Anti-CB2 Antibodies and Their Use in Flow Cytometry Assays for Measuring Cell Surface CB2 Expression

Abstract

The present invention relates to novel antibodies that bind to human CB2. Furthermore, the present invention relates to a method for detecting changes in cell surface CB2 expression in response to treatment with a CB2 agonist by flow cytometry. This method allows for the assessment and monitoring of target engagement of CB2 agonists, thereby aiding in the 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 in in vitro preclinical assays and clinical trials to monitor CB2 agonist-induced CB2 internalization as a target engagement biomarker. [Background technology]

[0002] Monoclonal antibodies (mAbs) are often raised against synthetic peptides derived from the predicted sequence of a target protein. Unfortunately, these antibodies, while strongly reactive with the peptide, often fail to recognize the native protein. 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, it is often necessary to use mammalian expression systems, as they are more likely to produce functional proteins with appropriate disulfide bonds and post-translational modifications. Although the introduction of affinity tags simplifies purification, obtaining recombinant proteins in their native conformation with sufficient yield and purity is still often difficult. This is most notably true for membrane and membrane-associated proteins, which can lose their native conformation during the purification process.

[0003] When attempting to generate mAbs capable of recognizing native proteins, it is important to use the target protein in its native conformation not only in the immunization step but also in the screening procedure. Many standard hybridoma screening protocols utilize recombinant proteins immobilized on solid supports, which can significantly alter the conformation of the protein.

[0004] To generate mABs that specifically recognize membrane-bound proteins in their native conformation, we applied a method that circumvents the need for purified recombinant proteins, utilizing antigens expressed on the surface of stably transfected mammalian cells for both mouse immunization and immunoassays (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. Currently, there are two known subtypes, termed cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 is primarily expressed in the central nervous system (i.e., the amygdala and hippocampus) and to a lesser extent in the periphery (liver, skeletal muscle, pancreas, and adipose tissue). CB2, encoded by the CNR2 gene, is primarily expressed in the periphery on cells of the immune system, such as macrophages and other subtypes of leukocytes (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, CB2 expression levels relative to mRNA levels differ between immune cell populations in both resting and activated states, potentially indicating an important role for CB2 in immune regulation and function. Furthermore, CB2 receptors are also expressed in the gastrointestinal system (Wright, KLet et al. Br J Pharmacol 2008,153(2),263-70) and are widely distributed in the brain, found primarily in microglia and not in 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 preclinical studies to investigate its role in health and disease. Results of these studies have implicated the ECS in a variety of pathophysiological processes (Pacher, P. et al. FEBS J 2013;280:1918-43).

[0007] CB1 is known to be involved in the psychoactive effects of cannabinoids, while receptor activation in peripheral tissues exhibits potent oxidative, inflammatory, and profibrotic activities. In contrast, CB2 activation has been shown to protect against tissue 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 nephropathies can be treated by CB2 agonism (Behl, T. et al. Diabetes Metab Res Rev 2016;32:251-9). Furthermore, activation of CB2 has been shown to be anti-inflammatory in the eye (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 regulating the endocannabinoid system through agonistic approaches may have numerous potential benefits in 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 that can respond upon ligand binding by internalizing the receptor into the endosomal compartment of the cell, thereby removing the respective 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 of CB2 agonist target engagement. There is a need for sensitive and specific assays to detect changes in CB2 cell surface expression to enable monitoring of drug effects in vitro, in vivo, and in clinical settings, and to support the association of CB2 target engagement with downstream PD and clinical effects in each indication. 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), the antibody comprising: a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 5, an HCDR2 of SEQ ID NO: 6, and an HCDR3 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, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10. In one embodiment, the antibody comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence 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 at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2.

[0011] In one embodiment, the antibody comprises an Fc domain. In a further embodiment, the Fc domain is an Fc domain of the IgG class, particularly the IgG3 subclass. In yet another embodiment, the Fc domain is a murine Fc domain.

[0012] In another embodiment, the antigen comprises a polypeptide sequence 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 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] The present invention further 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, comprising the steps of: a) detecting cell surface CB2 expression levels in a reference sample not treated with a CB2 agonist; b) detecting cell surface CB2 expression in the 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 CB2 agonist treatment indicates target engagement, and wherein cell surface CB2 expression is detected using an antibody according to the invention disclosed herein.

[0015] Further provided is a method for assessing a treatment response to a CB2 agonist in a subject by flow cytometry, the method comprising the steps of: a) detecting a cell surface CB2 expression level in a biological sample obtained from the subject before treatment with the CB2 agonist; b) detecting a cell surface CB2 expression level 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 invention disclosed herein.

[0016] In one aspect, cell surface expression of CB2 in the aforementioned methods is 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 the anti-human CB2 antibody.

[0017] In one embodiment, 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 embodiment, the subject is a human or a cynomolgus monkey.

[0019] In one embodiment, the biological sample is treated with a CB2 agonist in vitro, while in another embodiment, the subject has been treated with a CB2 agonist. In one embodiment, the biological sample is whole blood.

[0020] In a further embodiment, the dose-response relationship of the CB2 agonist is determined. In a further embodiment, the dose-response relationship of the test population aids in clinical dose selection for the target population.

[0021] Further provided is an antibody according to the invention for use in the aforementioned methods.

[0022] The use of an antibody according to the invention in the aforementioned methods is also encompassed by the present invention. [Brief explanation of the drawings]

[0023] [Figure 1] Specificity of CB2 antibodies analyzed by Western blot. Figure 1A: Western blot images for 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 1B: Molecular weight markers (MWM) used in Western blot experiments. Figure 1C: Sample loading scheme and sample description for Western blot. [Figure 2]Specificity of CB2 antibodies analyzed by immunoprecipitation. Figure 2A: Western blot analysis of samples and whole cell lysates (WB) after immunoprecipitation with anti-human CB2 antibody or PBS, respectively (detection: hCB2-2 / 9-HRPO). The arrow indicates the human CB2 band. Figure 2B: Immunoprecipitation analysis using a mouse-specific CB2 antibody on the input sample confirms the specificity of the mouse antibody (detection: mCB2-1 / 45-HRPO). The arrow indicates the mouse CB2 band. Figure 2C: Molecular weight markers (MWM) used in immunoprecipitation experiments. Figure 2D: Sample loading scheme and sample description. [Figure 3] Analysis of the specificity of the anti-hCB2 antibody 1 / 622 for hCB2. Flow cytometry analysis of increasing concentrations of the hCB2 antibody 1 / 622 on CHO cells transfected with hCB2 (light blocks) and hCB1 (dark blocks). Non-transfected CHO cells (black) served as a negative control. For detection, a F(ab)2 goat anti-mouse IgG3 PE antibody was used. [Figure 4] Flow cytometry analysis of hCB2 antibody 1 / 622 in hCB2-transfected CHO-DUKX parental cells and CHO-DUKX_HOMSA_CNR2_Clone_90_CRE-Luc cells. F(ab)2 goat anti-mouse IgG3 PE antibody was used for detection. Anti-human CB2 antibody 1 / 622 detected CB2 in hCB2-transfected CHO cells with a signal-to-background ratio of 10, while CHO-DUKX parental cells were negative. [Figure 5]Figures 5A and 5B: Comparison of different anti-hCB2 antibodies in detecting cell surface CB2 expression on B cells. Figure 5A: Flow cytometry analysis of increasing concentrations of hCB2 antibody 1 / 622 (black dots) and hCB2 clone 352110 (R&D Systems; dark rectangles). Fluorescence minus 1 (FMO) alone served as a control as well as a secondary PE-labeled antibody (0 μg antibody / sample). Figure 5B: Flow cytometry analysis of increasing concentrations of mouse IgG3 isotype control clone B10 (SBA; open dots), hCB2 antibody 1 / 622 (black dots), mouse IgG2b isotype control clone 26114 (R&D Systems; open rectangles), and hCB2 clone 352110 (R&D Systems; dark rectangles). The secondary PE-labeled antibody alone served as a 0 μg antibody / sample control. [Figure 6] Figures 6A, 6B, 6C, 6D, 6E, and 6F: Examples of gating strategies for flow cytometry assays for CB2 detection in T and B cells. Figure 6A: Single cells are identified to exclude forward scatter doublets. Figure 6B: From the single cell population, CD45+ lymphocytes are identified. Figure 6C: PBMCs are identified from the CD45+ lymphocyte population. Figure 6D: From the PBMC population, CD3+CD14- T cells and CD3-CD14- cells are identified. Figure 6E: From the CD3-CD14- gate, CD3-CD19+ B cells are identified. Figure 6F: CB2 PE is plotted on a histogram of CD3-CD19+ B cells. [Figure 7] Determination of IC50 for CB2 internalization on B cells in response to RG7774 in whole blood from healthy participants. Human whole blood was incubated with RG7774 for 1 hour at 37°C, followed by incubation with anti-human CB2 antibody 1 / 622. CB2 expression on the surface of B cells was analyzed using a lineage marker mix specific for B cells (CD45+CD3-CD14-CD19+) and T cells (CD45+CD3+CD14-). B / T cell CB2 MFI ratios were calculated and normalized to baseline. 0 μM was set as 100% baseline receptor expression. [Figure 8]Figures 8A, 8B, 8C, 8D, and 8E: Baseline median fluorescence intensity (MFI) ± SD of CB2 surface expression on different cell subpopulations in whole blood from diabetic patients (DP, triangles, type 1 or type 2) and matched healthy participants (HP, dots) at baseline. Figure 8A: Mean baseline MFI on B cells (CD45+CD3-CD14-CD19+). Figure 8B: Mean baseline MFI on basophils (CD45+CD3-CD14-CD19-CD193+IgE+). Figure 8C: Mean baseline MFI on monocytes (CD45+CD3-CD14+). Figure 8D: Mean baseline on T cell MFI (CD45+CD3+CD14-). Figure 8E: Mean baseline MFI on NK cells (CD45+CD3-CD14-CD19-CD193-IgE-CD16+CD56+). The mean MFI of each data point is based on repeated measurements. * indicates a significant change using one-way ANOVA analysis (p=0.0245 (p<0.05) with Tukey's multiple testing correction). n(HPs)=8, n(DP1)=4, n(DP2)=5. [Figure 9-1] Figures 9A, 9B, 9C, 9D, and 9E: Determination of IC50 values ​​for RG7774 in different cell subpopulations in whole blood from diabetic patients and matched healthy participants. Median fluorescence intensity (MFI) ± SD of CB2 surface expression in diabetic patients (open dots, dotted line) and age-matched healthy participants (closed dots, continuous line) after exposure to increasing concentrations of RG7774. The mean MFI for individual data points is based on repeated measurements. Figure 9A: B cells (CD45+CD3-CD14-CD19+). Figure 9B: Basophils (CD45+CD3-CD14-CD19-CD193+IgE+). Figure 9C: Monocytes (CD45+CD3-CD14+). Figure 9D: T cells (CD45+CD3+CD14-). Figure 9E: NK cells (CD45+CD3-CD14-CD19-CD193-IgE-CD16+CD56+). [Figure 9-2]Figures 9A, 9B, 9C, 9D, and 9E: Determination of IC50 values ​​for RG7774 in different cell subpopulations in whole blood from diabetic patients and matched healthy participants. Median fluorescence intensity (MFI) ± SD of CB2 surface expression in diabetic patients (open dots, dotted line) and age-matched healthy participants (closed dots, continuous line) after exposure to increasing concentrations of RG7774. The mean MFI for individual data points is based on repeated measurements. Figure 9A: B cells (CD45+CD3-CD14-CD19+). Figure 9B: Basophils (CD45+CD3-CD14-CD19-CD193+IgE+). Figure 9C: Monocytes (CD45+CD3-CD14+). Figure 9D: T cells (CD45+CD3+CD14-). Figure 9E: NK cells (CD45+CD3-CD14-CD19-CD193-IgE-CD16+CD56+). [Figure 10] Determination of in vitro IC50 for CB2 internalization on B cells in whole blood from cynomolgus monkeys. Cynomolgus monkey whole blood was incubated with RG7774 for 1 hour at 37°C, followed by incubation with anti-human CB2 antibody 1 / 622. A lineage marker mix specific for B and T cells was used to analyze CB2 expression on the surface of B cells. B / T cell CB2 MFI ratios were calculated and normalized to baseline. 0 μM was set as 100% baseline receptor expression. One out of five cynomolgus monkeys was a non-responder to RG7774 in vitro. [Figure 11] In vivo feasibility of a novel CB2 flow cytometry assay for monitoring in vitro pharmacodynamic responses to B cells upon oral administration. Six healthy cynomolgus monkeys were administered three different doses of RG7774 oral microsuspension, with two animals per dose group. Blood samples collected at different time points were incubated with anti-human CB2 antibody 1 / 622. CB2 expression on the surface of B cells was analyzed using a lineage marker mix specific for B and T cells. The B / T cell CB2 MFI ratio was calculated and normalized to baseline. 0 μM served as 100% baseline receptor expression. In the case of animal Mina, blood samples could only be collected up to 1 hour after the start of the experiment because this animal had to be removed from the experiment due to rectal prolapse. [Figure 12] A single-dose in vivo study monitoring the pharmacological effects of RG7774 on CB2 expression on B cells in cynomolgus monkeys upon oral administration. Three healthy cynomolgus monkeys were administered 100 mg / kg of the CB2 agonist RG7774. Blood was collected at different time points and then incubated with anti-human CB2 antibody 1 / 622. CB2 expression on the surface of B cells was analyzed using a lineage marker mix specific for B and T cells. The B / T cell CB2 MFI ratio was calculated and normalized to baseline. 0 μM served as 100% baseline receptor expression. One of the three cynomolgus monkeys was a non-responder to RG7774 in vivo. The 4- and 7-hour time points were excluded due to storage effects of the working antibody dilution (5 μg / sample) of anti-human CB2 Ab 1 / 622. This working dilution of the antibody is only stable on ice for 2 hours. [Figure 13] Arithmetic mean (+SD) of percent change from baseline in CB2 receptor target engagement on B cells in the blood (CFB) versus time after single oral doses of 0.75 mg to 300 mg of RG7774 in the fasted state (n=6 for active agent, n=2 for placebo per dose level). [Figure 14] Arithmetic mean (+SD) of percent change from baseline in CB2 receptor target engagement on B cells in the blood (CFB) versus time after a single oral dose of 100 mg RG7774 in the fasted and fed states. [Figure 15] Arithmetic mean (+SD) of percent change from baseline in CB2 receptor target engagement on B cells in the blood (CFB) versus time after multiple oral doses of 20 mg to 300 mg RG7774 once daily over 14 days. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition Unless otherwise defined below, terms are used herein as commonly used in the art.

[0025] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0026] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, each individual antibody in the population is identical and / or binds to the same epitope, excluding possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during production of the monoclonal antibody preparation. Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced 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 the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0027] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC, affinity chromatography, size exclusion chromatography) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some embodiments, the antibodies provided by the present invention are isolated antibodies.

[0028] 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.

[0029] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a native antibody.

[0030] "Antibody fragment" refers to a molecule other than an intact antibody that contains 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 specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003).

[0031] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light domain (CL), also called a light chain constant region. Immunoglobulin heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2) in the case of human immunoglobulins. Immunoglobulin light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

[0032] The terms "antigen-binding portion," "antigen-binding domain," or "antigen-binding portion of an antibody," as used herein, refer to a portion of an antibody comprising the area that specifically binds to and is complementary to part or all of an antigen. Thus, the term refers to the amino acid residues of an antibody responsible for antigen binding. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In particular, an antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). The antigen-binding portion of an antibody comprises amino acid residues from "complementarity-determining regions" or "CDRs." "Framework" or "FR" regions are variable domain regions other than the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N- to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. The CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and / or residues from "hypervariable loops." The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0033] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain whose sequence is hypervariable and which determines antigen-binding specificity, e.g., a "complementarity-determining region" ("CDR"). Typically, antibodies contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include: (a) Hypervariable loops occurring 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 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 (c) Antigen contacts occurring 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)). Examples include:

[0034] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. One skilled in the art will understand that the designations of CDRs can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0035] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the VH (or VL) in the following order: FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0036] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0037] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule (e.g., a three-dimensional structure composed of a contiguous stretch of amino acids or distinct regions of non-contiguous amino acids) to which an antigen-binding domain binds, forming an antigen-binding domain-antigen complex.

[0038] The term "epitope" refers to a protein determinant on an antigen, such as CB2, capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0039] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of 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 follows the EU numbering system, also known as the EU index, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc domain of an antibody is not directly involved in binding the antibody to an antigen. "Antibody Fc domain A" is a term well known to those skilled in the art and is defined based on papain cleavage of antibodies. Depending on the amino acid sequence of the constant region of the heavy chain, human and mouse antibodies or immunoglobulins are divided into classes: IgA, IgD, IgE, IgG and IgM, some of which are subclasses (isotypes), e.g., IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2 for human immunoglobulins, and IgG1, IgG 2a , IgG 2b , IgG 2c According to the heavy chain constant region, the different classes of immunoglobulins are called α (IgA), δ (IgD), ε (IgE), γ (IgG) and μ (IgM), respectively.

[0040] In one embodiment, the antibodies described herein are of the murine IgG class (i.e., IgG1, IgG 2a , IgG 2b , IgG 2cor of the IgG3 subclass). In one embodiment, the antibodies described herein are of the murine IgG3 subclass.

[0041] "Specifically bind" means that the binding is antigen-selective and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen (e.g., CB2) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analyzed with a BIAcore device) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0042] As used in this application, the term "amino acid" refers to the group of naturally occurring carboxy alpha-amino acids, including alanine (three letter code: ala, one 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).

[0043] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after sequence alignment and, if necessary, the introduction of gaps to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B, between a given amino acid sequence A and a given amino acid sequence B, or between a given amino acid sequence A and a given amino acid sequence B (or alternatively, a given amino acid sequence A having or comprising a certain % amino acid sequence identity between a given amino acid sequence B, a given amino acid sequence B, or a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. Of course, if 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 specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. By a nucleic acid or polynucleotide having a nucleotide sequence at least, e.g., 95%, "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, either individually among residues in the reference sequence or interspersed in one or more contiguous groups within the reference sequence, or anywhere between the residues in the reference sequence at their terminal positions. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0044] The antibodies described herein are preferably produced by recombinant means. Such methods are widely known in the state of the art and include protein expression in prokaryotic and eukaryotic cells, followed by isolation and purification of the antibody polypeptides, usually to a pharmaceutically acceptable degree of purity. For protein expression, nucleic acids encoding the light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is carried out in appropriate prokaryotic and eukaryotic host cells, such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibody is recovered from the cells (either from the supernatant or after cell lysis).

[0045] Recombinant production of antibodies is well known in the state of the art and is described, for example, in the reviews by 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, and Werner, RG, Drug Res. 48 (1998) 870-880.

[0046] The antibody may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification to eliminate other cellular components or other contaminants, such as other cellular nucleic acids or proteins, is carried out by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0047] Expression in NS0 cells is described, for example, in Barnes, LM, et al., Cytotechnology 32 (2000) 109-123 and 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 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, and Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK293) is described in Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83, and Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.

[0048] The heavy and light chain variable regions of the present invention are combined with promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and transcription termination sequences to form an expression vector construct. The heavy and light chain constructs are combined in a single vector and co-transfected, sequentially transfected, or separately transfected into host cells, which are then fused to form a single host cell that expresses both chains.

[0049] Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site, while eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0050] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accord with conventional practice.

[0051] Monoclonal antibodies are preferably separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA can be inserted into an expression vector, which can then be transfected into host cells, such as HEK293 cells, CHO cells, or myeloma cells, that do not otherwise produce immunoglobulin protein, resulting in the synthesis of recombinant monoclonal antibodies in the host cells.

[0052] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard to the number of times the cell has been transferred. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0053] As used herein, the terms "CB2" or "CB2R," unless otherwise specified, refer to any native 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 terms encompass "full-length," unprocessed CB2, as well as any form of CB2 resulting from processing within a cell. The terms also encompass naturally occurring variants of CB2, such as splice variants or allelic variants.

[0054] The terms "anti-CB2 antibody" and "α-CB2 antibody" refer to an antibody that can specifically bind to CB2 with sufficient affinity so that the antibody is useful as a diagnostic agent in targeting CB2.

[0055] "Specifically bind" means that the binding is antigen-selective and can be distinguished from undesired or nonspecific interactions. The ability of an antibody to bind to a specific antigen (e.g., CB2) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the binding of the 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 immune complex described herein specifically binds to CB2.

[0056] Conversely, an antibody that "freezes" a particular antigen is unable to bind that antigen with sufficient affinity to be useful as a diagnostic agent in targeting that antigen.

[0057] 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 the corresponding antigen of another species. For example, an antibody that binds to human CB2 can cross-react with cynomolgus monkey CB2, i.e., can bind to both human and cynomolgus monkey CB2. Thus, an antibody that does not "cross-react" with the corresponding antigen of another species does not bind to that antigen. For example, in one embodiment, an antibody that binds to human CB2 does not cross-react with mouse CB2, i.e., binds to human CB2 but not mouse CB2.

[0058] 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. "RG7774" refers to the compound [5-tert-butyl-3-(1-methyl-1H-tetrazol-5-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol, having the formula: [ka] It has.

[0059] CB2-specific antibodies In one embodiment, the anti-CB2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 5, an HCDR2 of SEQ ID NO: 6, and an HCDR3 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, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10. In one embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence 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 at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2.

[0060] The antibody can 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 certain embodiments, the antibody is a full-length antibody. In some embodiments, the antibody comprises an Fc domain composed of a first subunit and a second subunit. In some embodiments, the antibody is an immunoglobulin, particularly an IgG class, more particularly an IgG3 subclass immunoglobulin. In one embodiment, the Fc domain is a mouse Fc domain.

[0061] In some embodiments, the antigen comprises a polypeptide sequence 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 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.

[0062] CB2 target engagement (TE) assay Pharmacodynamic biomarkers are often used to evaluate the efficacy of new molecules and play an important role in decision-making during the development of new drugs. Using distal biomarkers, it is difficult to assess whether a molecule interacts with its target because converging signals can affect biomarker responses. Target engagement biomarkers proximal to the drug target, on the other hand, provide a more robust readout of the interaction between a drug and its target.

[0063] To investigate the pharmacodynamic effects of CB2 agonists, we developed a CB2 agonist target engagement assay. As other members of the G protein-coupled receptor family, activation of CB2 can lead to its internalization from the plasma membrane to endosomal compartments. Therefore, changes in cell surface expression of CB2 may indicate an appropriate readout of target engagement. We investigated a flow cytometry assay to detect cell surface CB2 expression. We demonstrated the suitability of the assay as a target engagement biomarker, thereby enabling testing of the pharmacodynamic effects of CB2 agonists in vitro and in vivo. This novel assay is a useful tool for preclinical and clinical evaluation of CB2 agonists.

[0064] This specification discloses 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 includes: a) detecting the cell surface CB2 expression level in a reference sample that is not treated with a CB2 agonist; b) detecting the cell surface CB2 expression in the 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.

[0065] Cell surface CB2 expression is detected using the antibodies disclosed herein.

[0066] Flow cytometry (FC) is a technique used to detect and measure physical and chemical properties, such as the expression of cell surface and intracellular molecules, of a cell population. A detection moiety is used to stain a cell population that expresses a molecule of interest. Flow cytometry methods are described, for example, in Handbook of Flow Cytometry Methods, J. Paul Robinson (Editor), Flow Cytometry - A Basic Introduction, Michael G Ormerod (2008), and Current Protocols in Cytometry (2010), Wiley. Generally, antibodies are 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. This second antibody then binds to the antibody that binds to the molecule of interest.

[0067] 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 the anti-human CB2 antibody. The detection moiety can be a fluorophore.

[0068] This assay can be used to examine the target engagement of various 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.

[0069] 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 a CB2 agonist. Alternatively, the assay can be performed on a biological sample from a subject that has been treated with a CB2 agonist. The biological sample can be whole blood.

[0070] CB2 target engagement assays can also be performed on cell lines expressing CB2.

[0071] This assay also allows for the determination of the dose-response relationship of CB2 agonists. The term "dose-response relationship" or "exposure-response relationship" refers to the relationship between the administered dose or exposure to a substance in the body and the measured effect (response). Target engagement in response to different doses of a CB2 agonist can be evaluated. Therefore, this assay facilitates the evaluation and monitoring of treatment responses to CB2 agonists.

[0072] Furthermore, this assay can be used to direct dose selection of a CB2 agonist. Determining the dose-response or exposure-response relationship for a test population can aid in dose selection for a target population. As used herein, the term "target population" refers to a group of subjects who can be treated with a CB2 agonist. The target population can be defined by characteristics such as disease, disease stage, age, and gender. As used herein, the term "test population" refers to a sample of subjects from the target population selected to represent the target population in a study. Thus, findings from the test population are applicable to the target population. The methods of the present disclosure can be utilized to determine the dose-response relationship of a CB2 agonist for a test population, thereby informing dose selection for the target population.

[0073] Furthermore, this assay allows for testing differences in target engagement between different populations. For example, differences in CB2 agonist target engagement between healthy subjects and diseased subjects or between young and elderly subjects can be analyzed. Therefore, this assay can assist in preclinical and / or clinical dose selection for different populations. Non-responders, i.e., subjects who do not respond to CB2 agonist treatment, can also be identified using the assay of the present disclosure. [Example]

[0074] Example 1: Generation of monoclonal antibodies specific to CB2 Our approach utilizes stably transfected mammalian cells (HEK293) that express recombinant antigens on their cell surface. The transfected cells are also used to measure seroconversion, hybridoma selection, and antibody characterization. By presenting the antigen in its native conformation for immunization and hybridoma selection, this procedure promotes the generation of antibodies capable of binding to the endogenous protein.

[0075] The CB2 immunogen was generated by expressing the CB2 N-terminal extracellular domain, followed by the bee venom melittin signal sequence, as a fusion with the mouse glycophorin A transmembrane domain and a His-tagged cytoplasmic domain. This construct allows for high expression levels with minimal adverse effects on the expressing cells, as well as correct orientation of the CB2 fragment relative to the membrane and subsequent native post-translational modifications (e.g., glycosylation). The pANITA3.1 plasmid (based on the plasmid described in Dreyer, Beauchamp, Matile, & Pluschke BMC Biotechnology 2010, 10:87) was used to generate the expression plasmid, and the resulting construct was transiently transfected into HEK293 cells. Cells were harvested 2 days post-transfection.

[0076] The expressed construct sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12, with the CB2-derived fragment underlined. The first 21 residues are removed by cellular signal sequence processing.

[0077] Human_CB2-pANITA3.1 (SEQ ID NO: 11) 1 MKFLVNVALV FMVVYISFIY ASMEECWVTE IANGSKDGLD SNPMKDYMIL 51 SGPQKAAADY KDDDDKLSPI QHDFPALVMI LIILGVMAGI IGTILLISYC 101 ISRMTKKSSV DIQSPEGGDN SVPLSSIEQT PNEESSNVSG GHHHHHH

[0078] Mouse_CB2-pANITA3.1 (SEQ ID NO: 12) 1 MKFLVNVALV FMVVYISFIY ASMEGCRETE VTNGSNGGLE FNPMKEYMIL 51 SSGQQAAADY KDDDDKLSPI QHDFPALVMI LIILGVMAGI IGTILLISYC 101 ISRMTKKSSV DIQSPEGGDN SVPLSSIEQT PNEESSNVSG GHHHHHH

[0079] Mice were immunized by repeated intravenous injection of live cells. As soon as the animals demonstrated a specific immune response, spleen cells were removed for fusion (Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497, 1975).

[0080] Positive clones (Table 1) were further characterized by immunofluorescence (IF, Table 2), Western blot (Table 3, Figure 1), and immunoprecipitation (Table 4, Figure 2) in full-length hCB2, hCB1, mCB2 stably transfected CHO cell lines in addition to the two pANITA constructs.

[0081] [Table 1]

[0082] For IF analysis, methanol-fixed cells were incubated with hCB2 and mCB2 clones, respectively, for 20 minutes in a humid chamber at 37°C. After rinsing twice in PBS and washing for 15 minutes, 125 μg / ml of FITC-conjugated goat anti-mouse IgG antibody (RAM / IgG(H+L) / FITC, Nordic Immunological Laboratories) diluted in PBS was added and incubated for 20 minutes in a humid chamber at 37°C. Cells were rinsed twice, washed for 15 minutes in PBS, mounted with mounting solution (50% PBS, 50% glycerol), and covered with a coverslip. Staining was evaluated by fluorescence microscopy using a Leica CTR500 fluorescence microscope and a Leica DFC300 FX digital camera.

[0083] [Table 2]

[0084] The antibody hCB2-1 / 622 showed high fluorescent signal intensity in two cell lines expressing human CB2, but no staining was observed in cells expressing human CB1, mouse CB2, or the negative control (Table 2).

[0085] For Western blot analysis, cells were harvested using enzyme-free dissociation buffer (Cell Dissociation Buffer Enzyme-Free Hanks' Basal, Gibco) and washed twice with PBS. 6 Cells were lysed in 0.1 ml of lysis buffer (1% NP40, 10% glycerol, 2 mM EDTA, 137 mM NaCl, 20 mM TrisHCl, pH 8, protease inhibitors) on ice for 10 minutes. Lysates were clarified by centrifugation at 20,000 g for 5 minutes. For SDS-PAGE, cell lysates were separated on a precast 4-12% gradient gel (NuPAGE® Novex 4-12% Bis-Tris Gel, Invitrogen) containing MES running buffer according to the manufacturer's instructions. Protein separation was visualized by colloidal blue staining. Proteins were electrophoretically transferred to a nitrocellulose membrane using a dry blotting system (iBlot, Invitrogen). After blocking the membrane in blocking buffer (5% milk in PBS) for 16 hours at 4°C, specific proteins were detected with appropriate dilutions of mAb in blocking buffer for 1 hour at approximately 20°C. The membrane was then washed four times for 5 minutes in blocking buffer and incubated for 1 hour at approximately 20° C. with anti-mouse IgG mAb conjugated to horseradish peroxidase (GAM / IgG (g chain) / HRP, Sigma, A9917) diluted 1:10,000 in blocking buffer. After washing again, the blot was developed using ECL Western blotting detection reagents (ECL Western blotting substrate, Pierce) to visualize the bands.

[0086] [Table 3]

[0087] Expression of the recombinant protein in pANITA3.1-transfected cells was confirmed by Western blot analysis using an anti-His tag antibody. Cells expressing the rat glucose-dependent insulinotropic polypeptide receptor (GIPR), which is also a G protein-coupled receptor, were used as a negative control.

[0088] Antibody hCB2-1 / 622 detected distinct bands for hCB2, but not mCB2, hCB1, or rat GIPR (Table 3).

[0089] For immunoprecipitation (IP), whole cell lysates were incubated with the respective antibodies, which were performed according to established protocols, followed by Western blotting as described herein above.

[0090] [Table 4]

[0091] IP experiments show that antibody hCB2-1 / 622 specifically binds to hCB2, but no cross-reactivity to mCB2 or rat GIPR was observed (Figure 2 and Table 4).

[0092] Further evidence for the specificity of the anti-human CB2 antibody 1 / 622 came from flow cytometry experiments using increasing levels of the antibody on untransfected, hCB1 or hCB2-transfected CHO K1.

[0093] Cells were washed once with 1x DPBS (Gibco, #14190-094) without CaCl2 or MgCl2. Accutase (Sigma, #A6964) was added, and the cells were returned to the incubator. Once detached, the cells were collected in a Falcon tube containing DMEM high glucose + GlutaMAX (Gibco, #31966-021) and 10% heat-inactivated FBS (Gibco, #16140-071). The cells were centrifuged at 250 x g and 4°C for 5 minutes, and the cell pellet was resuspended in cell staining buffer (BioLegend, #420201) and placed on ice until further use. The cells were then incubated with 0.1, 1, or 10 μg / ml hCB2-1 / 622 antibody for 30 minutes at 4°C. After incubation, the tube was centrifuged at 250 x g and 4°C for 5 minutes. The supernatant was completely removed, and the cells were washed twice with cold staining buffer. After centrifugation, the supernatant was completely removed. Human FcR blocking reagent (Miltenyi Biotech, catalog no. 130-059-901) was diluted in cold staining buffer according to the package insert. 50 μl was added to each sample, mixed, and incubated at 4°C for 10 minutes. Secondary antibody goat F(ab')2 anti-mouse IgG3PE (Southern Biotech, catalog no. 1102-09) was diluted 1:25 in cold staining buffer. 50 μl was added to each sample, mixed, and incubated in the dark at 4°C for 30 minutes. After incubation, the cells were washed twice with cold staining buffer, and the supernatant was completely removed after centrifugation. 200 μl of cold staining buffer was added to each tube, mixed carefully, and stored on ice or in the dark at 4°C until acquisition on a flow cytometer.

[0094] A dose-related increase in median fluorescence intensity (MFI) signal was detected only in the CHO K1 hCB2 cell line, whereas the MFI remained at background levels for CHO cells that were not transfected (CHO K1 control) or transfected with human CB1 protein (CHO K1 hCB1R, Figure 3).

[0095] Furthermore, a good signal-to-noise ratio for the detection of human CB2 in transfected versus parental non-transfected CHO-DUXX cells was established by flow cytometry (Figure 4), further confirming the specificity and suitability of antibody 1 / 622 for the specific detection of CB2.

[0096] Example 2: Comparison of anti-hCB2 antibody 1 / 622 with commercially available hCB2 antibodies Detection of baseline expression of cell surface CB2 on B cells was evaluated for clone 1 / 622 and compared to 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 increasing concentrations of each anti-human CB2 antibody.

[0097] The dose-dependent increase in median fluorescence intensity observed only for the hCB2-1 / 622 antibody supports the specific binding of this anti-human CB2 antibody compared to comparison conditions without primary anti-CB2 antibody (0 μg / sample) or FMO (fluorescence minus 1) control and control conditions in which the secondary fluorescently labeled antibody was omitted.

[0098] Example 3: Detection of changes in surface expression of CB2 on leukocytes by flow cytometry assay CB2 is a member of the G protein-coupled receptor family that can respond upon ligand (agonist) binding by internalization of the receptor into the endosomal compartment of the cell, thereby removing the respective 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). These changes in cell surface expression of CB2 on B cells in response to treatment with the CB2 agonist RG7774 were measured in whole blood samples from nine different working-age healthy human volunteers. Whole blood samples were treated in duplicate with eight compound concentrations, and the IC50 of RG7774 was determined using a newly developed flow cytometry assay.

[0099] Na-heparin whole blood samples were incubated in a 96-well plate containing RG7774 at 37°C in a 5% CO2 humidified incubator for 1 hour. Samples were removed from the 96-well plate, and 100 μl was transferred to labeled 5 ml polystyrene round-bottom tubes (Becton Dickinson, catalog no. 352052). Unlabeled anti-CB2 antibody clone 1 / 622 was diluted to 50 μg / ml in cell staining buffer (BioLegend, catalog no. 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 no. 55899) was diluted to 1x with distilled water and preheated to approximately 20°C. After the CB2 antibody incubation, 2 ml of prewarmed 1x BDPharmLyse lysis buffer was added to each tube, mixed, and incubated at approximately 20°C for 15 minutes. After incubation, the tubes were centrifuged at 250 x g and 4°C for 5 minutes. The supernatant was completely removed, and the cells were washed twice with cold cell staining buffer, followed by centrifugation and complete removal of the supernatant. Human FcR blocking reagent (Miltenyi Biotech, catalog no. 130-059-901) was diluted in cold cell staining buffer according to the package insert. 50 μl was added to each sample, mixed, and incubated at 4°C for 10 minutes. The secondary antibody goat F(ab')2 anti-mouse IgG3PE (Southern Biotech, catalog no. 1102-09) was diluted 1:25 in cold cell staining buffer. 50 μl was added to each sample, mixed, and incubated in the dark at 4°C for 30 minutes. After incubation, the cells were washed twice with cold cell staining buffer, and the supernatant was completely removed after centrifugation.A lineage marker mix was prepared using cold cell staining buffer containing the antibodies CD3 PerCp-Cy5.5 (BioLegend, catalog no. 344808), CD14 BV510 clone M5E2 (BioLegend, catalog no. 301842), CD16 APC (Becton Dickinson, catalog no. 561304), CD19 BV421 (BioLegend, catalog no. 302234), CD45 APC-Cy7 (BioLegend, catalog no. 304014), CD56 PE-Cy7, clone 5.1.H11 (BioLegend, catalog no. 362510), CD193 FITC (BioLegend, catalog no. 310720), and IgE Alexa Fluor 647, clone MHE-18 (BioLegend, catalog no. 325514). 100 μl of lineage marker mix was added to each sample, mixed, and incubated in the dark at 4°C for 20 minutes. After incubation, cells were washed twice with cold cell staining buffer, and the supernatant was completely removed after centrifugation. 200 μl of cold cell staining buffer was added to each tube, mixed carefully, and kept on ice or in the dark at 4°C until acquisition on the flow cytometer.

[0100] To examine the cell surface expression of CB2 on B cells, median fluorescence intensity (MFI) was measured using a 100-well platelet count (1000µL) of CD45 + CD3 - CD14 - CD19 + We gated around B cells (see gating strategy in Figure 6A-6E) and analyzed by acquiring a minimum of 2,000 events in this gate (Figure 6F). A lineage marker mix was used to identify 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 + Other cell populations such as CD45 T cells could be analyzed. T cells were always found to be CB2 negative. Therefore, for certain experiments, CD45 T cells were analyzed. + CD3 + CDC14 - T cells served as an internal negative control population.

[0101] To establish the IC50 value for RG7774, the ratio of CB2-PE MFI on B cells to CB2-PE MFI on T cells normalized to the mean of the ratios at 0 μM RG7774 was calculated according to the following formula:

number

[0102] Individual IC50 values ​​in healthy human participants for RG7774 ranged from 64.3 nM to 143.9 nM, with a mean of 96.5 ± SE 73.2 to 118.7 nM (Table 5).

[0103] [Table 5]

[0104] On average, the maximum reduction in CB2 surface expression on B cells in vitro was 55% above baseline (Figure 7), and the trend of the curves appeared comparable and similar for both genders (four female and five male donors). A reduction in CB2 cell surface expression under these experimental conditions could also be detected on basophils, but was not observed on T cells, monocytes, NK cells, eosinophils, or neutrophils (data not shown).

[0105] Example 4: CB2 expression in different cell subpopulations of diabetic patients and matched healthy participants To assess whether diabetic disease status (type 1 and / or type 2 diabetes) may affect CB2 expression on the surface of circulating blood cells, we compared CB2 expression levels at baseline (in the absence of RG7774) with expression levels on the same cell populations in demographically matched, age-matched healthy participants (Figure 8). Median fluorescence intensity (MFI) across all participants suggests that CB2 expression is generally low, with B cells and basophils showing the highest levels, while monocytes show approximately half of that expression, and T cells and NK cells show low or no expression, respectively. Furthermore, the data suggest that diabetic disease status results in a trend toward decreased CB2 expression on the surface of B cells and basophils, although this was only significant for CB2 expression on basophils from type 2 diabetic patients compared with healthy participants (one-way ANOVA analysis p = 0.0245, Tukey's multiple testing corrected p < 0.05). No such trend was observed when CB2 expression levels on monocytes were low.

[0106] Example 5: IC50 values ​​of RG7774 in different lymphocyte subsets in diabetic patients and matched healthy participants To understand the exposure-response relationship for drug-induced CB2 internalization in blood cell populations, particularly B cells and basophils, in whole blood from nine different diabetic type 1 and type 2 patients and eight demographically matched, age-matched healthy participants, treated in duplicate with eight compound concentrations, the IC50 of RG7774 was determined.

[0107] Human whole blood from diabetic patients and age-matched healthy participants was incubated with RG7774 for 1 hour at 37°C, followed by incubation with anti-human CB2 Ab1 / 622. CB2 expression on the surface of different blood cell types was analyzed using a lineage marker mix specific for B cells, basophils, T cells, NK cells, and monocytes. The MFI value at 0 μM RG7774 was set as 100% baseline receptor expression for each cell type.

[0108] RG7774-induced CB2 internalization was observed in B cells (Figure 9A) and basophils (Figure 9B), but not in monocytes (Figure 9C), T cells (Figure 9D), or NK cells (Figure 9E).

[0109] This observation was reproducible and the trends of the IC50 curves were comparable in diabetic patients of both genders and both diabetes subtypes and in matched healthy participants (Figure 9 and Table 6).

[0110] The maximum reduction (Emax) of CB2 surface expression on mean B cells and basophils in vitro was 50% or more above baseline in 16 of 17 patients (Table 6). For B cells, it resulted in an Emax of approximately 56% in diabetic patients and matched healthy controls, while for basophils, an average Emax of approximately 60% was observed in both groups. The value for subject 774USB16 was excluded because it was an outlier with an IC50 on B cells approximately 10-fold higher.

[0111] [Table 6]

[0112] [Table 7]

[0113] When comparing the mean IC50 ± SD values ​​of RG774 from diabetic and age-matched healthy controls with the IC50 obtained from healthy working-age participants (data from Example 3, Table 5), the data show a trend that diabetic patients and matched healthy participants may require approximately 2-3 times higher exposure to RG774 to achieve half-maximal internalization of CB2 on B cells compared to working-age healthy participants (Table 7, one-way anova with Tukey's multiple comparisons correction, p=0.0575). The mean age of the working-age population is lower than that of diabetic patients and matched healthy controls.

[0114] These in vitro data, obtained with a newly developed flow cytometry assay measuring CB2 receptor internalization on B cells and other blood cell types in response to RG7774, demonstrate that the pharmacodynamic response in elderly diabetic patients and demographically matched healthy participants is shifted to concentrations 2-3 fold higher than in young healthy participants, suggesting that higher exposures are required to achieve similar drug response effects in such participants, an important finding with implications for age-based dose selection.

[0115] Example 6: In vitro feasibility evaluation of the CB2 internalization assay in cynomolgus monkeys To establish whether the CB2 surface assay is cross-reactive in cynomolgus monkeys, blood obtained from five different healthy cynomolgus monkeys was tested in vitro, with individual IC50 values ​​and maximal reduction in CB2 surface expression for RG7774 in whole blood determined using eight different compound concentrations in duplicate (Figure 10, Table 8).

[0116] [Table 8]

[0117] The maximum decrease in CB2 surface expression on mean B cells in vitro was 43.8% ± 7.4% SD above baseline. Notably, one of the monkeys (Ikarus) did not demonstrate the expected pharmacodynamic response within the dose range tested, highlighting the potential for using this approach to identify individuals with inadequate or no drug response. No decrease in CB2 expression was observed on T cells, monocytes, or granulocytes (data not shown). This in vitro data confirmed the cross-reactivity of the CB2 flow cytometry assay in cynomolgus monkeys and justified experimental testing of the feasibility of this assay for monitoring target engagement in vivo following oral administration to cynomolgus monkeys.

[0118] Example 7: Feasibility evaluation of a newly developed CB2 flow cytometry assay to monitor CB2 internalization in vivo upon oral administration to cynomolgus monkeys The feasibility of applying the newly developed CB2 flow cytometry assay as an assay for monitoring the pharmacodynamic response to oral doses of RG7774 was evaluated in three groups of two cynomolgus monkeys. Upon oral administration of three different single doses of RG7774 microsuspension at 0.5, 5, and 100 mg / kg, respectively, blood samples from all animals were collected at selected time points: 0 h (pre-dose), 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 7 h, and 24 h after administration and subjected to CB2 flow cytometry analysis (Figure 11). A dose-dependent decrease in CB2 expression on B cells compared to individual baselines was observed, which persisted for approximately 24 h, strengthening confidence in the feasibility of the assay for clinical application.

[0119] Based on the increased confidence from the first in vivo study, a second cynomolgus monkey in vivo study was conducted to investigate pre- and post-dose time points to better understand the pharmacodynamic response to RG7774 over time.

[0120] In this in vivo cynomolgus monkey study, three cynomolgus monkeys were orally dosed with 100 mg / kg of RG7774. Blood samples were taken for TE assays at selected time points: -96 hours, -24 hours (both time points not shown), 0 hours (pre-dose), and 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 7 hours, 24 hours, 31 hours, 48 ​​hours, and 72 hours after dosing (Figure 12).

[0121] This study revealed a fast pharmacodynamic effect (within 15 minutes) and sustained effect of RG7774 on surface expression of CB2 on B cells, which could be reliably monitored in a dose-dependent manner over time, confirming the utility of the newly developed flow cytometry assay for clinical application as a target engagement assay.

[0122] Example 8: Successful implementation of the assay in a Phase I program confirms that this assay can be used in clinical practice, and the data will inform dose selection for clinical development. The clinical utility of the newly developed CB2 flow cytometry assay as a measure of target engagement in clinical practice was confirmed by its successful implementation in a Phase I clinical trial in healthy volunteers. In clinical practice, the B / T cell ratio was not used because it introduced higher variability compared to baseline-normalized B cell data alone.

[0123] In the first phase, oral administration of single ascending doses (SAD) of RG7774 ranging from 0.75 mg to 300 mg in the fasted state was evaluated. After oral administration of RG774, the onset of CB2 internalization was rapid, with the nadir (lowest CB2 surface expression) achieved 4 hours post-dose for doses below 10 mg RG7774 and 1 hour post-dose for doses of 10 mg to 30 mg RG7774 (Figure 13). At the two highest doses tested, 100 mg and 300 mg RG7774, the time to reach the nadir shifted as the area under the effect curve increased. The extent of CB2 internalization (i.e., CB2 target engagement) increased dose-dependently, with a slight increase at doses of 100 mg to 300 mg RG7774. This indicates successful target engagement of RG7774 upon oral administration. Additionally, the assay was used to inform stopping rules for dose escalation in the SAD portion of the first-in-human (FIH) trial.

[0124] The second part of the FIH study investigated the effect of food consumption (FE) by measuring cell surface CB2 expression by flow cytometry assay on B cells in the fasted (n=8) and fed (n=8) states, but did not reveal any relevant impact of food consumption on the pharmacodynamic (PD) effects of RG7774 (Figure 14).

[0125] Part 3 of the FIH study examined the effect of multiple ascending doses (MAD) of RG7774 from 20 mg to 300 mg administered once daily over 14 days on CB2 internalization (Figure 15).

[0126] The newly developed assay helped characterize the PD effects of a CB2 agonist (in this case, RG7774) in a clinical setting in healthy volunteers over 14 days of once-daily dosing, showing that steady state was reached during the observation period, no desensitization was observed over the 14-day period, and the induced drug effect in reducing CB2 surface receptor expression was reversible within approximately 1 week of stopping the drug, but was not yet fully reversed for the two highest doses.

[0127] [Table 9-1]

[0128] [Table 9-2]

Claims

1. An antibody comprising an antigen-binding moiety that binds to human cannabinoid receptor subtype 2 (CB2), comprising a heavy chain variable region (VH) including 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) including 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.

2. The antibody according to claim 1, comprising: (b) a heavy chain variable region (VH) having 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) having 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. FC domains are included, and optionally: a) The Fc domain is an Fc domain of the IgG class, particularly the IgG 3 subclass; and / or b) The antibody according to claim 1 or 2, wherein the Fc domain is a mouse Fc domain.

4. The antibody according to claim 1 or 2, 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.

5. The antibody according to claim 1 or 2, which cross-reacts with cynomolgus monkey CB2.

6. A method for detecting changes in cell surface CB2 expression in a biological sample obtained from a target in response to treatment with a CB2 agonist, by flow cytometry, a) A step of detecting the cell surface CB2 expression level in a reference sample that has not been treated with a CB2 agonist, b) A step of detecting cell surface CB2 expression in the sample after treatment with a CB2 agonist, c) a step of comparing the cell surface CB2 expression level in step b) with the cell surface CB2 expression level in step a), The decrease in cell surface CB2 expression in response to CB2 agonist treatment indicates target involvement. A method for detecting cell surface CB2 expression using the antibody described in claim 1.

7. A method for evaluating the treatment response to a CB2 agonist in a subject by flow cytometry, a) A step of detecting the cell surface CB2 expression level in a biological sample obtained from the subject before treatment with the CB2 agonist, b) A step of detecting the cell surface CB2 expression level in a biological sample obtained from the subject after treatment with the CB2 agonist, c) a step of comparing the cell surface CB2 expression level in step b) with the cell surface CB2 expression level in step a), A method for detecting cell surface CB2 expression using the anti-human CB2 antibody described in claim 1.

8. i) Cell surface expression of CB2 is a) The detection portion conjugated to the anti-human CB2 antibody, or b) A second antibody comprising a detection portion that binds to the anti-human CB2 antibody. Detected by; and / or ii) The method according to claim 6 or 7, wherein the CB2 agonist is (S)-1-[5-tert-butyl-3-(1-methyl-1H-tetrazole-5-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine-7-yl]-pyrrolidine-3-ol.

9. The method according to claim 6 or 7, wherein the subject is a human or a cynomolgus monkey.

10. The method according to claim 6 or 7, wherein the biological sample is treated in vitro with the CB2 agonist.

11. The method according to claim 6 or 7, wherein the subject is treated with the CB2 agonist.

12. The method according to claim 6 or 7, wherein the biological sample is whole blood.

13. The method according to claim 6 or 7, wherein the dose-response relationship of a CB2 agonist is determined, and optionally, the dose-response relationship of the test population supports the selection of a clinical dose for the target population.

14. A diagnostic agent comprising the antibody according to claim 1 or 2, for use in the method according to claim 6 or 7.

15. The use of the antibody according to claim 1 or 2 in the method according to claim 6 or 7.

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