Anti-CB2 antibodies and uses thereof for measuring cell surface CB2 expression in flow

Abstract

The present invention relates to novel antibodies that bind to human CB2. Furthermore, the present invention relates to a method of detecting a change in cell surface CB2 expression in response to treatment with a CB2 agonist by flow cytometry. The methods allow for the assessment and monitoring of target conjugation of CB2 agonists, thereby supporting preclinical and clinical development of CB2 agonists.

Description

Technical Field

[0001] The present invention relates to novel antibodies that specifically bind to human cannabinoid receptor 2 (CB2) and their use as target engagement biomarkers in preclinical assays in vitro as well as in clinical trials to monitor CB2 agonist-induced CB2 internalization. Background Art

[0002] Typically, monoclonal antibodies (mAbs) are produced against synthetic peptides derived from the predicted sequence of the target protein. Unfortunately, although these antibodies react strongly with peptides, they often fail to recognize native proteins. Another standard procedure for generating mAbs uses purified recombinantly expressed proteins. Prokaryotic expression systems are the most widely used expression hosts. However, when studying mammalian surface proteins, mammalian expression systems are usually required because they are more likely to produce functional proteins with appropriate disulfide bonds and post-translational modifications. Although the introduction of affinity tags simplifies purification, it is still generally difficult to obtain recombinant proteins in a native conformation with sufficient yield and purity. This is particularly applicable to membrane and membrane-associated proteins because they may lose their native structure during the purification process.

[0003] When attempting to generate mAbs that recognize native proteins, it is critical to use the target protein for its native identity not only during the immunization step but also for the screening procedure. Many standard hybridoma screening protocols utilize recombinant proteins immobilized on solid supports, which can significantly alter protein identity.

[0004] To generate mAbs that specifically recognize membrane-associated proteins in their native identity, we applied an approach that bypasses any need for purified recombinant proteins. This strategy utilizes antigens expressed on the surface of stably transfected mammalian cells to immunize mice and perform immunoassays such as testing seroconversion, hybridoma selection, and mAb characterization (Dreyer A. et al. BMC Biotechnology 2010, 10: 87).

[0005] Cannabinoid receptors are a class of cell membrane receptors belonging to the G protein-coupled receptor superfamily. There are currently two known subtypes, called cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 is mainly expressed in the central nervous system (i.e., cerebellar amygdala, hippocampus), and less in the periphery (liver, skeletal muscle, pancreas and fat). CB2 is encoded by the CNR2 gene and is mainly expressed peripherally on immune system cells (such as macrophages and other leukocyte subtypes) (Ashton, JC et al. Curr Neuropharmacol 2007, 5 (2), 73-80; Miller, AM et al. Br J Pharmacol 2008, 153 (2), 299-308; Centonze, D. et al. Curr Pharm Des 2008, 14 (23), 2370-42). Interestingly, the expression level of CB2 at the mRNA level is different between the immune cell populations under the resting state and the activated state, indicating that CB2 may play an important role in immunomodulation and function. In addition, CB2 receptors are also expressed in the gastrointestinal system (Wright, KL et al. Br J Pharmacol 2008, 153 (2), 263-70), and are widely distributed in the brain, mainly in microglia rather than neurons (Cabral, GA et al. Br J Pharmacol 2008, 153 (2): 240-51).

[0006] The discovery of the endocannabinoid system (ECS) in the central nervous system and various peripheral organs has prompted nonclinical studies to explore its role in health and disease. The results of these studies indicate the presence of the ECS in a variety of pathophysiological processes (Pacher, P. et al. FEBS J 2013; 280: 1918-43).

[0007] CB1 is known to be associated with the psychoactive effects of cannabinoids, but in peripheral tissues, receptor activation shows potent oxidative, inflammatory and profibrotic activity. In contrast, CB2 activation has been shown to protect tissues from damage (Cabral, GA et al. Expert Rev Mol Med 2009; 11: e3). For example, in animal models of chronic degenerative diseases (such as neurodegenerative inflammatory disorders, atherosclerosis and liver fibrosis), CB2 activation has been shown to reduce inflammatory, oxidative and fibrotic processes. Interestingly, in animal models, diabetes and other kidney diseases can be treated by CB2 agonism (Behl, T. et al. Diabetes Metab Res Rev 2016; 32: 251-9). In addition, CB2 activation has been shown to have ocular anti-inflammatory effects (Xu, H. et al. J Leukoc Biol 2007; 82: 532-41; Toguri, JT et al. Br J Pharmacol 2014; 171: 1448-61).

[0008] These data suggest that modulation of the endocannabinoid system by agonistic approaches may have a variety of potential benefits for acute, subchronic, and chronic conditions due to its anti-inflammatory, anti-fibrotic, and vascular health-promoting effects. For example, CB2 agonists may be beneficial in treating microvascular diabetic complications such as diabetic retinopathy (DR) and diabetic macular edema (DME) (Gruden, G. et al. Br J Pharmacol 2016; 173: 1116-27).

[0009] CB2 is a member of the G protein-coupled receptor family, which can respond to ligand binding by internalizing the receptor into the endosomal compartment of the cell, thereby removing the corresponding receptor from the cell surface and initiating downstream signaling effects (Calebiro, D. et al. Best Pract Res Clin Endocrinol Metab 2018: 32 (2): 83-91). Therefore, cell surface expression of CB2 may be suitable as a biomarker for CB2 agonist target engagement. There is a need for detecting changes in CB2 cell surface expression to enable monitoring of drug efficacy in vitro, in vivo and clinical settings, and to support sensitive and specific assays that link CB2 target engagement to downstream PD effects and clinical effects in corresponding indications. Summary of the invention

[0010] The invention disclosed herein provides an antibody comprising an antigen binding portion that binds to human cannabinoid receptor subtype 2 (CB2), wherein the antibody comprises: a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 5, HCDR 2 of SEQ ID NO: 6, and HCDR 3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9, and LCDR 3 of SEQ ID NO: 10. In one aspect, the antibody comprises: a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2.

[0011] In one aspect, the antibody comprises an Fc domain. In another aspect, the Fc domain is an IgG class Fc domain, particularly an IgG3 subclass Fc domain. In yet another aspect, the Fc domain is a mouse Fc domain.

[0012] In another aspect, the antibody comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4.

[0013] In one embodiment, the antibody cross-reacts with cynomolgus monkey CB2.

[0014] In addition, the present invention provides a method for detecting changes in cell surface CB2 expression in response to treatment with a CB2 agonist in a biological sample obtained from a subject by flow cytometry, the method comprising: a) detecting the cell surface CB2 expression level in a reference sample that has not been treated with a CB2 agonist, b) detecting the cell surface CB2 expression in a sample after treatment with a CB2 agonist, and c) comparing the cell surface CB2 expression level of step b) with the cell surface CB2 expression level of step a), wherein a decrease in cell surface CB2 expression in response to treatment with a CB2 agonist indicates target engagement, wherein the cell surface CB2 expression is detected using an antibody according to the present invention disclosed herein.

[0015] In addition, a method for assessing a subject's therapeutic response to a CB2 agonist by flow cytometry is provided, the method comprising: a) detecting the level of cell surface CB2 expression in a biological sample obtained from the subject before treatment with the CB2 agonist, b) detecting the level of cell surface CB2 expression in a biological sample obtained from the subject after treatment with the CB2 agonist, and c) comparing the cell surface CB2 expression level of step b) with the cell surface CB2 expression level of step a), wherein the cell surface CB2 expression is detected using an anti-human CB2 antibody according to the present invention disclosed herein.

[0016] In one aspect, the cell surface expression of CB2 in the above method is detected by: a) a detection moiety conjugated to the anti-human CB2 antibody, or b) a second antibody comprising a detection moiety that binds to the anti-human CB2 antibody.

[0017] In one aspect, the CB2 agonist used in the method is (S)-1-[5-tert-butyl-3-(1-methyl-1H-tetrazol-5-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-pyrrolidin-3-ol.

[0018] In another aspect, the subject is a human or a cynomolgus monkey.

[0019] In one aspect, the biological sample is treated with the CB2 agonist in vitro. In another aspect, the subject is treated with the CB2 agonist. In one aspect, the biological sample is whole blood.

[0020] In another aspect, a dose-response relationship for a CB2 agonist is determined. In another aspect, this dose-response relationship for a study population supports clinical dose selection for a target population.

[0021] Furthermore, there is provided an antibody according to the invention for use in the above methods.

[0022] The present invention also encompasses the use of an antibody according to the present invention in the above-mentioned method. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Specificity of CB2 antibodies analyzed by Western blot. Figure 1 A: Western blot images of antibodies hCB2-1 / 622, hCB2-2 / 9 and mCB2-1 / 45, PBS and comparison with total protein Colloidal Blue staining. The bottom row shows blots with longer exposure times compared to the top row. Figure 1 B: Molecular weight markers (MWM) used in Western blot experiments. Figure 1 C: Sample loading scheme and sample description for Western blot.

[0024] Figure 2 : Specificity of CB2 antibodies analyzed by immunoprecipitation. Figure 2 A: Western blot analysis of samples after immunoprecipitation with corresponding anti-human CB2 antibodies or PBS, and Western blot analysis of total cell lysates (WB) (detection: hCB2-2 / 9-HRPO). Arrows indicate human CB2 bands. Figure 2 B: Immunoprecipitation analysis using mouse specific CB2 antibody against input samples confirmed the specificity of the mouse antibody (detection: mCB2-1 / 45-HRPO). Arrows indicate mouse CB2 bands. Figure 2 C: Molecular weight markers (MWM) used in immunoprecipitation experiments. Figure 2 D: Sample loading scheme and sample description.

[0025] Figure 3 : Analysis of the specificity of anti-hCB2 antibody 1 / 622 for hCB2. Flow cytometry analysis was performed using increasing concentrations of hCB2 antibody 1 / 622 in CHO cells transfected with hCB2 (light bricks) and hCB1 (dark bricks). Untransfected CHO cells (black) were used as negative controls. Detection was performed using F(ab)2 goat anti-mouse IgG3 PE antibody.

[0026] Figure 4 : Flow cytometry analysis of hCB2 antibody 1 / 622 in CHO-DUKX parental cells and CHO-DUKX_HOMSA_CNR2_Clone_90_CRE-Luc cells transfected with hCB2. Detection was performed using F(ab)2 goat anti-mouse IgG3 PE antibody. Anti-human CB2 antibody 1 / 622 detected CB2 in CHO cells transfected with hCB2 with a signal to background ratio of 10 and negative CHO-DUKX parental cells.

[0027] Figure 5 A and Figure 5 B: Comparison of cell surface CB2 expression on B cells detected by different anti-hCB2 antibodies. Figure 5 A: Flow cytometric analysis using increasing concentrations of hCB2 antibody 1 / 622 (black dots) and hCB2 clone 352110 (R&D Systems; dark rectangles). Fluorescence minus one (FMO) was used as a control, as well as PE-labeled secondary antibody alone (0 μg antibody / sample). Figure 5 B: Using increasing concentrations of mouse IgG3 isotype control clone B10 (SBA; open dots), hCB2 antibody 1 / 622 (black dots), mouse IgG 2bIsotype control clone 26114 (R&D Systems; open rectangles) and hCB2 clone 352110 (R&D Systems; dark rectangles) were subjected to flow cytometry analysis. PE-labeled secondary antibody alone was used as a 0 μg antibody / sample control.

[0028] Figure 6 A. Figure 6 B. Figure 6 C. Figure 6 D. Figure 6 E. Figure 6 F: Example of gating strategy for flow cytometric assay of CB2 detection on T and B cells. Figure 6 A: Single cells were identified to exclude forward scatter doublets. Figure 6 B: Identification of CD45 from single cell populations + Lymphocytes. Figure 6 C: from CD45 + PBMCs were identified among the lymphocyte population. Figure 6 D: Identification of CD3 from PBMC population + CD14 - T cells and CD3 - CD14 - cell. Figure 6 E: from CD3 - CD14 - CD3 - CD19 + B cells. Figure 6 F: CB2 PE is drawn on CD3 - CD19 + B cells in the histogram.

[0029] Figure 7 : IC50 determination of CB2 internalization on B cells in whole blood of healthy participants in response to RG7774. Human whole blood was incubated with RG7774 for 1 hour at 37°C and then incubated with anti-human CB2 antibody 1 / 622. + CD3 - CD14 - CD19 + ) and T cells (CD45 + CD3 + CD14 - ) The expression of CB2 on the surface of B cells was analyzed with a cocktail of specific lineage markers. The B / T cell CB2 MFI ratio was calculated and normalized to the baseline. 0 μM was set as 100% baseline receptor expression.

[0030] Figure 8 A. Figure 8 B. Figure 8 C. Figure 8 D and Figure 8 E: Baseline median fluorescence intensity (MFI) ± SD of CB2 surface expression on different cell subsets in whole blood from diabetic patients (DP; triangles, type 1 or type 2) and matched healthy participants (HP; dots) at baseline. Figure 8 A: B cells (CD45 + CD3 - CD14 - CD19 + ) on the average baseline MFI. Figure 8 B: Basophils (CD45 + CD3 - CD14 - CD19 - CD193 + IgE + ) on the average baseline MFI. Figure 8 C: Monocytes (CD45 + CD3 - CD14 + ) on the average baseline MFI. Figure 8 D: T cells (CD45 + CD3 + CD14 - ) on the average baseline MFI. Figure 8 E: NK cells (CD45 + CD3 - CD14 - CD19 - CD193 - IgE - CD16 + CD56 + ). The mean baseline MFI on each data point is based on repeated measurements. * indicates a significant change using one-way ANOVA analysis (p=0.0245, with Tukey's multiple test correction (p<0.05). n(HP)=8, n(DP type 1)=4, n(DP type 2)=5.

[0031] Fig. 9 A. Fig. 9 B. Fig. 9 C. Fig. 9 D and Fig. 9E: IC50 values ​​of RG7774 determined in different cell subsets in whole blood of diabetic patients and matched healthy participants. Median fluorescence intensity (MFI) ± SD of CB2 surface expression in diabetic patients (open dots, dashed line) and age-matched healthy participants (solid dots, solid line) after exposure to increasing concentrations of RG7774. The average MFI of each data point is based on repeated measurements. Fig. 9 A: B cells (CD45 + CD3 - CD14 - CD19 + ). Fig. 9 B: Basophils (CD45 + CD3 - CD14 - CD19 - CD193 + IgE + ). Fig. 9 C: Monocytes (CD45 + CD3 - CD14 + ). Fig. 9 D: T cells (CD45 + CD3 + CD14 - ). Fig. 9 E: NK cells (CD45 + CD3 - CD14 - CD19 - CD193 - IgE - CD16 + CD56 + ).

[0032] Fig.10 : In vitro IC50 determination of CB2 internalization on B cells in whole blood from cynomolgus monkeys. Cynomolgus whole blood was incubated with RG7774 for 1 hour at 37°C and then incubated with anti-human CB2 antibody 1 / 622. The expression of CB2 on the surface of B cells was analyzed using a cocktail of lineage markers specific for B cells and T cells. The B / T cell CB2 MFI ratio was calculated and normalized to baseline. 0 μM was set as 100% baseline receptor expression. One of the five cynomolgus monkeys had no response to RG7774 in vitro.

[0033] Fig.11: In vivo feasibility of a novel CB2 flow cytometric assay to monitor pharmacodynamic responses on B cells after oral administration in vitro. Six healthy cynomolgus monkeys were given three different doses of oral microsuspension RG7774 with two animals per dose group. Blood drawn at different time points was incubated with anti-human CB2 antibody 1 / 622. The expression of CB2 on the surface of B cells was analyzed using a cocktail of lineage markers specific for B cells and T cells. The B / T cell CB2 MFI ratio was calculated and normalized to baseline. 0 μM was used as 100% baseline receptor expression. For animal Mina, the animal had to be removed from the study due to rectal prolapse, so blood samples could only be collected up to 1 hour after the start of the experiment.

[0034] Fig.12 : A single-dose in vivo study monitored the pharmacodynamic effect of RG7774 on CB2 expression on B cells in cynomolgus monkeys after oral administration. Three healthy cynomolgus monkeys were given 100 mg / kg of the CB2 agonist RG7774. Blood was drawn at different time points and then incubated with the anti-human CB2 antibody 1 / 622. The expression of CB2 on the surface of B cells was analyzed using a cocktail of lineage markers specific for B cells and T cells. The B / T cell CB2 MFI ratio was calculated and normalized to baseline. 0 μM was used as 100% baseline receptor expression. One of the three cynomolgus monkeys had no response to RG7774 in vivo. The 4 hour and 7 hour time points have been excluded due to the storage effect of the antibody working dilution (5 μg / sample) of anti-human CB2 Ab1 / 622. The working dilution of this antibody is only stable on ice for 2 hours.

[0035] Fig.13 : Arithmetic mean (+SD) percent change from baseline in CB2 receptor target engagement on B cells in blood (CFB) versus time following single oral dose administration of 0.75 mg to 300 mg RG7774 in the fasting state (n=6 active vs. 2 placebo at each dose level).

[0036] Fig.14 : Arithmetic mean (+SD) percent change from baseline in CB2 receptor target engagement on B cells in blood (CFB) versus time following a single oral dose of 100 mg RG7774 in the fasted and fed states.

[0037] Fig.15 : Arithmetic mean (+SD) percent change from baseline in CB2 receptor target engagement on B cells in blood (CFB) versus time following multiple oral doses of 20 mg to 300 mg RG7774 once daily for 14 days. DETAILED DESCRIPTION

[0038] definition

[0039] Unless otherwise defined below, the terms used herein are generally as used in the art.

[0040] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0041] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variant antibodies, each antibody included in the population is identical and / or binds to the same epitope, and the possible variant antibodies, for example, contain naturally occurring mutations or are produced during the production process of monoclonal antibody preparations, and such variants are usually present in trace amounts. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0042] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some aspects, the antibody is purified to a purity greater than 95% or 99%, and the purity is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC, affinity chromatography, size exclusion chromatography) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007). In some aspects, the antibodies provided by the present invention are isolated antibodies.

[0043] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0044] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure.

[0045] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003).

[0046] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons, which is composed of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH) (also referred to as a variable heavy chain domain or a heavy chain variable region), followed by three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL) (also referred to as a variable light chain domain or a light chain variable region), followed by a constant light chain (CL) domain (also referred to as a light chain constant region). For human immunoglobulins, the heavy chain of immunoglobulin can be assigned to one of five types, which are called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of immunoglobulin can be assigned to one of the following two types based on the amino acid sequence of its constant domain: called kappa (κ) and lambda (λ). Immunoglobulin is essentially composed of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.

[0047] The term "antigen binding portion", "antigen binding domain" or "antigen binding portion of an antibody" as used herein refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. The term therefore refers to the amino acid residues in an antibody that are responsible for antigen binding. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). The antigen binding portion of an antibody includes amino acid residues from a "complementarity determining region" or "CDR". The "framework" or "FR" region is those variable domain regions other than the hypervariable region residues defined herein. Therefore, the light chain variable domain and the heavy chain variable domain of an antibody include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus. In particular, the CDR3 of the heavy chain is the region that is most conducive to antigen binding and defines the characteristics of the antibody. CDR and FR regions are determined according to the standard definitions of Kabat et al. (Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and / or those residues from the "hypervariable loop". The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

[0048] As used herein, the term "hypervariable region" or "HVR" refers to the various regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, such as "complementarity determining regions" ("CDRs"). Generally speaking, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:

[0049] (a) hypervariable loops present 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));

[0050] (b) CDRs present 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 Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0051] (c) Antigenic contact points present 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)).

[0052] Unless otherwise indicated, CDRs are determined according to the method described in Kabat et al. (supra). Those skilled in the art will appreciate that CDR names may also be determined according to the method described in Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system.

[0053] "Framework" or "FR" refers to the variable domain residues excluding the complementarity determining regions (CDRs). The FR of a variable domain is generally composed of the following four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR sequences and FR sequences generally appear in the following order in VH (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0054] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0055] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule (e.g., a stretch of contiguous amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) to which an antigen binding domain binds, thereby forming an antigen binding domain-antigen complex.

[0056] The term "epitope" refers to a protein determinant of an antigen that is capable of specific binding to an antibody, such as CB2. Epitopes usually consist of chemically active surface macrogroups of molecules such as amino acids or sugar side chains, and usually epitopes have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former but not the latter is lost in the presence of denaturing solvents.

[0057] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the IgG heavy chain Fc region may be slightly different, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc domain of an antibody does not directly participate in the binding of the antibody to an antigen. "Antibody Fc domain" is a term well known to technicians and is defined based on the cleavage of antibodies by papain. According to the different amino acid sequences of the constant regions of their heavy chains, human and mouse antibodies or immunoglobulins can be divided into the following categories: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2 for human immunoglobulins, and IgG1, IgG4 and IgA2 for mouse immunoglobulins. 2a IgG 2b IgG 2c and IgG3. Depending on the heavy chain constant region, the different classes of immunoglobulins are called α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM).

[0058] In one embodiment, the antibodies described herein are of the murine IgG class (i.e., IgG1, IgG 2a IgG 2b IgG 2c In one embodiment, the antibodies described herein are of the murine IgG3 subclass.

[0059] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind a specific antigen (e.g., CB2) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0060] The term "amino acid" as used in this application refers to the group of naturally occurring carboxy α-amino acids including: alanine (three letter code: ala, single letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0061] "Percentage (%) of amino acid sequence identity relative to a reference polypeptide sequence is defined as after comparing a candidate sequence with a reference polypeptide sequence and introducing a space (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitution as a component of sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. The comparison for determining amino acid sequence identity percentage can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment on the full length of the compared sequence. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity%. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to US Copyright Office, Washington DC, 20559, with user documentation, where it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. In the case of using ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity % (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain amino acid sequence identity % with a given amino acid sequence B) of a given amino acid sequence A and a given amino acid sequence B is calculated as follows:

[0062] Multiply 100 by the fraction X / Y

[0063] Where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that in the case where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specifically indicated, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph. With respect to nucleic acids or polynucleotides having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted by another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, or may be interspersed individually among the residues of the reference sequence, or may be interspersed in one or more consecutive groups within the reference sequence. As a practical matter, known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2), may be used to routinely determine whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the invention.

[0064] Antibodies as described herein are preferably produced by recombinant means. Such methods are well known in the prior art and include expressing proteins in prokaryotic and eukaryotic cells, subsequently isolating antibody polypeptides, and generally purifying them to pharmaceutical purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast or Escherichia coli cells, and antibodies are recovered from cells (from supernatant or after cell lysis).

[0065] The recombinant production of antibodies is well known in the art and is reviewed, for example, in the following papers: Makrides, SC, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ, Mol. Biotechnol 16 (2000) 151-161; Werner, RG, Drug Res. 48 (1998) 870-880.

[0066] The antibody may be present in whole cells, cell lysates, or in partially purified or substantially pure form. Purification is performed to eliminate other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See Ausubel, Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987), et al.

[0067] Expression in NS0 cells is described, for example, in Barnes, LM et al., Cytotechnology 32 (2000) 109-123; Barnes, LM et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described, for example, in Durocher, Y. et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described, for example, in Orlandi, R. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described, for example, in Schlaeger, E.-J. and Christensen, K., Cytotechnology 30 (1999) 71-83; and Schlaeger, E.-J., J. Immunol. Methods 194 (1996) 191-199.

[0068] The heavy chain and light chain variable domains according to the present invention are combined with sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation and transcription termination to form an expression vector construct. The heavy chain and light chain expression constructs can be combined into a single vector, co-transfected, continuously transfected or transfected individually into a host cell and then fused to form a single host cell expressing the two chains.

[0069] Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0070] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". For example, the DNA of a presequence or secretory leader is operably linked to the DNA for a polypeptide (if it is expressed as a preprotein that participates in the secretion of the polypeptide); a promoter or enhancer is operably linked to a coding sequence that affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence that is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences connected are continuous and continuous and in the reading frame for the secretory leader sequence. However, enhancers do not have to be continuous. The connection is completed by connecting at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or linker is used according to conventional practice.

[0071] Monoclonal antibodies are suitable for separation from culture medium by conventional immunoglobulin purification methods (such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography). DNA and RNA encoding monoclonal antibodies are easy to be separated and sequenced by conventional methods. Hybridoma cells can be used as the source of such DNA and RNA. Once separated, DNA can be inserted into an expression vector, which is then transfected into a host cell such as HEK293 cell, CHO cell or myeloma cell that does not produce immunoglobulin in addition, to obtain the synthesis of recombinant monoclonal antibodies in the host cell.

[0072] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to intentional or unintentional mutations. Variant progeny having the same function or biological activity as screened in the original transformed cell are included.

[0073] Unless otherwise indicated, the term "CB2" or "CB2R" as used herein refers to any naturally occurring cannabinoid receptor subtype 2 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice). The term encompasses "full-length," unprocessed CB2, as well as any form of CB2 produced by processing in a cell. The term also encompasses naturally occurring variants of CB2, such as splice variants or allelic variants.

[0074] The terms "anti-CB2 antibody" and "α-CB2 antibody" refer to antibodies that are capable of specifically binding CB2 with sufficient affinity to allow the antibody to be useful as a diagnostic or pharmaceutical agent targeting CB2.

[0075] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind a specific antigen (e.g., CB2) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by SPR. The antibody contained in the immunoconjugate described herein specifically binds to CB2.

[0076] In contrast, an antibody that "does not bind" to an antigen does not bind to the antigen with sufficient affinity to render the antibody useful as a diagnostic agent targeting the antigen.

[0077] As used herein, the term "cross-reactivity" refers to the ability of an antibody that binds to an antigen of a particular species to also bind to a corresponding antigen of another species. For example, an antibody that binds to human CB2 may cross-react with cynomolgus monkey CB2, i.e., bind to both human and cynomolgus monkey CB2. Thus, an antibody that "does not cross-react" with a corresponding antigen of another species does not bind to that antigen. For example, in one aspect, an antibody that binds to human CB2 does not cross-react with mouse CB2, i.e., binds to human CB2 but not to mouse CB2.

[0078] The term "CB2 agonist" refers to a compound that binds to and thereby activates cannabinoid receptor 2. CB2 agonists are described, for example, in WO 2013 / 068306 A1. "RG7774" refers to a compound having the formula (S)-1-[5-tert-butyl-3-(1-methyl-1H-tetrazol-5-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-pyrrolidin-3-ol:

[0079]

[0080] Antibodies specific for CB2

[0081] In one embodiment, the anti-CB2 antibody comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 5, HCDR 2 of SEQ ID NO: 6, and HCDR 3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9, and LCDR 3 of SEQ ID NO: 10. In one embodiment, the antibody comprises: a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2.

[0082] The antibody may be any type of antibody or fragment thereof that retains specific binding to CB2, particularly human CB2. Antibody fragments include, but are not limited to, Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules. However, in specific embodiments, the antibody is a full-length antibody. In some embodiments, the antibody comprises an Fc domain comprising a first subunit and a second subunit. In some embodiments, the antibody is an immunoglobulin, particularly an IgG class immunoglobulin, more particularly an IgG3 subclass immunoglobulin. In one embodiment, the Fc domain is a mouse Fc domain.

[0083] In some embodiments, the antibody comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CB2 antibody is hCB2-1 / 622.

[0084] CB2 target engagement (TE) assay

[0085] Pharmacodynamic biomarkers are often used to assess the efficacy of new molecules and play a crucial role in decision making during new drug development. It is difficult to assess whether a molecule interacts with its target using distal biomarkers because convergent signals may affect the biomarker response. Target engagement biomarkers close to the drug target enable a more reliable readout of the interaction between a drug and its target.

[0086] To study the pharmacodynamic effects of CB2 agonists, the inventors developed a CB2 agonist target engagement assay. Like other members of the G protein-coupled receptor family, activation of CB2 may result in internalization of CB2 from the cell membrane to the endosomal compartment. Therefore, changes in CB2 cell surface expression may present a suitable readout in target engagement. A flow cytometric assay for detecting cell surface CB2 expression was studied. The inventors have demonstrated the applicability of this assay as a target engagement biomarker, thereby enabling the study of the in vitro and in vivo pharmacodynamic effects of CB2 agonists. This novel assay is a useful tool for preclinical and clinical evaluation of CB2 agonists.

[0087] Disclosed herein is a method for detecting changes in cell surface CB2 expression in response to treatment with a CB2 agonist in a biological sample by flow cytometry. The method comprises a) detecting cell surface CB2 expression levels in a reference sample that has not been treated with a CB2 agonist, b) detecting cell surface CB2 expression in a sample after treatment with a CB2 agonist, and c) comparing the cell surface CB2 expression level of step b) with the cell surface CB2 expression level of step a). A decrease in cell surface CB2 expression in response to treatment with a CB2 agonist indicates CB2 target engagement.

[0088] Cell surface CB2 expression was detected using antibodies as disclosed herein.

[0089] Flow cytometry (FC) is a technique for detecting and measuring the physical and chemical properties of cell populations, such as the expression of molecules on the cell surface and within the cell. The detection moiety is used to stain the cell population expressing the molecule of interest. The flow cytometry method is described in, for example, Handbook of Flow Cytometry Method, J. Paul Robinson (ed.); Flow Cytometry-A Basic Introduction, Michael G Ormerod (2008) and Current Protocols in Cytometry (2010), Wiley. Antibodies are usually used to stain cell surface proteins. The detection moiety can be conjugated to an antibody that binds to the molecule of interest. Alternatively, the detection moiety can be conjugated to a second antibody. The second antibody in turn binds to the antibody that binds to the molecule of interest.

[0090] Thus, in the methods disclosed herein, cell surface expression of CB2 can be detected by: a) a detection moiety conjugated to an anti-human CB2 antibody, or b) a second antibody comprising a detection moiety that binds to an anti-human CB2 antibody. The detection moiety can be a fluorophore.

[0091] This assay can be used to study target engagement of different CB2 agonists. In one embodiment, the CB2 agonist is (S)-1-[5-tert-butyl-3-(1-methyl-1H-tetrazol-5-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-pyrrolidin-3-ol.

[0092] The biological sample can be obtained from a subject. The subject can be a human or a cynomolgus monkey. The biological sample can be treated with a CB2 agonist in vitro, i.e., the biological sample is obtained from a subject and exposed to the CB2 agonist. Alternatively, the biological sample from a subject that has been treated with a CB2 agonist can be assayed. The biological sample can be whole blood.

[0093] CB2 target engagement assays can also be performed on cell lines expressing CB2.

[0094] The assay further enables determination of the dose-response relationship of a CB2 agonist. The term "dose-response relationship" or "exposure-response relationship" refers to the relationship between the dose administered in vivo or exposure to a substance and the measured effect (response). Target engagement in response to different doses of a CB2 agonist can be assessed. Thus, the assay helps to assess and monitor the therapeutic response to a CB2 agonist.

[0095] In addition, the assay can be used to guide dose selection of CB2 agonists. Determination of the dose or exposure-response relationship of a study population can support dose selection for a target population. As used herein, the term "target population" refers to a group of subjects that can be treated with a CB2 agonist. The target population can be defined by characteristics such as disease, disease stage, age, gender, etc. As used herein, the term "study population" refers to a sample of subjects from a target population that is selected to represent the target population within the study. Therefore, the findings of the study population should be applicable to the target population. The disclosed methods can be used to determine the dose-response relationship of a CB2 agonist for a study population, thereby informing dose selection for the target population.

[0096] In addition, the assay allows for the study of differences in target engagement between different populations. For example, differences in CB2 agonist target engagement in healthy subjects versus diseased subjects, or in young subjects versus elderly subjects can be analyzed. Thus, the assay can support preclinical and / or clinical dose selection for different populations. The disclosed assay can also be used to identify non-responders, i.e., subjects who do not respond to CB2 agonist treatment.

[0097] Examples

[0098] Example 1: Generation of monoclonal antibodies specific for CB2

[0099] Our method utilizes stably transfected mammalian cells (HEK293) that express recombinant antigens on their cell surface. Transfected cells are also used to measure seroconversion, hybridoma selection, and antibody characterization. By presenting antigens in their native conformation for immunization and hybridoma selection, this procedure promotes the production of antibodies capable of binding endogenous proteins.

[0100] The CB2 immunogen is produced by fusion expression of the CB2 N-terminal extracellular domain with the transmembrane and His-tagged cytoplasmic domain of mouse glycophorin A, preceded by a melittin signal sequence. This construct achieves high expression levels with minimal deleterious effects on the expressing cells; the correct orientation of the CB2 fragment relative to the membrane and subsequent natural post-translational modifications (e.g., glycosylation). The pANITA3.1 plasmid is used to generate an expression plasmid (based on the plasmid described in Dreyer, Beauchamp, Matile & Pluschke BMC Biotechnology 2010, 10: 87), and the resulting construct is transiently transfected into HEK293 cells, and the cells are harvested 2 days after transfection.

[0101] The sequences of the expressed constructs are shown in SEQ ID NO: 11 and SEQ ID NO: 12, where the CB2-derived fragment is underlined. The first 21 residues were removed by cellular signaling sequence processing.

[0102] Human_CB2-pANITA3.1 (SEQ ID NO: 11)

[0103] 1MKFLVNVALV FMVVYISFIY AS MEECWVTE IANGSKDGLD SNPMKDYMIL 51 QUR AAADYKDDDDKLSPIQHDFPALVMI LIILGVMAGIIGTILLISYC

[0104] 101ISRMTKKSSV DIQSPEGGDN SVPLSSIEQT PNEESSNVSG GHHHHHHmouse_CB2-pANITA3.1 (SEQ ID NO: 12)

[0105] 1MKFLVNVALVFMVVYISFIY AS MEGCRETE VTNGSNGGLE FNPMKEYMIL 51 SSGQQ AAADYKDDDDKLSPIQHDFPALVMI LIILGVMAGI IGTILLISYC

[0106] 101ISRMTKKSSV DIQSPEGGDN SVPLSSIEQT PNEESSNVSG GHHHHHH

[0107] Mice were immunized by repeated intravenous injections of live cells. Once the animals showed a specific immune response, spleen cells were removed for fusion ( G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497, 1975).

[0108] With the exception of the two pANITA constructs, positive clones (Table 1) were identified by immunofluorescence (IF; Table 2), Western blotting (Table 3, Table 4) on CHO cell lines stably transfected with full-length hCB2, hCB1, and mCB2. Figure 1 ) and immunoprecipitation (Table 4, Figure 2 ) for further characterization.

[0109] Table 1: mAb overview

[0110]

[0111] For IF analysis, methanol-fixed cells were incubated with the corresponding hCB2 and mCB2 clones at 37°C in a humid chamber for 20 min. After rinsing twice and washing in PBS for 15 min, 125 μg / ml FITC-conjugated goat anti-mouse IgG antibody (RAM / IgG (H+L) / FITC, Nordic Immunological Laboratories) diluted in PBS was added and incubated in a humid chamber for 20 min at 37°C. The cells were rinsed twice and washed in PBS for 15 min, mounted with mounting solution (50% PBS 50% glycerol) and covered with a coverslip. Staining was assessed by fluorescence microscopy on a Leica CTR500 fluorescence microscope and a Leica DFC300 FX digital camera.

[0112] Table 2: Summary of immunofluorescence results. “-” no fluorescence detected, “+”–“++++” fluorescence signal intensity, “nt” not tested.

[0113]

[0114]

[0115] Antibody hCB2-1 / 622 showed high fluorescence signal intensity in both cell lines expressing human CB2, whereas no staining was observed on cells expressing human CB1, mouse CB2, or negative control (Table 2).

[0116] For Western blot analysis, cells were collected using an enzyme-free dissociation buffer (enzyme-free Hanks' based cell dissociation buffer, Gibco) and washed twice with PBS. To prepare cell lysates, 10 6 Cells were lysed on ice for 10 min with 0.1 ml lysis buffer (1% NP40, 10% glycerol, 2 mM EDTA, 137 mM NaCl, 20 mM TrisHCl, pH 8, protease inhibitors). Lysates were clarified by centrifugation at 20'000 g for 5 min. For SDS-PAGE, MES running buffer was used on precast 4-12% gradient gels ( Novex 4-12% Bis-Tris Gel, Invitrogen) on the cell lysate. The separation of proteins was visualized by colloidal blue staining. The protein was electrophoretically transferred to a nitrocellulose membrane using a dry blotting system (iBlot, Invitrogen). After the membrane was blocked in a blocking buffer (5% milk in PBS) for 16 h at 4 ° C, specific proteins were detected using appropriately diluted mAbs in a blocking buffer at about 20 ° C for 1 hour. The membrane was then washed four times in a blocking buffer for 5 minutes and incubated with an anti-mouse IgG mAb (GAM / IgG (g-chain) / HRP, Sigma, A9917) conjugated to horseradish peroxidase diluted 1:10'000 in a blocking buffer at about 20 ° C for 1 h. After washing again, the blot was developed using an ECL protein blotting detection reagent (ECL Western Blotting Substrate, Pierce) to visualize the bands.

[0117] Table 3: Summary of Western blot results. "-" no signal, "+" - "+++++" signal intensity.

[0118]

[0119] The expression of recombinant proteins in pANITA3.1 transfected cells was confirmed by Western blot analysis using an anti-His tag antibody. Cells expressing rat glucose-dependent insulinotropic polypeptide receptor (GIPR), also a G protein-coupled receptor, were used as negative controls.

[0120] Antibody hCB2-1 / 622 detected a distinct band for hCB2, but not for mCB2, hCB1, or rat GIPR (Table 3).

[0121] For immunoprecipitation (IP), whole cell lysates were incubated with the corresponding antibodies. IP was performed according to the established protocol as described herein above and subsequent western blotting.

[0122] Table 4: Summary of immunoprecipitation results. “-” no signal, “+” - “++++” signal intensity

[0123]

[0124]

[0125] IP experiments showed that antibody hCB2-1 / 622 specifically bound to hCB2, but no cross-reaction with mCB2 or rat GIPR was found ( Figure 2 and Table 4).

[0126] Additional evidence for the specificity of the anti-human CB2 antibody 1 / 622 was generated by flow cytometry experiments using increased antibody levels on untransfected, hCB1 or hCB2 transfected CHO K1.

[0127] Wash cells 1x with 1x DPBS (Gibco, #14190-094) without CaCl2 and MgCl2. Add Accutase (Sigma, #A6964) and place the cells back in the incubator. Once the cells have detached, collect them into Falcon tubes containing DMEM high glucose + GlutaMAX (Gibco, #31966-021) and 10% heat-inactivated FBS (Gibco, #16140-071). Centrifuge the cells at 250x g and 4°C for 5 minutes, and resuspend the cell pellet in cell staining buffer (BioLegend, #420201) and place it on ice until further use. Incubate the cells with 0.1, 1 or 10 μg / ml hCB2-1 / 622 antibodies and incubate at 4°C for 30 minutes. After incubation, centrifuge the tubes at 250x g and 4°C for 5 minutes. Completely remove the supernatant, wash the cells twice with cold cell staining buffer, and completely remove the supernatant after centrifugation. Dilute the human FcR blocking reagent (Miltenyi Biotech, catalog number 130-059-901) with cold cell staining buffer according to the package insert. Add 50μl to each sample, mix and incubate at 4°C for 10 minutes. The secondary antibody goat F(ab')2 anti-mouse IgG3 PE (Southern Biotech, catalog number 1102-09) was diluted 1:25 in cold cell staining buffer. Add 50μl to each sample, mix and incubate at 4°C in the dark for 30 minutes. After incubation, wash the cells 2x with cold cell staining buffer and completely remove the supernatant after centrifugation. Add 200μl cold cell staining buffer to each tube, mix carefully and store on ice or in the dark at 4°C until acquisition on the flow cytometer.

[0128] A dose-related increase in the median fluorescence intensity (MFI) signal was detected only in the CHO K1 h CB2 cell line, whereas no increase was observed in cells not transfected (CHO K1 control) or transfected with human CB1 protein (CHO K1hCB1R; Figure 3 ) transfected CHO cells, the MFI remained below the background level.

[0129] In addition, a good signal-to-noise ratio for the detection of human CB2 in transfected versus parental untransfected CHO-DUXX cells was established by flow cytometry ( Figure 4 ), further confirming the specificity and applicability of antibody 1 / 622 for CB2-specific detection.

[0130] Example 2: Comparison of anti-hCB2 antibody 1 / 622 with commercially available hCB2 antibodies

[0131] Baseline expression of cell surface CB2 on clone 1 / 622 B cells was assessed and compared with a commercially available hCB2 antibody (R&D Systems; clone 352110) ( Figure 5 Flow cytometry experiments were performed as described in Example 3, but using untreated whole blood samples and using increasing concentrations of the corresponding anti-human CB2 antibodies.

[0132] The dose-dependent increase in median fluorescence intensity observed only in the hCB2-1 / 622 antibody supports specific binding of this anti-human CB2 antibody compared to comparator and control conditions without primary anti-CB2 antibody (0 μg / sample) or FMO (fluorescence minus one) controls, in which the fluorescently labeled secondary antibody was omitted.

[0133] Example 3: Detection of changes in surface expression of CB2 on leukocytes by flow cytometry

[0134] CB2 is a member of the G protein-coupled receptor family, which can respond by internalizing the receptor into the endosomal compartment of the cell after ligand (agonist) binding, thereby removing the corresponding receptor from the cell surface and initiating downstream signaling effects (Calebiro, D. et al. Best Pract Res Clin Endocrinol Metab 2018: 32 (2): 83-91). Such changes in CB2 cell surface expression on B cells in response to treatment with the CB2 agonist RG7774 were measured in whole blood samples from nine healthy human volunteers of different working ages. Whole blood samples were treated with eight concentrations of the compound in duplicate to determine the IC50 of RG7774 using a newly developed flow cytometric assay.

[0135] The Na heparin whole blood samples were incubated with RG7774 in a 96-well plate in a humidified incubator at 37°C for 1 hour. Samples were collected from the 96-well plate and 100 μl was transferred to a labeled 5 ml polystyrene round-bottom tube (Becton Dickinson, catalog number 352052). Unlabeled anti-CB2 antibody clone 1 / 622 was diluted to 50 μg / ml with cell staining buffer (BioLegend, catalog number 420201). 100 μl of diluted anti-CB2 antibody was added to each tube and incubated at 4°C for 30 minutes. For red blood cell lysis, 10x BD PharmLyse lysis buffer (Becton Dickinson, catalog number 55899) was diluted to 1x with distilled water and preheated to approximately 20°C. After CB2 antibody incubation, add 2ml of preheated 1x BD PharmLyse lysis buffer to each tube, mix and incubate at approximately 20°C for 15 minutes. After incubation, the tube was centrifuged at 250xg and 4°C for 5 minutes. The supernatant was completely removed, the cells were washed twice with cold cell staining buffer, and the supernatant was completely removed after centrifugation. According to the package insert instructions, human FcR blocking reagent (MiltenyiBiotech, catalog number 130-059-901) was diluted with cold cell staining buffer. 50μl was added to each sample, mixed and incubated at 4°C for 10 minutes. The secondary antibody goat F(ab')2 anti-mouse IgG3 PE (Southern Biotech, catalog number 1102-09) was diluted 1:25 in cold cell staining buffer. 50μl was added to each sample, mixed and incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed 2x with cold cell staining buffer, and the supernatant was completely removed after centrifugation. Lineage marker cocktail was prepared using cold cell staining buffer containing antibodies CD3 PerCp-Cy5.5 (BioLegend, catalog #344808), CD14 BV510 clone M5E2 (BioLegend, catalog #301842), CD16 APC (Becton Dickinson, catalog #561304), CD19 BV421 (BioLegend, catalog #302234), CD45 APC-Cy7 (BioLegend, catalog #304014), CD56 PE-Cy7, clone 5.1.H11 (BioLegend #362510), CD193 FITC (BioLegend, catalog #310720), and IgE Alexa Fluor 647, clone MHE-18 (BioLegend #325514). Add 100 μl of lineage marker mix to each sample, mix and incubate at 4 °C in the dark for 20 min.After incubation, wash cells 2x with cold cell staining buffer and completely remove supernatant after centrifugation. Add 200 μl cold cell staining buffer to each tube, mix carefully and keep on ice or at 4 °C in the dark until acquisition on the flow cytometer.

[0136] To determine the cell surface expression of CB2 on B cells, we stained the CD45 + CD3 - CD14 - CD19 + B cells were gated (see Figure 6 A-6E) and taking the minimum of 2000 events in this gate to analyze the median fluorescence intensity (MFI) ( Figure 6 F). In the case of a lineage marker cocktail, other cell populations can be analyzed, such as T cells (CD45 + CD3 + CD14 - ), monocytes (CD45 + CD3 - CD14 + ), basophils (CD45 + CD3 - CD14 - CD19 - CD193 + IgE + ), NK cells (CD45 + CD3 - CD14 - CD19 - CD193 - IgE - CD16 + CD56 + ), eosinophils (CD45 + CD16 - ) and neutrophils (CD45 + CD16 + ). It was found that T cells were always CB2 negative. Therefore, for some experiments, CD45 + CD3 + CDC14 - T cells served as an internal negative control population.

[0137] To determine the IC50 value of RG7774, the ratio of CB2-PE MFI on B cells to CB2-PE MFI on T cells was calculated according to the following equation, normalized to the mean of the ratios at 0 μM RG7774:

[0138]

[0139] Individual IC50 values ​​for RG7774 in healthy human participants ranged from 64.3 nM to 143.9 nM, with a mean of 96.5 ± SE 73.2-118.7 nM (Table 5).

[0140] Table 5: IC50 values ​​of RG7774 in healthy participants of working age.

[0141] Donor IC50[nM] R17.134 86.7 R17.135 88.2 R18.1 115.7 R18.2 64.3 R18.3 143.9 R18.4 98.4 R18.9 125.2 R18.13 81.6 R18.24 64.7

[0142] On average, the maximum reduction in CB2 surface expression on B cells in vitro was 55% above baseline ( Figure 7 ), and the curve trends were comparable and similar in both sexes (4 female and 5 male donors). A reduction in CB2 cell surface expression under these experimental conditions could also be detected on basophils, but not on T cells, monocytes, NK cells, eosinophils, and neutrophils (data not shown).

[0143] Example 4: CB2 expression on different cell subsets in diabetic patients and matched healthy participants

[0144] To assess whether diabetic status, either type 1 or type 2 diabetes, might have an effect on the expression of CB2 on the surface of circulating blood cells, CB2 expression levels at baseline (in the absence of RG7774) were compared to expression levels on the same cell populations from demographically and age-matched healthy participants ( Figure 8 ). The median fluorescence intensity (MFI) in all participants indicated that CB2 expression was generally low, but B cells and basophils showed the highest levels, while monocytes could show about half of the expression, and T cells and NK cells showed lower expression or no expression, respectively. In addition, the data indicate that the diabetic state causes a trend toward reduced CB2 expression on the surface of B cells and basophils, although this only reached significance in terms of CB2 expression on basophils in patients with type 2 diabetes compared to healthy participants (one-way ANOVA p=0.0245, p<0.05 corrected using Tukey's multiple test). This trend was not observed for lower CB2 expression levels on monocytes.

[0145] Example 5: IC50 values ​​of RG7774 in different lymphocyte subsets from diabetic patients and matched healthy subjects

[0146] To understand the exposure-response relationship of drug-induced CB2 internalization on blood cell populations, specifically B cells and basophils, whole blood from nine different patients with type 1 and type 2 diabetes and eight demographically and age-matched healthy participants was treated in duplicate with eight concentrations of the compound to determine the IC50 of RG7774.

[0147] Human whole blood from diabetic patients and age-matched healthy participants was incubated with RG7774 for 1 hour at 37°C and then incubated with anti-human CB2 Ab 1 / 622. CB2 expression on the surface of different blood cell types was analyzed using a cocktail of lineage markers specific for B cells, basophils, T cells, NK cells, and monocytes. For each cell type, the MFI value at 0 μM RG7774 was set as 100% baseline receptor expression.

[0148] In B cells ( Fig. 9 A) and basophils ( Fig. 9 B) RG7774-induced CB2 internalization was observed in monocytes ( Fig. 9 C) T cells ( Fig. 9 D) or NK cells ( Fig. 9 E) was not observed.

[0149] This observation was reproducible and the trends of the IC50 curves were comparable in diabetic patients and matched healthy participants, regardless of both sexes and both diabetes subtypes ( Fig. 9 and Table 6).

[0150] The maximum reduction (Emax) from baseline in CB2 surface expression on B cells and basophils in 16 of the 17 patients was ≥50% in vitro (Table 6). For B cells, the Emax for diabetic patients and matched healthy controls was approximately 56%, while for basophils, the Emax observed in both groups averaged approximately 60%. The value for subject 774USB16 was excluded because the IC50 on B cells was an outlier, with a value approximately 10x higher.

[0151] Table 6: IC50 and Emax values ​​for RG7774-induced CB2 internalization on B cells and basophils from diabetic patients (DM) and matched healthy participants (HP).

[0152]

[0153]

[0154] Table 7: Comparison of IC50 (CB2 on B cells) in healthy working age participants (Example 3; Table 5) and diabetic (DM) or age-matched healthy participants (HP).

[0155]

[0156] When the corresponding mean IC50±SD values ​​of RG774 from diabetic and age-matched healthy controls were compared to the IC50 obtained from working-age healthy participants (data from Example 3; Table 5), the data showed a trend that diabetic patients and matched healthy subjects may require approximately 2-3 times the exposure of RG774 to achieve half-maximal internalization of CB2 on B cells (Table 7; One-way Anova with Tukeys multiple comparison correction, p=0.0575). The mean age of the labor force population was lower than that of diabetic patients and healthy matched controls.

[0157] These in vitro data obtained with a newly developed flow cytometric assay measuring CB2 receptor internalization on B cells and other blood cell types in response to RG7774 indicate that the concentration variation of the pharmacodynamic response in elderly diabetic patients and demographically matched healthy participants is 2-3 times greater than that in younger healthy participants, which would indicate that such participants would require higher exposures to achieve similar drug response effects. This is a key observation that influences age-based dose selection.

[0158] Example 6: In vitro feasibility assessment of the CB2 internalization assay in cynomolgus monkeys.

[0159] To determine whether the CB2 surface assay is cross-reactive in cynomolgus monkeys, blood obtained from five different healthy cynomolgus monkeys has been tested in vitro and individual IC50 values ​​of RG7774 and maximal reduction of CB2 surface expression in whole blood have been determined in duplicate using 8 different concentrations of the compound ( Fig.10 , Table 8).

[0160] Table 8: IC50 (nM) of RG774 in five healthy cynomolgus monkeys.

[0161]

[0162]

[0163] The maximum reduction in CB2 surface expression on B cells in vitro was an average of 43.8% ± 7.4% SD compared to baseline. It is noteworthy that one of the monkeys (Ikarus) did not show the expected pharmacodynamic response within the tested dose range, which emphasizes the possibility of using this method to identify individuals with poor or no drug response. Reductions in CB2 expression have not been observed in T cells, monocytes, and granulocytes (data not shown). This in vitro data confirms the cross-reactivity of the CB2 flow cytometry assay in cynomolgus monkeys and demonstrates experimental testing of the feasibility of the assay for monitoring target engagement in vivo after oral administration in cynomolgus monkeys.

[0164] Example 7: Assessment of the feasibility of a newly developed CB2 flow cytometric assay for monitoring CB2 internalization in vivo following oral dosing in cynomolgus monkeys.

[0165] The feasibility of applying the newly developed CB2 flow cytometric assay as an assay for monitoring the pharmacodynamic response to oral administration of RG7774 was evaluated in three groups of two cynomolgus monkeys. After oral administration of three different single doses of 0.5, 5 and 100 mg / kg RG7774 microsuspension, blood samples were collected from all animals at selected time points: 0 (pre-dose) and 0.25, 0.5, 1, 2, 4, 7 and 24 h after dosing, and CB2 flow cytometric analysis was performed ( Fig.11 ). A dose-dependent reduction in individual baseline CB2 expression on B cells was observed, lasting approximately 24 hours and providing confidence in the feasibility of this assay for clinical application.

[0166] Based on the increased confidence from the first in vivo study, a second in vivo study in cynomolgus monkeys was conducted to explore pre- and post-dose time points to better understand the pharmacodynamic response of RG7774 over time.

[0167] In this in vivo cynomolgus monkey study, three cynomolgus monkeys were orally dosed with 100 mg / kg RG7774. Blood sampling for TE determination was performed at selected time points: -96, -24 (two time points not shown), 0 (pre-dose), and 0.25, 0.5, 1, 2, 4, 7, 24, 31, 48, and 72 h post-dose ( Fig.12 ).

[0168] This study revealed a rapid pharmacodynamic effect (within 15 min) and a persistent effect of RG7774 on CB2 surface expression on B cells that could be reliably monitored over time in a dose-dependent manner and confirmed the utility of the newly developed flow cytometric assay as a target engagement assay for clinical applications.

[0169] Example 8: Successful implementation of the assay in a Phase I program demonstrates that the assay can be used in a clinical setting - data inform dose selection for clinical development.

[0170] The utility of the newly developed CB2 flow cytometry assay as a measure of target engagement in the clinical setting has been demonstrated by the successful conduct of its Phase I clinical trial in healthy volunteers. In the clinical setting, the B / T cell ratio was not used because it introduces higher variability compared to baseline-normalized B cell data alone.

[0171] In a first step, single ascending doses (SADs) of RG7774 administered orally in the fasted state ranging between 0.75 mg and 300 mg were evaluated. Following oral administration of RG774, CB2 internalization began rapidly, reaching a nadir (minimum CB2 surface expression) at 4 hours post-dose for doses below 10 mg RG7774 and at 1 hour post-dose for doses between 10 mg and 30 mg RG7774 ( Fig.13 ). For the two highest tested doses of 100mg and 300mg RG7774, the time to reach the nadir varied with increasing area under the effect curve. The extent of CB2 internalization (i.e., CB2 target engagement) increased with dose, with a slight increase from 100mg to 300mg RG7774. This indicates successful target engagement of RG7774 following oral administration. In addition, this assay was used to inform the stopping rules for dose escalation in the SAD portion of the first-in-human (FIH) trial.

[0172] In the second part of the FIH study, the effect of food consumption (FE) was explored by measuring cell surface CB2 expression on B cells in the fasting (n=8) and fed states (n=8) by flow cytometry assay, which showed no relevant impact of food consumption on the pharmacodynamic (PD) effect of RG7774 ( Fig.14 ).

[0173] In the third part of the FIH study, the effect of multiple ascending doses (MAD) of RG7774 ranging from 20 mg to 300 mg once daily for 14 days on CB2 internalization was investigated. Fig.15 ).

[0174] The newly developed assay facilitated the characterization of the PD effects of a CB2 agonist (in this case RG7774) dosed once daily over 14 days in healthy volunteers in a clinical setting and demonstrated that steady-state was achieved during the observation period, that over 14 days, desensitization was not observed, and that the drug effect of induced reduction in CB2 surface receptor expression was largely reversible within 1 week after drug discontinuation, although the effect was not fully reversed at the two highest doses.

[0175]

[0176]

[0177]

Claims

1. An antibody comprising an antigen binding portion that binds to human cannabinoid receptor subtype 2 (CB2), wherein the antibody comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 5, HCDR 2 of SEQ ID NO: 6, and HCDR 3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9, and LCDR 3 of SEQ ID NO:

10.

2. The antibody of claim 1, wherein the antibody comprises: a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

2.

3. The antibody according to claim 1 or 2, wherein the antibody comprises an Fc domain. The antibody according to claim 3 , wherein the Fc domain is an IgG class Fc domain, in particular an IgG3 subclass Fc domain.

5. The antibody according to any one of claims 3 to 4, wherein the Fc domain is a murine Fc domain.

6. The antibody of any one of claims 1 to 5, comprising a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

4.

7. The antibody according to any one of claims 1 to 6, wherein the antibody cross-reacts with cynomolgus monkey CB2.

8. A method for detecting changes in cell surface CB2 expression in response to treatment with a CB2 agonist in a biological sample obtained from a subject by flow cytometry, the method comprising: a) detecting the expression level of cell surface CB2 in a reference sample that has not been treated with a CB2 agonist, b) detecting cell surface CB2 expression in a sample after treatment with a CB2 agonist, and c) comparing the expression level of CB2 on the cell surface of step b) with the expression level of CB2 on the cell surface of step a), wherein a decrease in cell surface CB2 expression in response to CB2 agonist treatment is indicative of target engagement, The antibody according to any one of claims 1 to 7 is used to detect cell surface CB2 expression.

9. A method for assessing a subject's therapeutic response to a CB2 agonist by flow cytometry, the method comprising: a) detecting the expression level of cell surface CB2 in a biological sample obtained from the subject before treatment with the CB2 agonist, b) detecting the level of cell surface CB2 expression in a biological sample obtained from the subject after treatment with the CB2 agonist, and c) comparing the expression level of CB2 on the cell surface of step b) with the expression level of CB2 on the cell surface of step a), The anti-human CB2 antibody according to any one of claims 1 to 7 is used to detect cell surface CB2 expression.

10. The method of claim 8 or 9, wherein the cell surface expression of CB2 is detected by: a) a detection moiety conjugated to the anti-human CB2 antibody, or b) a second antibody comprising a detection moiety that binds to said anti-human CB2 antibody.

11. The method of any one of claims 8 to 10, wherein the CB2 agonist is (S)-1-[5-tert-butyl-3-(1-methyl-1H-tetrazol-5-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-pyrrolidin-3-ol.

12. The method according to any one of claims 8 to 11, wherein the subject is a human or a cynomolgus monkey.

13. The method according to any one of claims 8 to 12, wherein the biological sample is treated in vitro with the CB2 agonist.

14. The method of any one of claims 8 to 12, wherein the subject is treated with the CB2 agonist.

15. The method according to any one of claims 8 to 14, wherein the biological sample is whole blood.

16. The method according to any one of claims 8 to 15, wherein a dose-response relationship of a CB2 agonist is determined.

17. The method of claim 16, wherein the dose-response relationship for the study population supports clinical dose selection for a target population.

18. The antibody according to any one of claims 1 to 7, for use in a method according to any one of claims 8 to 17.

19. Use of an antibody according to any one of claims 1 to 7 in a method according to any one of claims 8 to 17.

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