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 to CB2. Methods of making and using these CB2 antibodies are also provided.

Description

Cross-references to related applications

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

[0002] Reference to the electronic sequence list The contents of the electronic serial number (185982000440SEQLIST.xml; size: 533,491 bytes; and creation date: January 3, 2025) are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to an antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds to cannabinoid receptor type 2 (CB2); and methods for preparing and using the antibody. Background Technology

[0004] In mammals, two G protein-coupled receptors mediate cannabinoid signaling, which is present in both plants and animals. Cannabinoid receptor 1 (CB1) is most strongly expressed in neurons of the CNS and is the primary cause of the psychoactive effects of cannabinoids on the body (Nature, 346: 561-564, 1990). CB2 is mainly expressed in the spleen and hematopoietic cells, indicating that this receptor is primarily localized in the periphery (Nature, 365: 61-65, 1993; and reviewed in Pharmacol. Rev, 58(3): 389-462, 2006).

[0005] CB2 agonists are effective in various models of acute and chronic pain induced by 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), CB2 activation has been shown to suppress paclitaxel-induced CIPN in rats (J. Pharmacol. Exp. Ther. 327: 584–591, 2008). Furthermore, CB2 agonists suppress CIPN induced by all major classes of chemotherapeutic agents, including taxanes (i.e., paclitaxel mentioned above), vinblastine alkaloids (i.e., vincristine, Br. J. Pharmacol. 152:765-777, 2007), and platinum-derived (i.e., cisplatin, Mol Pain, 8:71) anticancer agents. CB2 agonists have also been shown to be effective in treating diabetic neuropathy in rats (Pharmacology. 82(3):193-200, 2008) and mouse models (Pain. June 2013;154(6):864-73; Psychopharmacology (Berl).233(11):2209-2219, 2016), further supporting the use of CB2 agonists in the treatment of a variety of pain indications.

[0006] CB2 activation exhibits strong anti-inflammatory activity as part of a natural negative feedback mechanism, in which the CB2 receptor is induced in damaged or virus-infected cells under inflammatory conditions (Gastroenterology. 129:437-453, 2005; Am. J. Pathol. 177:187-196, 2010; Br. J. Pharmacol. 151:1041-1048, 2007). CB2 activation subsequently inhibits the secretion of various inflammatory factors and signals, including TNFα, IL-6, IL-8, and CCL2 (reviewed in Cell. Mol. Life Sci. 73:4449-4470, 2016). Therefore, CB2 agonists can be used to treat inflammatory lung diseases (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 infections (Virulence 9:217–230, 2018).

[0007] CB2 receptor agonism also plays a role in mitigating the fibrotic process in multiple organs. For example, the 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 to alleviate 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 autoimmune responses (Am. J. Pathol. 177:187-196, 2010). In the liver, CB2 agonism in a mouse model protects the liver from liver fibrosis induced by carbon tetrachloride (CCl4) (Gastroenterology. 128:742-755, 2005) and alcohol (Hepatology. 54:1217-1226, 2011) and enhances liver regeneration after injury (Hepatology. 52:1046-1059, 2010). In addition to reducing inflammation, CB2 agonism also reduces myofibroblast proliferation and induces apoptosis, while myofibroblasts secrete collagen that forms fibrotic lesions (Gastroenterology. 128:742-755, 2005). Therefore, CB2 may exert beneficial effects on liver diseases (e.g., inflammation and fibrosis) through multiple mechanisms.

[0008] There is a need in this field to develop methods for the safe and effective use of CB2 receptor agonists with reduced risk of adverse events. CB2 small molecule agonists are rapidly cleared, exhibit significant CNS permeability, and carry a high risk of pharmacokinetic effects. Even the most peripherally restricted CB2 small molecules have 3%–5% CNS permeability (Pain. April 13, 2021). Most CB2 agonists exhibit some activity at CB1, 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), approximately 20% of treated patients reported some degree of dizziness, somnolence, or other central symptoms of CB1 activation (Higgins et al., 14th Congress of ECCO – Inflammatory Bowel Diseases; March 6–9, 2019; Copenhagen, Denmark). Furthermore, the short half-life of small molecules adds to compliance barriers, necessitating multiple daily dosings (J. Behav. Med. 31: 213–224, 2008). The antibody described herein addresses these needs in the art and offers related advantages.

[0009] All publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference in their entirety. Summary of the Invention

[0010] This application relates to an anti-CB2 antibody (hereinafter referred to as "CB2 antibody") comprising an immunoglobulin single variable domain (ISVD) that specifically binds to CB2; and methods for preparing and using the antibody.

[0011] In one aspect, this document provides a cannabinoid receptor type 2 (CB2) antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds to CB2, wherein the ISVD comprises a complementarity-determining region (CDR) 1, CDR2, and CDR3 of the ISVD containing the sequence of any of SEQ ID NO:500-899.

[0012] In some embodiments, the ISVD is a variable domain (VHH) of the heavy chain of a heavy chain antibody. In some embodiments, the ISVD is of camel origin. 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 NO:500-899, or a variant thereof having at least about 85% sequence identity with the amino acid sequence of any of SEQ ID NO:500-899.

[0014] In some embodiments, the ISVD comprises CDR1 containing the amino acid sequence of SEQ ID NO: 1, 4, or 7; CDR2 containing the amino acid sequence of SEQ ID NO: 10, 13, or 16; and CDR3 containing the amino acid sequence of SEQ ID NO: 19, 22, or 25; wherein 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 IgG1 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, wherein the numbering is based on the Kabat EU index. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:130.

[0016] In some implementations, the Fc region is an IgG1 Fc region containing the amino acid sequence of SEQ ID NO:126.

[0017] In some embodiments, the Fc region is a variant IgG1 Fc region exhibiting reduced effector function. In some embodiments, the variant IgG1 Fc region comprises amino acid substitutions for L234A, L235A, M252Y, S254T, and T256E, wherein the residues are numbered according to the EU index. In some embodiments, the variant IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:127. In some embodiments, the variant IgG1 Fc region comprises amino acid substitutions for L234A, L235A, and P329G, wherein the residues are numbered according to the EU index. In some embodiments, the variant IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:128. In some embodiments, the variant IgG1 Fc region comprises amino acid substitutions for L234A, L235A, M252Y, S254T, T256E, and P329G, wherein the residues are numbered according to the EU index. In some embodiments, the variant IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:129.

[0018] In some implementations, the antibody includes a hinge region.

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

[0020] In some embodiments, CB2 is human, mouse, rat, or cynomolgus monkey CB2. In some embodiments, CB2 is 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 to cannabinoid receptor type 1 (CB1).

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

[0023] A method for producing any of the above-described CB2 antibodies is further provided, the method comprising culturing the host cells under conditions conducive to the production of the CB2 antibody. In some embodiments, the method further comprises recovering the CB2 antibody produced by the host cells.

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

[0025] A further method for activating CB2 on a cell is provided, the method comprising contacting the cell with any of the aforementioned CB2 antibodies in an amount sufficient to activate CB2 on the cell. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

[0026] A further method is provided for treating an individual in need of a disease or condition, wherein CB2 activation can improve the disease or condition, the method comprising administering to the individual an effective amount of any of the aforementioned CB2 antibodies or the aforementioned pharmaceutical composition. In some embodiments, the disease or condition is selected from the group consisting of: chemotherapy-induced peripheral neuropathy (CIPN), diabetic peripheral neuropathy, respiratory infection, 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, CIPN is caused by a paclitaxel-based drug. In some embodiments, the paclitaxel-based 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] A method for treating cancer in an individual in need is further provided, comprising administering to the individual an effective amount of a chemotherapeutic agent and an effective amount of either of the aforementioned CB2 antibodies.

[0028] A further method for alleviating cytokine release syndrome in an individual in need is provided, comprising administering to the individual an effective amount of any of the aforementioned CB2 antibodies. In some embodiments, the method reduces the levels of IL-16 and / or IL-8 secreted by airway cells in the individual.

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

[0030] A further provided kit includes a CB2 antibody of any of the above-described CB2 antibodies or the above-described pharmaceutical composition, and instructions for use. Attached Figure Description

[0031] Figure 1The efficacy of CB2-mediated GI-cAMP signaling, as measured using the TR-FRET cAMP kit, was demonstrated after application of the specified antibodies (at two different concentrations for each specified antibody treatment) in RAW264.7 cells.

[0032] Figures 2A to 2F RAW264.7 cells cultured and treated with HU308, CB2 agonist antibody or isotype control (ABt269) are shown.

[0033] Figure 3 The specified antibody was added to RAW264.7 cells at an EC80 concentration and then titrated with the CB2 antagonist SR144528.

[0034] Figure 4A and Figure 4B ERK phosphorylation induced by the specified reagent in RAW264.7 cells is shown as evaluated by the TR-FRET pERK1 / 2 kit.

[0035] Figure 5 The recruitment of β-repressor protein 2 induced by the specified reagent in RAW264.7 cells is shown as evaluated by the HTRF β-repressor protein 2 recruitment assay kit.

[0036] Figures 6A to 6F The effects of the specified treatment on human liver slice model (hPCLS) cells, such as the secretion of collagen 1a1 (Col1a1) into the culture medium as assessed by ELISA, are shown. Figure 6A ) and through COL1A1 ( Figure 6B ), ACTA2 ( Figure 6C ), IL6 ( Figure 6D ), IL10 ( Figure 6E The mRNA expression levels of TNFα (FIG. 6F) were assessed. 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 Implementation

[0037] This disclosure is based, at least in part, on the discovery of novel antibodies (which may be synonymously referred to as "anti-CB2 antibodies" or "CB2 antibodies") that specifically bind to cannabinoid receptor type 2 (CB2) and their antigen-binding fragments. The CB2 antibody contains at least one immunoglobulin single variable domain (ISVD) that specifically binds to CB2. The ISVD contains a complementarity-determining region 1 (CDR1), a complementarity-determining region 2 (CDR2), and a complementarity-determining region 3 (CDR3) sequence. The ISVD may be a variable domain (VHH) of the heavy chain of a heavy chain antibody. The ISVD may be, for example, a camel-derived, chimeric, or humanized ISVD. The ISVD described herein specifically binds to CB2 (such as human, cynomolgus monkey, murine, and / or rat CB2) but has low or undetectable affinity for CB1 (such as human, cynomolgus monkey, murine, and / or rat CB1). The ISVD described herein is a selective agonist of CB2 that has no agonist activity or undetectable agonist activity against CB1. In some embodiments, the anti-CB2 antibody comprises an Fc fragment (e.g., human IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, or IgG4 Fc). Pharmaceutical compositions comprising these antibodies, kits, and treatments using these antibodies are also provided.

[0038] I. Definition The term "antibody" as used herein is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, four-chain antibodies (such as IgG antibodies), heavy chain antibodies, and their antibody fragments, provided they exhibit the desired antigen-binding activity, namely binding to CB2 (such as human CB2, cynomolgus monkey CB2, rat CB2, and / or mouse CB2). The term "four-chain antibody" as used herein refers 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 containing two heavy chains but lacking the two light chains typically found in four-chain antibodies. Cameloideas (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0039] As used herein, “agonist” means an antibody that stimulates the biological function and / or signal transduction of a target receptor. In the context of CB2 agonists, CB2 agonists can activate the endocannabinoid signaling pathway by activating CB2. CB2 agonist effects include, but are 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 known as "ISV" or "ISVD") is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with another variable domain (e.g., in the absence of the required VH / VL interaction between the VH and VL domains of a four-chain monoclonal antibody). Examples of ISVDs include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), shark IgNAR domains, single-domain antibodies (or dAbs) that are VH domains or derived from VH domains, and single-domain antibodies (or dAbs) that are VL domains or derived from VL domains. ISVDs based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. In some embodiments, the ISVD is a nanobodies. The term “nanobody” is generally defined as in WO 2008 / 020079 or WO2009 / 138519, and therefore generally refers in certain respects to VHH, humanized VHH, or camelified VH (such as camelified human VH), or generally refers to sequence-optimized VHH (such as those optimized for, for example, chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression).

[0041] An "antibody fragment" comprises a portion of an antibody, preferably the antigen-binding region or variable region of the antibody. Examples of antibody fragments include VHH, single-domain antibodies, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin (the variable domain, which contains the antigen-binding site). The constant domain contains the C of the heavy chain. H 1. C H 2 and C H 3. Structural domains (collectively referred to as C) H ) and light chain CHL (or C L ) structural domain.

[0042] The term "Fc region" or "crystallizable fragment region" used herein is used to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from an amino acid residue at position Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, compositions of complete antibodies may comprise antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations having mixtures with or without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting said group are identical except for possible small amounts of possibly naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies are highly specific (targeting a single antigenic site). Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to the specificity of monoclonal antibodies, they have the advantage that they are synthesized via hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibody intended for use according to this application can be prepared by a variety of techniques, including, for example, hybridoma methods (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 SpringHarbor Laboratory Press, 2nd edition, 1988); Hammerling et al. Monoclonal Antibodies and T - Cell Hybridomas 563-681 (Elsevier, NY, 1981), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technology (see, for example, Clackson et al., Nature352: 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 techniques for producing human antibodies or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, 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. Patent 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 a domain of the heavy or light chain of an antibody that participates in binding the antibody to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, screening libraries of complementary VL or VH domains derived from antibodies binding to a specific antigen can isolate antibodies binding to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0045] As used herein, the term "hypervariant region" or "HVR" refers to each of the highly sequence-variable regions ("complementarity-determining regions" or "CDRs") and / or regions forming structurally defined loops ("hypervariant loops") and / or regions containing antigen-contact residues ("antigen contacts"). Generally, 4-chain antibodies and their antigen-binding antibody fragments contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Typically, heavy-chain antibodies contain three HVRs (HVR1, HVR2, HVR3).

[0046] Many HVRs are in use and are covered herein. Exemplary HVRs of 4-chain antibodies and their antigen-binding antibody fragments described herein include: (a) hypervariable loops appearing at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917(1987)); (b) CDRs appearing at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al.) , Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts appearing at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and combinations of (d) (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 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., frame, “FR” residues) are numbered in this paper according to Kabat et al. (ibid.).

[0048] The amino acid residues of single-domain antibodies (such as VHH) can be determined based on the V... (The sentence is incomplete and requires more context to translate accurately.) H The domain was numbered according to a general numbering rule (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), which was applied to the VHH domain from camelids in the article Riechmann and Muyldermans, J. Immunol. Methods 2000 June 23; 240 (1-2):185-195. According to this numbering, VHH's FR1 contains amino acid residues at positions 1-30, VHH's CDR1 contains amino acid residues at positions 31-35, VHH's FR2 contains amino acids at positions 36-49, VHH's CDR2 contains amino acid residues at positions 50-65, VHH's FR3 contains amino acid residues at positions 66-94, VHH's CDR3 contains amino acid residues at positions 95-102, and VHH's FR4 contains amino acid residues at positions 103-113. In this regard, it should be noted—as discussed in the art regarding V... HAs is well known with the domain and the VHH domain—the total number of amino acid residues in each of these CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number).

[0049] "Frame" or "FR" residues are those variable domain residues other than HVR residues as defined in this paper.

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

[0051] "Humanized" antibodies are antibodies containing a minimal sequence derived from non-human antibodies. Generally, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of a recipient antibody are replaced by residues from the hypervariable region of a non-human species (donor antibody) possessing the desired antibody specificity, affinity, and capability. This non-human species could be camels, mice, rats, rabbits, or non-human primates. In some respects, a "humanized" antibody refers to a chimeric antibody containing amino acid residues from a non-human (e.g., camel) CDR and amino acid residues from a human FR. In some cases, the framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not present in either the recipient antibody or the donor antibody. These modifications are made to further enhance antibody performance. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the 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] Affinity-matured antibodies are antibodies with one or more alterations in one or more CDRs, such alterations increasing the antibody's affinity for the antigen compared to parent antibodies without these alterations. In some embodiments, affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using procedures known in the art. For example, random mutagenesis of CDRs and / or framework residues is described in, for example, the following literature: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. 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] The "percentage of amino acid sequence identity (%)" or "homology" of the peptide and antibody sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the compared peptide after sequence alignment and taking into account any conserved substitutions as part of sequence identity. Alignments for determining the percentage of amino acid sequence identity can be performed in various ways that are within the skill of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the sequence alignment computer program ALIGN-2 was used to generate the amino acid sequence identity % values. The ALIGN-2 sequence alignment computer program was written by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington DC, 20559, along with user documentation, and is registered under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be written for the UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and cannot be changed.

[0054] As used herein, the terms "specific binding," "specifically binding," or "specific to" a particular polypeptide or an epitope on a particular polypeptide target can refer to, for example, the K-axis of a molecule toward that target. D For at least about 10 -4 M, alternative site at least about 10 -5 M, alternative site at least about 10 -6 M, alternative site at least about 10 -7 M, alternative site at least about 10 -8 M, alternative site at least about 10 -9 M, alternative site at least about 10 -10 M, alternative site at least about 10 -11 M, alternative site at least about 10 -12 M or higher. In some embodiments, the term "specific binding" refers to a molecule binding to a specific polypeptide or an epitope on a specific polypeptide, without substantially binding to any other polypeptide or polypeptide epitope. K D Specific binding can be determined using methods known in the art, such as ELISA, surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining molecular binding compared to binding to a control molecule, which is typically a similarly structured molecule lacking binding activity. For instance, specific binding can be determined by competition with a target-like control molecule (e.g., an excess of an unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of the unlabeled target, specific binding is indicated.

[0055] As used herein, “treatment,” “treating,” or “treat” is defined as a method for achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by a disease; reducing the severity of the disease; stabilizing the disease (e.g., preventing or delaying the worsening of the disease); preventing or delaying the spread of the disease (e.g., metastasis); preventing or delaying the recurrence of the disease; delaying or slowing the progression of the disease; improving the disease state; providing remission (partial or complete remission) of the disease; reducing the dosage of one or more other medications required to treat the disease; delaying the progression of the disease; improving or enhancing quality of life; increasing weight gain; and / or prolonging survival. “Treatment” also encompasses alleviating the pathological consequences of a disease. The methods described herein cover any one or more of these aspects of treatment. In the context of pain treatment, such methods refer to pain relief. Pain relief includes, but is not limited to: reducing or improving the occurrence, sensation, perception and / or impact of pain and / or other symptoms; reducing the risk of pain in a subject; preventing the occurrence of pain; relieving pain, even if the pain subsides and / or relieving one or more symptoms of pain; reducing pain; weakening pain; limiting pain; reducing pain, including the duration and / or intensity of pain; alleviating pain; blocking pain; and inhibiting the spread of pain.

[0056] As used herein, the term "effective amount" or "therapeutic effective amount" of a substance (such as a CB2 antibody, construct, or composition) is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular condition or to achieve one or more specific desired outcomes. Effective amounts as used herein can vary depending on factors such as the patient's disease state, age, sex, and weight, and the substance's ability to elicit the desired response in an individual. An effective amount is also the amount by which any toxic or adverse effects of treatment are outweighed by the beneficial effects of treatment (i.e., the therapeutic index). Effective amounts can be administered in one or more doses. As understood in a clinical setting, effective amounts may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Therefore, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to have been administered in an effective amount if, in combination with one or more other agents, it achieves or results in a desired outcome.

[0057] "Individual" or "subject" is a mammal. Mammals include, but are not limited to: livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as rhesus monkeys and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0058] "Antibody effector function" refers to those biological activities attributable to the Fc region of the antibody (the native sequence Fc region or the Fc region of an amino acid sequence variant) and varies with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. "Reduced or minimized" antibody effector function means a reduction of at least 50% (or alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) compared to wild-type or unmodified antibodies. The determination of antibody effector function is readily available and measurable by those skilled in the art. In a preferred embodiment, antibody effector functions of complement binding, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity are affected. In some embodiments, effector function is eliminated by a glycosylation-eliminating mutation located in a constant region, such as an "effect-free mutation." In one lattice, the functional mutation of the effector is located in C H N297A or DANA mutations (D265A+N297A) in region 2. Shields et al. J. Biol. Chem. 276(9): 6591-6604 (2001). Alternatively, additional mutations leading to reduced or eliminated effector function include: K322A and L234A / L235A (LALA). Alternatively, mutations leading to reduced or eliminated effector function include: F234A and L235A (FALA) according to the EU index. Alternatively, mutations leading to reduced or eliminated effector function include: L234A, L235A, and P329G (LALAPG) according to the EU index. Alternatively, mutations leading to reduced or eliminated effector function include: M252Y, S254T, and T256E (YTE) according to the EU index. Alternatively, effector function can be reduced or eliminated by production techniques, such as expression in host cells that are not glycosylated (e.g., E. coli), or expression in host cells that produce glycosylation patterns that are ineffective or less effective in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem . 278(5): 3466-3473 (2003)).

[0059] As used herein, "pharmaceutical acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, for example, that can be incorporated into a pharmaceutical composition administered to a patient without causing any significantly undesirable biological effects or interacting in a harmful manner with any other component of the composition containing that material. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological and manufacturing testing and / or are included in the inactive ingredient guidance prepared by the US Food and Drug Administration.

[0060] Half maximum effective concentration (EC) 50 An antibody (or similar substance) is a measure of the effectiveness of a substance (such as an antibody) in inducing a specific biological or biochemical function. It indicates how much of a specific drug or other substance (such as an antibody) is needed, after a specified exposure time, to achieve the half-maximum response between baseline and maximum in a given biological process. This value is typically expressed as a molar concentration. 50 "IC" with antagonist drugs or other substances 50 "They are comparable. EC" 50 or IC 50 Measurements can be performed using bioassays such as ligand binding inhibition assays (competitive binding assays) via FACS analysis, cell-based cytokine release assays, or enhanced luminescent near-homogeneous assays (AlphaLISA).

[0061] An "isolated" nucleic acid molecule encoding the constructs, antibodies, or antigen-binding fragments thereof described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminant nucleic acid molecule typically associated with it in the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with any components related to the environment in which it is produced. The isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is in a form different from the form or environment in which it is found in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acids naturally present in cells that encode the polypeptides and antibodies described herein. The isolated nucleic acid includes nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located extrachromosomally or at chromosomal locations other than their natural chromosomal locations.

[0062] The term "control sequence" refers to the DNA sequence necessary for the expression of an operable coding sequence in a specific host organism. Control sequences suitable for prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0063] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to that sequence; or, if a ribosome binding site is positioned to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and in the reading phase. However, enhancers do not necessarily have to be contiguous. Ligation is accomplished by connecting at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0064] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. This term includes vectors in the form of self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell to which they have been introduced. Some vectors are capable of directing the expression of the nucleic acids they are operatively linked to. Such vectors are referred to herein as "expression vectors."

[0065] As used in this article, “transfection,” “transformation,” or “transduction” refers to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. This includes primary subject cells and their progeny.

[0066] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. The nucleic acid content of progeny cells cannot be exactly the same as that of parent cells, but may contain mutations. This article includes screened or selected mutant progeny that have the same function or biological activity as the initially transformed cells.

[0067] The terms "pharmaceutical formulation" or "pharmaceutical composition" refer to a formulation that is in a form that allows for the effective biological activity of the active ingredient and does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered. Such formulations are sterile. A "sterile" formulation is sterile or free of all live microorganisms and their spores.

[0068] The term "about" as used herein refers to the typical range of error for each value readily known to those skilled in the art. A reference to "about" a value or parameter herein includes (and describes) aspects of that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0069] It should be understood that aspects and implementations of this application include “comprising” aspects and implementations, “consisting of” aspects and implementations, and “substantially consisting of” aspects and implementations.

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

[0071] As used herein in phrases such as “A and / or B”, the term “and / or” is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein in phrases such as “A, B, and / or C”, the term “and / or” is intended to cover 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).

[0072] II. Anti-CB2 antibody This document describes antibodies that specifically bind to CB2 (i.e., "anti-CB2 antibody", "CB2 antibody", "antibody targeting CB2") in various embodiments. The CB2 antibodies described herein may comprise, contain, or be ISVDs, three CDRs derived from an immunoglobulin single variable domain (ISVD). For example, in some embodiments, the CB2 antibodies described herein comprise a binding domain comprising three CDRs derived from an ISVD taught herein, such as those listed in Table 3. In the following embodiments, the structure and functionality of the CB2 antibody are presented in a modular manner as an anti-CB2 ISVD. The description of a single anti-CB2 ISVD is not intended to limit the scope and is intended to cover CB2 antibodies that include, in whole or in part, three CDRs derived from an anti-CB2 ISVD or an anti-CB2 ISVD (e.g., CDRs as shown in Table 3).

[0073] The CB2 antibody described herein comprises 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 human CB1, such as human CB1. In some embodiments, the anti-CB2 ISVD does not excite human CB1, such as 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 (VHH) of the heavy chain of a heavy chain antibody. In some embodiments, the CB2 antibody comprises an ISVD and is of camel origin. In some embodiments, the CB2 antibody comprises a chimeric ISVD. In some embodiments, the CB2 antibody comprises a humanized ISVD. In some embodiments, the CB2 antibody comprises a partially humanized ISVD. In some aspects of the teachings provided herein, the antibody may be referenced with a leading zero (e.g., ABt285 = ABt0285).

[0074] In some embodiments, the ISVD comprises CDR1, CDR2, and / or CDR3 of an ISVD containing the amino acid sequence of any of SEQ ID NO:500-899. In some embodiments, the ISVD comprises CDR1, CDR2, and / or CDR3 of an ISVD containing the amino acid sequence of any of SEQ ID NO:500-899, wherein CDR1, CDR2, and CDR3 are defined according to the IMGT (for an interpretation of the IMGT, see Lefranc et al.). Dev. Comparat. Immunol. 27:55-77, 2003 and Ruiz et al., Nucleic Acids Res. 29(1):207-9, 2001). In some embodiments, the ISVD comprises CDR1, CDR2, and / or CDR3 of an ISVD containing the amino acid sequence of any of SEQ ID NO:500-899, wherein CDR1, CDR2, and CDR3 are defined according to Kabat. In some embodiments, the ISVD comprises CDR1, CDR2, and / or CDR3 of an ISVD containing the amino acid sequence of any of SEQ ID NO:500-899, wherein CDR1, CDR2, and CDR3 are defined according to Chothia.

[0075] In some embodiments, the CB2 antibody comprises the sequences named 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, as defined in Table 1 below.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_0 46, 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, M L_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_16 6. 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, M The ISVDs of CDR1, CDR2, and CDR3 for antibodies against L_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. 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 described herein are provided in Table 1 below. Those skilled in the art will readily identify the CDR or variable domain sequences of these antibodies using known algorithms.

[0077] The CB2 antibody described herein comprises an ISVD containing four frame regions (FRs). The CB2 antibody described herein may contain any suitable sequence for the frame regions. In some embodiments, FR1, FR2, FR3, and FR4 are defined according to IMGT.

[0078] In some embodiments, the CB2 antibody comprises an ISVD that specifically binds to CB2, wherein the ISVD comprises an amino acid sequence of any of SEQ ID NO: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 with any of SEQ ID NO:500-899.

[0079] In some embodiments, the CB2 antibody comprises the antibodies named 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, as defined in Table 1 belowML_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_0 47、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_14 8、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_1 57、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_1 66、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_ Amino acid sequence of CB2 antibody of 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.,

[0080] In some embodiments, the CB2 antibody described herein comprises an Fc region. In some embodiments, the CB2 antibody comprises an Fc region of IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In some embodiments, the CB2 antibody comprises an Fc region of IgG1. In some embodiments, the CB2 antibody comprises an Fc variant exhibiting reduced effector function. In some embodiments, this reduced effector function decreases ADCC activity. In some embodiments, the antibody comprises an IgG1 Fc region exhibiting reduced effector function. In some embodiments, the antibody comprises a variant IgG1 Fc region containing one or more variant amino acids. In some embodiments, the one or more variant amino acids in the variant IgG Fc region described herein may be additions, substitutions, or deletions. Such variant IgG1 Fc regions are relative to conserved Fc sequences, such as conserved human Fc sequences.

[0081] In some embodiments, the antibody comprises an IgG1 Fc region exhibiting reduced effector function, wherein the Fc region comprises amino acid substitutions for L234A and L235A, wherein the numbering is based on the Kabat EU index. 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 amino acid substitutions for F234A / L235A (FALA), wherein the numbering is based on the Kabat EU index. In some embodiments, the variant IgG1 Fc region comprises amino acid substitutions selected from the group consisting of (1) LALA, (2) YTE, and (3) LALAPG. In some embodiments, the variant IgG1 Fc region comprises the LALAPG variant. In some embodiments, the variant IgG1 Fc region comprises both the LALAPG variant and the YTE variant. In some embodiments, the variant IgG1 Fc region comprises both the LALA variant and the YTE variant. The LALAPG variants are designated L234A, L235A, and P329G, with residues numbered according to the EU index. The LALA variants are designated L234A and L235A, with residues numbered according to the EU index. The YTE variants are designated M252Y, S254T, and T256E, with residues numbered according to the EU index.

[0082] In some embodiments, the CB2 antibody comprises an Fc region containing the amino acid sequence of SEQ ID NO:130. In some embodiments, the CB2 antibody comprises an Fc region containing the amino acid sequence of SEQ ID NO:127. In some embodiments, the CB2 antibody comprises an Fc region containing the amino acid sequence of SEQ ID NO:128. In some embodiments, the CB2 antibody comprises an Fc region containing the amino acid sequence of SEQ ID NO:129. The CB2 antibody described herein may also 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 contains 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 that includes, from the N-terminus to the C-terminus, an ISVD, a peptide linker, a hinge region, and an Fc region. In some embodiments, the CB2 antibody does not include an Fc region. An exemplary hinge is provided: AESKYGPPCPPCP (SEQ ID NO:131) In some embodiments, the CB2 antibody comprises a polypeptide chain that includes, from the N-terminus to the C-terminus, an ISVD, a peptide linker and / or hinge region, and an Fc region.

[0083] 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 in Table 2.

[0084] In some embodiments, the CB2 antibody described herein comprises a CB2 antibody that competitively and specifically binds to CB2 against any of the other CB2 antibodies described herein. In some embodiments, competitive binding can be determined using an ELISA assay. In some embodiments, the CB2 antibody competitively and specifically binds to CB2 against a CB2 antibody comprising CDR1, CDR2, and CDR3 of ISVD sequences from any of SEQ ID NO:500-899, wherein CDR is defined according to any of Kabat, IMGT, or Chothia (for an explanation of IMGT, see Lefranc et al.). Dev. Comparat. Immunol.27:55-77, 2003 and Ruiz et al., Nucleic Acids Res. 29(1):207-9, 2001). In some embodiments, the CB2 antibody competitively and specifically binds to CB2 against the CB2 antibodies according to Table 1. In some embodiments, the K-value of the binding between these competing CB2 antibodies and CB2 is... D It is about 10 -7 M to approximately 10 -12 M (such as about 10) -8 M to approximately 10 -12 M, or approximately 10 -9 M to approximately 10 -11 M). In some embodiments, the CB2 antibody cross-reacts with CB2 derived from non-human mammals. In some embodiments, the competing CB2 antibody is camel-derived, chimeric, human, partially humanized, or fully humanized.

[0085] In some aspects, a cannabinoid receptor type 2 (CB2) antibody is provided, comprising an immunoglobulin single variable domain (ISVD) that specifically binds to CB2, wherein the ISVD comprises complementarity-determining regions (CDRs) 1, CDR2, and CDR3 of the ISVD containing the sequence of SEQ ID NO: 505. In some aspects, a cannabinoid receptor type 2 (CB2) antibody is provided, comprising an immunoglobulin single variable domain (ISVD) that specifically binds to CB2, wherein the ISVD comprises complementarity-determining regions (CDRs) 1, CDR2, and CDR3 of the ISVD containing the sequence of SEQ ID NO: 715. In some aspects, a cannabinoid receptor type 2 (CB2) antibody is provided, comprising an immunoglobulin single variable domain (ISVD) that specifically binds to CB2, wherein the ISVD comprises complementarity-determining regions (CDRs) 1, CDR2, and CDR3 of the ISVD containing the sequence of SEQ ID NO: 716. In some embodiments, the CB2 antibody comprises an ISVD containing 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 with 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 containing the sequence of SEQ ID NO:716, or a variant thereof having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO:716, wherein the CB2 antibody comprises CDR1, CDR2, and CDR3 of the ISVD containing the sequence of SEQ ID NO:716. In some embodiments, the ISVD is a variable domain (VHH) of the heavy chain of a heavy chain antibody. In some embodiments, the ISVD is of camel origin. In some embodiments, the ISVD is chimeric. In some embodiments, the ISVD is humanized.

[0086] In some embodiments, the CB2 antibody includes a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:1 as defined by IMGT, a CDR2 containing the amino acid sequence of SEQ ID NO:10 as defined by IMGT, and a CDR3 containing the amino acid sequence of SEQ ID NO:19 as defined by IMGT.

[0087] In some embodiments, the CB2 antibody includes a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:4 as defined by IMGT, a CDR2 containing the amino acid sequence of SEQ ID NO:13 as defined by IMGT, and a CDR3 containing the amino acid sequence of SEQ ID NO:22 as defined by IMGT.

[0088] In some embodiments, the CB2 antibody includes a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:7 as defined by IMGT, a CDR2 containing the amino acid sequence of SEQ ID NO:16 as defined by IMGT, and a CDR3 containing the amino acid sequence of SEQ ID NO:25 as defined by IMGT.

[0089] In some embodiments, the CB2 antibody includes a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:2 as defined by Kabat, a CDR2 containing the amino acid sequence of SEQ ID NO:11 as defined by Kabat, and a CDR3 containing the amino acid sequence of SEQ ID NO:20 as defined by Kabat.

[0090] In some embodiments, the CB2 antibody includes a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:5 as defined by Kabat, a CDR2 containing the amino acid sequence of SEQ ID NO:14 as defined by Kabat, and a CDR3 containing the amino acid sequence of SEQ ID NO:23 as defined by Kabat.

[0091] In some embodiments, the CB2 antibody includes a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:8 as defined by Kabat, a CDR2 containing the amino acid sequence of SEQ ID NO:17 as defined by Kabat, and a CDR3 containing the amino acid sequence of SEQ ID NO:26 as defined by Kabat.

[0092] In some embodiments, the CB2 antibody includes a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:3 as defined by Chothia, CDR2 containing the amino acid sequence of SEQ ID NO:12 as defined by Chothia, and CDR3 containing the amino acid sequence of SEQ ID NO:21 as defined by Chothia.

[0093] In some embodiments, the CB2 antibody includes a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:6 as defined by Chothia, CDR2 containing the amino acid sequence of SEQ ID NO:15 as defined by Chothia, and CDR3 containing the amino acid sequence of SEQ ID NO:24 as defined by Chothia.

[0094] In some embodiments, the CB2 antibody includes a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:9 as defined by Chothia, CDR2 containing the amino acid sequence of SEQ ID NO:18 as defined by Chothia, and CDR3 containing the amino acid sequence of SEQ ID NO:27 as defined by Chothia.

[0095] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:1 as defined by IMGT, CDR2 containing the amino acid sequence of SEQ ID NO:10 as defined by IMGT, and CDR3 containing the amino acid sequence of SEQ ID NO:19 as defined by IMGT.

[0096] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:4 as defined by IMGT, a CDR2 containing the amino acid sequence of SEQ ID NO:13 as defined by IMGT, and a CDR3 containing the amino acid sequence of SEQ ID NO:22 as defined by IMGT.

[0097] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:7 as defined by IMGT, a CDR2 containing the amino acid sequence of SEQ ID NO:16 as defined by IMGT, and a CDR3 containing the amino acid sequence of SEQ ID NO:25 as defined by IMGT.

[0098] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:2 as defined by Kabat, CDR2 containing the amino acid sequence of SEQ ID NO:11 as defined by Kabat, and CDR3 containing the amino acid sequence of SEQ ID NO:20 as defined by Kabat.

[0099] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:5 as defined by Kabat, CDR2 containing the amino acid sequence of SEQ ID NO:14 as defined by Kabat, and CDR3 containing the amino acid sequence of SEQ ID NO:23 as defined by Kabat.

[0100] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:8 as defined by Kabat, CDR2 containing the amino acid sequence of SEQ ID NO:17 as defined by Kabat, and CDR3 containing the amino acid sequence of SEQ ID NO:26 as defined by Kabat.

[0101] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:3 as defined by Chothia, CDR2 containing the amino acid sequence of SEQ ID NO:12 as defined by Chothia, and CDR3 containing the amino acid sequence of SEQ ID NO:21 as defined by Chothia.

[0102] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:6 as defined by Chothia, CDR2 containing the amino acid sequence of SEQ ID NO:15 as defined by Chothia, and CDR3 containing the amino acid sequence of SEQ ID NO:24 as defined by Chothia.

[0103] In some embodiments, the CB2 antibody comprises an ISVD containing a binding domain comprising CDR1 containing the amino acid sequence of SEQ ID NO:9 as defined by Chothia, CDR2 containing the amino acid sequence of SEQ ID NO:18 as defined by Chothia, and CDR3 containing the amino acid sequence of SEQ ID NO:27 as defined by Chothia.

[0104] CB2 Cannabinoid receptor type 2 (CB2) is a G protein-coupled receptor from the cannabinoid receptor family. In humans, it is composed of… CNR2The gene encodes a receptor for cannabinoid receptor type 1 (CB1). CB1 agonism is the primary cause of the psychoactive effects of cannabis, particularly tetrahydrocannabinol (THC). Due to the psychoactive effects of CB1 agonism, antibodies that specifically bind to CB2 but do not exhibit CB1-specific binding are required. Therefore, in one respect, the CB2 antibody presented herein is specific to CB2 and does not exhibit significant affinity for CB1. The CB2 receptor contains seven transmembrane domains, including a glycosylated N-terminus and an intracellular C-terminus.

[0105] The terms “cannabinoid receptor type 2,” “CB2,” “CB2 antigen,” and “CB2 epitope” are used interchangeably and include variants, isoforms, species homologs, and analogs of human CB2 that share at least one common epitope with CB2. In some embodiments, CB2 is human CB2, and CB2 antibodies specifically bind to human CB2. The CB2 gene and polypeptide sequence (e.g., human...) CNR2 Gene and polypeptide sequences are known in the art. An exemplary human CB2 sequence is provided with GenBank accession number P34972. In some embodiments, human CB2 may be at least about 90%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to human CB2 with GenBank accession number P34972. In some embodiments, the human CB2 sequence has no more than about 10 amino acid differences relative to the human CB2 sequence with GenBank accession number P34972. In some embodiments, human CB2 may exhibit no more than 5, 4, 3, 2, or 1 amino acid differences relative to human CB2 with GenBank accession number P34972. In some embodiments, the human CB2 sequence may differ from human CB2 with GenBank accession number P34972 due to, for example, conserved mutations or mutations in non-conserved regions, and the CB2 may have substantially the same biological function as human CB2 with GenBank accession number P34972.

[0106] In some embodiments, the CB2 antibody described herein specifically recognizes a CB2 polypeptide that shares at least about 90% amino acid sequence identity with human CB2 (Genbank accession number P34972). In some embodiments, the CB2 antibody may cross-react with CB2 from species other than humans (such as rat, murine, and / or cynomolgus monkey CB2) or other proteins structurally related to human CB2 (e.g., human CB2 homologs). In some embodiments, the CB2 antibody does not specifically bind to CB1.

[0107] biological activity The CB2 antibody described herein comprises an ISVD (e.g., from VHH or camelids) and has one or more of the following characteristics: (a) the ISVD is an agonist of CB2; (b) the ISVD does not agonize CB1; (c) the ISVD is present at a half-maximal effective concentration (EC50) of less than 55 nM. 50 (d) Reduce the level of trichodin-induced cyclic adenosine monophosphate (cAMP) in HEK293 cells overexpressing human CB2; (d) ISVD with EC50 levels less than 30 nM 50 (e) Reduced the level of trichodin-induced cyclic adenosine monophosphate (cAMP) in HEK293 cells overexpressing mouse CB2; and (f) ISVD nonspecifically bound to CB1; and (c) CB2 antibody at EC50 concentrations less than 850 nM. 50 HEK293 cells that overexpress human CB2.

[0108] The CB2 antibody described herein binds to CB2 with high affinity and specificity. In some embodiments, the CB2 antibody has a K0 of about 0.001 nM to about 10 nM (e.g., 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less) (inclusive of any value or range between these values). D Binding to human CB2. In some embodiments, the CB2 antibody is expressed at a concentration of about 0.001 nM to about 10 nM (e.g., 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less) or 0.001 nM or less (inclusive of any value or range between these values) with K. D The antibody is conjugated to cynomolgus monkey CB2. In some embodiments, the CB2 antibody is expressed at a concentration of approximately 0.001 nM to approximately 10 nM (e.g., 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less) (inclusive of any value or range between these values). D Binding to rat CB2. In some embodiments, the CB2 antibody is at a concentration of about 0.001 nM to about 10 nM (e.g., 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less) (inclusive of any value or range between these values) with K. D Combined with the rodent CB2.

[0109] In some embodiments, the CB2 antibody is a selective CB2 agonist. In some embodiments, the CB2 antibody is a selective CB2 agonist if it exhibits detectable CB2 agonist activity but not detectable CB1 agonist activity. In some embodiments, the CB2 antibody is a selective CB2 agonist if, as assessed by a reduction in cAMP production induced by laryngin in HEK293 cells overexpressing CB2 (for CB2 agonist assessment) or overexpressing CB1 (for CB1 agonist assessment), it exhibits detectable CB2 agonist activity but not detectable CB1 agonist activity. In some embodiments, the agonist activity is undetectable if the reduction in cAMP compared to the control group is not statistically significant (e.g., when p not < 0.05). In some embodiments, the CB1 agonist activity is undetectable if the measured level of activity differs by orders of magnitude between CB2 and CB1 agonist activity. In some implementations, the agonistic activity of the CB2 antibody (such as against CB1 or CB2) is evaluated at a concentration of about 1 µM.

[0110] In some embodiments, the CB2 antibody selectively binds to CB2 and non-selectively binds to CB1. In some embodiments, if the CB2 antibody has a K value 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 higher (inclusive of values ​​and ranges therein), D When CB1 is bound, the CB2 antibody does not selectively bind to CB1. In some embodiments, CB1 is human, rat, murine, or cynomolgus monkey CB1.

[0111] The CB2 antibody presented in this article targets human CB2, rat CB2, murine CB2, and / or cynomolgus monkey CB2. D and k off The assay can be performed using any method known in the art, including but not limited to, ELISA, fluorescence activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), and surface plasmon resonance (SPR). In some embodiments, the CB2 antibody provided herein targets human CB2, murine CB2, rat CB2, and / or cynomolgus monkey CB2. D and / or k off It is determined by SPR. In some implementations, the K of the CB2 antibody provided herein is... D and / or k off It was determined by surface plasmon resonance (SPR).

[0112] The properties of the CB2 antibody described herein can be evaluated using well-known methods (such as those used in the examples below). In some embodiments, the CB2 antibody does not bind (e.g., specifically binds) to CB1, such as human CB1.

[0113] The biological activity of the CB2 antibody described in this article can be measured by measuring its half-maximum effective concentration (EC50). 50 The half-maximum effective concentration (MCC) is used to determine the effectiveness of an antibody in binding to its target. For example, EC... 50 It can be used to indicate the effective concentration of CB2 antibody required to bind 50% of CB2 on the cell surface. EC 50 This also represents the plasma concentration required to achieve 50% of the maximum effect in vivo. EC 50 Measurements can be performed using assays known in the art, such as bioassays, including FACS binding assays, ligand binding inhibition assays (competitive binding assays) performed via FACS analysis, cell-based cytokine release assays, or enhanced luminescent near-homogeneous assays (AlphaLISA).

[0114] For example, the agonist activity of CB2 antibodies can be studied by measuring the decrease in cAMP levels induced by trichodin in HEK293 cells that overexpress human CB2 (but not CB1 if CB2 agonism is being assessed) or overexpress human CB1 (but not CB2 if CB1 agonism is being assessed), as described in Example 2 below. The results of this study demonstrate that CB2 antibodies can agonize CB2 but not CB1, and allow for comparison of CB2 antibodies with other CB1 or CB2 agonists.

[0115] The binding of CB2 antibody to CB2 agonists such as EC can be investigated by evaluating the binding of CB2 antibody to CB2-expressing stable HEK293 cells. 50 Similarly, the binding of CB2 antibody to CB1 agonist EC can be investigated by evaluating the binding of CB2 antibody to CB1-expressing HEK293 stable cells. 50 As discussed in Example 2 below, after incubating CB2 antibody with stable HEK293 cells expressing CB1 or CB2 in the presence of tuftin, cells were lysed by adding tracers and detection reagents. Following incubation, cAMP levels and ECMO levels were quantified using FRET. 50 The calculations. In some embodiments, the CB2 antibody described herein has no detectable agonist activity against CB1 (such as human, mouse, cynomolgus monkey, and / or rat CB1).

[0116] (II) Fusion Construction This application further provides an anti-CB2 fusion construct comprising any one of the CB2 antibody or antigen-binding fragments (e.g., ISVD or VHH) described herein, and a second polypeptide, such as a second antibody or its antigen-binding fragment, or an Fc fragment of an immunoglobulin. In some embodiments, the anti-CB2 construct comprises two or more polypeptides in addition to a CB2 antibody. One or more additional polypeptides may or may not alter or otherwise affect 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.

[0117] In some embodiments, the anti-CB2 construct comprises a second antibody or its antigen-binding fragment (such as ISVD, scFv, Fab, full-length antibody, etc.) that specifically recognizes the second epitope. In some embodiments, the second epitope is derived from CB2. In some embodiments, the second epitope is not derived from CB2. In some embodiments, the second antibody specifically recognizes the same epitope on CB2 as the CB2 antibody described herein. In some embodiments, the second antibody specifically recognizes a different epitope on CB2 than the CB2 antibody described herein.

[0118] In some embodiments, an anti-CB2 construct is provided comprising a plurality (e.g., 2, 3, 4 or more) of the CB2 antibodies described herein. In some embodiments, the plurality of CB2 antibodies are fused together via a linker (such as a peptide linker). The plurality of CB2 antibodies may be identical or different.

[0119] In some embodiments, the CB2 antibody comprises a second polypeptide that, compared to the CB2 antibody alone, enhances the half-life, solubility, and / or absorption of the CB2 antibody, reduces the immunogenicity or toxicity of the CB2 antibody, eliminates or mitigates adverse side effects of the CB2 antibody, imparts other advantageous properties to the CB2 antibody, and / or reduces other undesirable properties of the CB2 antibody. Some non-limiting examples of such polypeptides include serum proteins, such as human serum albumin (HSA; see, for example, WO 2000 / 027435) or hapten molecules (e.g., haptens recognized by circulating antibodies, see, for example, WO 98 / 22141). It has been shown that fragments of immunoglobulins (such as V...) H Linking a domain to serum albumin or a fragment thereof can increase the antibody's half-life (see, for example, WO00 / 27435 and WO 01 / 077137). Therefore, in some embodiments, the CB2 antibody comprises a CB2 antibody optionally fused to serum albumin or a fragment thereof via a suitable linker (such as a peptide linker). In some embodiments, the serum albumin contains at least domain III (see PCT / EP2007 / 002817).

[0120] CB2 only heavy chain antibody (HCAb) In some embodiments, the CB2 antibody is a heavy-chain-only antibody (HCAb) comprising the CB2 ISVD described herein. In some embodiments, the CB2 ISVD is combined with one or more C... H 2 and / or C H 3. Structural domains, such as Fc fragment fusion. In some implementations, C H 2 and / or C H The 3-domain is derived from human immunoglobulin. In some embodiments, the CB2 antibody binds to C via a peptide linker. H 2 and / or C H 3. Domain fusion. C H 2 and / or C H The 3-domain can increase the half-life of CB2 antibodies in vivo.

[0121] Therefore, in some embodiments, an isolated CB2 HCAb is provided comprising a CB2 ISVD (meaning an ISVD specifically binding to CB2) 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 IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc fragment is a human Fc, such as human IgG1 (hIgG1) Fc, hIgG2 Fc, or hIgG4 Fc. In some embodiments, the Fc fragment is effector-free, and its antibody effector functions, such as ADCC, CDC, and / or ADCP (antibody-dependent phagocytosis), are reduced, minimized, or eliminated. In some embodiments, the effector-free Fc comprises a fragment located at C... HThe Fc fragment contains an N297A or DANA mutation (D265A+N297A) in region 2. In some embodiments, the effectorless Fc contains K322A and L234A / L235A (LALA) mutations. In some embodiments, the effectorless Fc contains F234A and L235A (FALA) mutations. In some embodiments, the Fc fragment is an effectorless IgG1 Fc, such as an effectorless hIgG1 Fc. In some embodiments, the Fc fragment is human IgG4 Fc (S228P). In some embodiments, the Fc fragment is IgG4 Fc (FALA). In some embodiments, the CB2 HCAb is monomeric. In some embodiments, the CB2 HCAb is dimer. In some embodiments, the CB2 HCAb is multispecific and multivalent (such as bispecific and divalent), for example, containing two or more different CB2 antibodies as described herein. In some implementations, the CB2 HCAb is monospecific and multivalent (e.g., bivalent), such as containing two or more copies of the same CB2 ISVD. An exemplary IgG4 Fc is provided: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:130) An example IgG1 Fc sequence is also provided: EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:126) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:127) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:128) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:129) 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 (G4S)3 (SEQ ID NO:132).

[0122] Therefore, in some embodiments, an isolated CB2 HCAb is provided, comprising an ISVD that specifically recognizes CB2, wherein the CB2 antibody comprises CDR1, CDR2 and CDR3 of an ISVD sequence from any of SEQ ID NO:500-899, and wherein the CB2 ISVD is fused to the Fc fragment of an immunoglobulin.

[0123] In some embodiments, an isolated CB2 HCAb is provided comprising an ISVD that specifically recognizes CB2, wherein the ISVD comprises an amino acid sequence of any of SEQ ID NO:500-899 or a variant thereof having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of SEQ ID NO:500-899, and wherein the CB2 antibody is fused to the Fc fragment of an immunoglobulin. In some embodiments, an isolated CB2 HCAb is provided comprising an ISVD that specifically recognizes CB2, wherein the ISVD comprises an amino acid sequence of any of SEQ ID NO:500-899, and wherein the CB2 antibody is fused to the Fc fragment of an immunoglobulin. In some embodiments, the CB2 antibody is fused to the Fc fragment via a peptide linker. In some embodiments, the CB2 HCAb is monomeric. In some implementations, the Fc fragment is human IgG1 Fc, human IgG1 Fc without effector function, hIgG2 Fc, human IgG4 Fc, IgG4 Fc (F234A / L235A), or IgG1 (L234A / L235A, where the numbering is based on the EU index).

[0124] Also provided is an isolated CB2 HCAb that competitively and specifically binds to CB2 with either the isolated CB2 HCAb or CB2 antibody described herein.

[0125] peptide linkers Various domains and components in a CB2 antibody construct, such as the CB2 ISVD (meaning an ISVD that specifically binds to CB2), the Fc fragment, the first antigen-binding moiety, and the second antigen-binding moiety, can be fused together via suitable linkers (such as peptide linkers). The length, flexibility, and / or other characteristics of one or more peptide linkers used in a CB2 antibody may have some influence on properties including, but not limited to, affinity, specificity, or affinity for one or more specific antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not spatially interfere with each other. In some embodiments, the peptide linker contains flexible residues (such as glycine and serine) to allow adjacent domains to move freely relative to each other. For example, a glycine-serine duplex may be a suitable peptide linker.

[0126] The peptide linker can have any suitable length. In some embodiments, the length of the peptide linker is at least about 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. In some embodiments, the length of the peptide linker is no more than about 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. In some embodiments, the length of the peptide linker is any one 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, 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.

[0127] 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 an antibody containing only the heavy chain can be used as the linker. See, for example, WO1996 / 34103. In some embodiments, the linker is a (G4S)3 linker (SEQ ID NO:132).

[0128] (III) Antibody variants In some embodiments, consider amino acid sequence variants of the CB2 antibody provided herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the precursor condition is that the final construct possesses the desired properties, such as antigen binding.

[0129] a) Substitution, insertion, deletion, and variants In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” in Table 4. More substantial variations are provided under the heading “Exemplary Substitutions” in Table 4 and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or modified ADCC or CDC.

[0130] Amino acids can be grouped according to their common side chain characteristics: (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0131] Non-conservative replacement would require replacing members of one of these categories with members of another category.

[0132] In some embodiments, the CB2 antibody provided herein comprises an ISVD containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the amino acid sequences in SEQ ID NO: 500-899. In some embodiments, the ISVD sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the CB2 antibody containing this sequence retains its ability to bind CB2 (e.g., human CB2, murine CB2, rat CB2, and / or cynomolgus monkey CB2). In some embodiments, a total of 1 to 10 amino acids in any of the SEQ ID NO: 500-899 have been substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR). In some embodiments, the CB2 antibody comprises the ISVD group shown in any of SEQ ID NO:500-899, including post-translational modifications of that sequence.

[0133] One type of substitution variant involves substituting one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant chosen for further research will have certain biological properties modified (e.g., improved) relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will have some biological properties of the parent antibody substantially retained. An exemplary substitution variant is an affinity-matured antibody that can be readily generated.

[0134] A useful method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a group of residues or target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0135] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a peptide that extends the serum half-life of the antibody.

[0136] b) Glycosylation variants In some implementations, the isolated CB2 antibody provided herein has been modified to increase or decrease the degree of glycosylation in the construct. Adding or removing glycosylation sites from the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0137] In cases where a CB2 antibody contains an Fc region (e.g., a CB2 antibody-Fc fusion protein (e.g., HCAb)), the carbohydrates to which it is attached can be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to the C-terminal region of the Fc region via N-bonds. H 2. Asn297 of the structural domain. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharide in the CB2 antibody of this application may be modified to create antibody variants with certain improved properties.

[0138] In some embodiments, CB2 antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the glycan chain relative to the sum of all glycan structures attached to Asn 297 (e.g., complex, heterogeneous, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (EU number of the Fc region residue) in the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297 due to minor sequence variations in the antibody, i.e., between position 294 and position 300. Such fucoidylated variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Publicly disclosed examples involving "defucosylated" or "fucosylated" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 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; WO2005 / 053742; WO2002 / 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 defucosylation antibodies include Lec13 CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11); and gene knockout cell lines, such as α-1,6-fucosyltransferase gene. FUT8Gene knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al. Biotechnol. Bioeng ., 94(4):680-688 (2006); and WO2003 / 085107).

[0139] Further, CB2 antibody variants with bisected oligosaccharides are provided, for example, wherein the 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, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having 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, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0140] c) Fc region variants In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the CB2 antibody provided herein, thereby generating an Fc region variant. This Fc region variant may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) located at one or more amino acid positions.

[0141] In some embodiments, this application covers CB2 antibodies containing Fc region variants with some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the CB2 antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore potentially lacks ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet. Annu. Rev. Immunol Table 2 on page 464 of 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al.). Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I et al. Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al.) J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox 96). ® Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells that can be used for this type of assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, assays can be performed in vivo, such as in animal models, e.g., Clynes et al. Proc. Nat'l Acad. Sci. USA The ADCC activity of the molecule of interest was assessed in the animal models disclosed in 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al.). J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB, et al.). Int'l. Immunol. 18(12):1759-1769 (2006)).

[0142] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitutions of residues 265 and 297 to alanine (US Patent No. 7,332,581). Fc mutants are described in Schlothauer et al. Protein Eng Des Sel October 2016; 29(10):457-466; Pejchal et al. Antibodies . 2023; 12(3):54;Dall'Acqua et al. J Biol Chem August 18, 2006; 281(33):23514-24; Ramdani et al. International Journal of Molecular Sciences These references are hereby incorporated by way of citation in . 2022; 23(17):9604.

[0143] Certain antibody variants with improved or weakened binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al.) J. Biol. Chem. 9(2): 6591-6604 (2001). In some embodiments, the CB2 antibody variant includes an Fc region having one or more amino acid substitutions that reduce ADCC activity, such as substitutions at positions 298, 333, and / or 334 (EU numbers of residues) in the Fc region.

[0144] In some implementations, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie, etc. J. Immunol. 164: 4178-4184 (2000).

[0145] In some embodiments, CB2 antibodies are provided that comprise a variant Fc region with one or more amino acid substitutions that increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). This increased half-life and improved binding to the neonatal Fc receptor (FcRn) (which is responsible for transferring maternal IgG to the fetus) (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. Antibodies binding to FcRn (24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain one or more substituted Fc regions that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more Fc region residues, for example, substitution at Fc region residue 434 (US Patent No. 7,371,826).

[0146] See also Duncan and Winter. Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which relate to other instances of Fc region variants.

[0147] In some respects, the CB2 antibodies covered in this article may include one or more modifications that occur during production, such as C-terminal lysine cleavage of the Fc region.

[0148] CB2 antibodies containing any Fc variant or combination thereof described herein are considered.

[0149] d) Antibody derivatives In some embodiments, the CB2 antibody provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used in a therapeutic manner under defined conditions.

[0150] In some embodiments, a conjugate of the CB2 antibody with a non-protein portion that can be selectively heated by exposure to radiation is provided. In some embodiments, the non-protein portion is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). Radiation may have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat non-protein portions to the temperature at which cells near the antibody-non-protein portion are killed.

[0151] In some embodiments, the CB2 antibody provided herein may be further modified to include one or more bioactive proteins, peptides, or fragments thereof. As used interchangeably herein, "bioactive" or "biologically active" means exhibiting biological activity in vivo to perform a specific function. For example, it may mean combining with a specific biomolecule such as a protein, DNA, etc., and then promoting or inhibiting the activity of that biomolecule. In some embodiments, the bioactive protein or fragment thereof includes proteins and peptides administered to a patient as active pharmaceutical substances to prevent or treat a disease or ailment, as well as proteins and peptides used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, and proteins and peptides administered to a patient to prevent disease, such as vaccines. In some embodiments, the bioactive protein or fragment thereof has immunostimulatory / immunomodulatory, membrane transport, or enzymatic activity. In some embodiments, the bioactive protein, peptide, or fragment thereof is an enzyme, hormone, growth factor, cytokine, or mixture thereof. In some embodiments, the bioactive protein, peptide, or fragment may specifically recognize a target peptide (such as an antigen or other protein).

[0152] In some embodiments, the bioactive protein or fragment thereof that may be included in the CB2 antibody described herein is a protein-binding protein. In some embodiments, the bioactive protein or fragment thereof that may be included in the CB2 antibody described herein is an antibody mimic, which is a small engineered protein containing an antigen-binding domain similar to an antibody (Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and contain a single polypeptide. Exemplary antibody mimics that may be included in the CB2 antibody described herein may include, but are not limited to: designed ankyrin repeat proteins (DARPin; containing 3-5 fully synthetic ankyrin repeat sequences flanked by N-terminal and C-terminal cap domains), affinity multimers (avimer; a high-affinity protein containing multiple A domains, each with low affinity for the target), or anticarrier proteins (lipocarrier-based scaffolds with four accessible loops, the sequence of each loop being randomized). In some embodiments, the bioactive protein or fragment thereof that may be included within the CB2 antibody described herein is an armadillo repeat protein (e.g., β-catenin, α-input protein, globin, adenomatous polyposis protein (APC)), which comprises an armadillo repeat unit (a characteristic repeating amino acid sequence of approximately 40 residues in length). Each armadillo repeat sequence consists of a pair of α-helices forming a hairpin structure. Multiple copies of this repeat form form a so-called α-solenoid structure. Armadillo repeat proteins are capable of binding different types of peptides in a constant binding mode dependent on the peptide backbone, without requiring specific conserved side chains or interactions with the free N-terminus or C-terminus of the peptide. Combined with the inherent modularity of repeat proteins, the possibility of recognizing peptide residues by residue makes armadillo repeat proteins promising candidates for designing universal scaffolds for peptide binding.

[0153] In some embodiments, the bioactive protein or fragment thereof that may be included in the CB2 antibody described herein is a ligand, such as lymphokines and cytokines that interact with specific cell receptors. Lymphokines are low-molecular-weight proteins secreted by T cells when they are stimulated to grow by antigens or lectins.

[0154] III. Pharmaceutical Composition This application further provides pharmaceutical compositions comprising any one of the CB2 antibodies described herein and optionally a pharmaceutically acceptable carrier. The pharmaceutical compositions can be prepared by mixing the CB2 antibody described herein with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)) in lyophilized or aqueous form.

[0155] The pharmaceutical composition is preferably stable, wherein the CB2 antibody described herein substantially retains its physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed herein. Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, MarcelDekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at selected temperatures over selected time periods. For rapid screening, the formulation can be stored at 40°C for 2 weeks to 1 month, during which time its stability can be measured. If the formulation is to be stored at 2°C–8°C, it should generally be stable at 30°C or 40°C for at least 1 month, and / or stable at 2°C–8°C for at least 2 years. If the formulation is to be stored at 30°C, it should generally be stable at 30°C for at least 2 years, and / or stable at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. In some embodiments, the stable formulation of the CB2 antibody described herein may contain less than about 10% (preferably less than about 5%) of the CB2 antibody present in the formulation in aggregate form.

[0156] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); preservatives, isotonic agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents, such as EDTA and / or nonionic surfactants.

[0157] Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; and proteins such as serum albumin, gelatin, or immunoglobulins. Proteins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; 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).

[0158] Buffers are used to control the pH within a range that optimizes therapeutic efficacy, especially if stability is pH-dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffers used in this application include both organic and inorganic acids and their salts. Examples include citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, and acetates. Additionally, buffers may include histidine and trimethylamine salts such as Tris.

[0159] Preservatives are added to slow microbial growth and are typically present in the range of 0.2%–1.0% (w / v). The addition of preservatives can, for example, facilitate the production of multipurpose (multi-dosage) formulations. Suitable preservatives used in this application include octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium halide (e.g., benzalkonium chloride, benzalkonium bromide, benzalkonium iodide), benzyl chloride; thimerosal, phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0160] Tensile agents (sometimes called "stabilizers") are present to adjust or maintain the liquid tension in a composition. When used with large, charged biomolecules such as proteins and antibodies, tensile agents are often referred to as "stabilizers" because they can interact with charged groups on the side chains of amino acids, thereby reducing the likelihood of intermolecular and intramolecular interactions. Tensile agents can be present in any amount from 0.1% to 25% by weight, preferably from 1% to 5%, taking into account the relative amounts of other components. Preferred tensile agents include polyhydroxy sugar alcohols, preferably trihydroxy or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol.

[0161] Additional excipients include agents that can act as one or more of the following: (1) build-up agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include: polyhydroxy sugar alcohols (listed 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, sorbitol, xylose, ribose, ribitol, inositol, galactose, galactitol, glycerol, cyclic alcohols (e.g., inositol), polyethylene glycol, etc. Alcohols; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; 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); trisaccharides, such as raffinose; and polysaccharides, such as dextrin or dextran.

[0162] The presence of nonionic surfactants or detergents (also known as "wetting agents") helps dissolve the therapeutic agent and protects the therapeutic protein from agitation-induced aggregation. This also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in the 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.

[0163] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamer (184, 188, etc.), and PURONIC. ® Polyols, TRITON ®Polyoxyethylene sorbitol monoether (TWEEN®-20, TWEEN®-80, etc.), polydocanol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzyl chloride.

[0164] For pharmaceutical compositions to be used in vivo, they must be sterile. Sterility can be achieved through filtration using a sterile filter membrane. The pharmaceutical compositions described herein are typically placed in containers with sterile access points, such as intravenous solution bags or vials with stoppers that can be pierced by a subcutaneous needle.

[0165] The route of administration is consistent with known and accepted methods, such as by single or multiple bolus injections or by infusion over a long period of time in an appropriate manner, for example by injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, or intra-articular routes, local application, inhalation, or by continuous or prolonged release.

[0166] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antagonists, in the form of shaped articles such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly(-)-3-hydroxybutyric acid.

[0167] The pharmaceutical compositions described herein may also contain more than one active compound, preferably those compounds having complementary activities that do not adversely affect each other, depending on the specific indication being treated. Alternatively, or additionally, the composition may contain cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, or growth inhibitors. Such molecules are suitably combined in amounts effective for the intended purpose.

[0168] The active ingredient can also be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules) prepared by cohesive techniques or interfacial polymerization, encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or encapsulated in crude emulsions. Such techniques are disclosed in… Remington's Pharmaceutical Sciences In the 18th edition.

[0169] In some embodiments, the pharmaceutical composition is contained in single-use vials, such as single-use sealed vials. In some embodiments, the pharmaceutical composition is contained in reusable vials. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is stored at low temperatures.

[0170] IV. Treatment Methods The CB2 antibodies and their compositions (such as pharmaceutical compositions) described herein can be used in a variety of applications, such as in diagnostics, molecular assays, and therapies.

[0171] In some embodiments, a method for activating CB2 on cells is provided, the method comprising contacting the cells with any of the CB2 antibodies described herein in an amount sufficient to activate CB2 on the cells. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, activating CB2 on cells can treat or improve a disease or ailment. Activation of CB2 in a subject can trigger acute and sustained analgesic effects.

[0172] In some embodiments, a method of treating a subject for a disease or ailment is provided, wherein CB2 activation improves the disease or ailment, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein. In some embodiments, the disease or ailment is chemotherapy-induced peripheral neuropathy (CIPN). In some embodiments, the disease or ailment is a respiratory infection. In some embodiments, the disease or ailment is liver fibrosis. In some embodiments, the disease or ailment is cold sensitivity. In some embodiments, the disease or ailment is inflammatory bowel disease. In some embodiments, the disease or ailment is endometriosis.

[0173] Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of chemotherapy used to treat cancer. CIPN is often associated with neurotoxic chemotherapeutic agents. CIPN is a painful, dose-limiting side effect that limits treatment tolerability, reduces patient outcomes, decreases treatment adherence, and diminishes quality of life. Paclitaxel-based drugs (such as paclitaxel) are commonly associated with CIPN. The CB2 antibody described in this article may be used to treat or improve CIPN, including paclitaxel-induced CIPN.

[0174] In some embodiments, a method is provided for treating chemotherapy-induced peripheral neuropathy (CIPN) in a subject receiving chemotherapy, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein. In some embodiments, the chemotherapy agent is a paclitaxel-based drug. In some embodiments, the paclitaxel-based drug is paclitaxel. In some embodiments, the chemotherapy agent is vincristine. In some embodiments, the chemotherapy agent is cisplatin.

[0175] In some embodiments, a method of treating cancer in a subject of need is provided, the method comprising administering to the subject an effective amount of a chemotherapy drug and an effective amount of a pharmaceutical composition comprising any one of the CB2 antibodies described herein. In some embodiments, the chemotherapy drug is a paclitaxel-based drug. In some embodiments, the paclitaxel-based drug is paclitaxel. In some embodiments, the chemotherapy drug is vincristine. In some embodiments, the chemotherapy drug is cisplatin. In some embodiments, the chemotherapy drug causes chemotherapy-induced peripheral neuropathy (CIPN).

[0176] A significant proportion of cancer patients receiving neurotoxic chemotherapy experience chemotherapy-induced peripheral neuropathy (CIPN). CIPN can affect a patient's health-related quality of life, treatment adherence, appetite, and may exacerbate pre-existing pain, among other negative effects. These effects combined may limit the dose of neurotoxic chemotherapy a patient can tolerate, where higher doses might otherwise be more effective in treating the indicated cancer. CIPN can be measured by any suitable means known in the art, including through questionnaire completion or the application of diagnostic protocols. Exemplary questionnaires and protocols for CIPN include the Functional Assessment of Cancer Therapy / Gynecologic Oncology Group Neurotoxicity Questionnaire (FACT / GOG-Ntx), the Chemotherapy-Induced Peripheral Neuropathy Assessment Tool (CIPNAT), the European Organization for Research & Treatment in Cancer Quality of LifeQuest-CIPN 20 (EORTC QLQ-CIPN 20), the Modified Total Neuropathy Score (mTNS), the Clinical Version of the Total Neuropathy Score (TNSc), and the 5-item Simplified Total Neuropathy Score (TNSr 5-item). Other measures can be used to assess the symptoms of CIPN, including the Fullerton Advanced Balance Scale (FAS), 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 Forward Extension Test, Short Physical Performance Battery (SPPB), and Groove Pegboard Test. The methods described in this article consider any suitable means of assessing CIPN.

[0177] In some embodiments, a method for improving chemotherapy-induced peripheral neuropathy (CIPN) is provided, the method comprising administering an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein. In some embodiments, CIPN is caused by a paclitaxel-based drug. In some embodiments, the paclitaxel-based drug is paclitaxel. In some embodiments, the chemotherapy agent is vincristine. In some embodiments, the chemotherapy agent is cisplatin.

[0178] In some embodiments, a method for treating chemotherapy-related side effects in a cancer patient is provided, the method comprising administering to the patient an effective amount of any of the CB2 antibodies described herein. In some embodiments, the side effect is CIPN or a CIPN-related side effect. In some embodiments, the method further comprises administering a chemotherapy drug. In some embodiments, the chemotherapy drug is a paclitaxel class. In some embodiments, the paclitaxel class is paclitaxel. In some embodiments, the chemotherapy drug is vincristine. In some embodiments, the chemotherapy drug is cisplatin.

[0179] In some embodiments, a method for improving the quality of life of a subject receiving chemotherapy is provided, the method comprising administering to the patient an effective amount of any of the CB2 antibodies described herein. Quality of life can be assessed by any suitable means known in the art, including a Quality of Life Questionnaire (QoLQ). QoLQ can be self-reported and / or agent-reported. The method described herein takes into account any suitable QoLQ. In some embodiments, the method further comprises administering a chemotherapy drug. In some embodiments, the chemotherapy drug is a paclitaxel class. In some embodiments, the paclitaxel class is paclitaxel. In some embodiments, the chemotherapy drug is vincristine. In some embodiments, the chemotherapy drug is cisplatin.

[0180] In some embodiments, a method for improving compliance in a subject receiving chemotherapy is provided, the method comprising administering to the patient an effective amount of any of the CB2 antibodies described herein. In some embodiments, the method further comprises administering a chemotherapy drug. In some embodiments, the chemotherapy drug is a taxane. In some embodiments, the taxane is paclitaxel. In some embodiments, the chemotherapy drug is vincristine. In some embodiments, the chemotherapy drug is cisplatin.

[0181] In some embodiments, a method is provided for increasing the dose of a chemotherapy regimen administered to a subject, the method comprising administering to the subject an effective amount of any 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 a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a taxane. In some embodiments, the taxane is paclitaxel. In some embodiments, the chemotherapeutic agent is vincristine. In some embodiments, the chemotherapeutic agent is cisplatin.

[0182] In some embodiments, a method for treating an individual suffering from a respiratory infection is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein. In some embodiments, the respiratory infection is an influenza infection, such as an influenza infection caused by influenza A virus or influenza B virus. In some embodiments, the influenza infection is caused by influenza A virus subtype H1N1. In some embodiments, the respiratory infection is caused by a coronavirus such as SARS-CoV-2 (where the respiratory infection is COVID-19).

[0183] In some embodiments, a method is provided to alleviate cytokine release syndrome in an individual in need, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein. In some embodiments, the method reduces the levels of IL-16 and / or IL-8 secreted by airway cells in the individual. In some embodiments, the method reduces 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 reduces 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 some implementations, the method reduces the secretion of IL-8 and IL-16 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, as well as values ​​and ranges therein.

[0184] In some embodiments, a method for treating liver fibrosis is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein. Liver fibrosis can be assessed by any suitable means known in the art, including by liver biopsy or non-invasive 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 of the CB2 antibodies described herein, thereby reducing the levels of one or more of COL1A1, ACTA2, IL6, IL10, or TNFα.

[0185] In some embodiments, a method for treating cold sensitivity and / or touch-evoked pain is provided, the method comprising administering to an individual in need an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein. Cold sensitivity and / or atypical pain can be assessed by any suitable method known in the art, including questionnaires and / or patient-reported ranking of cold sensitivity or atypical pain.

[0186] In some embodiments, a method for treating inflammatory bowel disease is provided, the method comprising administering to an individual in need an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein.

[0187] In some embodiments, a method for treating endometriosis is provided, comprising administering to an individual in need an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein.

[0188] In some embodiments, a method for treating diabetic peripheral neuropathy (DPN) is provided, comprising administering to an individual in need an effective amount of a pharmaceutical composition comprising any of the CB2 antibodies described herein.

[0189] The dosage and desired drug concentration of the pharmaceutical composition of this application may vary depending on the specific intended use. Determining the appropriate dosage or route of administration is entirely within the skill of a person skilled in the art. Animal studies provide reliable guidance for determining effective dosages for human therapy. Interspecies scaling of effective dosages can be performed following the principles established in the following literature: Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” Toxicokinetics and New Drug Development Edited by Yacobi et al., Pergamon Press, New York, 1989, pp. 42-46.

[0190] When administered in vivo using the CB2 antibody described herein (including pharmaceutical compositions containing the CB2 antibody described herein), the normal dose can vary from about 10 ng / kg mammalian body weight to about 100 mg / kg mammalian body weight, depending on the route of administration. Within the scope of this application, different formulations will be effective for different treatments and different conditions, and administration intended to treat a specific organ or tissue may require delivery in a manner different from that used for another organ or tissue. Furthermore, the dose can be administered by single or multiple administrations or by continuous infusion. For repeated administration over several days or longer, treatment will continue until the disease symptoms are suppressed as desired, depending on the condition. However, other dosing regimens may be useful. The progress of this therapy can be easily monitored using routine techniques and assays.

[0191] In some embodiments, the pharmaceutical composition is administered once (e.g., by bolus injection). In some embodiments, the pharmaceutical composition is administered multiple times (such as any number of times, such as 2, 3, 4, 5, 6, or more). If multiple administrations are performed, these administrations can be made via the same or different routes and can be performed at the same site or at alternative sites. The pharmaceutical composition can be administered once daily to once a year. The interval before administration can be any from about 24 hours to a year. The interval may also be irregular (e.g., as the tumor progresses). In some embodiments, there are no interruptions in the dosing schedule. A person skilled in the medical field can easily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0192] The pharmaceutical compositions of this application (including, but not limited to, reconstituted formulations and liquid formulations) are administered to an individual in need of treatment, preferably a human, according to known methods (such as intravenous administration as a bolus or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intravenous (iv), intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes). Reconstituted formulations can be prepared by dissolving the lyophilized CB2 antibody described herein in a diluent to disperse the protein therein. Exemplary pharmaceutically acceptable (safe and non-toxic to human administration) diluents suitable for use in this application include, but are not limited to, sterile water, water for sterile injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile physiological saline, Ringer's solution or dextran solution, or aqueous solutions of salts and / or buffers.

[0193] In some embodiments, the pharmaceutical composition is administered to an individual via subcutaneous (i.e., subcutaneous) administration. For this purpose, a syringe can be used to inject the pharmaceutical composition. However, other devices for administering the pharmaceutical composition are also available, such as injection devices; injection pens; auto-injector devices, needle-free devices; and subcutaneous patch delivery systems. In some embodiments, the pharmaceutical composition is administered to an individual intravenously. In some embodiments, the pharmaceutical composition is administered to an individual via infusion (such as intravenous infusion). Infusion techniques for immunotherapy are known in the art (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)).

[0194] V. Preparation Method The CB2 antibody described herein can be prepared using any method known in the art or as described herein. In some embodiments, a method for producing a CB2 antibody is provided, the method comprising: (a) culturing a host cell containing an isolated nucleic acid or vector encoding the CB2 antibody described herein under conditions that efficiently express the encoded CB2 antibody; and (b) obtaining the expressed CB2 antibody from the host cell. In some embodiments, step (a) further comprises producing a host cell containing an isolated nucleic acid or vector encoding the CB2 antibody described herein.

[0195] Methods for preparing antibodies containing ISVDs are known in the art. See, for example, Els Pardon et al., Nature. Protocol , 2014; 9(3): 674. sdAb (such as V H H) can be obtained using methods known in the art, such as by making Camelidae Hybridomas are obtained by immunizing a species (such as a camel or llama) with the virus, or by cloning a library of single-domain antibodies using molecular biology techniques known in the art and then selecting the single clone of the unselected library via ELISA or by using phage display.

[0196] To recombinantly produce ISVDs, the nucleic acid encoding the antibody is isolated and inserted into a reproducible vector for further cloning (DNA amplification) or expression. The DNA encoding a single-domain antibody can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to the 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. Typically, the preferred host cell is of prokaryotic or eukaryotic (usually mammalian) origin.

[0197] In some embodiments, a vector (e.g., an expression vector) comprising the nucleic acid described herein is provided. In some embodiments, a host cell comprising such nucleic acid or vector is provided. In some embodiments, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells, Expi293 cells, or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In some embodiments, the host cell is prokaryotic, such as Escherichia coli cells. In some embodiments, a method for preparing a CB2 antibody is provided, wherein the method comprises culturing a host cell (as provided above) comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody, and optionally, recovering the antibody from the host cell (or host cell culture medium).

[0198] To recombinantly produce the CB2 antibody, a nucleic acid encoding an antibody, such as that described above, is isolated and inserted into a vector for further cloning and / or expression in host cells. This nucleic acid can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0199] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be produced in bacteria, for example, particularly where glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Volume 248 (BKC Lo, ed., HumanaPress, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli. After expression, the antibody can be separated from bacterial paste in soluble fractions and can be further purified.

[0200] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in antibodies with partially or fully human glycosylation patterns. See Gerngross. Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0201] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous cells have been identified that can be used in conjunction with insect cells (particularly for transfecting fall armyworm). Spodoptera frugiperda Baculovirus strains (cells).

[0202] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (which describe PLATNIBODIES™ technology for producing antibodies in transgenic plants).

[0203] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 cell line (COS-7) transformed with SV40; human embryonic kidney lines (such as those by Graham et al., etc.). J. Gen Virol. 293 or 293 cells as described in 36:59 (1977); young hamster kidney cells (BHK); mouse Sertoli cells (e.g., Mather, Biol. Reprod. TM4 cells as described in 23:243-251 (1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); such as those by Mather et al. Annals, NY Acad. Sci. TRI cells as described in 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, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, No. 248 roll (BKC Lo, editor, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0204] VI. Products and medicine boxes Further provided are kits and articles comprising the isolated CB2 antibody described herein, a pharmaceutical composition comprising any CB2 antibody described herein, an isolated nucleic acid or vector encoding the antibody, or an isolated host cell comprising the isolated nucleic acid or vector encoding the CB2 antibody described herein. In some embodiments, a kit is provided comprising any of the pharmaceutical compositions described herein, and preferably includes instructions for its use.

[0205] The medicine box of this application is housed in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The medicine box may optionally provide additional components, such as cushioning agents and explanatory information. Therefore, this application also provides articles of manufacture including vials (e.g., sealed vials), bottles, wide-mouth jars, flexible packaging, etc.

[0206] Articles may include containers and labels or packaging inserts located on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials such as glass or plastic. Typically, the container contains a composition that is effective in treating the disease or condition described herein (such as cancer) and may have a sterile access point (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The label or packaging insert indicates that the composition is intended to treat the specific condition of an individual. The label or packaging insert will also include instructions on how to administer the composition to an individual. The label may indicate reconstitution and / or usage instructions. Containers containing pharmaceutical compositions may be multi-use vials, which allow for repeated administration of the reconstituted formulation (e.g., 2 to 6 doses). Packaging inserts refer to the instructions for use typically included in the packaging of commercially available therapeutic products, containing information about indications, use, dosage, administration, contraindications and / or warnings related to the use of such therapeutic products. Additionally, the product may include a second container containing pharmaceutically acceptable buffer solutions, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may also include other materials that are commercially and user-appropriate, including additional buffers, diluents, filters, needles, and syringes.

[0207] The medicine box or product may include a pharmaceutical composition and instructions for use in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies, containing multiple unit doses.

[0208] Example The following embodiments are intended to illustrate the invention only and should not be construed as limiting the invention in any way. The following embodiments and detailed descriptions are provided by way of illustration rather than limitation.

[0209] Example 1. Machine learning prediction of CB2 agonist antibodies.

[0210] Historically, antibody discovery and design have relied on laborious bioscanning techniques, accidental discovery of screening libraries, or complex, rationally engineered methods. When effective, these methods have often proven time-consuming, expensive, and limited in scope, and sometimes they fail to find any hits on difficult targets. The recently emerging protein language models (PLMs)—based on large language models (LLMs) originally developed for natural language—have ushered in a paradigm shift, revolutionizing how we explore, manipulate, and generate antibody sequences. Trained on massive datasets of antibody sequences and related properties such as binding, stability, and immunogenicity, PLMs now offer an enticing prospect of accelerating antibody discovery and design with unprecedented speed, accuracy, and creativity.

[0211] PLM has never been applied before to the problem of generating antibodies (i.e., "functional antibodies") that not only specifically bind to targets but also modulate the target's signaling activity to achieve therapeutic effects. PLM is primarily trained to predict the next amino acid (AA) in a given AA sequence or to fill in a masked set of AAs in a larger sequence. By training on a large corpus of observed sequences, PLM can learn the relationship between sequence and structure. Given functional information about sequences, a key obstacle to pre-training PLM in discovering and ultimately designing functional antibodies is the lack of large-scale, high-quality functional datasets.

[0212] Abalone Bio’s Functional Antibody Selection Technology (FAST) platform generates large-scale, high-quality functional datasets of antibody sequence-function relationships for targets such as GPCRs. Data generated from agonist antibody selection activities targeting receptor CB2 are merged. A pre-trained antibody PLM is fine-tuned into a binary classifier with antibody functions from our functional dataset. The trained model is used to score sequences on a validation and test set not yet presented to the model. Candidate sequences are then selected, and the model scores them as having a high probability of being functional antibodies. A subset of these high-probability functional sequences is tested by generating antibody proteins and testing their function using mammalian cell assays, and many sequences active in CB2 assay cell lines are identified.

[0213] Data preprocessing and scoringNGS data from the CB2 agonist discovery campaign were processed using the commercial service PipeBio. The discovery campaign started with a pool of sequences L, had four rounds of enrichment (E1-E4), and had parallel low-strictness (LS) branches (E1 LS, E2 LS, etc.) after round 1.

[0214] The original reads from the clusters were then merged, annotated, and transformed into “sequences” based on the unique CDR3 amino acid sequence, and the correct sequences were filtered out. A growth score was then defined for each sequence. Each sequence count was normalized to the total reads in the experiment using the formula log(cdr3_reads) - log(total_reads). A sequence was labeled a “grower” (i.e., a functional antibody promoting the growth of the FAST strain) if a) the normalized count in E2 was 20% greater than the normalized count in E1, b) the normalized count in low-rigidity round 2 (E2 LS) was 20% greater than the normalized count in the library (L), and c) the total count observed in HS 1, 2, and LS 1 plus L001 > 100. This scoring method yielded approximately 1% of the sequences labeled as growers.

[0215] Modeling LoRA was used to fine-tune BERT on classification tasks (grown or non-grown objects). Approximately 5% (133K out of 26M) of the model parameters were trained / fine-tuned.

[0216] An antibody named AB101 with an ISVD sequence was generated from the FAST platform: EVQLVESGGGLVQPGGSLRLSCAASGSIFSIMAMAWYRQAPGKEREWVSGIDTGGGTYYAESVKGRFTISNDNSKNTAYLQMNSLKPEDTAVYYCSGAIKYGSGRFDIKNYWGQGTQVTVSS (SEQ ID NO:55) First, the data was split into training and test sets in a single run. Sequences with the highest hits AB101 and CDR3 differing from AB101's CDR3 by less than 2 amino acids were excluded. Sequences with <80% similarity to the hV gene were also excluded. A weighted (inverse class frequency) sampler and AdamW were used for training, without hyperparameter optimization. ROC-AUC and mean precision score were calculated to evaluate the model, with a calculated ROC-AUC of 0.7 and an APS of 0.25.

[0217] The data is then divided into large development sets and small validation sets (including AB101). K-fold cross-validation and fine-tuning are implemented. The small validation dataset (approximately 10% of the sequences, including all sequences containing AB101 and its related sequences) is defined as above. The remaining large development sequence set is then randomly divided into 10 equal-sized folds. For each fold, training is performed on 90% of the training data and prediction on 10%. For each of the 10 models, training is performed as described above on the training set of each fold of the model.

[0218] The validation test set sequences were then scored by calculating and averaging the probabilities of being growth products (i.e., functional antibodies) for each of the 10 k-fold models. The probabilities of functional antibodies for each sequence in each fold test set were also scored using the individual fold models. Two hundred sequences from the validation test set (ML_V) and two hundred sequences from each of the individual k-fold test sets were provided for further analysis. These antibodies are summarized in Table 1.

[0219] Example 2. In vitro activation of an antibody that predicted the ML of the CB2 receptor inhibited cAMP signaling in RAW264.7 mouse macrophages.

[0220] The positive control agonist CB2 antibody ABt281 has the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGSIFSIMAMAWYRQAPGKEREWVSGIDTGGGTYYAESVKGRFTISNDNSKNTAYLQMNSLKPEDTAVYYCSGAIKYGSGRFDIKNYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:202) The positive control agonist CB2 antibody ABt285 has the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGSIFSIMAMAWYRQAPGKEREWVSGIDTGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCSGAIKYGSGRFDIKNYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:203) ABt269 is an anti-GFP VHH-Fc antibody used as a control, which has the following sequence: QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO:204) RAW264.7 cells were cultured and treated with the CB2 agonist HU308 (onternabez) or CB2 positive control agonist antibodies (ABt281, ABt285) or isotype controls (Abt269) or ABhit089-128 (see Table 1). ABhit089-128 represents 37 of the top 40 predicted agonist antibodies based on ML (Example 1). For both antibody concentrations (1 μM and 500 nM), the efficacy of CB2-mediated GI-cAMP signaling was measured using a cAMP-Glo ​​assay (Promega, V1501), where luminescence was inversely proportional to cAMP concentration. Application of HU308, ABt281, or ABt285 increased the luminescence signal in a dose-dependent manner. A low response was observed with the negative control antibody ABt269. Several ABhit antibodies showed dose-dependent changes in luminescence consistent with CB2 agonist activation. Figure 1 and Table 5) below.

[0221] Example 3. In vitro activation of ML-predicted agonist antibodies against the CB2 receptor inhibited cAMP signaling in RAW264.7 mouse macrophages, and CB2 antagonist treatment demonstrated the specificity of CB2 activation by blocking agonist activation.

[0222] RAW264.7 cells were cultured and treated with the CB2 agonists HU308, ABhit095, ABhit104, or ABhit114. The efficacy of CB2-mediated GI-cAMP signaling was measured using a cAMP-Glo ​​assay (Promega), where luminescence was inversely proportional to cAMP concentration. The application of HU308 and all ABhit antibodies increased luminescence in a dose-dependent manner. Figure 2A ).

[0223] Then, similar experiments were performed, in which RAW264.7 cells were cultured and treated with the cAMP activator NKH477 to stimulate cAMP production. Then, CB2 agonist HU308 or a CB2-specific agonist antibody (such as...) was used. Figures 2B to 2F Cells were treated with either the CB2-mediated GI-cAMP signaling efficacy shown in the illustration or the isotype control (Abt269). The luminescence efficacy was measured using the cAMP-Glo ​​assay (Promega), where luminescence was inversely proportional to cAMP concentration.

[0224] The use of CB2 agonists reduced NKH477-stimulated cAMP production (Table 6 below), while ABt269 did not affect cAMP production.

[0225] exist Figure 3 In this study, HU308 and ABhit antibodies were added to RAW264.7 cells at EC80 concentrations, followed by titration with the CB2 antagonist SR144528 to determine the increased CB2 specificity of luminescence. HU308, ABhit095, ABhit104, and ABhit114 showed dose-dependent luminescence reductions in antagonist-dependent amounts, indicating CB2-specific activation by HU308 and ABhit antibodies.

[0226] Example 4. In vitro activation of the CB2 receptor: Activation of pERK1 / 2 signaling in RAW264.7 mouse macrophages.

[0227] RAW264.7 cells were cultured, starved of serum for 4 hours, and exposed to the CB2-specific agonist HU-308 and CB2-specific agonist antibodies (e.g., ...). Figure 4A and Figure 4B (as shown in the figure) or the isotype control (ABt269). CB2-mediated pERK1 / 2 signaling efficacy was measured using the TR-FRET pERK1 / 2 kit (LANCE Ultra pERK1 / 2 kit, Perking Elmer, TRF4000). TR-FRET signaling was increased in a dose-dependent manner with the application of HU308 or CB2 agonist antibodies, while no ERK phosphorylation was observed with the isotype control ABt269 (Figure 4 and Table 6 below).

[0228] Example 5. ML-predicted CB2 agonist antibody recruits B-repressor protein 2 in RAW264.7 mouse macrophages.

[0229] RAW264.7 cells were cultured and transfected with B-Arr2 plasmid (20 ng, Revvity, PWTBARR2) and pcDNA3 plasmid (130 ng) using Lipofectamine 2000. Transfected cells were treated with the CB2-specific agonist HU308, CB2 agonist antibodies (ABhit094, ABhit0124, ABhit0125), or an allotype control (ABt269). CB2-mediated B-Arr2 recruitment was measured using the TR-FRET HB-Arr2 Recruitment Kit (HTRF B-Arr2 Recruitment Kit, Revvity, 62BDBARR2PEB). Application of HU308 or CB2 agonist antibodies increased TR-FRET signaling.

[0230] Table 6. CB2 agonist-mediated reduction in NKH477-stimulated cAMP production

[0231] Example 6. ML-predicted CB2 agonist antibodies reduce inflammatory and fibrotic factors in a precisely diced human liver slice model.

[0232] This precisely dissected human liver slice model (hPCLS) preserves all cell types within their native structures, thus uniquely capturing endogenous cell-cell interactions. PCLS from deidentified resected samples of two injured human male livers were used to test the activity of ABhit_0125 (100 nM and 1 μM), ABt269 isotype control (1 μM), and Alk5i (positive control) after 24 and 48 hours of incubation. Alk5i is a TGF-β type I receptor kinase (ALK5) inhibitor (CAS446859-33-2).

[0233] Human precisely dissected liver sections (hPCLS) are generated from the discarded remnants of surgically removed human livers. hPCLS generation is performed on resection margins with normal appearance around HCCs. The ischemic time between hepatectomy and PCLS generation is 3–4 hours. Cores with a diameter of 8 mm are generated from intact liver sections (200 μm thick) and collected from buffer trays using a soft brush. The sections are then transferred to six-well tissue culture plates (3 sections / well) filled with 37°C warm William's E GlutaMAX medium supplemented with 25 mM glucose and 50 μg / ml gentamicin. The sections are pre-incubated for 24 hours to allow the tissues to reach equilibrium. After pre-incubation, fresh medium containing any of the following is added: ABhit_0125 (100 nM and 1 μM), ABt269 isotype control (1 μM), and Alk5i (10 μM), followed by further incubation for 24 and 48 hours.

[0234] For efficacy assessment, at the end of drug treatment for each condition, the secretion of collagen 1a1 (Col1a1) into the culture medium was assessed by ELISA. Figure 6A At 48 hours, compared with the isotype control ABt269, 1 μM ABhit_0125 and Alk5i significantly reduced Col1a1.

[0235] In addition, total mRNA was extracted using tissue sections at the end of drug treatment for each condition. Collagen 1a1 (COL1A1) was quantified using RT-qPCR. Figure 6B ), α-smooth muscle actin (ACTA2, Figure 6C ), interleukin-6 (IL-6, Figure 6D Interleukin-10 (IL10) Figure 6E ) and tumor necrosis factor α (TNFα, Figure 6FmRNA levels. Treatment with ABhit_0125 resulted in a statistically significant reduction in these inflammatory or fibrotic factors, including COL1A1, ACTA2, IL6, IL10, and TNFα; however, no significant changes were observed when treated with ABt269 (an isotype control antibody).

Claims

1. A cannabinoid receptor type 2 (CB2) antibody comprising an immunoglobulin single variable domain (ISVD) that specifically binds to CB2, wherein the ISVD comprises a complementarity-determining region (CDR) 1, CDR2, and CDR3 of the ISVD containing a sequence of any one of SEQ ID NO:500-899.

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

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

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 according to any one of claims 1 to 5, wherein the ISVD comprises an amino acid sequence selected from the group consisting of SEQ ID NO:500-899, or a variant thereof having at least about 85% sequence identity with the amino acid sequence of any one of SEQ ID NO:500-899.

7. The CB2 antibody according to any one of claims 1 to 6, wherein the ISVD comprises CDR1 containing the amino acid sequence of SEQ ID NO: 1, 4 or 7; CDR2 containing the amino acid sequence of SEQ ID NO: 10, 13 or 16; and CDR3 containing the amino acid sequence of SEQ ID NO: 19, 22 or 25; wherein the CDR1, the CDR2 and the 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 according to any one of claims 1 to 9, wherein the antibody further comprises an Fc region.

11. The CB2 antibody of claim 10, wherein the Fc region is the Fc region of IgG1 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, wherein the numbering is based on the Kabat EU index.

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 IgG1 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 IgG1 Fc region exhibiting reduced effector function.

17. The CB2 antibody of claim 16, wherein the variant IgG1 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 IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:

127.

19. The CB2 antibody of claim 16, wherein the variant IgG1 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 IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:

128.

21. The CB2 antibody of claim 16, wherein the variant IgG1 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 IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:

129.

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

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

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

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

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

28. The CB2 antibody according to any one of claims 1 to 27, wherein the CB2 antibody does not stimulate CB1.

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

30. The CB2 antibody according to any one of claims 1 to 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 a CB2 antibody as described in any one of claims 1 to 30.

32. An expression vector comprising the nucleic acid as described in claim 31.

33. A host cell comprising the nucleic acid as described in claim 31 or the expression vector as described in claim 32.

34. A method for generating a CB2 antibody, comprising culturing a host cell as described in claim 33 under conditions for generating the CB2 antibody.

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

36. A pharmaceutical composition comprising a CB2 antibody as described in any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

37. A method of activating CB2 on cells, comprising contacting the cells with an amount of CB2 antibody as claimed in any one of claims 1 to 30 sufficient to activate CB2 on the cells.

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

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

40. A method for treating an individual in need suffering from a disease or ailment, wherein activating CB2 improves said disease or ailment, the method comprising administering to said individual an effective amount of the CB2 antibody as claimed in any one of claims 1 to 30.

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

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

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

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

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

46. ​​The method of claim 45, wherein the disease or ailment 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 for alleviating cytokine release syndrome in an individual in need, comprising administering to the individual an effective amount of the CB2 antibody as described in any one of claims 1 to 30.

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

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

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

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

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

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