Cannabinoid receptor type 2 antibodies and uses thereof

Abstract

The present application provides CB2 antibodies and constructs comprising an immunoglobulin single variable domain (ISVD) that specifically binds a CB2. Also provided are methods of making and using these CB2 antibodies.

Description

CANNABINOID RECEPTOR TYPE 2 ANTIBODIES AND USES THEREOFCROSS-REFERENCE TO REAL TED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 618,357 filed on January 7, 2024, U.S. Provisional Application No. 63 / 645,441 filed on May 10, 2024, and U.S. Provisional Application No. 63 / 735,223 filed on December 17, 2024, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (185982000440SEQLIST.xml; Size: 533,491 bytes; and Date of Creation: January 3, 2025) is herein incorporated by reference in its entirety.FIELD

[0003] The present invention relates to antibodies comprising an immunoglobulin single variable domain (ISVD) that specifically bind a cannabinoid receptor type 2 (CB2), and methods of making and using thereof.BACKGROUND

[0004] In mammals, there are two G-protein coupled receptors that mediate the signaling of cannabinoids, which are found in both plants and animals. Cannabinoid Receptor 1 (CB1) is expressed most strongly in the neurons of the CNS and is primarily responsible for the psychotropic effects of cannabinoids on the body (Nature, 346: 561-564, 1990). CB2 is expressed predominantly in the spleen and hematopoietic cells, demonstrating a predominantly peripheral localization for this receptor (Nature, 365: 61-65, 1993; and reviewed in Pharmacol. Rev, 58(3): 389-462, 2006).

[0005] CB2 agonists are effective in multiple models of acute and chronic pain resulting from chemical, mechanical and thermal pain stimuli (reviewed in Pharmacol. Ther. 95: 127-135 2002; Pharmacol. Rev. 58(3): 389-462, 2006). In chemotherapy-induced neuropathic pain (CIPN), it has been shown that CB2 activation suppresses paclitaxel-induced CIPN in rats (J. Pharmacol. Exp. Ther. 327: 584-591, 2008). Moreover, CB2 agonists suppressed CIPN due to all majorclasses of chemotherapeutic agents, including taxane (i.e., paclitaxel-cited above), vinca alkaloid (i.e., vincristine, Br. J. Pharmacol. 152:765-777, 2007) and platinum-derived (i.e., cisplatin, Mol Pain, 8:71) anti-cancer agents. CB2 agonists have also shown to be an effective treatment of diabetic neuropathy in both rat (Pharmacology. 82(3): 193-200, 2008) and mouse models (Pain. 2013 Jun; 154(6): 864-73; Psychopharmacology (Berl). 233(11):2209-2219, 2016), further supporting the use of CB2 agonists in the treatment of multiple pain indications.

[0006] CB2 activation has strong anti-inflammatory activity that is part of a natural negative feedback mechanism, where CB2 receptor is induced in injured or virally-infected cells in inflammatory settings (Gastroenterology. 129:437-453, 2005; Am. J. Pathol. 177: 187-196, 2010; Br. J. Pharmacol. 151 : 1041-1048, 2007). CB2 activation subsequently represses secretion of multiple inflammatory factors and signals, including TNFa, IL-6, IL-8, CCL2 (reviewed in Cell. Mol. Life Sci. 73:4449-4470, 2016). Accordingly, CB2 agonists would be useful for the treatment of inflammatory conditions of the lung (Biomed Res. Int. 2014: 971750, 2014) atherosclerosis (Eur. J. Pharmacol. 649:285-292,2010), inflammatory bowel disease (World J. Gastroenterol. 22:9515-9524, 2016), and viral infection (Virulence 9:217-230, 2018).

[0007] CB2 receptor agonism also plays a role in mitigating fibrotic processes in multiple organs. For example, receptor agonist JWH-133 has been shown to reduce leukocyte infiltration and dermal thickening in bleomycin-induced experimental fibrosis (Arthritis Rheum 60: 1129- 1136, 2009) and alleviates bleomycin-induced pulmonary fibrosis in mice (Oncotarget.8: 103486-103498, 2017). The CB2 receptor has been identified as a potential target for the treatment of systemic sclerosis because it controls both skin fibroblast proliferation and the autoimmune reaction (Am. J. Pathol. 177: 187-196, 2010). In the liver, CB2 agonism in mouse models protect against liver fibrosis caused by carbon tetrachloride (CC14) (Gastroenterology. 128:742-755, 2005) and alcohol (Hepatology. 54: 1217-1226, 2011), and enhance liver regeneration after injury (Hepatology. 52: 1046-1059, 2010). In addition to reducing inflammation, CB2 agonism reduces the proliferation and induces apoptosis of myofibroblasts, which secrete collagen, which forms the fibrotic lesions (Gastroenterology. 128:742-755, 2005). Therefore, CB2 may beneficially impact liver disease through multiple mechanisms, i.e., inflammation and fibrosis.

[0008] There is need in the art for developing methods of using CB2 receptor agonists in safe and effective therapies and to reduce the risk of adverse events. CB2 small molecule agonists are rapidly cleared, can have significant CNS permeability, and are a high risk for drug metabolism effects. Even the most peripherally-restricted CB2 small molecules have 3-5% CNS penetrance (Pain. Apr 13, 2021). Most CB2 agonists exhibit some activity at CB1, and thus CNS-mediated side effects are a concern. For example, in a Phase II study of Olorinab - a CB2 small molecule agonist developed for Irritable Bowel Syndrome — 20% of treated patients reported some degree of dizziness, somnolence, or other central signs of CB1 activation (Higgins, et al 14th Congress of ECCO - Inflammatory Bowel Diseases; March 6-9, 2019; Copenhagen, Denmark). Additionally, a compliance handicap is added by the short half-lives of small molecules, which imposes a need for multiple daily doses (J. Behav. Med. 31 : 213-224, 2008). The antibodies described herein satisfy these needs in the art and provide related advantages as well.

[0009] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.BRIEF SUMMARY

[0010] The present application relates to anti-CB2 antibodies (hereinafter referred to as “CB2 antibodies”) comprising an immunoglobulin single variable domain (ISVD) that specifically binds a CB2, and methods of making and using thereof.

[0011] In one aspect, provided herein is a cannabinoid receptor type 2 (CB2) antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds a CB2, wherein the ISVD comprises a complementarity determining region (CDR) 1, a CDR2, and a CDR3 of the ISVD comprising the sequence of any one of SEQ ID NOs:500-899.

[0012] In some embodiments, the ISVD is a variable domain of the heavy chain of a heavy chain antibody (VHH). In some embodiments, the ISVD is camelid. In some embodiments, the ISVD is chimeric. In some embodiments, the ISVD is humanized.

[0013] In some embodiments, the ISVD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:500-899, or a variant thereof having at least about 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 500-899.

[0014] In some embodiments, the ISVD comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, 4, or 7; a CDR2 comprising an amino acid sequence of SEQ ID NO: 10, 13, or16; and a CDR3 comprising an amino acid sequence of SEQ ID NO: 19, 22, or 25; wherein the CDR1, CDR2 and CDR3 are defined according to IMGT. In some embodiments, the ISVD comprises SEQ ID NO:505, SEQ ID NO:715, or SEQ ID NO:716. In some embodiments, the ISVD comprises SEQ ID NO:716.

[0015] In some embodiments, the CB2 antibody comprises an Fc region. In some embodiments, the antibody comprises an Fc region of an IgGl or IgG4. In some embodiments, the antibody comprises a variant IgG4 Fc region exhibiting reduced effector function. In some embodiments, the Fc region comprises amino acid substitutions F234A and L235A, with numbering according to the EU index of Kabat. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 130.

[0016] In some embodiments, the Fc region is an IgGl Fc region comprising the amino acid sequence of SEQ ID NO: 126.

[0017] In some embodiments, the Fc region is a variant IgGl Fc region exhibiting reduced effector function. In some embodiments, the variant IgGl Fc region comprises amino acid substitutions L234A, L235A, M252Y, S254T, and T256E, wherein the residues are numbered according to the EU index. In some embodiments, the variant IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the variant IgGl Fc region comprises amino acid substitutions L234A, L235A, and P329G, wherein the residues are numbered according to the EU index. In some embodiments, the variant IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the variant IgGl Fc region comprises amino acid substitutions L234A, L235A, M252Y, S254T, T256E, and P329G, wherein the residues are numbered according to the EU index. In some embodiments, the variant IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 129.

[0018] In some embodiments, the antibody comprises a hinge region.

[0019] In some embodiments, the ISVD is fused to the Fc region via a peptide linker.

[0020] In some embodiments, the CB2 is human, mouse, rat or cynomolgus monkey CB2. In some embodiments, the CB2 is a human CB2.

[0021] In some embodiments, the CB2 antibody is a CB2 agonist. In some embodiments, the CB2 antibody does not agonize CB1. In some embodiments, the ISVD does not specifically bind a cannabinoid receptor type 1 (CB1).

[0022] Further provided is an isolated nucleic acid encoding any one of the CB2 antibodies described above. Further provided is an expression vector comprising said nucleic acid. Further provided is a host cell comprising said nucleic acid or said expression vector.

[0023] Further provided is a method of producing any one of the CB2 antibodies described above, comprising culturing the host cell described above under conditions where the CB2 antibody is produced. In some embodiments, the method further comprises recovering the CB2 antibody produced by the host cell.

[0024] Further provided is a pharmaceutical composition comprising any one of the CB2 antibodies described above and a pharmaceutically acceptable carrier.

[0025] Further provided is a method of agonizing CB2 on a cell, comprising contacting the cell with an amount of any one of the CB2 antibodies described above that is sufficient for activating CB2 on the cell. In some embodiments, the method is in vitro. In some embodiments, the method is in vivo.

[0026] Further provided is a method of treating an individual in need thereof having a disease or condition, wherein agonizing CB2 ameliorates the disease or condition, comprising administering to the individual an effective amount of any one of the CB2 antibodies described above or the pharmaceutical composition described above. In some embodiments, the disease or condition is selected from the group consisting of chemotherapy induced peripheral neuropathy (CIPN), diabetic peripheral neuropathy, respiratory infections, liver fibrosis, cold sensitivity, inflammatory bowel disease, and endometriosis. In some embodiments, the disease or condition is chemotherapy induced peripheral neuropathy (CIPN). In some embodiments, the CIPN is caused by a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the disease or condition is a respiratory infection. In some embodiments, the respiratory infection is caused by a virus selected from the group consisting of influenza viruses and coronaviruses. In some embodiments, the respiratory infection is caused by SARS-CoV-2. In some embodiments, the respiratory infection is caused by H1N1 influenza. In some embodiments, the disease or condition is liver fibrosis. In some embodiments, the disease or condition is diabetic peripheral neuropathy.

[0027] Further provided is a method of treating a cancer in an individual in need thereof, comprising administering to the individual an effective amount of a chemotherapeutic agent, and an effective amount of any one of the CB2 antibodies described above.

[0028] Further provided is a method of reducing a cytokine release syndrome in an individual in need thereof, comprising administering to the individual an effective amount of any one of the CB2 antibodies described above. In some embodiments, the method decreases IL- 16 and / or IL-8 level secreted by airway cells in the individual.

[0029] In some embodiments of any of the methods described above, the individual is a human.

[0030] Further provided is a kit comprising the CB2 antibody of any one of the CB2 antibodies described above or the pharmaceutical composition described above, and instructions for use.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows CB2 mediated GI-cAMP signaling efficacy measured using TR-FRET cAMP kit after application of indicated antibodies in RAW264.7 cells (at two different concentrations for each indicated antibody treatment).

[0032] FIGs. 2A-2F show RAW264.7 cells cultured and treated with HU308, CB2 agonist antibodies or isotype control (ABt269).

[0033] FIG. 3 shows indicated antibodies added to RAW264.7 cells at EC80 concentrations and then titrated with CB2 antagonist SRI 44528.

[0034] FIGs. 4A and 4B show ERK phosphorylation induced by indicated agents in RAW264.7 cells as assessed by a TR-FRET pERKl / 2 kit.

[0035] FIG. 5 shows recruitment beta-arrestin2 induced by indicated agents in RAW264.7 cells as assessed by a HTRF beta-arrestin2 recruitment detection kit.

[0036] FIGs. 6A-6F show the effect of the indicated treatments in human liver slice model (hPCLS) cells as assessed by secretion of collagenlal (Collal) into media as assessed by ELISA (FIG. 6A), and by mRNA expression levels for COL1 Al (FIG. 6B), ACTA2 (FIG. 6C), IL6 (FIG. 6D), IL 10 (FIG. 6E), and TNFa (FIG. 6F). Two-way ANOVA; Dunnett’s multiple comparison test; mean + / - standard deviation; *p<0.05, **p<0.005, ***p<0.0005,****p<0.0001, ns=not significant.DETAILED DESCRIPTION

[0037] The present disclosure is based, at least in part, on the discovery of novel antibodies which specifically bind a cannabinoid receptor type 2 (CB2), which may be referred to synonymously as “anti-CB2 antibodies” or “CB2 antibodies,” and antigen-binding fragments thereof. The CB2 antibodies comprise at least an immunoglobulin single variable domain (ISVD) that specifically binds a CB2. The ISVD comprises a complementarity determining region 1 sequence (CDR1), a complementarity determining region 2 sequence (CDR2), and a complementarity determining region 3 sequence (CDR3). The ISVD may be a variable domain of the heavy chain of a heavy chain antibody (VHH). The ISVD may be, for example, a camelid, chimeric, or humanized ISVD. The ISVDs described herein may specifically bind CB2 (such as a human, cynomolgus, murine, and / or rat CB2), but have low or undetectable affinity for CB1 (such as a human, cynomolgus, murine, and / or rat CB1). The ISVDs described herein are selective agonists of CB2, which have no or undetectable agonist activity against CB1. In some embodiments, the anti-CB2 antibody comprises an Fc fragment (e.g., a human IgGl Fc, effectorless IgGl Fc, IgG2 Fc, or IgG4 Fc). Also provided are pharmaceutical compositions comprising these antibodies, kits, and methods of treatment using the antibodies.I. Definitions

[0038] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, 4-chain antibodies (such as IgG antibodies), heavy chain antibodies, and antibody fragments thereof so long as they exhibit the desired antigen-binding activity, z.e., binding to CB2 (such as a human CB2, cynomolgus CB2, rat CB2, and / or a mouse CB2). The term “4-chain antibody” is used herein to refer to an antibody or antigen-binding fragment having two heavy chains and two light chains. The term “heavy chain antibody,” also known as “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises two heavy chains, but lacks two light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0039] As used herein, an “agonist” means an antibody stimulates a biological function and / or signaling of a target receptor. In the context of a CB2 agonist, the CB2 agonist is capable of activating the endocannabinoid signaling pathway through activation of CB2. CB2 agonismincludes, but is not limited to, (a) stimulating or activating CB2 and (b) enhancing, increasing, promoting, inducing, or prolonging the activity, function, or presence of CB2 signaling or downstream signaling products.

[0040] The term “immunoglobulin single variable domain” (also referred to as “ISV” or “ISVD”) is generally used to refer to immunoglobulin variable domains (which may be heavy chain or light chain domains, including VH, VHH or VL domains) that can form a functional antigen-binding site without interaction with another variable domain (e.g., without a VH / VL interaction as is required between the VH and VL domains of a 4-chain monoclonal antibody). Examples of ISVDs include Nanobodies (including a VHH, a humanized VHH and / or a camelized VHs such as camelized human VHs), shark IgNAR domains, single-domain antibodies (or dAbs) that are VH domains or that are derived from a VH domain, single-domain antibodies (or dAbs) that are VL domains or that are derived from a VL domain. ISVDs that are based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. In some embodiments, an ISVD is a Nanobody. The term “Nanobody” is generally as defined in WO 2008 / 020079 or WO 2009 / 138519, and thus in a specific aspect generally denotes a VHH, a humanized VHH or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as, e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximum expression).

[0041] “Antibody fragments” comprise a portion of an antibody, preferably the antigen binding or variable region of the antibody. Examples of antibody fragments include VHHs, singledomain antibodies, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); singlechain antibody molecules; and multispecific antibodies formed from antibody fragments. The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CHI, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0042] The term “Fc region” or “fragment crystallizable region” herein is used to define a C- terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C- terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0043] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, z.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or posttranslation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinantDNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or humanlike antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0044] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0045] The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and / or form structurally defined loops (“hypervariable loops”) and / or contain the antigen-contacting residues (“antigen contacts”). Generally, 4-chain antibodies and antigen-binding antibody fragments thereof comprise six HVRs: three in the VH(Hl, H2, H3), and three in the VL (LI, L2, L3). Generally, heavy-chain antibodies comprise three HVRs (HVR1, HVR2, HVR3).

[0046] A number of HVR delineations are in use and are encompassed herein. Exemplary HVRs for 4-chain antibodies and antigen-binding antibody fragments thereof herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49- 56 (L2), 26-35 (Hl), 26-35b (Hl), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0047] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., framework, “FR,” residues) are numbered herein according to Kabat et al., supra.

[0048] The amino acid residues of a single-domain antibody (such as VHH) can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Cam elids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that — as is well known in the art for VH domains and for VHH domains — the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actualsequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).

[0049] “Framework” or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.

[0050] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is identical to or derived from a particular source or species, while the remainder of the heavy and / or light chain is identical to or derived from a different source or species.

[0051] “Humanized” antibodies are antibodies that contain minimal sequence derived from the non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as camelid, mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In certain aspects, a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human (e.g., camelid) CDRs and amino acid residues from human FRs. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0052] An “affinity-matured” antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In some embodiments, an affinity- matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity- matured antibodies are produced by procedures known in the art. For example, random mutagenesis of CDR and / or framework residues is described by, for example: Barbas et al. ProcNat. Acad. Sci. USA 91 :3809-3813 (1994); Schier c / a / . Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0053] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No.TXU5 10087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0054] The term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for -4 example, by a molecule having a KD for the target of at least about 10 M, alternatively at least about 105M, alternatively at least about 106M, alternatively at least about 107M, alternatively at least about 108M, alternatively at least about 109M, alternatively at least about 1010M, -11 -12 alternatively at least about 10 M, alternatively at least about 10 M, or greater. In some embodiments, the term “specific binding” refers to binding where a molecule binds a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. KD can be determined by methods known in the art, such asELISA, surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.

[0055] As used herein, “treatment,” treating,” or “treat” are defined as an approach for obtaining beneficial or desired results including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, stabilizing a disease (e.g., preventing or delaying the worsening of a disease), preventing or delaying the spread (e.g., metastasis) of a disease, preventing or delaying the recurrence of a disease, delay or slowing the progression of a disease, ameliorating a disease state, providing a remission (partial or total) of a disease, decreasing the dose of one or more other medications required to treat a disease, delaying the progression of a disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of a disease. The methods herein contemplate any one or more of these aspects of treatment. With respect to treatment of pain, such methods refer to reduction of pain. Reduction of pain includes, but is not limited to, reducing or ameliorating the generation of, sensation of, perception of, and / or effects of pain and / or its other symptoms; reducing the risk of occurrence of pain in a subject; impeding the initiation of pain; relieving pain, i.e., causing regression of the pain and / or relieving one or more symptoms of pain; alleviating pain; lessening of pain; limiting pain; reducing pain including duration and / or intensity of pain; mitigating pain; blocking pain; and inhibiting the propagation of pain.

[0056] As used herein, the term “effective amount” or “therapeutically effective amount” of a substance (such as a CB2 antibody, construct, or composition) is at least the minimum concentration required to bring about a measurable improvement or prevention of a particular disorder or to achieve particular desired result or results. An effective amount herein may vary according to factors such as the disease state, the age, sex, and weight of the patient, and theability of the substance to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects (i.e., a therapeutic index). An effective amount can be administered in one or more administrations. As is understood in the clinical context, an effective amount may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable results may be or is achieved.

[0057] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and nonhuman primates such as rhesus and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0058] “Antibody effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation. “Reduced or minimized” antibody effector function means that which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodified antibody. The determination of antibody effector function is readily determinable and measurable by one of ordinary skill in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody dependent cytotoxicity are affected. In some embodiments, effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., “effectorless mutation.” In one aspect, the effectorless mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001). Alternatively, additional mutations resulting in reduced or eliminated effector function include: K322A and L234A / L235A (LALA). Alternatively, mutations resulting in reduced or eliminated effector function include: F234A and L235A (FALA) according to EUindex. Alternatively, mutations resulting in reduced or eliminated effector function include: L234A, L235A, and P329G (LALAPG) according to EU index. Alternatively, mutations resulting in reduced or eliminated effector function include: M252Y, S254T, and T256E, (YTE) according to EU index. Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli.) or in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003).

[0059] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

[0060] Half maximal effective concentration (ECso) is a measure of the effectiveness of a substance (such as an antibody) which induces a specific biological or biochemical function. It indicates how much of a particular drug or other substance (such as an antibody) is needed to achieve a response halfway between the baseline and maximum after a specified exposure time in a given biological process. The values are typically expressed as molar concentration. ECso is comparable to an “ICso” for an antagonist drug or other substance. ECso or ICso can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay), cell based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0061] An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than inthe form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0062] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0063] Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0064] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0065] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected,transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0066] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0067] The term “pharmaceutical formulation” of “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. A “sterile” formulation is aseptic or free from all living microorganisms and their spores.

[0068] Reference to “about” a value or parameter herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0069] It is understood that aspects and embodiments of the present application include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0070] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0071] The term “and / or” as used herein a phrase such as “A and / or B” is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used herein a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).II. Anti-CB2 antibodies

[0072] Described herein, in various embodiments, are antibodies which specifically bind CB2 (i.e., “anti-CB2 antibodies,” “CB2 antibodies,” “antibodies targeting CB2”). The CB2 antibodies described herein may comprise three CDRs from an immunoglobulin single variable domain (ISVD), or comprise an ISVD, or may be ISVDs. For instance, in some embodiments, the CB2 antibody described herein comprises a binding domain comprising three CDRs from an ISVD taught herein, e.g., such as listed in Table 3. In the embodiments below, the structure and functionality of CB2 antibodies are presented in a modular fashion as anti-CB2 ISVDs. The recitation of an anti-CB2 ISVD alone is not intended to be limiting in scope, and encompasses, in a non-limiting fashion, CB2 antibodies which include, in whole or in part, three CDRs from an anti-CB2 ISVD or an anti-CB2 ISVD e.g., CDRs as shown in Table 3).

[0073] The CB2 antibodies described herein comprise an anti-CB2 ISVD. In some embodiments, the anti-CB2 ISVD specifically recognizes human CB2. In some embodiments, the anti-CB2 ISVD does not specifically recognize a CB1, such as a human CB1. In some embodiments, the anti-CB2 ISVD does not agonize a CB1, such as a human CB1. In some embodiments, the anti-CB2 ISVD specifically recognizes an epitope within human CB2. In some embodiments, the CB2 antibody comprises an ISVD that is a variable domain of the heavy chain of a heavy chain antibody (VHH). In some embodiments, the CB2 antibody comprises an ISVD and is a camelid. In some embodiments, the CB2 antibody comprises an ISVD that is chimeric. In some embodiments, the CB2 antibody comprises an ISVD that is humanized. In some embodiments, the CB2 antibody comprises an ISVD that is partially humanized. In some aspects of the teachings provided herein, the antibodies may be referenced with a leading zero (e.g. ABt285=ABt0285).

[0074] In some embodiments, the ISVD comprises a CDR1, CDR2, and / or CDR3 of an ISVD comprising the amino acid sequence of any one of SEQ ID NOs:500-899. In some embodiments, the ISVD comprises a CDR1, CDR2, and / or CDR3 of an ISVD comprising the amino acid sequence of any one of SEQ ID NOs:500-899, wherein the CDR1, CDR2, and CDR3 are defined according to IMGT (for explanation of IMGT, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003 and Ruiz et al., Nucleic Acids Res. 29(l):207-9, 2001). In some embodiments, the ISVD comprises a CDR1, CDR2, and / or CDR3 of an ISVD comprising the amino acidsequence of any one of SEQ ID NOs:500-899, wherein the CDR1, CDR2, and CDR3 are defined according to Kabat. In some embodiments, the ISVD comprises a CDR1, CDR2, and / or CDR3 of an ISVD comprising the amino acid sequence of any one of SEQ ID NOs: 500-899, wherein the CDR1, CDR2, and CDR3 are defined according to Chothia.

[0075] In some embodiments, the CB2 antibody comprises an ISVD comprising a CDR1, a CDR2, and a CDR3 of an antibody designated ML T 001, ML T 002, ML T 003, ML T 004,ML_T_005, ML_T_006, ML_T_007, ML_T_008, ML_T_009, ML_T_010, ML T 011, ML_T_012, ML_T_013, ML_T_014, ML_T_015, ML_T_016, ML_T_017, ML_T_018, ML T 019, ML_T_020, ML_T_021, ML_T_022, ML_T_023, ML_T_024, ML_T_025, ML_T_026, ML_T_027, ML_T_028, ML_T_029, ML_T_030, ML_T_031, ML_T_032, ML T 033, ML T 034, ML T 035, ML T 036, ML T 037, ML T 038, ML T 039, ML_T_040, ML_T_041, ML_T_042, ML_T_043, ML_T_044, ML_T_045, ML_T_046, ML_T_047, ML_T_048, ML_T_049, ML_T_050, ML T 051, ML_T_052, ML_T_053, ML_T_054, ML T 055, ML T 056, ML T 057, ML T 058, ML T 059, ML T 060, ML T 061, ML T 062, ML T 063, ML T 064, ML T 065, ML T 066, ML T 067, ML T 068, ML T 069, ML_T_070, ML_T_071, ML_T_072, ML_T_073, ML_T_074, ML T 075, ML_T_076, ML_T_077, ML_T_078, ML_T_079, ML_T_080, ML T 081, ML T 082, ML T 083, ML_T_084, ML_T_085, ML_T_086, ML_T_087, ML_T_088, ML T 089, ML_T_090, ML T 091, ML_T_092, ML_T_093, ML_T_094, ML_T_095, ML T 096, ML T 097, ML T 098, ML T 099, ML T 100, ML T 101, ML T 102,ML_T_103, ML T 104, ML T 105, ML T 106, ML T 107, ML T 108, ML T 109,ML_T_110, ML_T_111, ML_T_112, ML_T_113, ML_T_114, ML_T_115, ML_T_116, ML_T_117, ML_T_118, ML_T_119, ML T 120, ML T 121, ML T 122, ML T 123,ML T 124, ML T 125, ML T 126, ML T 127, ML T 128, ML T 129, ML T 130,ML T 131, ML T 132, ML T 133, ML T 134, ML T 135, ML T 136, ML T 137,ML T 138, ML T 139, ML_T_140, ML_T_141, ML_T_142, ML_T_143, ML_T_144, ML T 145, ML T 146, ML T 147, ML T 148, ML T 149, ML T 150, ML T 151,ML T 152, ML T 153, ML T 154, ML T 155, ML T 156, ML T 157, ML T 158,ML T 159, ML T 160, ML T 161, ML T 162, ML T 163, ML T 164, ML T 165,ML T 166, ML T 167, ML T 168, ML T 169, ML T 170, ML T 171, ML T 172,ML T 173, ML_T_174, ML T 175, ML T 176, ML T 177, ML T 178, ML T 179, ML T 180, ML T 181, ML T 182, ML T 183, ML T 184, ML T 185, ML T 186, ML T 187, ML T 188, ML T 189, ML T 190, ML T 191, ML T 192, ML T 193, ML T 194, ML T 195, ML T 196, ML T 197, ML T 198, ML T 199, ML T 200, ML_V_001, ML_V_002, ML_V_003, ML_V_004, ML_V_005, ML_V_006, ML_V_007, ML_V_008, ML_V_009, ML_V_010, ML_V_011, ML_V_012, ML_V_013, ML_V_014, ML_V_015, ML_V_016, ML_V_017, ML_V_018, ML_V_019, ML_V_020, ML_V_021, ML_V_022, ML_V_023, ML_V_024, ML_V_025, ML_V_026, ML_V_027, ML_V_028, ML_V_029, ML_V_030, ML_V_031, ML_V_032, ML_V_033, ML_V_034, ML_V_035, ML_V_036, ML_V_037, ML_V_038, ML_V_039, ML_V_040, ML_V_041, ML_V_042, ML_V_043, ML_V_044, ML_V_045, ML_V_046, ML_V_047, ML_V_048, ML_V_049, ML_V_050, ML_V_051, ML_V_052, ML_V_053, ML_V_054, ML_V_055, ML_V_056, ML_V_057, ML_V_058, ML_V_059, ML_V_060, ML_V_061, ML_V_062, ML_V_063, ML_V_064, ML_V_065, ML_V_066, ML_V_067, ML_V_068, ML_V_069, ML_V_070, ML_V_071, ML_V_072, ML_V_073, ML_V_074, ML_V_075, ML_V_076, ML_V_077, ML_V_078, ML_V_079, ML_V_080, ML_V_081, ML_V_082, ML_V_083, ML_V_084, ML_V_085, ML_V_086, ML_V_087, ML_V_088, ML_V_089, ML_V_090, ML_V_091, ML_V_092, ML_V_093, ML_V_094, ML_V_095, ML_V_096, ML_V_097, ML_V_098, ML_V_099, ML_V_100, ML_V_101, ML_V_102, ML_V_103, ML_V_104, ML_V_105, ML_V_106, ML_V_107, ML_V_108, ML_V_109, ML_V_110, ML_V_111, ML_V_112, ML_V_113, ML_V_114, ML_V_115, ML_V_116, ML_V_117, ML_V_118, ML_V_119, ML_V_120, ML_V_121, ML_V_122, ML_V_123, ML_V_124, ML_V_125, ML_V_126, ML_V_127, ML_V_128, ML_V_129, ML_V_130, ML_V_131, ML_V_132, ML V 133, ML_V_134, ML V 135, ML_V_136, ML_V_137, ML_V_138, ML_V_139, ML_V_140, ML_V_141, ML_V_142, ML_V_143, ML_V_144, ML_V_145, ML_V_146, ML_V_147, ML_V_148, ML_V_149, ML_V_150, ML_V_151, ML_V_152, ML_V_153, ML_V_154, ML V 155, ML V 156, ML_V_157, ML_V_158, ML_V_159, ML_V_160, ML_V_161, ML_V_162, ML V 163, ML_V_164, ML_V_165, ML_V_166, ML_V_167, ML_V_168, ML_V_169, ML_V_170, ML_V_171, ML_V_172, ML_V_173, ML_V_174, ML V 175, ML_V_176, ML_V_177, ML_V_178, ML_V_179, ML_V_180, ML_V_181, ML_V_182,ML V 183, ML_V_184, ML_V_185, ML_V_186, ML_V_187, ML_V_188, ML_V_189, ML_V_190, ML_V_191, ML_V_192, ML_V_193, ML_V_194, ML_V_195, ML_V_196, ML_V_197, ML_V_198, ML_V_199, or ML_V_200 as defined in Table 1, below. In some embodiments, the CDRs are defined according to Kabat. In some embodiments, the CDRs are defined according to Chothia. In some embodiments, the CDRs are defined according to IMGT.

[0076] The sequences of the ISVDs of the exemplary CBS antibodies noted herein are provided in Table 1, below. A skilled person in the art would readily identify the CDRs or variable domain sequences of these antibodies using known algorithms.

[0077] The CB2 antibodies described herein comprise an ISVD comprising four framework regions (FRs). The CB2 antibodies described herein may comprise any suitable sequences for the framework regions. In some embodiments, the FR1, FR2, FR3 and FR4 are defined according to IMGT.

[0078] In some embodiments, the CB2 antibody comprises an ISVD which specifically binds CB2, wherein the ISVD comprises an amino acid sequence of any one of SEQ ID NOs:500-899, or a variant thereof having at least 85%, such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 500-899.

[0079] In some embodiments, the CB2 antibody comprises the sequence of amino acids of the CB2 antibody designated ML_T_001, ML_T_002, ML_T_003, ML_T_004, ML_T_005, ML_T_006, ML_T_007, ML_T_008, ML_T_009, ML_T_010, ML T 011, ML_T_012,ML_T_013, ML T 014, ML T 015, ML T 016, ML T 017, ML T 018, ML T 019,ML_T_020, ML_T_021, ML_T_022, ML_T_023, ML_T_024, ML_T_025, ML_T_026, ML T 027, ML T 028, ML T 029, ML T 030, ML T 031, ML T 032, ML T 033,ML_T_034, ML T 035, ML_T_036, ML_T_037, ML_T_038, ML_T_039, ML_T_040,ML_T_041, ML_T_042, ML_T_043, ML_T_044, ML_T_045, ML_T_046, ML_T_047, ML_T_048, ML_T_049, ML_T_050, ML T 051, ML_T_052, ML_T_053, ML_T_054, ML T 055, ML T 056, ML_T_057, ML_T_058, ML_T_059, ML_T_060, ML T 061, ML_T_062, ML T 063, ML T 064, ML T 065, ML T 066, ML T 067, ML T 068, ML T 069, ML_T_070, ML_T_071, ML_T_072, ML_T_073, ML_T_074, ML_T_075,ML_T_076, ML_T_077, ML_T_078, ML_T_079, ML_T_080, ML T 081, ML_T_082, ML T 083, ML_T_084, ML T 085, ML T 086, ML T 087, ML T 088, ML T 089, ML_T_090, ML T 091, ML_T_092, ML_T_093, ML_T_094, ML_T_095, ML_T_096, ML_T_097, ML T 098, ML_T_099, ML_T_100, ML T 101, ML_T_102, ML_T_103, ML_T_104, ML_T_1O5, ML T 106, ML T 107, ML T 108, ML T 109, ML T 110, ML_T_111, ML_T_112, ML_T_113, ML_T_114, ML_T_115, ML_T_116, ML_T_117, ML T 118, ML T 119, ML T 120, ML T 121, ML T 122, ML T 123, ML T 124,ML T 125, ML T 126, ML T 127, ML T 128, ML T 129, ML T 13O, ML T 131,ML T 132, ML T 133, ML T 134, ML T 135, ML T 136, ML T 137, ML T 138,ML T 139, ML_T_140, ML T 141, ML_T_142, ML_T_143, ML_T_144, ML T 145, ML T 146, ML T 147, ML T 148, ML T 149, ML T 15O, ML T 151, ML T 152,ML T 153, ML T 154, ML T 155, ML T 156, ML T 157, ML T 158, ML T 159,ML T 160, ML T 161, ML T 162, ML T 163, ML T 164, ML T 165, ML T 166,ML T 167, ML T 168, ML T 169, ML T 170, ML T 171, ML T 172, ML T 173,ML_T_174, ML T 175, ML T 176, ML T 177, ML T 178, ML T 179, ML T 18O,ML T 181, ML T 182, ML T 183, ML T 184, ML T 185, ML T 186, ML T 187,ML T 188, ML T 189, ML T 190, ML T 191, ML T 192, ML T 193, ML T 194,ML T 195, ML T 196, ML T 197, ML T 198, ML T 199, ML T 200, ML V 001,ML_V_002, ML_V_003, ML_V_004, ML_V_005, ML_V_006, ML_V_007, ML_V_008, ML_V_009, ML_V_010, ML_V_011, ML_V_012, ML_V_013, ML_V_014, ML_V_015, ML_V_016, ML_V_017, ML_V_018, ML_V_019, ML_V_020, ML_V_021, ML_V_022, ML_V_023, ML_V_024, ML_V_025, ML_V_026, ML_V_027, ML_V_028, ML_V_029, ML_V_030, ML_V_031, ML_V_032, ML_V_033, ML_V_034, ML_V_035, ML_V_036, ML_V_037, ML_V_038, ML_V_039, ML_V_040, ML_V_041, ML_V_042, ML_V_043, ML_V_044, ML_V_045, ML_V_046, ML_V_047, ML_V_048, ML_V_049, ML_V_050, ML_V_051, ML_V_052, ML_V_053, ML_V_054, ML_V_055, ML_V_056, ML_V_057, ML_V_058, ML_V_059, ML_V_060, ML_V_061, ML_V_062, ML_V_063, ML_V_064, ML_V_065, ML_V_066, ML_V_067, ML_V_068, ML_V_069, ML_V_070, ML_V_071, ML_V_072, ML_V_073, ML_V_074, ML_V_075, ML_V_076, ML_V_077, ML_V_078, ML_V_079, ML_V_080, ML_V_081, ML_V_082, ML_V_083, ML_V_084, ML_V_085,ML_V_086, ML_V_087, ML_V_088, ML_V_089, ML_V_090, ML_V_091, ML_V_092, ML_V_093, ML_V_094, ML_V_095, ML_V_096, ML_V_097, ML_V_098, ML_V_099, ML_V_100, ML_V_101, ML_V_102, ML_V_103, ML_V_104, ML_V_105, ML_V_106, ML_V_107, ML_V_108, ML_V_109, ML_V_110, ML_V_111, ML_V_112, ML_V_113, ML_V_114, ML_V_115, ML_V_116, ML_V_117, ML_V_118, ML_V_119, ML_V_120, ML_V_121, ML V 122, ML_V_123, ML_V_124, ML_V_125, ML_V_126, ML_V_127, ML_V_128, ML_V_129, ML_V_130, ML_V_131, ML_V_132, ML_V_133, ML_V_134, ML V 135, ML V 136, ML_V_137, ML_V_138, ML_V_139, ML_V_140, ML_V_141, ML_V_142, ML_V_143, ML_V_144, ML_V_145, ML_V_146, ML_V_147, ML_V_148, ML_V_149, ML_V_150, ML_V_151, ML_V_152, ML_V_153, ML_V_154, ML_V_155, ML V 156, ML V 157, ML_V_158, ML_V_159, ML_V_160, ML_V_161, ML_V_162, ML V 163, ML_V_164, ML_V_165, ML_V_166, ML_V_167, ML_V_168, ML_V_169, ML_V_170, ML_V_171, ML_V_172, ML_V_173, ML_V_174, ML_V_175, ML_V_176, ML_V_177, ML_V_178, ML_V_179, ML_V_180, ML_V_181, ML_V_182, ML_V_183, ML_V_184, ML V 185, ML_V_186, ML_V_187, ML_V_188, ML_V_189, ML_V_190, ML_V_191, ML_V_192, ML_V_193, ML_V_194, ML_V_195, ML_V_196, ML_V_197,ML_V_198, ML_V_199, or ML_V_200 as defined in Table 1, below.

[0080] The CB2 antibodies described herein, in some embodiments, comprise an Fc region. In some embodiments, the CB2 antibody comprises an Fc region of an IgGl, IgG2, IgG3, or IgG4, or a variant thereof. In some embodiments, the CB2 antibody comprises an Fc region of an IgGl. In some embodiments, the CB2 antibody comprises an Fc region of an IgG4. In some embodiments, the CB2 antibody comprises an Fc variant that exhibits reduced effector function. In some embodiments, the reduced effector function reduces ADCC activity. In some embodiments, the antibody comprises an IgGl Fc region exhibiting reduced effector function. In some embodiments, the antibody comprises a variant IgGl Fc region comprising one or more variant amino acids. In some embodiments, the one or more variant amino acids of the variant IgG Fc regions described herein may be an addition, a substitution, or a deletion. Such variant IgGl Fc regions are relative to a conserved Fc sequence, e.g., a conserved human Fc sequence.

[0081] In some embodiments, the antibody comprises an IgGl Fc region exhibiting reduced effector function, wherein the Fc region comprises the amino acid substitutions L234A and L235A, with numbering according to the EU index of Kabat. In some embodiments, the antibody comprises an IgG4 Fc region exhibiting reduced effector function. In some embodiments, the antibody comprises an IgG4 Fc region exhibiting reduced effector function, wherein the Fc region comprises the amino acid substitutions F234A / L235A (FALA), with numbering according to the EU index of Kabat. In some embodiments, the variant IgGl Fc region comprises the amino acid substitutions selected from the group consisting of (1) LALA, (2) YTE, and (3) LALAPG. In some embodiments, the variant IgGl Fc region comprises the LALAPG variants. In some embodiments, the variant IgGl Fc region comprises the LALAPG variants and the YTE variants. In some embodiments, the variant IgGl Fc region comprises the LALA variants and the YTE variants. The LALAPG variants are known as L234A, L235A, P329G, wherein the residues are numbered according to the EU index. The LALA variants are known as L234A and L235A, wherein the residues are numbered according to the EU index. The YTE variants are known as M252Y, S254T, and T256E, wherein the residues are numbered according to the EU index.

[0082] In some embodiments, the CB2 antibody comprises an Fc region, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the CB2 antibody comprises an Fc region, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the CB2 antibody comprises an Fc region, wherein the Fc regioncomprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the CB2 antibody comprises an Fc region, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 129. The CB2 antibodies described herein may further comprise a hinge region and / or a peptide linker. In some embodiments, the CB2 antibody further comprises a hinge region. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the ISVD is fused to the Fc region via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 132). In some embodiments, the CB2 antibody comprises a polypeptide chain comprising from the N-terminus to the C-terminus: the ISVD, a peptide linker, a hinge region, and an Fc region. In some embodiments, the CB2 antibody does not comprise an Fc region. An exemplary hinge is provided:AESKYGPPCPPCP (SEQ ID NO: 131)

[0083] In some embodiments, the CB2 antibody comprises a polypeptide chain comprising from the N-terminus to the C-terminus: an ISVD, a peptide linker and / or hinge region, and an Fc region.

[0084] In some embodiments, the CB2 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:28-39. Exemplary polypeptide chains are shown inTable 2

[0085] In some embodiments, the CB2 antibody describes herein comprises a CB2 antibody that specifically binds to CB2 competitively with any one of the other CB2 antibodies described herein. In some embodiments, competitive binding may be determined using an ELISA assay. Insome embodiments, the CB2 antibody specifically binds to a CB2 competitively with a CB2 antibody comprising a CDR1, a CDR2, and a CDR3 from the ISVD sequence of any one of SEQ ID NOs: 500-899, wherein the CDRs are defined according to any one of Kabat, IMGT, or Chothia (for explanation of IMGT, see Lefranc el al.. Dev. Comparat. Immunol. 27:55-77, 2003 and Ruiz et al., Nucleic Acids Res. 29(l):207-9, 2001). In some embodiments, the CB2 antibody specifically binds to a CB2 competitively with a CB2 antibody according to the CB2 antibodies of Table 1. In some embodiments, the KD of the binding between the competing CB2 antibodies and CB2 is about 10'7M to about 10'12M (such as about 10'8M to about 10'12M, or about 10'9M to about 10'11M). In some embodiments, the CB2 antibody cross-reacts with a CB2 from a non-human mammal. In some embodiments, the competing CB2 antibody is a camelid, chimeric, human, partially humanized, or fully humanized.

[0086] In some aspects, provided is a cannabinoid receptor type 2 (CB2) antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds a CB2, wherein the ISVD comprises a complementarity determining region (CDR) 1, a CDR2, and a CDR3 of the ISVD comprising the sequence of SEQ ID NO:505. In some aspects, provided is a cannabinoid receptor type 2 (CB2) antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds a CB2, wherein the ISVD comprises a complementarity determining region (CDR) 1, a CDR2, and a CDR3 of the ISVD comprising the sequence of SEQ ID NO:715. In some aspects, provided is a cannabinoid receptor type 2 (CB2) antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds a CB2, wherein the ISVD comprises a complementarity determining region (CDR) 1, a CDR2, and a CDR3 of the ISVD comprising the sequence of SEQ ID NO:716. In some embodiments, the CB2 antibody comprises an ISVD comprising the sequence of SEQ ID NO:505, SEQ ID NO:715, or SEQ ID NO:716, or a variant thereof having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO:505, SEQ ID NO:715, or SEQ ID NO:716. In some embodiments, the CB2 antibody comprises an ISVD comprising the sequence of SEQ ID NO:716, or a variant thereof having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO:716, wherein the CB2 antibody comprises a CDR1, a CDR2, and a CDR3 of the ISVD comprising the sequence of SEQ ID NO:716. In some embodiments, the ISVD is a variable domain of the heavy chain of a heavy chain antibody (VHH). In some embodiments, the ISVD iscamelid. In some embodiments, the ISVD is chimeric. In some embodiments, the ISVD is humanized.

[0087] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 defined according to IMGT, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10 defined according to IMGT, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19 defined according to IMGT.

[0088] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NON defined according to IMGT, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 defined according to IMGT, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22 defined according to IMGT.

[0089] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:7 defined according to IMGT, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 defined according to IMGT, and a CDR3 comprising the amino acid sequence of SEQ ID NO:25 defined according to IMGT.

[0090] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:2 defined according to Kabat, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11 defined according to Kabat, and a CDR3 comprising the amino acid sequence of SEQ ID NO:20 defined according to Kabat.

[0091] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:5 defined according to Kabat, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14 defined according to Kabat, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23 defined according to Kabat.

[0092] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:8 defined according to Kabat, a CDR2 comprising the amino acid sequence of SEQ ID NO: 17 defined according to Kabat, and a CDR3 comprising the amino acid sequence of SEQ ID NO:26 defined according to Kabat.

[0093] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NON defined according to Chothia, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 defined according to Chothia, and a CDR3 comprising the amino acid sequence of SEQ ID NO:21 defined according to Chothia.

[0094] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:6 defined according to Chothia, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15 defined according to Chothia, and a CDR3 comprising the amino acid sequence of SEQ ID NO:24 defined according to Chothia.

[0095] In some embodiments, the CB2 antibody comprises a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:9 defined according to Chothia, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 defined according to Chothia, and a CDR3 comprising the amino acid sequence of SEQ ID NO:27 defined according to Chothia.

[0096] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:1 defined according to IMGT, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10 defined according to IMGT, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19 defined according to IMGT.

[0097] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NON defined according to IMGT, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 defined according to IMGT, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22 defined according to IMGT.

[0098] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:7 defined according to IMGT, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 defined according to IMGT, and a CDR3 comprising the amino acid sequence of SEQ ID NO:25 defined according to IMGT.

[0099] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:2 defined according to Kabat, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11 defined according to Kabat, and a CDR3 comprising the amino acid sequence of SEQ ID NO:20 defined according to Kabat.

[0100] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:5 definedaccording to Kabat, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14 defined according to Kabat, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23 defined according to Kabat.

[0101] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:8 defined according to Kabat, a CDR2 comprising the amino acid sequence of SEQ ID NO: 17 defined according to Kabat, and a CDR3 comprising the amino acid sequence of SEQ ID NO:26 defined according to Kabat.

[0102] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:3 defined according to Chothia, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 defined according to Chothia, and a CDR3 comprising the amino acid sequence of SEQ ID NO:21 defined according to Chothia.

[0103] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:6 defined according to Chothia, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15 defined according to Chothia, and a CDR3 comprising the amino acid sequence of SEQ ID NO:24 defined according to Chothia.

[0104] In some embodiments, the CB2 antibody comprises an ISVD comprising a binding domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV defined according to Chothia, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18 defined according to Chothia, and a CDR3 comprising the amino acid sequence of SEQ ID NO:27 defined according to Chothia.CB2

[0105] The cannabinoid receptor type 2 (CB2) is a G protein-coupled receptor from the cannabinoid receptor family. In humans, it is encoded by the CNR2 gene. It is closely related to the cannabinoid receptor type 1 (CB1). CB1 agonism is primarily responsible for the psychotropic effects of cannabis, particularly tetrahydrocannabinol (THC). Due to the psychoactive effect of CB 1 agonism, antibodies which specifically bind CB2 but which do not exhibit specific binding to CB1 are desirable. Thus, in one aspect, the CB2 antibodies provided herein are specific for CB2 and do not exhibit significant affinity and do not specifically bind CB1. The CB2 receptor comprises seven transmembrane-spanning domains, contains a glycosylated N-terminus, and an intracellular C-terminus.

[0106] The terms “cannabinoid receptor type 2”, “CB2”, “CB2 antigen”, and “CB2 epitope” are used interchangeably, and include variants, isoforms, species homologs of human CB2, and analogs having at least one common epitope with CB2. In some embodiments, the CB2 is human CB2, and the CB2 antibody specifically binds human CB2. CB2 gene and polypeptide sequences (e.g., human CNR2 gene and polypeptide sequences) are known in the art. An exemplary human CB2 sequence is provided at GenBank Accession Number P34972. In some embodiments, a human CB2 may be at least about 90%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the human CB2 of Genbank Accession Number P34972. In some embodiments, a human CB2 sequence has no more than about 10 amino acid differences from the human CB2 of Genbank Accession Number P34972. In some embodiments, the human CB2 may display no more than 5, 4, 3, 2, or 1 amino acid difference from the human CB2 of Genbank Accession Number P34972. In some embodiments, a human CB2 sequence may differ from the human CB2 of Genbank Accession Number P34972 by having, for example, conserved mutations or mutations in non-conserved regions and the CB2 has substantially the same biological function as the human CB2 of Genbank Accession Number P34972.

[0107] In some embodiments, the CB2 antibodies described herein specifically recognize a CB2 polypeptide having at least about 90% amino acid sequence identity to the human CB2 of Genbank Accession Number P34972. In some embodiments, the CB2 antibody may crossreact with CB2 from species other than human (such as rat, murine, and / or cynomolgus CB2), orother proteins which are structurally related to human CB2 (e.g., human CB2 homologs). In some embodiments, the CB2 antibody does not specifically bind CB 1.Biological activities

[0108] The CB2 antibodies described herein comprise an ISVD (e.g., a VHH or a camelid), and has one or more of the following characteristics: (a) the ISVD is an agonist of a CB2; (b) the ISVD does not agonize a CB1; (c) the ISVD reduces forskolin-induced cyclic adenosine monophosphate (cAMP) level in HEK293 cells overexpressing human CB2 with a half maximal effective concentration (ECso) that is less than 55 nM; (d) the ISVD reduces forskolin-induced cyclic adenosine monophosphate (cAMP) level in HEK293 cells overexpressing mouse CB2 with an ECso that is less than 30 nM; (e) the ISVD does not specifically bind a CB1; and (f) the CB2 antibody binds HEK293 cells overexpressing human CB2 with an ECso that is less than 850 nM.

[0109] The CB2 antibody described herein binds CB2 with high affinity and specificity. In some embodiments, the CB2 antibody binds human CB2 with a KD of about 0.001 nM to about 10 nM (e.g., lOnM or less, InM or less, O.lnM or less, 0.01 nM or less, or 0.001 nM or less), including any value or range in between these values. In some embodiments, the CB2 antibody binds cynomolgus CB2 with a KD of about 0.001 nM to about 10 nM (e.g., lOnM or less, InM or less, 0. InM or less, 0.01 nM or less), or 0.001 nM or less, including any value or range in between these values. In some embodiments, the CB2 antibody binds rat CB2 with a KD of about 0.001 nM to about 10 nM (e.g., lOnM or less, InM or less, O. lnM or less, 0.01 nM or less, or 0.001 nM or less), including any value or range in between these values. In some embodiments, the CB2 antibody binds murine CB2 with a KD of about 0.001 nM to about 10 nM (e.g., lOnM or less, InM or less, O. lnM or less, 0.01 nM or less, or 0.001 nM or less), including any value or range in between these values.

[0110] In some embodiments, the CB2 antibody is a selective CB2 agonist. In some embodiments, a CB2 antibody is a selective CB2 agonist if it exhibits detectable CB2 agonist activity and does not exhibit detectable CB1 agonist activity. In some embodiments, a CB2 antibody is a selective CB2 agonist if it exhibits detectable CB2 agonist activity and does not exhibit detectable CB1 agonist activity as assessed by reduction of forskolin-induced cyclicadenosine monophosphate (cAMP) production in HEK293 cells overexpressing CB2 (for CB2 agonism assessment) or overexpressing CB1 (for CB1 agonism assessment). In some embodiments, agonist activity is not detectable if the reduction in cAMP as compared to a control is not statistically significant (such as when p is not < 0.05). In some embodiments, CB1 agonist activity is not detectable if the degree of measured activity is an order of magnitude different between CB2 agonism and CB1 agonism. In some embodiments, the CB2 antibody agonism activity (such as for CB1 or CB2) is assessed at a concentration of about 1 pM.[OHl] In some embodiments, the CB2 antibody selectively binds CB2 and does not selectively bind CB1. In some embodiments, the CB2 antibody does not selectively bind CB1 if the CB2 antibody binds a CB1 with a KD of greater than about 1,000 nM, such as greater than about 1,000 nM, 2,000 nM, 3,000 nM, 4,000 nM, 5,000 nM, or more, including values and ranges therebetween. In some embodiments, the CB1 is a human, rat, murine, or cynomolgus CB1.

[0112] The KD and koff of the CB2 antibodies provided herein for human CB2, rat CB2, murine CB2, and / or cynomolgus CB2 can be determined by any method known in the art, including, but not limited to, e.g., ELISA, fluorescence activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), and surface plasmon resonance (SPR). In some embodiments, the KD and / or koff of a CB2 antibody provided herein for human CB2, murine CB2, rat CB2, and / or cynomolgus CB2 is determined via SPR. In some embodiments, the KD and / or koff of a CB2 antibody provided herein is determined by surface plasmon resonance (SPR).

[0113] The characteristics of the CB2 antibodies described herein can be assessed using well known methods, e.g., methods used in the Examples below. In some embodiments, the CB2 antibody does not bind (e.g., specifically bind) to a CB1, such as a human CB1.

[0114] The biological activity of the CB2 antibodies described herein can be determined by measuring its half maximal effective concentration (ECso), which is a measure of the effectiveness of an antibody in binding to its target. For example, the ECso can be used to indicate the effective concentration of a CB2 antibody needed to bind 50% CB2 on a cell surface. ECso also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. ECso can be measured by assays known in the art, for example, bioassays such as FACS binding analysis, inhibition of ligand binding by FACS analysis (competition bindingassay), cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0115] For example, agonist activity of CB2 antibodies can be studied by measuring reduction in forskolin-induced cAMP levels in HEK293 cells over-expressing human CB2 (but not CB1, if assessing CB2 agonism) or human CB1 (but not CB2, if assessing CB1 agonism), as described in Example 2, below. The results of the study can demonstrate the ability of the CB2 antibody to agonize CB2, to not agonize CB1, and to compare the CB2 antibody to other CB1 or CB2 agonists.

[0116] The agonist ECso of binding to CB2 by the CB2 antibodies can be studied by evaluating binding of the CB2 antibody to CB2-expressing HEK293 stable cells. Similarly, agonist ECso of binding to CB1 by the CB2 antibodies can be studied by evaluating binding of the CB2 antibody to CB1 -expressing HEK293 stable cells. As discussed in Example 2, below, after incubating the CB2 antibodies with the CB1 -expressing or CB2-expressing HEK293 stable cells in the presence of forskolin, cells may be lysed by addition of tracer and detection reagents. After incubating, FRET allows quantification of cAMP levels and calculation of ECso. In some embodiments, there is not detectable agonist activity of CB1 (such as a human, mouse, cynomolgus, and / or rat CB1) by the CB2 antibodies described herein.(II) Fusion constructs

[0117] The present application further provides anti-CB2 fusion constructs comprising any one of the CB2 antibodies or antigen-binding fragments (e.g., ISVD or VHH) described herein and a second polypeptide, such as a second antibody or antigen binding fragment thereof, or an Fc fragment of an immunoglobulin. In some embodiments, the anti-CB2 construct comprises two or more polypeptides other than the CB2 antibody. The additional polypeptide(s) may or may not change or otherwise influence the biological properties of the CB2 antibody, and may or may not add further functionality to the CB2 antibody. In some embodiments, the second polypeptide confers one or more desired properties or functionalities to the CB2 antibody.

[0118] In some embodiments, the anti-CB2 construct comprises a second antibody or antigen binding fragment thereof (such as ISVD, scFv, Fab, full-length antibody, etc.) that specifically recognizes a second epitope. In some embodiments, the second epitope is from a CB2. In some embodiments, the second epitope is not from a CB2. In some embodiments, the second antibodyspecifically recognizes the same epitope on a CB2 as the CB2 antibody described herein. In some embodiments, the second antibody specifically recognizes a different epitope on CB2 as the CB2 antibody described herein.

[0119] In some embodiments, there is provided an anti-CB2 construct comprising a plurality (such as 2, 3, 4, or more) of CB2 antibodies described herein. In some embodiments, the plurality of the CB2 antibodies are fused to each other via a linker (such as a peptide linker). The plurality of the CB2 antibodies can be the same or different.

[0120] In some embodiments, the CB2 antibody comprises a second polypeptide that enhances the half-life, solubility, and / or absorption, reduces immunogenicity or toxicity, eliminates or attenuates undesirable side effects, confers other advantageous properties to and / or reduces other undesired properties of the CB2 antibody, compared to the CB2 antibody alone. Some nonlimiting examples of such polypeptides include serum proteins, such as human serum albumin (HSA; see e.g. WO 2000 / 027435) or haptenic molecules (e.g. haptens that are recognized by circulating antibodies, see e.g. WO 98 / 22141). It was shown that linking fragments of immunoglobulins (such as VH domains) to serum albumin or fragments thereof may increase antibody half-life (see e.g. WO 00 / 27435 and WO 01 / 077137). Thus, in some embodiments, the CB2 antibody comprises a CB2 antibody fused to serum albumin or a fragment thereof, optionally via a suitable linker (such as peptide linker). In some embodiments, the serum albumin comprises at least domain III (see PCT / EP2007 / 002817).CB2 heavy chain-only antibody (HCAb)

[0121] In some embodiments, the CB2 antibody is a heavy chain-only antibody (HCAb) comprising a CB2 ISVD described herein. In some embodiments, the CB2 ISVD is fused to one or more CH2 and / or CH3 domains, e.g., an Fc fragment. In some embodiments, CH2 and / or CH3 domains are derived from human immunoglobulins. In some embodiments, the CB2 antibody is fused to the CH2 and / or CH3 domains via a peptide linker. The CH2 and / or CH3 domains may increase the half-life of the CB2 antibody in vivo.

[0122] Thus, in some embodiments, there is provided an isolated CB2 HCAb comprising a CB2 ISVD (meaning an ISVD which specifically binds a CB2) described herein fused to an Fc fragment of an immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the CB2 HCAb comprises an Fc fragment of IgG, such as IgGl, IgG2, IgG3, or IgG4. In someembodiments, the Fc fragment is a human Fc, such as human IgGl (hlgGl) Fc, h!gG2 Fc, or hIgG4 Fc. In some embodiments, the Fc fragment is effectorless, with reduced, minimized, or eliminated antibody effector functions such as ADCC, CDC, and / or ADCP (antibody-dependent cellular phagocytosis). In some embodiments, the effectorless Fc comprises an N297A or DANA mutation (D265A+N297A) in the CH2 region. In some embodiments, the effectorless Fc comprises K322A and L234A / L235A (LALA) mutations. In some embodiments, the effectorless Fc comprises F234A and L235A (FALA) mutations. In some embodiments, the Fc fragment is an effectorless IgGl Fc, such as effectorless hlgGl Fc. In some embodiments, the Fc fragment is a human IgG4 Fc (S228P). In some embodiments, the Fc fragment is an IgG4 Fc (FALA). In some embodiments, the CB2 HCAb is monomeric. In some embodiments, the CB2 HCAb is dimeric. In some embodiments, the CB2 HCAb is multispecific and multivalent (such as bispecific and bivalent), e.g., comprising two or more different CB2 antibodies described herein. In some embodiments, the CB2 HCAb is monospecific and multivalent (e.g., bivalent), e.g., comprising two or more copies of the same CB2 ISVD. An exemplary IgG4 Fc is provided:APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 130)

[0123] Exemplary IgGl Fc sequences are also provided: EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<V SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 126)EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 127)EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<V SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 128)EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<V SNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 129)

[0124] In some embodiments, the CB2 antibody and the Fc fragment are fused to each other via a peptide linker. In some embodiments, the peptide linker is a (G4S)3 (SEQ ID NO: 132).

[0125] Thus, in some embodiments, there is provided an isolated CB2 HCAb comprising an ISVD specifically recognizing CB2, wherein the CB2 antibody comprises a CDR1, a CDR2, and a CDR3 from the ISVD sequence of any one of SEQ ID NOs: 500-899, and wherein the CB2 ISVD is fused to an Fc fragment of an immunoglobulin.

[0126] In some embodiments, there is provided an isolated CB2 HCAb comprising an ISVD specifically recognizing CB2, wherein the ISVD comprises the amino acid sequence of any one of SEQ ID NOs: 500-899 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to any one of SEQ ID NOs:500-899, and wherein the CB2 antibody is fused to an Fc fragment of an immunoglobulin. In some embodiments, there is provided an isolated CB2 HCAb comprising an ISVD specifically recognizing CB2, wherein the ISVD comprises the amino acid sequence of any one of SEQ ID NOs:500-899, and wherein the CB2 antibody is fused to an Fc fragment of an immunoglobulin. In some embodiments, the CB2 antibody is fused to the Fc fragment via apeptide linker. In some embodiments, the CB2 HCAb is monomeric. In some embodiments, the Fc fragment is a human IgGl Fc, effectorless human IgGl Fc, hIgG2 Fc, human IgG4 Fc, IgG4 Fc (F234A / L235A), or IgGl (L234A / L235A, with numbering according to the EU index of Kabat).

[0127] Also provided are isolated CB2 HCAbs that specifically binds to CB2 competitively with any one of the isolated CB2 HCAbs or CB2 antibodies described herein.Peptide linkers

[0128] The various domains and components, such as CB2 ISVDs (meaning ISVDs which specifically bind a CB2), Fc fragment, the first antigen binding portion, and the second antigen binding portion in the CB2 antibody construct may be fused to each other via a suitable linker, such as a peptide linker. The length, the degree of flexibility and / or other properties of the peptide linker(s) used in the CB2 antibody may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0129] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0130] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodiesmay be used as the linker. See, for example, WO 1996 / 34103. In some embodiments, the linker is a (G4S)3 linker (SEQ ID NO: 132).(Ill) Antibody variants

[0131] In some embodiments, amino acid sequence variants of the CB2 antibody provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a) Substitution, insertion, deletion and variants

[0132] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs.Conservative substitutions are shown in Table 4 under the heading of “Preferred substitutions.” More substantial changes are provided in Table 4 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or modified ADCC or CDC.

[0133] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0134] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0135] In some embodiments, the CB2 antibody provided herein comprises a ISVD comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 500-899. In some embodiments, an ISVD sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the CB2 antibody comprising that sequence retains the ability to bind CB2 (e.g., a human CB2, a murine CB2, a rat CB2, and / or acynomolgus CB2). In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in any of SEQ ID NOs: 500-899. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, the CB2 antibody comprises the ISVD set for in any one of SEQ ID NOs: 500-899, including post-translational modifications of that sequence.

[0136] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated.

[0137] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0138] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.b) Glycosylation variants

[0139] In some embodiments, an isolated CB2 antibody provided herein is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0140] Where the CB2 antibody comprises an Fc region (e.g., CB2 antibody -Fc fusion protein (e.g., HCAb), the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in a CB2 antibody of the present application may be made in order to create antibody variants with certain improved properties.

[0141] In some embodiments, CB2 antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g, complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L ); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865;WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; W02005 / 053742;W02002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch.Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 Al, Presta, L; and WO 2004 / 056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane- Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., BiotechnoL Bioeng., 94(4):680- 688 (2006); and W02003 / 085107).

[0142] CB2 antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function.Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). c) Fc region variants

[0143] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the CB2 antibodies provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

[0144] In some embodiments, the present application contemplates a CB2 antibody comprising an Fc region variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the CB2 antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retainsFcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’lAcad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 ( ee Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006)).

[0145] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581). Fc mutants are described in Schlothauer et al. Protein Eng Des Sei. 2016 Oct;29(10):457-466; Pejchal et al. Antibodies. 2023; 12(3):54;Dall' Acqua et al. J Biol Chem. 2006 Aug 18;281(33):23514-24. Ramdani et al. International Journal of Molecular Sciences. 2022; 23(17):9604, which are hereby incorporated by reference.

[0146] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0147] In some embodiments, a CB2 antibody variant comprises an Fc region with one or more amino acid substitutions which decreases ADCC activity, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0148] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol.164: 4178-4184 (2000).

[0149] In some embodiments, there is provided a CB2 antibody comprising a variant Fc region comprising one or more amino acid substitutions which increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).

[0150] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[0151] In certain aspects, the CB2 antibody encompassed herein may comprise one or more modifications that occur during production, e.g., a C-terminal lysine cleavage of an Fc region.

[0152] CB2 antibodies comprising any of the Fc variants described herein, or combinations thereof, are contemplated. d) Antibody derivatives

[0153] In some embodiments, a CB2 antibody provided herein may be further modified to comprise additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limitedto, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3 -di oxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0154] In some embodiments, conjugates of a CB2 antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

[0155] In some embodiments, a CB2 antibody provided herein may be further modified to comprise one or more biologically active protein, polypeptides or fragments thereof. “Bioactive” or “biologically active”, as used herein interchangeably, means showing biological activity in the body to carry out a specific function. For example, it may mean the combination with a particular biomolecule such as protein, DNA, etc., and then promotion or inhibition of the activity of such biomolecule. In some embodiments, the bioactive protein or fragments thereof include proteins and polypeptides that are administered to patients as the active drug substance for prevention of or treatment of a disease or condition, as well as proteins and polypeptides that are used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, as well as proteins and polypeptides that are administered to a patient to prevent a disease such as a vaccine. In some embodiments, the bioactive protein or fragments thereof have immune-stimulatory / immune-regulatory, membrane transport, or enzymatic activities. In some embodiments, the biologically active protein, polypeptides or fragments thereof is an enzyme, a hormone, a growth factor, a cytokine, or a mixture thereof. In some embodiments, the biologically active protein, polypeptides or fragments can specifically recognize a target peptide (such as antigen, or other proteins).

[0156] In some embodiments, the bioactive protein or fragments thereof that can be comprised within the CB2 antibody described herein is a protein-binding protein. In some embodiments, the bioactive protein or fragments thereof that can be comprised within the CB2 antibody described herein is an antibody mimetics, which are small engineered proteins comprising antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and comprise a single polypeptide. Exemplary antibody mimetics that can be comprised within the CB2 antibody described herein can be, but are not limited to, a Designed ankyrin repeat protein (DARPin; comprising 3-5 fully synthetic ankyrin repeats flanked by N- and C-terminal Cap domains), an avidity multimer (avimer; a high-affinity protein comprising multiple A domains, each domain with low affinity for a target), or an Anticalin (based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized). In some embodiments, the bioactive protein or fragments thereof that can be comprised within the CB2 antibody described herein is an Armadillo repeat protein (e.g., P-catenin, a-importin, plakoglobin, adenomatous polyposis coli (APC)), which comprises armadillo repeat units (characteristic, repetitive amino acid sequence of about 40 residues in length). Each Armadillo repeat is composed of a pair of alpha helices that form a hairpin structure. Multiple copies of the repeat form what is known as an alpha solenoid structure. Armadillo repeat proteins are able to bind different types of peptides, relying on a constant way of binding of the peptide backbone without requiring specific conserved side chains or interactions with free N- or C-termini of a peptide. The possibility of recognizing a peptide residue by residue, combined with the intrinsic modularity of a repeat protein, makes the armadillo repeat proteins promising candidates for the design of a generic scaffold for peptide binding.

[0157] In some embodiments, the biologically active protein or fragments thereof that can be comprised within the CB2 antibody described herein is a ligand, such as lymphokines andcellular factors which interact with specific cellular receptor. Lymphokines are low molecular weight proteins which are secreted by T cells when antigens or lectins stimulate T cell growth.III. Pharmaceutical compositions

[0158] Further provided by the present application are pharmaceutical compositions comprising any one of the CB2 antibodies described herein, and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing a CB2 antibody described herein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.

[0159] The pharmaceutical composition is preferably to be stable, in which the CB2 antibody described herein essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40°C for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8°C, generally the formulation should be stable at 30°C or 40°C for at least 1 month, and / or stable at 2-8°C for at least 2 years. Where the formulation is to be stored at 30°C, generally the formulation should be stable for at least 2 years at 30°C, and / or stable at 40°C for at least 6 months. For example, the extent of aggregation during storage can be used as an indicator of protein stability. In some embodiments, the stable formulation of CB2 antibody described herein may comprise less than about 10% (preferably less than about 5%) of the CB2 antibody present as an aggregate in the formulation.

[0160] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers (e.g. sodium chloride), stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and / or non-ionic surfactants.

[0161] Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ or polyethylene glycol (PEG).

[0162] Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use in the present application include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

[0163] Preservatives are added to retard microbial growth, and are typically present in a range from 0.2%-1.0% (w / v). The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Suitable preservatives for use in the present application include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0164] Tonicity agents, sometimes known as “stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight,preferably 1% to 5%, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0165] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); tri saccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0166] Non-ionic surfactants or detergents (also known as “wetting agents”) are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation- induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0167] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0168] In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0169] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.

[0170] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3 -hydroxybutyric acid.

[0171] The pharmaceutical compositions herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0172] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0173] In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.IV. Methods of treating

[0174] The CB2 antibodies and compositions (such as pharmaceutical compositions) thereof described herein are useful for a variety of applications, such as in diagnosis, molecular assays, and therapy.

[0175] In some embodiments, provided is a method of agonizing CB2 on a cell, comprising contacting the cell with an amount of any one of the CB2 antibodies described herein that is sufficient for activating CB2 on the cell. In some embodiments, the method is in vitro. In some embodiments, the method is in vivo. In some embodiments, agonizing CB2 on a cell treats or ameliorates a disease or condition. Agonism of a CB2 in a subject can trigger acute and prolong antinociceptive effects.

[0176] In some embodiments, there is provided a method of treating a disease or condition in a subject, wherein agonizing CB2 ameliorates the disease or condition, comprising administering to the subject an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. In some embodiments, the disease or condition is chemotherapy induced peripheral neuropathy (CIPN). In some embodiments, the disease or condition is a respiratory infection. In some embodiments, the disease or condition is liver fibrosis. In some embodiments, the disease or condition is cold sensitivity. In some embodiments, the disease or condition is inflammatory bowel disease. In some embodiments, the disease or condition is endometriosis.

[0177] Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of chemotherapy for the treatment of cancer. CIPN is usually associated with neurotoxic chemotherapeutic agents. CIPN is a painful, dose-limiting side effect that can limit treatment tolerance, reduce patient outcomes, reduce treatment compliance, and reduce patient quality oflife. Taxol drugs, such as paclitaxel, as commonly associated with CIPN. The CB2 antibodies described herein are useful for treating or ameliorating CIPN, including CIPN induced by taxol drugs.

[0178] In some embodiments, there is provided a method of treating a chemotherapy induced peripheral neuropathy (CIPN) in a subject receiving a chemotherapeutic agent, comprising administering to the subject an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. In some embodiments, the chemotherapeutic agent is a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the chemotherapeutic is vincristine. In some embodiments, the chemotherapeutic is cisplatin.

[0179] In some embodiments, provided is a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of a chemotherapeutic to the subject and an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. In some embodiments, the chemotherapeutic is a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the chemotherapeutic is vincristine. In some embodiments, the chemotherapeutic is cisplatin. In some embodiments, the chemotherapeutic causes chemotherapy induced peripheral neuropathy (CIPN).

[0180] A large plurality of subjects having cancer treated with neurotoxic chemotherapies experience chemotherapy-induced peripheral neuropathy. CIPN can impact subject health- related quality of life, treatment adherence, appetite, and can exacerbate preexisting pain, among other negative effects. Together, these effects can limit the dose of a neurotoxic chemotherapy which can be tolerated by the subject, where a higher dose may otherwise be more effect in treating the indicated cancer. CIPN may be measured by any suitable means known in the art, including by administration of a questionnaire or application of a diagnostic protocol. Exemplary questionnaires and protocols for CIPN include the Functional Assessment of Cancer Therapy / Gynecologic Oncology Group Neurotoxicity questionnaire (FACT / GOG-Ntx), Chemotherapy- induced Peripheral Neuropathy Assessment Tool (CIPNAT), European Organization for Research & Treatment in Cancer Quality of Life Quest-CIPN 20 (EORTC QLQ-CIPN 20), Modified Total Neuropathy Score (mTNS), Total Neuropathy Score, clinical version (TNSc), 5- item reduced Total Neuropathy Score (TNSr 5-item). Other measures may be used to assess the symptoms of CIPN, including the Fullerton Advanced Balance Scale (FABS), Timed Up and Go(TUG), Activities Specific Balance Confidence Scale (ABC), Balance Evaluation Systems Test (BESTest), Berg Balance Scale (BBS), Repeated Sit to Stand test, Functional Reach test, Short Physical Performance Battery (SPPB), and a grooved peg board test. Any suitable means of assessing CIPN are contemplated by the methods described herein.

[0181] In some embodiments, provided is a method of ameliorating chemotherapy induced peripheral neuropathy (CIPN), the method comprising administering an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. In some embodiments, the CIPN is caused by a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the chemotherapeutic is vincristine. In some embodiments, the chemotherapeutic is cisplatin.

[0182] In some embodiments, provided is a method of treating a side effect associated with a chemotherapy in a cancer patient, the method comprising administering to the patient an effective amount of any one of the CB2 antibodies described herein. In some embodiments, the side effect is CIPN or a side effect associated with CIPN. In some embodiments, the method further comprises administering the chemotherapeutic. In some embodiments, the chemotherapeutic is a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the chemotherapeutic is vincristine. In some embodiments, the chemotherapeutic is cisplatin.

[0183] In some embodiments, provided is a method of improving the quality of a life of a subject receiving a chemotherapy, the method comprising administering to the patient an effective amount of any one of the CB2 antibodies described herein. Quality of life may be assessed by any suitable means known in the art, including a quality of life questionnaire (QoLQ). The QoLQ may be self-reported and / or proxy reported. Any suitable QoLQ is contemplated by the methods described herein. In some embodiments, the method further comprises administering the chemotherapeutic. In some embodiments, the chemotherapeutic is a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the chemotherapeutic is vincristine. In some embodiments, the chemotherapeutic is cisplatin.

[0184] In some embodiments, provided is a method of improving the compliance of a subject receiving a chemotherapy, the method comprising administering to the patient an effective amount of any one of the CB2 antibodies described herein. In some embodiments, the methodfurther comprises administering the chemotherapeutic. In some embodiments, the chemotherapeutic is a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the chemotherapeutic is vincristine. In some embodiments, the chemotherapeutic is cisplatin.

[0185] In some embodiments, provided is a method of increasing the dose of a chemotherapy regimen being administered to a subject, the method comprising administering to the subject an effective amount of any one of the CB2 antibodies described herein, wherein the dose of the chemotherapy regimen is increased after administration of the CB2 antibody. In some embodiments, the method further comprises administering the chemotherapeutic. In some embodiments, the chemotherapeutic is a taxol drug. In some embodiments, the taxol drug is paclitaxel. In some embodiments, the chemotherapeutic is vincristine. In some embodiments, the chemotherapeutic is cisplatin.

[0186] In some embodiments, provided is a method of treating an individual having a respiratory infection, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. In some embodiments, the respiratory infection is an influenza infection, such as an influenza infection caused by an influenza A virus or an influenza B virus. In some embodiments, the influenza infection is caused by an influenza A virus subtype H1N1. In some embodiments, the respiratory infection is caused by a coronavirus, such as SARS-CoV-2 (wherein the respiratory infection is COVID-19).

[0187] In some embodiments, provided is a method of reducing a cytokine release syndrome in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. In some embodiments, the method decreases IL- 16 and / or IL-8 level secreted by airway cells in the individual. In some embodiments, the method decreases IL-16 secretion by airway cells by about 20% or more, such as about 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and 90% or more, and values and ranges therebetween. In some embodiments, the method decreases IL-8 secretion by airway cells by about 20% or more, such as about 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and 90% or more, and values and ranges therebetween. In someembodiments, the method decreases IL-8 and IL-16 secretion by airway cells by about 20% or more, such as about 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and 90% or more, and values and ranges therebetween.

[0188] In some embodiments, provided is a method of treating liver fibrosis, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. Liver fibrosis may be assessed by any suitable means known in the art, including by liver biopsy or noninvasive means such as elastography and / or serum biochemistry. In some embodiments, the method comprises administering to the individual an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein, thereby reducing the levels of one or more of COL1A1, ACTA2, IL6, IL10 or TNFa.

[0189] In some embodiments, provided is a method of treating cold sensitivity and / or allodynia, the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. Cold sensitivity and / or allodyniua may be assessed by any suitable means known in the art, including a questionnaire and / or patient-reported ranking of cold sensitivity or allodynia.

[0190] In some embodiments, provided is a method of treating inflammatory bowel disease, the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein.

[0191] In some embodiments, provided is a method of treating endometriosis, the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein.

[0192] In some embodiments, provided is a method of treating diabetic peripheral neuropathy (DPN), the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein.

[0193] Dosages and desired drug concentrations of pharmaceutical compositions of the present application may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for humantherapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi el al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0194] When in vivo administration of the CB2 antibody described herein (including pharmaceutical compositions comprising a CB2 antibody described herein) are used, normal dosage amounts may vary from about 10 ng / kg up to about 100 mg / kg of mammal body weight depending upon the route of administration. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0195] In some embodiments, the pharmaceutical composition is administered for a single time (e.g. bolus injection). In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times). If multiple administrations, they may be performed by the same or different routes and may take place at the same site or at alternative sites. The pharmaceutical composition may be administered daily to once per year. The interval between administrations can be about any one of 24 h to a year. Intervals can also be irregular (e.g. following tumor progression). In some embodiments, there is no break in the dosing schedule. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0196] The pharmaceutical compositions of the present application, including but not limited to reconstituted and liquid formulations, are administered to an individual in need of treatment, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intravenous (i.v.), intra-articular, intrasynovial, intrathecal,oral, topical, or inhalation routes. A reconstituted formulation can be prepared by dissolving a lyophilized CB2 antibody described herein in a diluent such that the protein is dispersed throughout. Exemplary pharmaceutically acceptable (safe and non-toxic for administration to a human) diluents suitable for use in the present application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution, or aqueous solutions of salts and / or buffers.

[0197] In some embodiments, the pharmaceutical compositions are administered to the individual by subcutaneous (i.e. beneath the skin) administration. For such purposes, the pharmaceutical compositions may be injected using a syringe. However, other devices for administration of the pharmaceutical compositions are available such as injection devices; injector pens; auto-injector devices, needleless devices; and subcutaneous patch delivery systems. In some embodiments, the pharmaceutical compositions are administered to the individual intravenously. In some embodiments, the pharmaceutical composition is administered to an individual by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)).V. Methods of preparation

[0198] The CB2 antibody described herein may be prepared using any methods known in the art or as described herein. In some embodiments, there is provided a method of producing a CB2 antibody, comprising: (a) culturing a host cell comprising an isolated nucleic acid or vector encoding the CB2 antibody described herein under conditions effective to express the encoded CB2 antibody; and (b) obtaining the expressed CB2 antibody from said host cell. In some embodiments, the method of step (a) further comprises producing a host cell comprising the isolated nucleic acid or vector encoding the CB2 antibody described herein.

[0199] Methods of preparing antibodies comprising ISVDs are known in the art. See, for example, Els Pardon et al., Nature Protocol, 2014; 9(3): 674. sdAbs (such as VHHS) may be obtained using methods known in the art such as by immunizing a Camelid species (such as camel or llama) and obtaining hybridomas therefrom, or by cloning a library of single-domainantibodies using molecular biology techniques known in the art and subsequent selection by ELISA with individual clones of unselected libraries or by using phage display.

[0200] For recombinant production of the ISVD, the nucleic acids encoding the antibodies are isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the single-domain antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

[0201] In some embodiments, a vector (e.g., expression vector) comprising a nucleic acid described herein are provided. In some embodiments, a host cell comprising such nucleic acid or vector is provided. In some embodiments, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell, Expi293 cell, or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, the host cell is prokaryotic, e.g. an E. coli cell. In some embodiments, a method of making a CB2 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0202] For recombinant production of a CB2 antibody, a nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0203] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression ofantibody fragments in E. cold). After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0204] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0205] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0206] Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0207] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR' CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255- 268 (2003).VI. Articles of manufacture and kits

[0208] Further provided are kits and articles of manufacture comprising any of the isolated CB2 antibodies described herein, pharmaceutical compositions comprising any of the CB2 antibodies describes herein, isolated nucleic acids or vectors encoding thereof, or isolated host cells comprising the isolated nucleic acids or vectors encoding the CB2 antibodies described herein. In some embodiments, a kit is provided which comprises any one of the pharmaceutical compositions described herein and preferably provides instructions for its use.

[0209] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0210] The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition which is effective for treating a disease or disorder (such as cancer) described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the particular condition in an individual. The label or package insert will further comprise instructions for administering the composition to the individual. The label may indicate directions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It mayfurther include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0211] The kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.EXAMPLES

[0212] The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.Example 1. Machine-learning-predicted agonist antibodies of CB2.

[0213] Historically, the discovery and design of antibodies relied on laborious biopanning techniques, serendipitous screening libraries, or complex rational engineering approaches. When effective, these methods often proved time-consuming, expensive, and limited in scope, and sometimes they fail to find any hits for difficult targets. The recent emergence of protein language models (PLMs)-models based on large language models (LLMs) originally developed for natural language-has ushered in a paradigm shift, revolutionizing the way we explore, manipulate, and generate antibody sequences. PLMs, trained on vast datasets of antibody sequences and associated properties such as binding, stability, and immunogenicity, now offer the tantalizing prospect of accelerating antibody discovery and design with unprecedented speed, precision, and creativity.

[0214] PLMs have not been previously applied to the problem of generating antibodies that go beyond specifically binding a target to also modulating the target’s signaling activity for a therapeutic effect, i.e., “functional antibodies”. PLM’s are primarily trained either to predict the next amino acids (AAs) given a sequence of AAs or to fill-in a set of AA’s masked in a larger sequence. By training on a large corpora of observed sequences, PLM’s can learn sequence to structure relationships. Given functional information about sequences, PLM’s pre-trained on the key roadblock for LPLMs in the discovery and ultimately design of functional antibodies is the lack of large-scale, high-quality functional dataset.

[0215] Abalone Bio’s Functional Antibody Selection Technology (FAST) platform is capable of generating large-scale, high-quality functional datasets of antibody sequence-function relationships against targets such as GPCRs. The data generated from an agonist antibody selection campaign against the receptor CB2 were combined. A pre-trained antibody PLM was fine-tuned to be a binary classifier of antibody functionality with our functional datasets. The trained models were used to score validation and test set sequences the models had not been presented. Candidate sequences were then selected that the model scored as a high probability of being a functional antibody. A subset of these high probability functional sequences was tested by producing antibody proteins and testing their function with mammalian cell assays, and a number of sequences were identified that have activity in CB2 assay cell lines.

[0216] Data pre-processing and scoring. NGS data from the CB2 agonist discovery campaign were processed using a commercial service, PipeBio. The discovery campaign started with a pool of sequences L, had 4 rounds of enrichment (E1-E4), and after round 1 had a parallel low stringency (LS) branches (El LS, E2 LS, etc.)

[0217] Clustered raw reads were then merged, annotated, and turned into “sequences” based on unique CDR3 amino acid sequence, and filtered for correct sequences. A growth score was then defined for each sequence. Each sequence count was normalized by total reads in experiment using the formula log(cdr3_reads) - log(total_reads). A sequence was labeled as “grower” (i.e., a functional antibody that promotes growth in the FAST strain) if a) the normalized counts in E2 were 20% greater than in El, and b) normalized counts in low-stringency round 2 (E2 LS) were 20% greater than in the library (L), and c) total observed counts across HS 1, 2 and LS 1 and L001 > 100. This scoring method yielded about 1% of sequences labelled as growers.

[0218] Modeling. LoRA was used to set up fine-tuning BERT on a classification task- grower or not-. ~5% (133K out of the 26M) of model parameters were trained / fine-tuned.

[0219] An antibody designated AB 101 was generated from the FAST platform, having the ISVD sequence:EVQLVESGGGLVQPGGSLRLSCAASGSIFSIMAMAWYRQAPGKEREWVSGIDTGGGTYY AESVKGRFTISNDNSKNTAYLQMNSLKPEDTAVYYCSGAIKYGSGRFDIKNYWGQGTQ VTVSS (SEQ ID NO:55)

[0220] First, a single split of data into training and test sets was performed. The top hit, AB 101, and any sequences with CDR3s that had less than 2 amino acids difference from ABlOl’sCDR3, was excluded. Also excluded were sequences with < 80% similarity to hV gene. A weighted (inverse to class frequency) sampler and AdamW was used for training, with no hyperparameter optimization. ROC-AUC and average precision score were calculated to evaluate the model, and an ROC-AUC of 0.7 and APS of 0.25 was calculated.

[0221] Data was then split into large development + small validation (including AB 101) sets multiple times. K-folds cross-validation and fine-tuning was implemented. The small validation data set was defined as above (-10% of the sequences, including all sequences including AB 101 and its related sequences) and then the remaining large development sequence set was randomly split into 10 equally sized folds. For each fold, training was on 90% and predict on held out 10%. For each of the 10 models, training was as above on the model’s individual fold’s training set.

[0222] The validation test set sequences were then scored by calculating probabilities of being a grower, i.e., a functional antibody, using each of the 10 k-fold models and averaging them.Functional antibody probabilities were also scored for sequences in each of the folds’ test sets with that fold’s model. 200 sequences from the validation test sequences (ML_V) and 200 from the individual k-fold test set sequences are provided for further analysis. These antibodies are summarized in Table 1.Example 2. In vitro activation by ML-predicted agonist antibodies of CB2 receptor inhibits cAMP signaling in RAW264.7 mouse macrophage cells.

[0223] The positive control agonist CB2 antibody ABt281 has the sequence:EVQLVESGGGLVQPGGSLRLSCAASGSIFSIMAMAWYRQAPGKEREWVSGIDTG GGTYYAESVKGRFTISNDNSKNTAYLQMNSLKPEDTAVYYCSGAIKYGSGRFDI KNYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:202)

[0224] The positive control agonist CB2 antibody ABt285 has the sequence:EVQLVESGGGLVQPGGSLRLSCAASGSIFSIMAMAWYRQAPGKEREWVSGIDTG GGTYYADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCSGAIKYGSGRFDIKNYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:203)

[0225] ABt269 is an anti-GFP VHH-Fc antibody used as a control, having the sequence: QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMS SAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW GQGTQVTVSSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:204)

[0226] RAW264.7 cells were cultured and treated with CB2 agonist HU308 (ontemabez) or CB2 positive control agonist antibodies (ABt281, ABt285) or isotype control (Abt269) or ABhitO89- 128 (see Table 1). ABhitO89-128 represent 37 of the top 40 predicted agonist antibodies based upon ML (Example 1). For two concentrations of antibody (1 pM and 500 nM), CB2 mediated GI-cAMP signaling efficacy was measured using cAMP-Glo assay (Promega, V1501), whereby luminescence is inversely proportional to cAMP concentration. Application of HU308, ABt281 or ABt285 increases luminescence signal, in dose dependent manner. Low response is observed with negative control antibody ABt269. Several ABhit antibodies show dose dependent changes in luminescence consistent with CB2 agonism (FIG. 1 and Table 5, below).Example 3. In vitro activation by ML-predicted agonist antibodies of CB2 receptor inhibits cAMP signaling in RAW264.7 mouse macrophage cells and CB2 antagonist treatment demonstrates specificity of CB2 activation by blocking agonist activation.

[0227] RAW264.7 cells were cultured and treated with CB2 agonists HU308, ABhitO95, ABhitlO4, or ABhitl 14. CB2 mediated GI-cAMP signaling efficacy was measured using the cAMP-Glo assay (Promega), whereby luminescence is inversely proportional to cAMP concentration. Application of HU308 and all AB hit antibodies increased luminescence in a dose dependent manner (FIG. 2A).

[0228] A similar experiment was then conducted, wherein RAW264.7 cells were cultured and treated with cAMP activator NKH477 to stimulate cAMP production. The cells were then treated with CB2 agonist HU308 or CB2 specific agonist antibodies (as indicated in legends for FIGs. 2B-F) or isotype control (Abt269). CB2 mediated GI-cAMP signaling efficacy was measured using the cAMP-Glo assay (Promega), whereby luminescence is inversely proportional to cAMP concentration.

[0229] Application of CB2 agonists decreased NKH477 stimulated cAMP production (Table 6, below), whereas ABt269 did not affect cAMP production.

[0230] In FIG. 3, HU308 and ABhit antibodies were added to RAW264.7 cells at EC80 concentrations and then titrated with CB2 antagonist SR144528 to determine CB2 specificity of increased luminescence. HU308, ABhitO95, ABhitlO4 and ABhitl 14 showed antagonist dose dependent decreases in luminescence, showing CB2-specific activation by HU308 and ABhit antibodies.Example 4. In vitro activation of CB2 receptor activates pERKl / 2 signaling in RAW264.7 mouse macrophage cells.

[0231] RAW264.7 cells were cultured, serum starved for 4 hours, and exposed to CB2-specific agonist HU-308, CB2-specific agonist antibodies (as indicated in FIGs. 4A and 4B), or isotype control (ABt269). CB2-mediated pERKl / 2 signaling efficacy was measured by using TR-FRET pERKl / 2 kit (LANCE Ultra pERKl / 2 kit, Perking Elmer, TRF4000). Application of HU308, or CB2 agonist antibodies increased TR-FRET signal in a dose dependent manner, while no ERK phosphorylation was observed with the isotype control, ABt269 (FIG. 4 and Table 6, below).Example 5. ML predicted CB2 agonist antibodies recruit B-Arrestin 2 in RAW264.7 mouse macrophage cells.

[0232] RAW264.7 cells were cultured and transfected with B-Arr2 plasmid (20ng, Revvity, PWTBARR2) and pcDNA3 plasmid (130ng) via lipofectamine 2000. Transfected cells were treated with CB2 specific agonist HU308, CB2 agonist antibodies (ABhitO94, ABhitO124, ABhitO125) or isotype control (ABt269). CB2 mediated B-Arr2 recruitment was measured using TR-FRET B-Arr2 recruitment kit (HTRF B-Arr2 recruitment kit, Revvity, 62BDBARR2PEB). Application of HU308 or CB2 agonist antibodies increases TR-FRET signal.Table 6. CB2 agonist-mediated decrease of NKH477 stimulated cAMP productionExample 6. ML predicted CB2 agonist antibody reduces inflammatory and fibrotic factors in precision cut human liver slice model.

[0233] The precision cut human liver slice model (hPCLS) preserves all cell types within their native architecture to uniquely capture endogenous cell-cell interactions. PCLS from de- identified resection samples of two injured, human male livers were used to test the activity of ABhit_0125 (lOOnM and IpM), ABt269 isotype control (IpM), and Alk5i (positive control) following 24-hour and 48-hour incubation. Alk5i is a TGF-beta type I receptor kinase (ALK5) inhibitor (CAS 446859-33-2).

[0234] Human precision-cut liver slices (hPCLS) were be generated from discarded remnants of surgically resected human livers. Normal-appearing resection margins surrounding HCC were selected for hPCLS generation. The ischemic time between post-hepatectomy and generated PCLS was 3-4 hours. Cores of 8 mm diameter were generated from which intact liver slices (200 pm thickness) and collected from the buffer tray using a soft bristle brush and transferred to six well tissue culture plate (3 slices / well) filled with 37°C warm William’s E GlutaMAX media supplemented with 25 mM glucose and 50 pg / ml gentamycin. The slices were pre-incubated for 24 hours to equilibrate the tissues. After pre-incubation, fresh medium was added containing either ABhit_0125 (lOOnM and IpM), ABt269 isotype control (IpM), and Alk5i (lOpM) for an additional 24 and 48 hours.

[0235] For efficacy assessment, at the end of drug treatment from each condition, secretion of collagenlal (Collal) into the media was assessed by ELISA (FIG. 6A). At 48-hours the 1 pM ABhit_0125 and Alk5i significantly reduced Collal compared ABt269, the isotype control.

[0236] Also, at the end of drug treatment from each condition, slices were used for total mRNA extraction. Collagenlal (COL1A1, FIG. 6B), alpha smooth muscle actin (ACTA2, FIG. 6C), interleukin 6 (IL6, FIG. 6D), interleukin 10 (IL 10, FIG. 6E) and tumor necrosis factor alpha (TNFa, FIG. 6F) mRNA levels were quantified using RT-qPCR. For COL1 Al, ACTA2, IL6, IL10 and TNFa treatment with ABhit_0125 resulted in statistically significant reduction of theseinflammatory or fibrotic factors, however, no significant change was observed upon treatment with ABt269 (isotype control antibody).Ill

Claims

CLAIMSWhat is claimed is:

1. A cannabinoid receptor type 2 (CB2) antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds a CB2, wherein the ISVD comprises a complementarity determining region (CDR) 1, a CDR2, and a CDR3 of the ISVD comprising the sequence of any one of SEQ ID NOs:500-899.

2. The CB2 antibody of claim 1, wherein the ISVD is a variable domain of the heavy chain of a heavy chain antibody (VHH).

3. The CB2 antibody of claim 2, wherein the ISVD is camelid.

4. The CB2 antibody of claim 2, wherein the ISVD is chimeric.

5. The CB2 antibody of claim 2, wherein the ISVD is humanized.

6. The CB2 antibody of any one of claims 1-5, wherein the ISVD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 500-899, or a variant thereof having at least about 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs:500-899.

7. The CB2 antibody of any one of claims 1-6, wherein the ISVD comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, 4, or 7; a CDR2 comprising an amino acid sequence of SEQ ID NO: 10, 13, or 16; and a CDR3 comprising an amino acid sequence of SEQ ID NO: 19, 22, or 25; wherein the CDR1, CDR2 and CDR3 are defined according to IMGT.

8. The CB2 antibody of claim 7, wherein the ISVD comprises SEQ ID NO:505, SEQ ID NO:715, or SEQ ID NO:716.

9. The CB2 antibody of claim 8, wherein the ISVD comprises SEQ ID NO:716.

10. The CB2 antibody of any one of claims 1-9, wherein the antibody further comprises an Fc region.

11. The CB2 antibody of claim 10, wherein the Fc region is an Fc region of an IgGl or IgG4.

12. The CB2 antibody of claim 11, wherein the Fc region is a variant IgG4 Fc region exhibiting reduced effector function.

13. The CB2 antibody of claim 12, wherein the Fc region comprises amino acid substitutions F234A and L235A, with numbering according to the EU index of Kabat.

14. The CB2 antibody of claim 12 or 13, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 130.

15. The CB2 antibody of claim 11, wherein the Fc region is an IgGl Fc region comprising the amino acid sequence of SEQ ID NO: 126.

16. The CB2 antibody of claim 11, wherein the Fc region is a variant IgGl Fc region exhibiting reduced effector function.

17. The CB2 antibody of claim 16, wherein the variant IgGl Fc region comprises amino acid substitutions L234A, L235A, M252Y, S254T, and T256E, wherein the residues are numbered according to the EU index.

18. The CB2 antibody of claim 17, wherein the variant IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 127.

19. The CB2 antibody of claim 16, wherein the variant IgGl Fc region comprises amino acid substitutions L234A, L235A, and P329G, wherein the residues are numbered according to the EU index.

20. The CB2 antibody of claim 19, wherein the variant IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 128.

21. The CB2 antibody of claim 16, wherein the variant IgGl Fc region comprises amino acid substitutions L234A, L235A, M252Y, S254T, T256E, and P329G, wherein the residues are numbered according to the EU index.

22. The CB2 antibody of claim 21, wherein the variant IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 129.

23. The CB2 antibody of any one of claims 10-22, wherein the antibody further comprises a hinge region.

24. The CB2 antibody of any one of claims 10-23, wherein the ISVD is fused to the Fc region via a peptide linker.

25. The CB2 antibody of any one of claims 1-24, wherein the CB2 is human, mouse, rat or cynomolgus monkey CB2.

26. The CB2 antibody of claim 25, wherein the CB2 is a human CB2.

27. The CB2 antibody of any one of claims 1-26, wherein the CB2 antibody is a CB2 agonist.

28. The CB2 antibody of any one of claims 1-27, wherein the CB2 antibody does not agonizeCB1.

29. The CB2 antibody of any one of claims 1-28, wherein the ISVD does not specifically bind a cannabinoid receptor type 1 (CB1).

30. The CB2 antibody of any one of claims 1-29, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:28-39.

31. An isolated nucleic acid encoding the CB2 antibody of any one of claims 1-30.

32. An expression vector comprising the nucleic acid of claim 31.

33. A host cell comprising the nucleic acid of claim 31 or the expression vector of claim 32.

34. A method of producing a CB2 antibody, comprising culturing the host cell of claim 33 under conditions where the CB2 antibody is produced.

35. The method of claim 34, further comprising recovering the CB2 antibody produced by the host cell.

36. A pharmaceutical composition comprising the CB2 antibody of any one of claims 1-30 and a pharmaceutically acceptable carrier.

37. A method of agonizing CB2 on a cell, comprising contacting the cell with an amount of the CB2 antibody of any one of claims 1-30 that is sufficient for activating CB2 on the cell.

38. The method of claim 37, wherein the method is in vitro.

39. The method of claim 37, wherein the method is in vivo.

40. A method of treating an individual in need thereof having a disease or condition, wherein agonizing CB2 ameliorates the disease or condition, comprising administering to the individual an effective amount of the CB2 antibody of any one of claims 1-30.

41. The method of claim 40, wherein the disease or condition is selected from the group consisting of chemotherapy induced peripheral neuropathy (CIPN), diabetic peripheral neuropathy, respiratory infections, liver fibrosis, cold sensitivity, inflammatory bowel disease, and endometriosis.

42. The method of claim 41, wherein the disease or condition is chemotherapy induced peripheral neuropathy (CIPN).

43. The method of claim 42, wherein the CIPN is caused by a taxol drug.

44. The method of claim 43, wherein the taxol drug is paclitaxel.

45. A method of treating a cancer in an individual in need thereof, comprising administering to the individual an effective amount of a chemotherapeutic agent, and an effective amount of the CB2 antibody of any one of claims 1-30.

46. The method of claim 45, wherein the disease or condition is a respiratory infection.

47. The method of claim 46, wherein the respiratory infection is caused by a virus selected from the group consisting of influenza viruses and coronaviruses.

48. The method of claim 46, wherein the respiratory infection is caused by SARS-CoV-2.

49. The method of claim 46, wherein the respiratory infection is caused by H1N1 influenza.

50. A method of reducing a cytokine release syndrome in an individual in need thereof, comprising administering to the individual an effective amount of the CB2 antibody of any one of claims 1-30.

51. The method of claim 50, wherein the method decreases IL-16 and / or IL-8 level secreted by airway cells in the individual.

52. The method of claim 41, wherein the disease or condition is liver fibrosis.

53. The method of claim 41, wherein the disease or condition is diabetic peripheral neuropathy.

54. The method of any one of claims 40-53, wherein the individual is a human.

55. A kit comprising the CB2 antibody of any one of claims 1-30 or the pharmaceutical composition of claim 36, and instructions for use.

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