 variants was observed.

[0312] We further characterized the mechanism of PA13R3-P1C4 humanized variant antibody antagonism, in particular whether such antibodies behave as CB1 inverse agonists or neutral antagonists. Rimonabant (SR141716A), a known CB1 inverse agonist, and AM6545, a known CB1 neutral antagonist, were used as reference compounds.

[0313] To characterize whether PA13R3-P1C4 humanized variant antibodies acts as an inverse agonist or neutral antagonist, a cAMP assay was performed in the absence of exogenous agonist. Forskolin, a nonspecific adenylyl cyclase activator, was added to assay media to elevate basal cAMP levels to within the limits of detection. The cAMP agonist functional assay (Cisbio) was performed on white 384-well low volume plates (Greiner). Eight thousand cells/well in serum free Ham's F12 media of human CB1 expressing TRex-CHO cells were seeded to the plate followed by incubating mAb or control compounds at various concentrations at 22 °C for 10 minutes. Five μΜ forskolin (Sigma Aldrich) was added to the cells and incubated for 30 minutes at 22 °C. After a 30 minute incubation at 22 °C, 5 cAMP-d2 (1 :39 dilution with conjugate and lysis buffer provided by Cisbio) and 5 μΐ, anti-cAMP cryptate (1 :9 dilution with conjugate and lysis buffer provided by Cisbio) was added to the cells and incubated for 1 hour. FRET signal was detected with the Envision multilabel plate reader (Perkin Elmer) at anti-cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism.

[0314] We compared the activity of humanized variants PlC4-h2-IgG2 and PlC4-h2-IgG4 with rimonabant, AM6545 and the P2A12-IgGl negative control antibody in 1.5 μΜ forskolin stimulated TRex-CHO CB1 cells (Figure 16A) as well as 5 μΜ forskolin stimulated TRex-CHO CB1 cells (Figure 16B). The results show that PlC4-h2-IgGl, PlC4-h2-IgG2, PlC4-h2-IgG4, and rimonabant dose-dependently increased cAMP levels, which are indicative of an inverse agonist mechanism. In contrast, the CB1 neutral antagonist AM6545 and the P2A12-IgGl negative control antibody did not affect cAMP levels.

[0315] Schild plot analysis was performed to determine the equilibrium dissociation constant (K _B ), which is the measure of the binding affinity of the antagonist for its receptor independent of the nature and concentration of agonist used. Dose response curves of CB1 agonists CP55,940 and WIN55,212 in cAMP HTRF antagonist assays were determined in the presence of various concentrations of PlC4-h2-IgG4.

[0316] Figures 17A-D show the effect of increasing PlC4-h2-IgG4 concentrations on CP55,940 and WIN55,212 induced CB1 activity by cAMP assay (respectively). The dose ratio (R) was calculated based on EC50 of CP55,940 or WIN55,212 by which concentration of CB1 agonists (CP55,940 or WIN55,212) needs to be increased by to obtain the same response in the presence of PlC4-h2-IgG4 as was obtained in its absence. Tables 14 and 15 below shows the Schild slope and equilibrium dissociation constant (K _B ) measured from 4 different experiments.

Table 14. Schild slope and equilibrium dissociation constant CP55,940

Example 19. ERK activation assay for PA13R3-P1C4 humanized variants

[0317] We showed in Example 10 and Figure 5A that parental antibody PA13R3-P1C4 blocks WIN55,212 induced ERK activation. To confirm that PA13R3-P1C4 humanized variants also block WIN55,212 induced ERK activation we tested the ability of these antibodies to inhibit WIN55,212 induced ERK phosphorylation.

[0318] Two days before the experiment, Trex-CHO CB1 receptor-expressing cells were seeded at 500,000 cells/well into 6-well plates. ^g/mL tetracycline was used to induce CB1 receptor expression after 24 hours. Cells were serum starved for at least two hours before the experiment. Purified IgGs at 300 nM were added to the culture media, after 30 minutes, cells were stimulated with CB1 receptor agonist WIN55,212 (100 nM) for 10 and 15 minutes. Cell lysates were harvested and the level of ERK activation was determined by western blot. Anti- ERK and Anti-phospho-specific ERK antibodies were obtained from Cell Signaling Inc.

[0319] As shown in Figure 18A treatment with CB1 agonist WIN55,212 increased the level of phosphorylated ERK suggesting that the CB1 receptor signals through the ERK pathway. Pre- treatment with CB1 -specific antagonist rimonabant inhibited the WIN55,212-induced ERK activation. Similar to rimonabant, pre-treatment with anti-CBl antibodies PlC4-h2-IgGl and PlC4-h4-IgGl inhibited WIN55,212 induced ERK activation (Figure 18A). PlC4-hO-IgGl did not inhibit WIN55,212 induced ERK activation. This is consistent with the cAMP antagonist assay, which showed that PlC4-hO-IgGl is less potent than other humanized P1C4 variants and the lack of effect on blocking WIN55,212 induced ERK activation was hypothesized.

[0320] The effect of PlC4-h2 in IgG2 and IgG4 frameworks on WIN55,212 activated ERK pathway was also examined. Similar to chimeric PA13R3-P1C4 and humanized PlC4-h2-IgGl, pre-treatment with CB1 antibodies PlC4-h2-IgG2 and PlC4-h2-IgG4 blocked WIN55,212 induced ERK phosphorylation (Figure 18B). Non-GPCR targeting mAb P2A12 did not block WIN55,212 induced ERK activation. These results indicate that these Fc frameworks do not affect the antagonist characteristics of PA13R3-P1C4.

Example 20. CB1 receptor internalization study for PA13R3-P1C4 humanized variants [0321] Flow cytometry was used to characterize the activity of PA13R3-P1C4 and humanized variant antibodies in a CB 1 receptor internalization assay in the presence or absence of agonist or antagonist compounds.

[0322] On the day of the experiment, cells were serum starved for 2 hours. Cells were then pre-incubated with CBl antibody (300 nM), AM6545 (CBl neutral antagonist) and negative control (BRIL binder) for half an hour. CBl agonist (1 μΜ WIN55,212) was then added to the culture media for 1 hour to induce receptor internalization. Surface expression of CBl was stained with anti-CBl N-terminus mouse monoclonal antibody from R&D and the mean fluorescence intensity (MFI) was determined using flow cytometry (Guava).

[0323] CBl expression in TRex-293 cells was induced by tetracycline as evident by the increase in surface staining using a mouse monoclonal antibody targeting the CBl N-terminus (Figure 19 A, Panel A, dotted trace). Treatment with tetracycline and CBl agonist WIN55,212 reduced surface staining indicating the loss of CBl on cell surface through internalization (Figure 19A, Panel A, dashed trace). Pre-treatment with CBl -specific antagonist rimonabant (Figure 19 A, Panel B, solid black trace) inhibited the agonist-induced reduction in cell surface CBl staining. Similar to rimonabant, pre-treatment with anti-CBl antibodies (PA13R3-P1C4 (Figure 19 A, Panel D, solid black trace), PlC4-h2-IgGl (Figure 19 A, Panel F, solid black trace) and PlC4-h4-IgGl (Figure 19 A, Panel G, solid black trace)) inhibited WIN55,212 induced CBl receptor internalization. PlC4-hO-IgGl (Fig. 19A, Panel E, solid black trace) and negative control antibody P2A12-IgGl (Figure 19A, Panel C, solid black trace) did not inhibit WIN55,212 induced CBl internalization. Consistent with the cAMP antagonist and ER activation assays, PlC4-hO-IgGl is less potent than other humanized P1C4 variants and the lack of effect on blocking WIN55,212 induced receptor internalization may be due to the high off-rate of PlC4-hO-IgGl .

[0324] Among the different human IgG subclasses , the Fc regions of IgG2 and IgG4 subclasses bind poorly to effector molecules, such as activating FcyRs and to complement lq (Clq), resulting in lower effector function activity. As such, PlC4-h2 was cloned into human Fc frameworks IgG2 and IgG4 as for our therapeutic applications activation of immune effector functions is undesired (discussed in Example 17). Humanized variants PlC4-h2-IgGl, PlC4-h4- IgGl, PlC4-h2-IgG2 and PlC4-h2-IgG4 were then assayed for blockade of CBl receptor internalization. As above, CBl expression in TRex-293 cells was induced by tetracycline as was evident by the increase in surface staining using a mouse monoclonal antibody targeting the CBl N-terminus (Figure 19B, Panel A, dotted trace). Treatment with tetracycline and CBl agonist WIN55,212 reduced surface staining indicating the loss of CBl on cell surface through internalization (Figure 19B, Panel A, dashed trace). Pre-treatment with CBl -specific antagonist rimonabant (Figure 19B, Panel B, solid black trace) inhibited the agonist-induced reduction in cell surface CBl staining. Similar to rimonabant, pre-treatment with anti-CBl antibodies (PA13R3-P1C4 (Figure 19B, Panel D, solid black trace), PlC4-h2-IgGl (Figure 19B Panel F, solid black trace) and PlC4-h4-IgGl (Figure 19B, Panel G, solid black trace)) inhibited WIN55,212 induced CBl receptor internalization. PlC4-hO-IgGl (Figure 19B, Panel E, solid black trace) and negative control antibody P2A12-IgGl (Figure 19B, Panel C, solid black trace) did not inhibit WIN55,212 induced CBl internalization.

Example 21. Binding of humanized PA13R3-P1C4 antibody variants by flow cytometry

[0325] The binding affinity of PA13R3-P1C4 humanized variants was determined by flow cytometry using TRex CHO cells stably transfected with the CBl . TRex CHO parental cells, and TRex-CHO cells stably transfected with tetracycline inducible human CBl, human CB2 or mouse CBl expression constructs were harvested. To determine binding of test antibodies, one hundred microliters of 1 x 10 ⁶ cells/mL of cells were incubated with test antibodies with a range of concentrations between 1 μΜ and 1.3 nM for 30 minutes on ice. Cells were then centrifuged at 4°C at 1600 rpm for 3 minutes; supernatant was aspirated and cells were washed with 200 μΐ, FACS buffer. The washing procedure was repeated twice. After the final wash, cells were re- suspended in FACS buffer containing PE-conjugated anti-human Fc secondary antibody (1 :200 dilutions) and incubated at 4°C for 30 minutes. Cells were washed with 200 μΐ, FACS buffer twice and analyzed by flow cytometry (Guava). Data analysis and measurement of binding affinity (K _D ) was performed using GraphPad Prism software. The mean dissociation constant (K _D ) of PA13R3-P1C4 humanized variants was determined by averaging at least 4 different experiments using at least 2 different batches of protein (Figure 20A-D and Table 16). Table 16. Mean dissociation constant (K _D ) of PA13R3-P1C4 humanized variants

* p < 0.05, **p < 0.01, *** p<0.001.

[0326] Humanized variants except h2-IgG2, h2-IgG2/4 and h4-IgG2/4 showed statistically significant increased binding affinity to human CB1 relative to the parental chimeric antibody PA13R3-P1C4. The mean dissociation constant for each species was determined using at least two different protein preparations (except PlC4-h2-IgGl which has only one protein preparation). Dissociation constants of each species were comparable between different protein preparations (Table 16). The binding affinities of PlC4-h2 and PlC4-h4 variants were similar. However, a slight trend, although not statistically significant, of higher binding affinities of IgGl variants compared to IgG2, IgG4 and IgG2/4 variants was observed.

[0327] PlC4-hO-IgGl has the lowest apparent dissociation constant measured (K _D 24 nM). However, the maximum FACS signal (mean fluorescence intensity) of PlC4-hO-IgGl did not reach as high as the other P1C4 variants. This may indicate a higher off-rate of PlC4-hO-IgGl . [0328] Binding selectivity and cross-reactivity of PA13R3-P1C4 and its humanized variants was also characterized by flow cytometry using TRex CHO cells stably transfected with the GPCRs of interest. Specifically, binding selectivity for CBl over CB2 was determined. Cross reactivity to mouse CBl was also determined. For detecting expression of mouse CBl, 100 μΐ, of 1 x 10 ⁶ cells/mL of cells were incubated with anti-CBl rabbit polyclonal antibody (Santa Cruz) at 1 μg/100 on ice for 30 min. Mouse monoclonal anti-CB2 antibody from R&D Systems was used to confirm the expression of CB2. 100 of 1 x 10 ⁶ cells/mL of cells were incubated with anti-CB2 antibody at 0.5 μg/100 μί on ice for 30 minutes. After incubation, cells were then centrifuged at 4°C at 1600 rpm for 3 minutes; supernatant was aspirated and cells were washed with 200 μΐ, FACS buffer. The procedure was repeated twice. After the final wash, cells were re-suspended in FACS buffer containing FITC-conjugated anti-rabbit secondary antibody (1 :200 dilutions) for mouse CBl detection and PE-conjugated anti-mouse IgG secondary antibody (1 :200 dilutions) for CB2 expression and incubated at 4°C for 30 minutes. After 30 a minute incubation with secondary antibody, cells were then centrifuged at 4°C at 1600 rpm for 3 minutes; supernatant was aspirated to remove excess secondary antibodies. Cells were washed with 200 μΐ, FACS buffer twice and analyzed by flow cytometry (Guava). Binding of purified IgG full concentration curves ranging from 1 μΜ to 1.3 nM was determined. Data analysis and measurement of binding affinity (K _D ) was performed using GraphPad Prism software.

[0329] Binding selectivity and cross-reactivity of humanized PA13R3-P1C4 variants to human CBl versus human CB2 and mouse CBl are shown in Figures 20E-M. Similar to PA13R3-P1C4, the humanized variants bound selectively to human CBl vs human CB2. No substantial binding to mouse CBl was observed at concentrations up to 1 μΜ, indicating lack of cross-reactivity with this species despite high amino acid identity between human and mouse CBl in the extracellular domains (97% identity).

Example 22. Swapping of ELC2 effect on FACS binding

[0330] To test the effect of ECL2 mutations to PlC4's ability to bind to CBl expressed on cell surface, we built a CBl cell expression construct by site-directed mutagenesis. Briefly, 2 oligos, Apollo_ECL2_h2m_F (CTGCAATCTGTTTGCTCAGACATTTTCCCACTCAT TGATGAAACCTACCT) (SEQ ID NO: 826) and Apollo_ECL2_h2m_R (GGAAAATGTCT GAGCAAACAGATTGCAGTTTCTTGCAGTTCCAGCCCAGG) (SEQ ID NO: 827) were used as primers in a PCR reaction using pcDNA4TO-human CBl as template. The reaction introduced E- K and H- L mutations in ECL2, making the human CBl ECL2 sequence identical to murine CBl ECL2. The 50 PCR reaction mixture contained Ι ΟμΙ _^ 5 ^X PCR buffer, 2μΕ dNTPs (lOmM each), 0.25μΕ each of forward and reverse primers (ΙΟΟμΜ stock), 50 ng of template DNA, Ι μΙ, DMSO, and Ι μΙ, Phusion polymerase (NEB). The PCR cycles were 95 °C for 30 seconds, 55 °C 1 minute, 72 °C 7 minutes, and repeated for 16 times. After the PCR reaction was finished, Ι μΙ, Dpnl (201Ι/μΕ) was added into PCR product. The PCR product was incubated at 37 °C for 1 hour before 1 μΐ _^ it was transformed into Dh5a E. coli. The resulting transformants were plated and single colonies were sequenced to verify the mutation. The resulting construct was named as pcDNA4TO-human/mouse ECL2 swapped CBl-IRES-GFP.

[0331] To confirm the sites E->K and H->L in ECL2 were critical for PA13R3-P1C4 CBl binding, transiently transfected TRex-CHO cells with human CB l , mouse CBl or human/mouse ECL2 swapped expression constructs were used to investigate the binding of PlC4-h4-IgGl by flow cytometry.

[0332] TRex-CHO cells were grown in Ham's F12 culture media supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin and 10 μg/ml blasticidine. The day before transfection, cells were passaged and seed at 0.5 x 10 ⁶ cell in 2 ml culture media per well in a 6- well plate. On the day of transfection, cells were transfected with pcDNA4TO-human CBl , pcDNA4TO-mouse CBl-IRES-GFP, pcDNA4TO-human/mouse ECL2 swapped CB l-IRES- GFP or pcDNA4TO-GFP negative control using Lipofectamine 2000 following Life Technologies' instructions. The day after transfection, cells were treated with 1 μg/ml tetracycline for 24 hours to induce CBl expression. On the day of experiment, media were aspirated; cells were washed with DPBS once and incubated with cell dissociation buffer for 5 minutes. Cells were collected and centrifuged at 1600 rpm for 3 minutes. Supernatants were aspirated and cells were washed with DPBS. Cell counts were determined using Bio-Rad T10 cell counter. Cells were centrifuged at 1600 rpm for 3 minutes; supernatant was aspirated to remove DPBS and resuspended at 1 x 10 ⁶ cells/mL FACS buffer (3% FBS in DPBS, 0.09% sodium azide). To determine binding of PA13R3-P1C4 mAb to human CBl, mouse CBl , human/mouse ECL2 swapped CBl and control cells, 100 μΐ, of 1 x 10 ⁶ cells/mL of cells were incubated with PlC4-h4-IgGl, R&D CBl mAb or P2A12 for 30 minutes on ice. Secondary PE- conjugated anti-human mAb or PE-conjugated anti-mouse mAb was diluted 1 :200 fold in FACS buffer. Cells only stained with secondary antibody were used as negative control. After incubation with secondary antibody, cells were washed with 200 μΐ of FACS buffer twice and analyzed by flow cytometry (Guava).

[0333] As shown in Figure 21, PlC4-h4-IgGl bound to human CBl , but not mouse CBl or human/mouse ECL2 swapped CBl . The only difference between human CBl and human/mouse ECL2 swapped CBl are at the ECL2 residues E- K and H- L. The flow cytometry binding results indicated that ECL2 are critical for P1C4 binding. R&D CBl monoclonal antibody was used as positive control showing the expression of CBl . P2A12 and pcDNA4TO-GFP were used as staining control and empty vector control respectively.

Example 23. ADCC and CDC Effector Function Analysis

[0334] To confirm the presumed absence of effector function of humanized P1C4 variants PlC4-h2-IgG2 and PlC4-h2-IgG4 antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed using Daudi cells.

[0335] For ADCC assay, Daudi target cell concentration was adjusted to 12.5 x 10 ⁴ cells/mL with ADCC medium. 80 of cell suspension (1 x 10 ⁴ viable cells) was added to each well of a round-bottom 96-well plate. Twenty μΕ of antibody, serially diluted in ADCC medium, was dispensed to each well in triplicate. The final concentrations of Rituximab and Anti-hel-hlgGl were: 0.128 ng/mL, 0.64 ng/mL, 3.2 ng/mL, 16 ng/mL, 80 ng/mL, 0.4 μg/mL, 2 μg/mL; the final concentration of Anti-hel-hIgG2, Anti-hel-hIgG4, PlC4-h2-IgG2 and PlC4-h2-IgG4 were: 3.2 ng/mL, 16 ng/mL, 80 ng/mL, 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL. The plate was incubated at 22-25 °C for 30 minutes. PBMC concentration was adjusted with ADCC medium so that by adding ΙΟΟμί of PBMC cells to the target cells, the Effector: Target cell ratio were 25: 1 and 50: 1, respectively. The plate was centrifuged at 250 x g for 4 minutes and then incubated at 37 °C. 45 minutes prior to harvesting the supernatant, 20 μΐ _^ of lysis solution from the CytoTox 96 kit (Promega) was added to maximum lysis control wells. After a 5 hour incubation, the plate was centrifuged at 250 x g for 4 minutes. 50 of supernatant from each well was transfer to a clean flat-bottom 96 well assay plate. 50 of substrate from CytoTox 96 kit (Promega) was added to each well and mixed for 30 seconds. The plate was incubated at room temperature for 30 minutes before 50 of stop solution was added to each well and mixed for 30 seconds. The absorbance was read at 490 nm. For data analysis: the % lysis is calculated using GraphPad Prism 5.0 to generate the IC ₅₀ , as follows:

(Experimental release) - (Target + PBMC)

% Lysis = 100%

(Max lysis) - Ave (Target only)

[0336] Rituximab induced ADCC effect in Daudi cells with the IC50 of 1.19 ng/mL at 50: 1 E/T ratio, of 0.92 ng/mL at 25: 1 E/T ratio. As shown in Figure 22A-C the P1C4 Fc variants antibodies had no ADCC effect at 50:1 and 25: 1 E/T ratio in Daudi cells.

[0337] For CDC assay Daudi target cells were harvested and cell concentration was adjusted to 80 x 10 ⁴ cells/mL with cell culture medium. To a flat-bottom 96-well white plate, 25 μΕΛνεΙΙ of cells was added. Then, 12.5 μΕΛνεΙΙ of P1C4, Rituximab, Anti-HEL-hlgGl, and Anti-HEL- hIgG2, serially diluted in ADCC medium, were added to each well in duplicate. The final concentrations of Rituximab and Anti-hel-hlgGl were: 0.128 ng/mL, 0.64 ng/mL, 3.2 ng/mL, 16 ng/mL, 80 ng/mL, 0.4 μg/mL, 2 μg/mL; the final concentration of Anti-hel-hIgG2, Anti-hel- hIgG4, PlC4-H2-IgG2 and PlC4-H2-IgG4 were: 3.2 ng/mL, 16 ng/mL, 80 ng/mL, 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL. The plates were incubated in the hood for 15 minutes. Human complement, diluted with ADCC medium, was added to the plates containing cells at 12.5 μΕΛνεΙΙ to reach a final concentration of 10%. For the maximum lysis wells, 5 μΐ _^ of lysis solution was added. The final volume of all the wells was adjusted to 50 μΐ, with ADCC medium. The plates were incubated at 37 °C for 2 hours before 50 μΕΛνεΙΙ of CTG solution was added to the cells. The plates were shaken on a microplate shaker for 2 minutes at a speed of 200 and then incubated at room temperature for 10 minutes. The luminescence signal was red with Envision. For data analysis, % cytotoxicity and IC ₅₀ was calculated using GraphPad Prism 5.0 as follows:

Experimental - Ave (cell+complement)

% Cytotoxicity = x 100%

Ave (Maxi lysis) - Ave (cell only)

[0338] The CDC effect of antibodies was tested in Daudi cells, cell lysis of Daudi by PBMC was accessed by cell titer Glo assay, the final concentration of complement was 10%. Rituximab induced CDC effect in Daudi cells with IC50 of 399 ng/niL. As shown in Figure 22D the P1C4 Fc variants antibodies had no CDC effect in Daudi cells.

Example 24. Recognition of denatured CB1 protein by P1C4 antibodies

[0339] A study was performed to examine whether PA13R3-P1C4 and its humanized variant antibodies recognize epitope(s) on denatured CB1 protein (His tagged, N/C-termini truncated CB1) by western blot analysis. Purified human CB1 protein (750 ng per lane) was mixed with SDS reducing buffer containing beta-mercaptoethanol (Double Helix). The denatured CB1 recombinant protein was loaded onto 12% reducing SDS-PAGE gels and protein was separated at 120V for one hour, then electro-transferred to PVDF membranes (pre-soaked in methanol) at 300 niA for 70 minutes. Membranes were blocked with 5% NFDM/PBS-T for one hour at 22 °C followed by immune-blotting with test antibodies (2 μg/mL) in 5% NFDM/PBS-T overnight at 4 °C. Commercial mouse anti-His antibody and mouse anti-CBl antibody (R&D Systems) were used as positive controls, and rabbit anti-CBl antibody (Cayman), which recognizes a C-terminal region that was deleted from the recombinant CB1 protein used in this study, served as a negative control.

[0340] After overnight incubation, membranes were washed with 0.5% Tween-20 PBS (PBS-T) three times at 22 °C. Secondary antibodies AP-conjugated anti-human IgG (1 :5000) or anti-mouse IgG or anti-rabbit IgG in 5% NFDM/PBS-T were added to respective membranes and incubated for 1 hour at 22 °C. Membranes were washed three times with PBS-T each for 5 minutes. After the final wash, signal was developed by incubating with NBT/BCIP substrate solution at 22 °C and the reaction was stopped by washing the membrane under running water.

[0341] Western blot results showed that positive control antibodies were able to detect denatured CB l protein with the correct apparent molecular weight of 63 kDa (Figure 23B, Lanes 9 and 10). No band was detected by the negative control C-terminal specific antibody (Figure 23B, Lane 11), by PA13R3-P1C4 (Figure 23 A, Lane 2) or by humanized variant antibodies (Figure 23 A, Lanes 3 to 6). Anti-human Fc secondary antibody used in the experiment was able to detect purified human IgG (Figure 23A, Lane 1). The results indicate that PA13R3-P1C4 and humanized variant antibodies could not recognize denatured and linearized CBl protein. Together with the flow cytometry experiments, these results confirm that PA13R3-P1C4 and humanized variant antibodies recognize conformational but not linear epitopes.

Example 25. Chimeric and humanized P1C4 Fab cAMP and FACS binding

[0342] To determine PA13R3-P1 C4 Fab binding affinity, full binding curves were generated on CBl receptor by testing a range of concentrations using TRex-CHO cells stably transfected with tetracycline inducible CBl expression construct by flow cytometry. Three-fold serial dilutions from 3 μΜ to 0.1 μΜ were prepared. FITC-conjugated anti-human antibody was used to detect PA13R3-P1C4 Fab. PA13R3-P1C4 Fab dose dependently bound TRex-CHO CB l cells (Figure 24A and Table 17).

[0343] A cAMP functional assay was performed to measure the antagonism of P1C4 Fab. The cAMP functional assay (Cisbio) was performed on white 384-well low volume plate (Greiner). 8000 cells/well of stably expressed CBl TRex CHO cells were seeded to the plate followed by incubating P1C4 Fab at varies concentrations at room temperature for 10 minutes. 5 μΜ of forskolin (Sigma Aldrich) and 9 nM of the cannabinoid CP55940 (Sigma Aldrich) were added to the cell stimulation mixture to and incubated for 30 minutes at room temperature to activate CBl . After the 30 minutes incubation, 5 μΐ _^ of cAMP-d2 (1 :39 dilution with conjugate and lysis buffer provided by Cisbio) and 5 μΣ, of anti-cAMP cryptate (1 :9 dilution with conjugate and lysis buffer provided by Cisbio) were added to the cell stimulation and incubated for an hour. FRET signal was detected with Envision multilabel plate reader (Perkin Elmer) at anti- cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism. The results are shown in Figure 24B and Table 17. The mean ± SD of IC50S and dissociation constants (Kd) shown were measured from at least 2 different experiments.

Table 16. IC50 and disassociation constants of PA13R3-P1C4 Fab

Example 26. Biophysical Characterization of P1C4 Humanized Variants

[0344] We characterize the stability and solubility of PA13R3-P1C4 humanized Fc variants by conducting tests to measure stability under low and high pH and to test maximum solubility. We also characterized the stability of these molecules under accelerated conditions, in human and non-human primate serum, after multiple freeze/thaw cycles and under conditions of pH shift.

[0345] In order to characterize the pH stability of the P1C4 humanized variants 200 of each antibody (~5 mg/mL) and PBS control were prepared and introduced into a Pur-A-Lyzer Maxi 12000 Dialysis cassette (Sigma, Cat# PURX12015-1KT). Proteins were dialyzed against 2L of pH 3 buffer (0.1 M acetic acid adjusted to pH 3 by NaOH) or pH 9 buffer (0.2 M glycine adjusted to pH 9 by NaOH) respectively at 4 °C overnight. Protein samples were recovered from dialysis cassettes into pre-weighted empty Eppendorf tubes and checked for visible precipitation. The sample volume was measured by weight. To confirm the success of dialysis, 3 of dialyzed sample was taken and checked by pH paper. Fifty μΕ samples were removed for SEC analysis. The remaining samples were kept in a 40 °C incubator for 48 hours. After that, samples were checked for visible precipitation again. Six μΐ, of 48 hour-incubated sample was injected into TSK G3000SWXL SEC column and the protein concentration calculated by SEC peak area (after 40 °C incubation). To determine protein recovery rate, the following formula was used: Before Dialysis (Vol. x Cone.)/ After Dialysis (Vol. x Cone.) x 100%.

[0346] No precipitation of the 4 IgGs was observed after dialysis to pH 3 and pH 9 buffers followed by a 48 hour incubation at 40 °C. For all four IgGs, recovery rates were about 71-83%. Low recovery rate was likely due to sample remaining in dialysis cassette. Monomer IgGs calculated by SEC profile were more than 99% for all 4 dialyzed IgGs. IgGs in pH 3 buffer after a 48 hour incubation at 40 °C showed wider SEC peaks, suggesting higher heterogeneity of IgGs after long incubation at pH 3 buffer. The cAMP and CBl -expressing cell-binding activity was measure according Examples 11 and 7 and the results are summarized in Tables 18 and 19, respectively. The results showed that under pH 3, the cAMP activity of PlC4-h2-IgG4, P1C4- h4-IgG2 and PlC4-h4-IgG4 decreased, as did PlC4-h4-IgG2 under pH 9.

Table 18. pH stability of P1C4 humanized variants: solubility

^• SH ton .

¾C4-H2-¾C>2 3 4.5 200 ¾ 7:5 m 27.88 >99

?iC4-H2- ¾64 3 5.2 200 -.00 m 7183

3 4.8 200 a.ss 17? 7 ·; 07 • ^• 99

? 4 H4- ¾G4 3 5.5 200 2.9* 232 ^" 5 53 99

¾C4 H2- ¾G2 S 4,8 200 4.23- 27S SO 92 •• ^• 99

¾C -H2-;s<3 * S.2 200 4.6 i a 80.50 ϊ9

¾C4-H4-;s<32 9 4.6 2(H1 4.26 179 si8¾

¾C4-H4-¾54 S S.S 2¾ ≤ vv 138 81S2 >

......EL... tc-i-r:, (wg/^U Sessv? f rati; %

4.8 200 3.79 ■·' .' ^■ SS •>99

? C-i- 3 5,2 200 4,05 m 7183 S9

¾C4- H4-s<22 3 4.6 200 3.35 172 74.S7

¾C4-H4-ii¾4 3 5,5 20S 2.59 2¾2 75.SS ^■

9 4.6 175 80.S2 §:?

S S.2 m 4.6 182 80.50 >s¾

?iC4-H4-¾S2 s 4.5 200 4.25 1?S S2.SS >§*

?iC4-H4-¾S4 s 5.5 200 4.7? m S1.32 99 Table 19. H stability of P1C4 humanized variants: cAMP activity

[0347] The solubility of P1C4 humanized variants was characterized by concentrating 400 μΐ. IgG (~5 mg/mL), by centrifugal filtration (Amicon Ultra-0.5mL 30K) at 14000 x g at 4 °C down to -100 μί. 200 μΐ, more IgG was added into centrifugal filters and concentrated at 14000 x g at 4 °C down to -100 μΐ _^ . After that another 200 μΐ _^ IgG was added into centrifugal filters and concentrate at 14000 x g at 4 °C down to -100 μΐ. (from total 800 μΐ. to 100 μΐ,). The concentrated protein was inspected for visible precipitation by pipetting up and down. The concentration was then continued at 14000 x g at 4 °C until the volume was less than 50 μΐ _^ . The centrifugal filters were reversed and set in pre-weighted empty tube. The centrifuge filters were spun down at 1000 x g for 5 minutes at 4 °C to collect the concentrated sample. The tubes were weighed to obtain sample volumes. If precipitation was visible, the tubes were spun down at 14000 x g for 10 minutes at 4 °C. Supernatants were removed into new 1.5 mL Eppendorf tubes and incubate at room temperature for 24 hours. Afterwards, precipitation was spun down by centrifugation at 14000 x g for 10 minutes at 4 °C and supernatants were moved into new 1.5 mL Eppendorf tubes. For SEC characterization, 6 μΐ _^ supernatant of concentrated sample was injected into TSK G3000SWXL SEC column and the protein concentration calculated by SEC peak area. The protein recovery rate was calculated as: Before Cone. (Vol. x Cone.)/ After Cone. (Vol. x Cone.) x 100 %.

[0348] Four Fc variants were concentrated to the protein concentration higher than 85 mg/mL. The protein recovery rates were more than 99%. Slight visible precipitation of PlC4-h2- IgG4 at 93.9 mg/mL before and after 24 hour incubation at room temperature was observed. No visible precipitation for other 3 IgGs at concentration higher than 85 mg/mL after 24 hour incubation at room temperature was observed. Monomer IgGs calculated by SEC profile are more than 96% for all 4 concentrated IgGs compared to the starting level at -99%. The data are summarized in Table 20.

Table 20. Solubility of P1C4 humanized variants

[0349] The accelerated stability of P1C4 humanized variants was also assessed. In a 1.5 mL Eppendorf tube, 100 protein sample (~5mg/ml) was placed. The tube was sealed with Parafilm. Two tubes were set up, one was kept at 4 °C and the other was kept at 40 °C for 33 days. An aliquot of 20 μΐ was removed to check visible precipitation and for SEC analysis. For SEC analysis, 6 sample was injected into TSK G3000SWXL SEC column and the protein concentration was measured by SEC peak area. The protein recovery rate was calculated as following: Before Incubation (Vol. x Cone.)/ After Incubation (Vol. Cone.) x 100 %.

[0350] No precipitation was observed after a 33 day incubation at 4 °C and 40 °C. Monomer IgGs calculated by SEC profile were more than 98% for all 4 IgGs at both 4 °C and 40 °C. The recovery rate calculated by SEC profile was more than 96% for all 4 IgGs at both 4 °C and 40 °C (Table 21). No change in potency of P1C4 Fc variant was observed after 33 days at 4 °C and 40 °C, except PlC4-h4-IgG2, which showed IC ₅₀ outside of the referenced range indicating there is a slight reduce in its potency. (Table 22).

Table 21. Accelerated stability: Solubility

Table 22. Accelerated stability: Potency

[0351] Serum stability of P1C4 humanized variants was also characterized. Human serum was obtained from Sigma. Non-Human Primate serum was collected by Crown Bioscience. In an Eppendorf tube, 950 serum was mixed with 50 IgG at final cone. 250 μg/ml (-1.67 μΜ) and incubate at 37 °C. Samples of 200 μΐ _^ were taken at time points 0, 24, 48, and 72 hours for flow cytometry binding assay using CBl -expressing cells. The starting concentration for IgG tested was 500 nM and the samples were serial diluted 3 fold for flow cytometry assays to determine binding K _D . The results, listed in Table 23 and 24, showed no changes in affinity after 24 hours incubation with human or NHP serum at 37 °C. No significant change in K _D was observed for the samples except PlC4-h2-IgG2, which showed a decrease of CBl -expressing cell-binding affinity after 48 hours incubation with human and NHP sera. In addition, PlC4-h4- IgG2 also showed reduced CBl - expressing cell-binding affinity after 48 hours. Table 23. Human serum stability of P1C4 humanized variants, 24 hours

Table 24. NHP serum stability of P1C4 humanized variants

[0352] The freeze/thaw stability of P1C4 humanized variants was characterized as follows. A 100 aliquot from frozen stocks of each humanized P1C4 Fc variants was thawed in a 22 °C water bath, then rapid frozen by liquid nitrogen. The frozen sample was kept at -80 °C for at least 20 minutes before it was thawed in a 22 °C water bath again. The samples went through 10 such freeze/thaw cycles. Visual inspection was used to check for precipitation. A 20 μΐ, aliquot was removed from the sample for SEC analysis at freeze/thaw cycle 1, 5, and 10. For SEC characterization, 6 of incubated sample was injected into TSK G3000SWXL SEC column. The protein concentration was measured by SEC peak area. The protein recovery rate was calculated by using formula: Before F/T (Cone.)/ After F/T (Cone.) x 100 %.

[0353] The results showed that after 10 freeze/thaw cycles monomer IgGs were higher than 97% for all 4 IgG variants tested. The protein recovery rate was more than 96% for all IgGs. P1C4-H2-IG4 showed slight turbidity after 1st freeze thaw cycle, but no significant precipitation or decrease of protein concentration was observed. The results are summarized in Tables 25-28. The protein samples recovered after 5 freeze/thaw cycles were also tested for function using the cAMP antagonist assay, and the calculated IC ₅₀ results showed no significant changes in potency with the numbers falling within the normal range (Table 29).

Table 25. Freeze / thaw stability of PlC4-H2-IgG2

Table 26. Freeze / thaw stability of PlC4-H2-IgG4

Table 27. Freeze / thaw stability of PlC4-H4-IgG2

Table 28. Freeze / thaw stability of PlC4-H4-IgG4

Table 29. Freeze / thaw stability of humanized variants: cAMP activity after 5 cycles

[0354] To examine stability of 4 P1C4 Fc variants under pH shift, 0.2 mL MabSelect resin was packed into 10 mL column and equilibrated with 10 mL DPBS pH 7.4. 150 IgG sample (~ 5 mg/mL) was loaded onto column. The column was washed with 1.6 mL DPBS. The IgG was then eluted with 1.6 mL sodium citrate (pH ~3.5). The concentration of the protein was measured. The protein was then concentrated to ~3 mg/mL for functional assay. No visible precipitation was observed. These samples were spun down at 14000 x g for 10 minutes at 4 °C. The supernatant was transferred to a new eppendorf tube and the protein concentration was measured. This sample was also checked for aggregation by SEC and functionally tested by cAMP antagonist assay. The SEC profiles showed that monomer IgG percentage is more than 95% after the pH is shifted to pH 3.5. The cAMP antagonist results are listed in Table 30 and showed that PlC4-h2-IgG4, PlC4-h4-IgG2 and PlC4-h4-IgG4 were stable at pH 3.5, with the IC50S measured falling within the acceptable range. PlC4-h2-IgG2 behaved differently at pH 3.5, with a lower IC50 value compared to the acceptable range. This result is similar to that in the pH stability test. Table 30. Stability of P1C4 humanized variants under pH shift

Example 27. CDR Mutagenesis of PA13R3-P1C4

[0355] To investigate the role of each amino acid position on each CDR, site-directed saturation mutagenesis was conducted on every CDR position of chimeric P1C4 Fab. Mutagenic primers containing NNS or a specific codon were synthesized, dissolved in Tris-EDTA buffer and diluted to 10 μΜ working stock. Twenty five PCR reactions were set up in 96-well plates using high fidelity DNA polymerase. The resulting PCR products were treated with 0.8 Dpnl (20 υ/μί) in each well 37 °C for 5 hours. The Dpnl -treated PCR product (2 μΕ) was transformed into 30 μΐ E. coli Dh5a competent cells. DNA was isolated from the transformants by miniprep and sequenced to identify desired mutations. Plasmid DNA from desired clones was used to transform E. coli BL21 (CodonPlus) competent cells. Single colonies were used for Fab protein expression.

[0356] For Fab expression, colonies were picked into 96-well plate containing 100 μΐ SB medium and cultured overnight. On the next day, 10 μΐ, from each well was used to inoculate 500 μΐ _^ ZYM medium with 50 μg/mL Kanamycin in deep well 96 well plate. The plate was sealed with breathable plate sealer and shaken 36 hours at 25 °C in shaker at 450rpm.

[0357] To prepare samples for ELISA, the deep well 96-well plate was centrifuged at 3000 rpm in the Beckman table-top for 20 minutes (-2050 rcf) at 4 °C. 100 μΐ _^ supernatant was transferred from the expression plate into a dilution plate containing 200 μΐ _^ PBS, pH 7.4 and mixed well. [0358] Fab expression was measured by ELISA. 96-well half-well ELISA plates (Corning, 3690) were directly coated with 2 μg/mL anti-his antibody (Sigma HI 029 2mg/ml) at 50 μΐ/well overnight at 4 °C. The plate was then washed 6 times with 150 PBST. Each well was then blocked with 175 μΕ/well of 3% milk/PBS, pH 7.4 for 1 hour at 22 °C. The plate was then washed 6 times with PBST before 25 μΕΛνεΙΙ Fab containing-culture medium diluted 3 times in PBS, pH 7.4 was added to each well and incubated for 1 hour at 22 °C. The plate was again washed 6 times with PBST before 50 μΐ/well of HRP labeled anti-human Fab antibody (0293 Sigma) was added at 1 : 10000 dilution in 3% milk and incubated at 22 °C for 1 hour. To develop the ELISA the plate was washed 6 times with PBST and 50 μΐ _^ / well of TMB substrate were added. The reaction was stopped by adding 50 μΐ, / well 1 N HC1 and the OD ₄₅₀ was read on the BioTek reader.

[0359] For ELISA measuring iCAPS-binding, 96-well half-well ELISA plates were coated with 2 μg/mL, 25 μΕΛνεΙΙ of streptavidin in PBS, pH 7.4 overnight at 4°C. The plate was then washed 6 times with 150 μΐ _^ PBST with rotation in between. Then 25 μΕΛνεΙΙ of 5ug/mL CB1 iCAPS was added to the plate for 1 hour incubation. After washing 6 times with PBST, each well was blocked with 175 μΕ/well of 3% milk/PBS pH 7.4 for 1 hour at 22 °C. The plate was then washed 6 times with PBST before 25 μΕΛνεΙΙ Fab containing-culture medium diluted 3 times by PBS pH 7.4 was added to each well and incubated for 1 hour at 22 °C. The plate was again washed 6 times with PBST before 50 μΐ/well anti-human Fab antibody (0293 Sigma) was added at 1 : 10000 dilution by 3% milk and incubated at 22 °C for 1 hour. To develop the plate, the plate was washed 6 times with PBST, and 50 μΐ, I well of TMB substrate were added. The reaction was stopped by adding 50 μΐ, I well 1 N HC1 and the OD ₄₅₀ was read on the BioTek reader.

[0360] Each clone was assayed in triplicate. To calculate the relative binding to iCAPS normalized by Fab expression, the ELISA signal for iCAPS binding was divided by Fab expression ELISA data, and compared to the data from parental clone. Allowable changes were defined as clones that retained at least 50% specific iCAPS binding activity compared to the parental clones. These allowable changes are summarized in Table 31 and Table 32. The CDR sequences of the indicated mutations can be found in Table 34. Table 31. Allowable mutations PA13R3-P1C4 Heavy Chain CDRs

Table 32. Allowable mutations PA13R3-P1C4 Light Chain CDRs

Example 28. Immuno-staining of CB1 in liver tissue samples by PlC4-h2-IgG4 antibody conjugated to HRP.

[0361] PlC4-h2-IgG4 antibody was labelled with the Lightning-Link HRP conjugation kit from (Innova Bioscience, 701-0010) according to the manufacturer's instructions. Slides of Parafilm treated human liver samples were treated with Clearene Solvent (Leica Biosystems) for 5 minutes and then with decreasing concentration ethanol to a final 50% concentration. The slides were then placed into methanol/hydrogen peroxide for 15 minutes after which they were briefly washed in PBS. Slides were then treated with citric saline (Vector, H-3300) with heating for antigen retrieval before being placed in pre-warmed of PBS (350ml) and Trypsin (2.45ml) in a 37 °C water bath for 20 minutes. After washing with PBS, the slides were blocked with casein (Vector, SP-5020) for 1 hour at room temperature. After blocking 1 : 100 diluted HRP conjugated PlC4-h2-IgG4 or isotype control antibody was added and incubated overnight at 4 °C. The slides were then washed with PBS, and treated with 3 drops of Vector ABC Tertiary (Vector, PK-7100) at 22 °C for 45 minutes. PBS was used to wash the slides for 3 times and DAB mix (Vector, SK- 4100) was added to the slides for 5-10 minutes before they were again washed briefly with PBS. Following this, the slides were counter stained with Meyers Haematoxylin (TCS Biosciences, HS315) for 1 minute followed by treatment in water, increasing concentration of ethanol (50% to 100%)), 2 rounds of Clearene Solvent before they were mounted in Pertex (Leica Biosystems).

[0362] The results showed positive CBl -specific staining in macrophage, hepatocytes, and hepatic myofibroblasts in early NASH (Figure 25, left panel), NASH fibrosis (Figure 25, middle panel) and late fibrosis (Figure 25, right panel) samples. No staining was observed with isotype controlled irrelevant antibodies (Figure 26) or normal tissue samples (Figure 27).

Example 29. Measuring effects of anti-CBl antibody on genetic markers of fibrosis in primary human hepatic stellate cells

[0363] Primary hepatic stellate cells (HSCs) were isolated from liver tissue obtained from 3 healthy donors. After 2-3 passages in DMEM + 10% FBS, the cells were activated on plastic and placed in medium with 0.5% serum overnight. The cells were then treated for 6 or 24 hours with rimonabant (a CBl antagonist), PlC4-h2-IgG4 and non- functional control antibodies at various concentrations. Inhibition of pro-fibrotic gene signatures, including a-SMA, Pro-collagen A1(I), TIMP1, and TGFp, were measured by RT-PCR, and the data were plotted.

[0364] The results showed that there was a significant decrease in Pro-collagen A1(I) expression when the HSCs were treated with PlC4-h2 antibodies, but not with non- functional control and PBS (Figure 28). There was also a significant decrease in TGFP (Figure 29) and TIMP1 (Figure 30) expression compared to PBS and non-functional antibody controls. In addition, the decrease in a-SMA expression was also significant in cells treated with PlC4-IgGl or IgG4 compared to that in PBS or non- functional binder treated cells (Figure 31).

Example 30: Quantification of anti-CBl antibody in cynomolgus monkey cerebral spinal fluid (CSF) [0365] Cynomolgus monkeys (2 male and 2 female/group) were treated with PlC4-h2-IgG4 according to the treatment scheme in Table 33, and CSF was collected at the indicated time points. PlC4-h2-IgG4 was quantified by ELISA coating 96 well plates with an anti-ID antibody at 1 ug/mL followed by the addition of CSF and detection with an HRP-conjugated anti-IgG antibody (Abeam) and color reagent.

[0366] As shown in Table 33, the antibody was only detected at very low levels if at all. In the highest dose group, less than 0.1% of the injected dose is detectable in the CSF indicating that antibody exposure of CNS is very low.

Table 33. Quantification of anti-CBl antibody in cynomolgus monkey CSF

BLQ=below limit of quantification

Example 31. Measuring effects of anti-CBl antibody on metabolic and cardiovascular factors in cynomolgus monkeys

[0367] The RIO program demonstrated the cardiometabolic effects of rimonabant treatment using several factors. See, e.g., Pi-Sunyeret al, 2006, J Am Coll Cardio, 147:362A. As such, the effect of the anti-CBl antibodies disclosed herein is also evaluated for effects on similar cardiometabolic factors. [0368] Obese cynomolgus and rhesus monkeys are treated with the anti-CBl antibody P1C4- h2-IgG4 at 3 mg/kg or 0.3 mg/kg with weekly dosing s.c. For negative controls, a set of primates are injected with either (1) pharmaceutical carrier only; or (2) a control antibody known not to bind CB 1. Effects on primate food intake, body weights, insulin sensitivity, triglyceride levels, and other cardiovascular risk factors are observed.

Primates treated with the anti-CBl antibody PlC4-h2-IgG4 at 3 mg/kg or 0.3 mg/kg are shown to exhibit reduced triglyceride levels and other cardiovascular risk factors. Primates treated with the anti-CBl antibody PlC4-h2-IgG4 at 3 mg/kg or 0.3 mg/kg are also shown to exhibit improved insulin sensitivity. Primates injected with either control antibody or carrier-only are observed without any improvement in these factors.

[0369] Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the invention. All references cited herein are incorporated herein by reference in their entireties for all purposes.

Table 34. Sequences of allowable mutations within PA13R3-P1C4 CDRs

Example 32. Affinity Maturation of anti-CBl antibody PlC4-h2-IgG4

[0370] In order to obtain more potent variants of PlC4-h2-IgG4 an affinity maturation campaign was carried out by constructing three large yeast display libraries containing randomized CDR residues and using magnetic-activated cell sorting (MACS) and FACS to select improved binders. For additional details see, for example, Wang et al, 2011, J Biol Chem. 286(51): 44218-44233. The design of the libraries was based on an analysis of the variability of CDR residues in a large database of natural human antibodies of the same germline families as PlC4-h2-IgG4 (VH1 family member 1-69 and Vk3 family member 3-20). The CDR residues that were selected for randomization are underlined below in Table 35.

Table 35. Residues randomized in P1C4 affinity maturation libraries

[0371] The three yeast display libraries were designed to carry mutations in (1) the light chain only, (2) the heavy chain only, or (3) in both the light and heavy chains. The theoretical diversity of each library was 6.5 x 10 ³ , 1.4 x 10 ⁵ and 9.0 x 10 ⁸ and the actual size of each library was 1.5 x 10 ⁵ , 5.0 x 10 ⁷ and 2.3 x 10 ⁹ respectively.

[0372] After several rounds of MACS and FACS selection, with progressively lower concentrations of CBl iCAPS, four clones were identified that showed potentially higher affinity for CBl . All four clones had changes only in the light chain CDRs, and carried unchanged parental heavy chains. The sequences of the novel light chains are shown below in Table 36 with the altered residues underlined. Table 36. Sequences of Affinity Matured P1C4 Clones

[0373] The individual residues found to be altered in the light chains of the affinity matured P1C4 variants are summarized below in Table 37.

Table 37. Summary of Amino Acid Changes in Affinity Matured Clones

[0374] The four affinity matured P1C4 variants that were identified via the yeast display selections were then reformatted from the yeast display vector to full length IgG4 (bearing a hinge mutation at S228P) and tested for improved binding affinity and antagonist function. Each antibody was expressed in 293 cells and purified by Protein A affinity chromatography. Parental PlC4-h2-IgG4 was also expressed and purified in an identical manner to ensure a comparable control was available.

[0375] Improvement in binding affinity of the four affinity matured P1C4 variants was assessed by flow cytometry on TRex-CHO cells expressing full length native human CB1, with TRex-CHO parental cells serving as the negative control. Briefly, ΙΟΟμΙ of lxl0 ⁶ cells/ml of cells were incubated with P1C4 affinity matured variants and parental P1C4 IgGs in 3 -fold serial dilutions starting from ΙμΜ to 1.3nM for 30 minutes on ice. After being washed with 200 of FACS buffer twice, cells were incubated with secondary antibody for 30 minutes on ice. Cells were washed with 200 μΐ _^ of FACS buffer twice and transferred to BD Falcon 5 mL tube and analyzed by FACS. Data analysis and measurement of binding affinity (K _D ) was performed using GraphPad Prism software. The results are summarized in Tables 38. Binding affinity was enhanced approximately 2-3 fold for all four clones in comparison to parental PlC4-h2-IgG4.

Table 38. Summary of dissociation constants of P1C4 affinity matured clones

[0376] Functional activity of the affinity matured P1C4 variants was quantified by cAMP antagonist assay using a commercially available cAMP kit based on a competitive immunoassay using cryptate-labeled anti-cAMP antibody and d2-labeled cAMP (Cisbio). Eight thousand cells/well of human CB1 expressing TRex-CHO cells were seeded to a white 384-well plate followed by incubating mAb or control compound at various concentrations at 22°C for 10 minutes. Five μΜ forskolin (Sigma Aldrich) and 9 nM CP55,940 (Sigma Aldrich) were added to the cells and incubated for 30 minutes at 22°C to activate CB1 signaling. After 30 minutes incubation at 22°C, 5 cAMP-d2 and 5 anti-cAMP cryptate were added to the cells and incubated for 1 hour. FRET signal was detected with Envision multilabel plate reader (Perkin Elmer) at anti-cAMP cryptate excitation at 620 nm and emission at 665 nm. Data analysis was performed using GraphPad Prism. The results are summarized in Table 39. Similar to the enhancement observed for binding affinity, functional potency was enhanced up to 2-3 fold in some affinity matured clones (RY-HL-LC - A 12_F AC S4 and RY-LC -B 12_F AC S3) in comparison to parental PlC4-h2-IgG4.

Table 39. Summary of IC50 of P1C4 affinity matured clones