Cannabinoid Receptor 2 Activating Antibodies: A Promising Therapeutic Strategy for Macrophage-Driven Fibro-Inflammatory Diseases

Cannabinoid Receptor 2 Activating Antibodies: A Promising Therapeutic Strategy for Macrophage-Driven Fibro-Inflammatory Diseases | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Cannabinoid Receptor 2 Activating Antibodies: A Promising Therapeutic Strategy for Macrophage-Driven Fibro-Inflammatory Diseases Raghavender Reddy Gopireddy , Dipankar Bhattacharya , Swastik Sen , Monica A. Schwartz , View ORCID Profile Scott L. Friedman , Richard C. Yu , Toshi Takeuchi , View ORCID Profile Lauren J. Schwimmer doi: https://doi.org/10.1101/2025.07.11.664477 Raghavender Reddy Gopireddy a Abalone Bio, Inc ., 1250 45 th St. Suite 520, Emeryville, CA 94608 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dipankar Bhattacharya b Division of Liver Diseases, Icahn School of Medicine at Mount Sinai , Box 1123, 1425 Madison Ave. Room 1170, New York, NY 10029, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Swastik Sen a Abalone Bio, Inc ., 1250 45 th St. Suite 520, Emeryville, CA 94608 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Monica A. Schwartz a Abalone Bio, Inc ., 1250 45 th St. Suite 520, Emeryville, CA 94608 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Scott L. Friedman b Division of Liver Diseases, Icahn School of Medicine at Mount Sinai , Box 1123, 1425 Madison Ave. Room 1170, New York, NY 10029, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Scott L. Friedman Richard C. Yu a Abalone Bio, Inc ., 1250 45 th St. Suite 520, Emeryville, CA 94608 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Toshi Takeuchi a Abalone Bio, Inc ., 1250 45 th St. Suite 520, Emeryville, CA 94608 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lauren J. Schwimmer a Abalone Bio, Inc ., 1250 45 th St. Suite 520, Emeryville, CA 94608 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lauren J. Schwimmer For correspondence: lschwimmer{at}abalonebio.com Abstract Full Text Info/History Metrics Preview PDF Abstract Fibrotic diseases, often fueled by chronic inflammation, represent a major unmet medical need. A critical driver of this process is the activation of macrophages, which secrete pro-inflammatory cytokines and chemokines, leading to immune cell recruitment and collagen deposition by myofibroblasts. Agonizing the cannabinoid receptor 2 (CB2), expressed on macrophages, offers a potential therapeutic avenue to suppress this activation. Despite widespread interest in CB2 modulation, challenges remain in the development of small molecule agonists, including insufficient specificity and poor drug-like properties. Antibodies are highly specific with favorable pharmacokinetics and bioavailability, but GPCR agonist antibodies have been difficult to discover. Here we present first-in-class CB2 agonist antibodies, AB120 and AB150. These antibodies are CB2 specific, G-alpha biased, and effectively decrease the expression of macrophage activation markers and key pro-inflammatory cytokines, including IL-6, IL-1β and TNF-α. Furthermore, using an ex vivo human precision-cut liver slice (hPCLS) model of liver fibrosis, we observe that treatment with these CB2 agonist antibodies not only reduces inflammatory markers but also decreases collagen expression, indicating a potential to halt or reverse fibrosis. These novel CB2-specific agonist antibodies hold promise as therapeutics for a range of fibrotic and inflammatory conditions driven by chronic macrophage activation and demonstrate the potential of GPCR antibody agonists to drug this challenging class of targets. Significance Statement Fibrotic diseases driven by chronic inflammation lack effective treatments. CB2 agonism modulates macrophage activation to reduce pro-inflammatory cytokines suggesting a novel avenue of treatment, but small molecule CB2 agonists have faced substantial limitations due to off-target effects. We introduce two novel CB2-specific agonist antibodies with potent anti-inflammatory and anti-fibrotic effects in vitro and in a human ex vivo liver fibrosis model. These antibodies provide a promising new therapeutic strategy and highlight the advantages of antibodies as GPCR agonist drugs. Introduction The cannabinoid receptor 2 (CB2) has received notable therapeutic interest for a range of conditions, particularly those involving inflammation and immune modulation. 1 – 5 However, small molecule agonist development has been hindered by the high lipophilicity of the molecules, resulting in poor pharmacokinetics, and by cross-reactivity to CB1, which is highly expressed in the central nervous system (CNS) and associated with adverse psychoactive effects. 6 – 8 Antibodies are stable, well-expressed, have excellent pharmacokinetic properties, and have exquisite specificity toward their targets. 9 Therefore, a CB2 antibody agonist is poised to overcome the major challenges encountered by small molecule agonists. Here, we functionally characterize the first reported CB2 antibody agonists, AB120 and AB150, 10 , 11 which demonstrate selective CB2 activation and anti-inflammatory properties. CB2 is a class A G-protein coupled receptor (GPCR) that is primarily expressed in lymphocytes and myeloid cells, including macrophages, microglia, and peripheral sensory neurons. 12 , 13 CB2 couples to G i/o and modulates various intracellular signal transduction pathways, including inhibition of cAMP production, ERK1/2 phosphorylation and β-arrestin-2 recruitment. 8 , 14 , 15 These signals ultimately modulate expression of anti-inflammatory and anti-fibrotic genes. 16 – 22 CB2 activation induces macrophage autophagy and suppresses macrophage activation, 23 – 25 thereby reducing expression of pro-inflammatory cytokines and chemokines including IL6, TNF-α, IL-1β and CCL4. Chronic macrophage activation can lead to fibrosis in many tissues, including the liver. 22 , 26 Therefore, CB2 agonists are well situated to treat chronic inflammation and fibrotic diseases in which macrophages contribute to the pathology. Selective small molecule CB2 agonists have been pursued for treating various conditions such as inflammatory pain, neuropathy, and liver fibrosis. 4 , 5 , 8 , 22 , 27 While these compounds primarily target CB2, their development has been hindered by the off-target effects on CB1, 16 , 28 as the high conservation of the ligand binding pocket makes it challenging to produce highly specific small molecules. 29 Specificity for CB2 is crucial, as even low CB1 receptor occupancy (4%-14%) can lead to activation of the receptor and subsequent inhibition of locomotor activity in mice. 30 Off-target activation of CB1 is undesirable as CB1 is widely expressed in the CNS, where activation leads to the psychoactive effects associated with the recreational use of cannabinoids including increased appetite. 31 In addition, activation of CB1 can be pro-fibrotic, countering the biological effect of the CB2 agonism. 32 , 33 Due to off-target liabilities of some small molecule drugs, highly specific and selective antibody drugs are attractive alternatives and are now an important component of the therapeutic landscape. 34 The CB2 orthosteric binding pocket is highly conserved, while the low amino acid sequence conservation of the surface available residues between CB1 and CB2 (26% identity) makes it unlikely that CB2 agonist antibodies would cross-react to CB1. Thus, antibodies that target the extracellular loops of CB2 and activate the receptor allosterically have the potential to be more specific than small molecules. Here we demonstrate in vitro cAMP, β-arrestin and ERK1/2 signaling of our CB2 agonist antibodies, AB120 and AB150, in mouse and human macrophage cell lines. This activity is specific to the CB2 receptor and biased toward cAMP over β-arrestin and ERK1/2. We also show that these CB2 agonist antibodies reduce macrophage activation and decrease secretion of pro-inflammatory and pro-fibrotic proteins from cell lines and precision cut human liver slices (hPCLS). AB120 and AB150 have potential therapeutic applications in a variety of inflammatory conditions where macrophage biology is implicated, including peripheral neuropathies and fibrotic diseases. Results We performed in vitro assays with the CB2 agonist VHH-Fc chimeric antibodies AB120 and AB150 using mouse (RAW264.7) and human (THP-1 differentiated with PMA, Sup Fig 1 ) macrophage cell lines, which express both CB1 and CB2 35 to determine the functional activity and selectivity for CB2 and not CB1. The cannabinoid receptors are primarily G i coupled, whereby activation leads to inhibition of adenylate cyclase and reduction of cAMP production. We utilized a bioluminescence-based high-throughput assay to measure cAMP, in which the cell lines were stimulated with NKH477 (a forskolin derivative) to produce cAMP and treated with AB120, AB150, HU-308 (CB2 small molecule agonist), 8 , 27 or AB100 (negative isotype control antibody). Importantly, both, AB120 and AB150, but not AB100, potently inhibit cAMP production in both mouse and human macrophage cell lines. The EC 50 and E max of both antibodies are comparable to HU-308, which was chosen as the CB2 agonist benchmark due to its >100-fold CB2 selectivity over CB1 and balanced activity across multiple signaling pathways. 8 ( Figure 1A and D , Table 1 ). This agonist activity is inhibited by SR144528, 36 , 37 a CB2 selective antagonist ( Figure 1B and E ), but not by SR141716A, 38 a CB1 selective antagonist ( Figure 1C and F ), which can inhibit ACEA, a CB1 selective agonist. These data demonstrate that the effect on cellular cAMP levels by AB120 and AB150 is specific to CB2 receptor agonism. Download figure Open in new tab Supplementary Figure1. THP-1 Cells differentiation by PMA. THP-1 cells were incubated for 24h without (A) or with 200ng/mL PMA (B) and replaced the media. Images were captured at 10x magnification after 5 days by using phase-contrast inverted microscope. Scale bar −10μm Download figure Open in new tab Fig.1 Dose response curve for CB2 specific agonist-dependent cAMP inhibition in RAW26.7 (A-C) cells and THP1-cells (D-F). CB2 specific agonist (HU-308), CB1 specific agonist (ACEA), and CB2 specific agonist antibodies (AB120, AB150) decreases NKH477 induced cAMP production as detected via cAMP-Glo luminescence (Promega), where cAMP concentration is inversely proportional to luminescence (A and D). Additionally, CB2 selective antagonist, SR144528, inhibits HU308, AB120 and AB150 agonist activity (B and E). CB1 selective antagonist, SR141716A, only inhibits CB1 selective agonist, ACEA, activity (C and F). Data are represented as mean ± SD from two independent experiments. View this table: View inline View popup Download powerpoint Table 1. Efficacy and Potency profile of CB2 agonists CB2 agonism may also induce recruitment of β-arrestin-2, leading to internalization and downregulation of CB2, 39 potentially limiting the in vivo effect of the antibodies. Therefore, it is important to characterize this signaling pathway. A TR-FRET based high-throughput assay was used to assess β-arrestin-2 recruitment by measuring the interaction between β-arrestin-2 and AP2, which interacts with β-arrestin-2 only after engagement with CB2. We found that, although AB120 and AB150 have similar potencies to HU-308 for β-arrestin-2 recruitment on both human and mouse macrophages ( Figure 2 and Table 1 ), both are partial agonists with lower E max compared to HU-308 in this pathway. This demonstrates that both antibodies are also biased agonists, as the potencies (EC 50 ) for the G-protein dependent cAMP inhibition are >10-fold more than that for β-arrestin-2 recruitment. Download figure Open in new tab Fig. 2 CB2 agonist antibodies-promoted β-Arrestin-2 recruitment detected by the TR-FRET assay. Dose response curve of β-Arrestin-2 recruitment by CB2 agonist antibodies in RAW264.7 (A) and THP-1 derived cells (B). Cells were transfected with 20ng of human β-Arrestin-2 expressing plasmid and 130ng empty pcDNA3 plasmid followed by measurement of β-Arrestin-2 recruitment assay. Data are represented as mean ± SD from two independent experiments. CB2 agonists are also known to signal through a third pathway by inducing phosphorylation of ERK1/2 through G βγ. 40 Therefore, we measured the amount of ERK1/2 phosphorylation induced by CB2 agonists using a TR-FRET based assay. The potencies (EC 50 ) of both AB120 and AB150 are similar to HU308 in both RAW264.7 and differentiated THP-1 cells and to that of the β-arrestin-2 recruitment ( Figure 3 and Table 1 ). However, in contrast to β-arrestin-2 recruitment, both antibodies have a similar E max to HU-308. Download figure Open in new tab Fig.3 CB2 agonist antibodies-promoted ERK1/2 phosphorylation detected by the LANCE Phospho-ERK1/2 TR-FRET assay. (A and B) show dose dependent ERK1/2 phosphorylation by CB2 agonist antibodies in RAW264.7 and THP-1 derived cells. Data are represented as mean ± SD from two independent experiments. Reduction of cAMP via CB2 agonism is known to prevent the activation of PKA, thereby reducing the expression of pro-inflammatory genes. 41 , 42 Therefore, we next sought to study the downstream consequences of the initial signaling events in macrophages. Macrophages are a primary component of the innate host defense immune response and are involved in tissue repair and homeostasis. Depending on external stimuli, macrophages can be induced to express markers that are traditionally characterized as M1, pro-inflammatory, or M2, anti-inflammatory states. 19 , 23 , 26 , 43 The M1/M2 paradigm is currently recognized as an oversimplification of the actual variation in activated macrophages; however, here it serves as a useful approach to distinguish functional effects of CB2 agonism in isolated macrophage cell lines. Considering this, an ideal CB2 agonist antibody would reduce expression of pro-inflammatory cytokines and chemokines from M1 macrophages. M2 macrophages are more complicated as they are primarily considered to be anti-inflammatory, however, upon chronic activation, M2 macrophages secrete TGF-β, which is profibrotic. 44 – 47 Therefore, an ideal CB2 agonist may be one that inhibits activation of both M1 and M2 macrophages or one that biases the polarization toward M2 over M1 in the context of macrophage cell lines. Here we demonstrate that CB2 agonism by AB120 and AB150 affects mRNA expression in mouse and human macrophage cell lines that have been stimulated with LPS or IL-4 to induce M1 or M2-like states, respectively. For RAW264.7 or differentiated THP-1 cells stimulated with LPS, CB2 agonist antibodies reduce the expression of M1 macrophage associated, pro-inflammatory genes ( TNF-α, NOS2, CCL4, IL-6, and IL-1β ) compared to untreated LPS stimulated and unstimulated cells ( Figure 4 , Table 2 ). Treatment of these same cell lines with IL-4 to induce M2 polarization, increases the expression of M2 macrophage associated genes ( Arg1, Mrc2, Mgl1, and Clec7A ) ( Figure 4 , Table 2 ). CB2 agonists HU308, AB120, and AB150 prevent the upregulation of these genes in RAW264.7 cells. However, in differentiated THP-1 cells, while HU308 and AB120 prevented the IL4 induced expression of Arg1, Mrc2, Mgl1, and Clec7A, AB150 only prevented the upregulation of Arg1 and not Mrc2, Mgl1, or Clec7A . AB100, the isotype control, had no effect on induced M1 or M2 macrophage gene expression. Overall, these CB2 agonists influenced the M1 and M2 macrophage gene expression biasing it toward an anti-inflammatory and anti-fibrotic state with more M2 associated gene expression than M1. Download figure Open in new tab Fig.4 mRNA expression of M1 and M2 macrophage genes in RAW264.7 and THP-1 derived cells. Cells were incubated with LPS (100ng/ml) or IL-4 (5ng/ml). Data are represented as mean ± SD from two independent experiments. Statistical significance is defined as: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 View this table: View inline View popup Download powerpoint Table 2. Protein markers of M1 and M2 macrophage activation and their function CB2 agonists have the potential to treat fibrosis by limiting macrophage activation, thereby reducing expression of pro-inflammatory and pro-fibrotic genes. 22 , 61 , 62 To demonstrate the potential of AB120 and AB150 to reduce the expression of fibrotic markers, human precision cut liver slices (hPCLS) from de-identified resection samples of human liver were treated with AB120, AB150, AB100, or Alk5i (a TGF-βRI kinase inhibitor and a positive control for this assay). This ex vivo fibrosis model preserves all cell types and tissue architecture, enabling a more comprehensive study of endogenous cell-type interactions in the liver. 63 , 64 Forty-eight (48) hours after treatment, mRNA expression of Col1A1, Acta2, IL6, IL10, and TNF-α and collagen secretion were measured via rtPCR and ELISA. Interestingly, both AB120 and AB150 reduced the gene expression of these fibrosis and inflammation markers, as well as the secretion of collagen as measured by ELISA ( Figure 5 , Table 2 ). As Col1A1 and Acta2 are markers of hepatic stellate cell (HSC) activation and collagen is primarily expressed from these stellate cells, these data demonstrate that, while AB120 and AB150 directly affect macrophage activation, they also have downstream effects on other cell types that contribute to the fibrotic process. Download figure Open in new tab Fig.5 CB2 antibodies reduced expression of fibrogenic and inflammatory genes (B-F) and secretion of collagen (A) in human precision cut liver slices. For B-F, each point is the mean of mRNA expression normalized to RPL13A expression for an individual patient sample (n=3-6) measured in triplicate via rtPCR, with the bar representing the overall mean ± SD. For A, collagen in the culture medium was quantified via ELISA and depicted as mean ± SD of a single measurement per patient sample (n=3-6). Statistical significance is defined as: * P < 0.05 Discussion While CB2 agonism shows therapeutic potential in a number of diseases, most small molecule CB2 agonists are highly lipophilic, have short in vivo half-lives, and have unwanted activity on CB1. 8 To address this, we have developed the first two CB2 agonist VHH-Fc antibodies, AB120 and AB150, which were discovered using a function-based selection. 10 Antibodies that are GPCR agonists are extremely rare due to limitations of traditional antibody discovery techniques that optimize for and produce high affinity binders, which usually do not affect target function beyond inhibition. In fact, to date only seven GPCR agonist antibodies have been previously reported in the literature. 65 – 72 Antibody drugs have excellent safety and efficacy track records in management of chronic conditions (e.g., the top-selling drug adalimumab [Humira] and many others 73 ). Antibodies are often more target specific, with fewer off-target effects and have longer half-lives and duration of action than small molecule or peptide-based drugs. VHH domains have been clinically validated (i.e. Ablynx’s Cablivi) and their small paratope size has been hypothesized to be more compatible with GPCR epitope size than the VH/VL paratope of conventional monoclonal antibodies. 74 Antibodies also have the advantage of peripheral restriction, which lowers potential CNS effects compared to small molecule agonists, particularly considering the CNS-biased expression of CB1 and the psychotropic effects of activation. 6 AB120 and AB150 are VHH-Fc antibodies, which consists of the VHH linked to a modified human IgG1 Fc with reduced effector function. This VHH fusion to the Fc extends the antibody half-life in vivo. Importantly, we have identified molecules with desired biases of downstream signaling. GPCRs, including CB2, are known to engage multiple pathways upon activation. 8 , 75 , 76 The degree of pathway activation, or biased signaling, has downstream biological effects, which can determine the ultimate pharmacology of an agonist. Here we demonstrate cAMP biased CB2 agonism by AB120 and AB150 on mouse and human macrophage cell lines. These antibodies potently decrease NKH477 stimulated cAMP production similarly to HU-308, a CB2 selective small molecule agonist. This decrease in cAMP production ultimately reduces expression of pro-inflammatory genes by CREB phosphorylation and inhibiting NF-κB induced transcription. 42 , 77 In contrast, AB120 and AB150 recruit β-arrestin-2 with lower potency and efficacy. As β-arrestin-2 recruitment can lead to internalization and down-regulation of CB2, 7 , 39 this signaling bias is advantageous for a CB2 agonist therapeutic. By signaling through the G i /G o -protein pathway, with less engagement of β-arrestin, more CB2 will remain on the cell surface, enabling continued action of AB120 and AB150. Previous reports also demonstrate that stimulation of CB2 leads to ERK-mediated cellular activation and anti-inflammatory effects in monocytes/macrophages. 40 , 78 , 79 In agreement with these reports, we showed that AB120 and AB150 antibodies induce ERK1/2 phosphorylation in mouse and human macrophage cells suggesting CB2 agonist antibodies engage with CB2 and are coupled to the activation of the ERK-1/2 kinase cascade through a classical upstream mechanism involving G i /G o -protein βγ−subunits. 40 Also important is the specificity of CB2 agonist therapeutics. Given that many small molecule CB2 agonists have residual CB1 activity, specificity of AB120 and AB150 for CB2 is important to avoid CB1 induced off-target pro-inflammatory and CNS effects. We demonstrate this specificity as the inhibition of cAMP production by AB120 and AB150 is counteracted by a CB2 antagonist, but not by a CB1 antagonist, in macrophage cell lines that endogenously express both CB2 and CB1. CB2 receptors are predominantly distributed in the peripheral immune system, including macrophages, with little expression in the CNS, which makes this receptor an attractive therapeutic target for inflammation and fibrosis. Macrophages can both promote and resolve inflammation depending on their activation state and environment. In the liver, the macrophage population is heterogeneous including tissue resident macrophages (i.e. Kupffer cells) and monocyte derived macrophages. 80 Under normal conditions, these cells maintain tissue homeostasis by balancing pro- and anti-inflammatory responses of macrophages to promote immunity to viruses, bacteria and parasites while maintaining healthy tissue through phagocytosis. Upon chronic assault with toxins, viruses, or high-fat diet, macrophages respond by polarizing to an M1 macrophage state and secreting pro-inflammatory cytokines and chemokines (CCL4, TNF-α, IL-6, IL-1β) that attract additional immune cells (i.e. neutrophils, monocytes, and NK cells), and produce reactive oxygen species (NOS2). Within the liver, these cytokines also activate hepatic stellate cells (HSC), leading to enhanced extracellular matrix (ECM) protein deposition including collagen, resulting in increased fibrosis. IL-10 is an anti-inflammatory cytokine that can counteract the effects of pro-inflammatory cytokines, however high IL-10 concentration is indicative of excess inflammation and has been linked to severity of liver cirrhosis. 81 , 82 Chronic inflammation drives fibrosis that does not resolve, affecting organ function. 81 In metabolic dysfunction-associated fatty liver disease (MAFLD), there is dysbiosis in the gastrointestinal tract that leads to increased gut permeability and higher levels of endotoxin/LPS in the serum of patients with MAFLD. 83 LPS has been shown to stimulate the Kupffer cell (KC) secretion of multiple cytokines and chemokines, including TNF-α, IL-1β, IL-6 and the chemokine CCL4. 84 Increased levels of these proteins leads to fibrogenic activity from hepatic stellate cells (HSC), promoting the progression of liver disease in metabolic dysfunction-associated steatohepatitis (MASH). Conversely, reduction in the levels of these proteins, as seen with AB120 and AB150 in vitro and ex vivo, should decrease HSC activation and fibrosis in MASH. TNF-α engages HSC to increase secretion of TIMP-1, a potent contributor to liver fibrosis. 85 , 86 Moreover, activation of the IL-6 pathway in conjunction with TNF-α leads to strongly pro-inflammatory effects 87 that can synergistically trigger chronic inflammation and fibrosis in the liver. IL-1β is another potent cytokine that is increased in LPS treated macrophages that can act on multiple cell types to promote release of chemokines such as CCL2 and CCL5 for further immune cell recruitment, and can directly activate HSC to increase fibrotic activity. 88 , 89 The increase in CCL4 chemokine secretion with LPS serves as a chemoattractant for multiple immune cell populations, including natural killer cells, monocytes, T cells and dendritic cells, which respond to CCL4 gradients in response to tissue damage. 90 Furthermore, CCR5, which is a receptor for CCL4, is expressed on HSC, and activation of this receptor can directly promote hepatic fibrosis. 91 Together, these data demonstrate that KC secretion of these multiple cytokines and chemokines can serve as a focal point for the activation of HSC and promotion of the fibrotic phenotype. Consistent with this mechanism, we show LPS-driven increase of TNF-α, IL-1β, IL-6 and the chemokine CCL4 in two macrophage cell lines. Importantly, we have demonstrated that CB2 agonist antibodies, AB120 and AB150, decreased the expression of these LPS-induced pro-inflammatory cytokines and chemokines below baseline levels in these mouse and human macrophage cell lines. Similar reduction in inflammatory cytokines was seen with primary human tissue in hPCLS, consistent with the proposed model where reduction in these cytokines, will also reduce hepatic stellate cell activation and subsequently reduce collagen expression and deposition. This model is further supported by the reduced expression of smooth muscle actin and alpha-1 type I collagen observed upon treatment with AB120 and AB150 in the hPCLS model. On the other hand, treatment of macrophages with IL4 promotes expression of anti-inflammatory M2 macrophage markers. However, when chronically activated, M2 macrophages can be pro-fibrotic due to increased expression of TGF-β. 50 , 92 On mouse macrophages, AB120 and AB150 prevented IL-4 induced expression of M2 macrophage markers, but did not decrease the expression from baseline. However, for the differentiated THP-1 cells, the effect of these CB2 agonist antibodies was varied, with AB120 preventing the IL-4 induced M2 marker upregulation and AB150 only preventing Arg1 upregulation. Overall, using the simplified M1/M2 model, these antibodies bias macrophage polarization to favor anti-inflammatory over pro-inflammatory gene expression. While more complex in vivo, this biased polarization is an advantage for these antibodies in treating liver fibrosis and resolving the inflammatory state. In conclusion, we identified the first agonist antibodies for CB2 - one of only eight GPCRs for which antibody agonists have been discovered - and in vitro studies validate our hypothesis that our first-in-class CB2 agonist antibodies signal through G-alpha biased signaling and reduce expression of pro-inflammatory cytokines and macrophage activation. The effect on in vitro signaling, gene expression and macrophage activation directly translates to gene expression in human liver tissue, where reduction in HSC activation, inflammation and fibrosis markers are also observed in the hPCLS studies. These data make a strong case for AB120 and AB150 as promising therapeutics for liver fibrosis. In addition to liver fibrosis, macrophages drive other fibrotic disease including pulmonary and renal fibrosis. 22 CB2 is also expressed on peripheral neurons and microglial cells; therefore, these CB2 agonists have potential applications for treating peripheral neuropathies induced by chemotherapy or diabetes. 93 – 95 Based on the functional biology demonstrated by AB120 and AB150 in this report, we are pursuing further evaluation of these antibodies for therapeutic use in multiple indications, including liver fibrosis and peripheral neuropathies. Materials and Methods Cell lines, reagents and ligands Mouse macrophage cell line (RAW264.7) and Human leukemia monocytic cell line (THP-1) cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (DMEM) and RPMI-1640 medium from ATCC supplemented with 10% FBS, 20mM HEPES and 1X antibiotic and antimycotic, respectively. All cells were maintained at 37 °C in the presence of 5% CO 2 . 3-Isobutyl-1-methylxanthine, LPS and IL-4 are from Sigma-Aldrich. RO-1276, NKH 477, HU-308, SR 155528, ACEA, SR141716A (rimonabant), and Phorbol 12-myristate 13-acetate (PMA) are from Tocris. Antibody cloning and production VHH nucleotide sequences for AB100, AB120, and AB150 were procured as gene fragments from IDT and cloned into the TGEX-Fc expression vector (Antibody Design Labs) using NEBuilder HiFi DNA assembly (New England Biolabs). For each antibody, plasmid DNA was transfected into Expi 293F cells (ThermoFisher Scientific) with Expi 293 transfection reagent using the manufacturer’s protocol and incubated at 37 0 C with 80% CO2 for 5 days. The protein A resin, MabSelect Sure (Cytiva), was used to purify the VHH-Fc fusions from the cell culture supernatant through gravity flow. The purified antibodies were dialyzed into 1X PBS (pH 7.4). Differentiation of THP-1 and primary human monocyte-derived macrophages THP-1 cells were seeded at 0.25×10 6 cells/mL and stimulated with 200ng/mL phorbol 12-myristate 13-acetate (PMA, Tocris) for 24 hrs. Then, culture media was removed, and fresh media was added. After 96 hrs, THP-1 cells are fully matured into macrophages and firmly attached to the bottom of the culture vessel ( Supplementary Figure 1 ). Cells can be now used for subsequent experiments. 96 Microscope image acquisition THP-1 cells were seeded on 6 well plate and differentiated with PMA. A Nikon Eclipse TE300 Inverted Microscope equipped with a Hg short arc HBO lamp (OSRAM) was used to acquire images. The phase-contrast images were collected using a 10x objective lens. The images were collected before and five days after PMA treatment. Luminescence cAMP assay cAMP-Glo Max assay kit (Promega, V1682) was used to measure cAMP signals in Raw264.7 and THP-1 cells. Briefly, 10000 cells per well seeded on Poly-D-lysine (Sigma Aldrich) coated 384 well view plate (Revvity) and incubate 16-18 hours. Test compounds were added to the wells, briefly centrifuged, and incubated for 30 min at 37°C. After 30 min, cAMP detection reagent (Glo one) with PKA was added to cells, briefly centrifuged, and incubated 20 min at ambient temperature (RT). In the final step, kinase Glo reagent was added to the cells, briefly centrifuged, and incubated for 10 min at RT. The luminescence signal was measured by using Synergy HTX multi-mode microplate reader (Bio Tek). The data were fit with a non-linear regression of variable slope using GraphPad Prism software (version 10.0) TR-FRET Phospho-ERK Detection assay The TR-FRET Phospho-ERK assay was carried using LANCE Ultra phospho-ERK1/2 (Thr202/TYR204) detection kit (Revvity, TRF400C) following manufacturer’s protocol. Briefly, 80,000 cells were seeded on 96 well clear bottom plates and incubated overnight at 37°C. The next day, cells were serum starved for 4 hrs. followed by removal of the serum free medium. Test compounds were then added to the plate and incubated 5 min at RT with shaking (200rpm). Later, test compounds were removed and lysis buffer added to the plate and incubated for 30 minutes at RT with shaking (200 rpm). Cell lysates (15ul) were transferred to 384 Optiview detection plates (Revvity) and premixed Eu-labeled anti-ERK1/2 (T202-Y204) antibodies and ULight labeled anti-ERK1/2 antibodies were added to the plate and incubated 1hr at RT. TR-FRET signal was measured with a Victor2 multi plate reader. The ratio ((em.655/em615)*10,000) was calculated and the data fit with a non-linear regression of variable slope using GraphPad Prism software (version 10.0). β-arrestin-2 recruitment assay The β-arrestin-2 recruitment assay was performed using HTRF β-arrestin 2 Recruitment Detection Kit (Revvity) following manufacturer’s protocol. Briefly, cells were seeded on 96 well culture plate and incubated for 24 hrs at 37 °C. After 24hrs, β-arrestin (20ng) and empty pcDNA3 (150ng) plasmids were transfected to the cells by using lipofectamine 2000 transfection kit (Thermo Fisher Scientific) and incubated 24hr at 37°C incubator. After 24hrs, the media was removed, and test compounds were added to the plate and incubated for 30min. Later, the media was removed, and stabilization buffer was added to the plate and incubate for 15 min at RT. Stabilization buffer was removed, and the plate was washed 3x with wash buffer. Later, pre-mixed d2 and Eu cryptate antibodies were added to the cells and incubated overnight at RT. Next day, HTRF signal was measured by using a Victor2 multi plate reader. The ratio ((em.655/em615)*10,000) was calculated and fit the data with a non-linear regression of variable slope using GraphPad Prism software (version 10.0). Quantitative PCR Macrophage gene expression quantification was adapted from previous publications. 19 , 23 Cells were seeded on 6 well culture plate and incubate 24hrs at 37°C incubator. Next, cells were serum starved for 24hrs and added test compounds to the cells and incubated 16 to 18hr at 37°C. After 18hrs, LPS (100ng/ml) or IL-4 (10ng/ml) was added to the cells, followed by 8hrs and 16hrs incubation, respectively. After incubation, cells were lysed and RNA extracted from collected samples using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. mRNA was reverse transcribed using Super script IV VILO master mix kit (Invitrogen). Finally, quantitative PCR (qPCR) was performed on AriaMx RT-PCR system (Agilent), using Power SYBR Green Universal Master mix (Applied Biosystems). Samples were measured in triplicates. The qRT-PCR data were processed and analyzed by the 2 -ΔΔCT method. The sequences of the primer pairs used are listed in Supplementary Table 1 . 19 Statistical analyses were performed with GraphPad Prism software (version 10.0) by using two-way ANOVA with Dunnett’s multiple comparisons test. Statistical significance is defined as: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 View this table: View inline View popup Download powerpoint Supplemental Table 1 Precision cut liver slices The technique for human precision-cut liver slices (hPCLS) was previously described, 64 with minimal modification. The protocol relies on fully de-identified liver samples and thus formal review by the Institutional Review Board (IRB) at the School of Medicine at Mount Sinai is not required. Briefly, de-identified patient liver samples surrounding HCC or metastatic regions were transported to the lab in Belzer UW Cold Storage Solution (UW Solution). An 8 mm diameter core was obtained, and 200 μm slices were generated from each core in the presence of UW solution, with continuous supplies of 95% O2 and 5% CO2. The slices were cultured in complete media containing WE-GlutaMAX, supplemented with 25 mM glucose, 15% Cellartis Hepatocyte Maintenance Media (Takara Bio), 1X insulin-transferrin-selenium, 100 nM dexamethasone, 2% FBS, and 50 μg/ml gentamycin. After 24 hours of initial culture, the media were replaced with fresh complete media containing either IgG or two different doses of antibodies for an additional 48 hours. Conditioned media was collected for ELISA measurement of secreted Col1a1. Slices were also collected for total RNA extraction and measurement of mRNA expression. Data was normalized to treatment with isotype control (AB100). Statistical analyses were performed with GraphPad Prism software (version 10.0) by using two-way ANOVA with Dunnett’s multiple comparisons test for mRNA expression and ordinary one-way ANOVA with Dunnett’s multiple comparisons test for secreted Col1a1. Statistical significance is defined as: * P < 0.05 Data Availability The authors declare that all the data supporting the findings of this study are available within the paper and its Supplemental Data. Author Contributions Raghavender Reddy Gopireddy – Investigation, methodology, formal analysis, visualization, writing – original draft, writing-review and editing Dipankar Bhattacharya – Investigation, formal analysis Swastik Sen – Investigation, resources, writing – original draft Monica Schwartz – Investigation, supervision, writing-review and editing Scott L. Friedman – Methodology, funding acquisition, writing-review and editing Richard Yu – Conceptualization, funding acquisition, methodology, writing-review and editing Toshi Takeuchi – Supervision, methodology, writing-review and editing Lauren Schwimmer – Supervision, formal analysis, methodology, funding acquisition, writing – original draft, writing – review and editing Funding Sources This research was primarily supported by NIH SBIR grants, R43DK125191 and R44DK125191, to Abalone Bio, with Scott Friedman as a co-investigator. This research was additionally supported by NIH SBIR grants, R43CA241513 and R44CA241513, and NSF SBIR grants, 1747391 and 1853147. Research performed at Icahn School of Medicine at Mount Sinai was also partially funded by NIH grant R01DK128289. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors used Gemini 2.5, ChatGTP Teams, and Claude Sonnet 4 to review the readability and language of the text. After using these tools, the authors edited the text as needed and take full responsibility for the content of the published article. The text was not directly edited by these AI technologies. Acknowledgements We thank Rodrigo Baltanas, Brett Robinson, Gustavo Pesce, Sameer Soi, Stephanie Lopez and Allison Cooke for their work to develop Abalone Bio’s antibody discovery platform and to discover CB2 agonist antibodies. We also thank Ken Mackie and Amey Dhopeshwarkar at Indiana University for their support in functional assessment of CB2 agonist antibodies. Finally, we thank Alejandro Colman-Lerner for his continued support and yeast genetics expertise. Funder Information Declared National Institute of Diabetes and Digestive and Kidney Diseases, https://ror.org/00adh9b73 , R43DK125191 , R44DK125191 , R01DK128289 National Cancer Institute, https://ror.org/040gcmg81 , R43CA241513 , R44CA241513 National Science Foundation , 1747391 , 1853147 Footnotes Minor revisions were made to correct errors and omissions in the text. Abbreviations CB1 Cannabinoid Receptor 1, CB2 Cannabinoid Receptor 2, CNS Central nervous system, CREB cAMP-response element-binding protein, ECM Extracellular matrix, ERK Extracellular signal-regulated kinase, GPCR G-protein coupled receptor, HSC Hepatic stellate cells, hPCLS human precision cut liver slices, LPS Lipopolysaccharide, MAFLD Metabolic dysfunction-associated fatty liver disease, MASH Metabolic dysfunction-associated steatohepatitis, PKA Protein kinase A, TGFβ Transforming growth factor beta, TR-FRET Time-Resolved Fluorescence Resonance Energy Transfer References 1. ↵ Xu , K. et al. Microglial Cannabinoid CB2 Receptors in Pain Modulation . Int. J. Mol. Sci . 24 , 2348 ( 2023 ). OpenUrl CrossRef PubMed 2. Turcotte , C. , Blanchet , M.-R. , Laviolette , M. & Flamand , N . The CB2 receptor and its role as a regulator of inflammation . Cell. Mol. Life Sci. CMLS 73 , 4449 – 4470 ( 2016 ). OpenUrl PubMed 3. Mallat , A. , Teixeira-Clerc , F. & Lotersztajn , S. Cannabinoid signaling and liver therapeutics . J. Hepatol . 59 , 891 – 896 ( 2013 ). OpenUrl CrossRef PubMed 4. ↵ Castro , J. et al. Olorinab (APD371), a peripherally acting, highly selective, full agonist of the cannabinoid receptor 2, reduces colitis-induced acute and chronic visceral hypersensitivity in rodents . Pain 163 , e72 – e86 ( 2022 ). OpenUrl CrossRef PubMed 5. ↵ Guindon , J. & Hohmann , A. G . Cannabinoid CB2 receptors: a therapeutic target for the treatment of inflammatory and neuropathic pain . Br. J. Pharmacol . 153 , 319 – 334 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 6. ↵ Busquets-Garcia , A. , Bains , J. & Marsicano , G . CB1 Receptor Signaling in the Brain: Extracting Specificity from Ubiquity . Neuropsychopharmacology 43 , 4 – 20 ( 2018 ). OpenUrl CrossRef PubMed 7. ↵ Atwood , B. K. , Wager-Miller , J. , Haskins , C. , Straiker , A. & Mackie , K . Functional Selectivity in CB2 Cannabinoid Receptor Signaling and Regulation: Implications for the Therapeutic Potential of CB2 Ligands . Mol. Pharmacol . 81 , 250 – 263 ( 2012 ). OpenUrl Abstract / FREE Full Text 8. ↵ Soethoudt , M. et al. Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity . Nat. Commun . 8 , 13958 ( 2017 ). 9. ↵ Lu , R. M. et al. Development of therapeutic antibodies for the treatment of diseases . J. Biomed. Sci . 27 , 1 ( 2020 ). 10. ↵ Yu , R. , Pesce , C. G. , Baltanas , R. & Robison , B . Yeast display of proteins in the periplasmic space , U S20210198806A 1. ( 2021 ). 11. ↵ Baltanás , R. et al. Cannabinoid Receptor Type 2 Antibodies and Uses Thereof , WO2025128847 . ( 2025 ). 12. ↵ Lynn , A. B. & Herkenham , M . Localization of cannabinoid receptors and nonsaturable high-density cannabinoid binding sites in peripheral tissues of the rat: implications for receptor-mediated immune modulation by cannabinoids . J. Pharmacol. Exp. Ther . 268 , 1612 – 1623 ( 1994 ). OpenUrl Abstract / FREE Full Text 13. ↵ Atwood , B. K. & Mackie , K . CB2: a cannabinoid receptor with an identity crisis . Br. J. Pharmacol . 160 , 467 – 479 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 14. ↵ Bouaboula , M. et al. Signaling Pathway Associated with Stimulation of CB2 Peripheral Cannabinoid Receptor . Eur. J. Biochem . 237 , 704 – 711 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 15. ↵ Shoemaker , J. L. , Ruckle , M. B. , Mayeux , P. R. & Prather , P. L . Agonist-Directed Trafficking of Response by Endocannabinoids Acting at CB2 Receptors . J. Pharmacol. Exp. Ther . 315 , 828 – 838 ( 2005 ). OpenUrl Abstract / FREE Full Text 16. ↵ Pertwee , R. G . Pharmacology of cannabinoid CB1 and CB2 receptors . Pharmacol. Ther . 74 , 129 – 180 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 17. Mukherjee , S. et al. Species comparison and pharmacological characterization of rat and human CB2 cannabinoid receptors . Eur. J. Pharmacol . 505 , 1 – 9 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 18. Julien , B. et al. Antifibrogenic role of the cannabinoid receptor CB2 in the liver . Gastroenterology 128 , 742 – 755 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 19. ↵ Guillot , A. et al. Cannabinoid receptor 2 counteracts interleukin-17-induced immune and fibrogenic responses in mouse liver: Guillot et al. Hepatology 59 , 296 – 306 ( 2014 ). OpenUrl CrossRef PubMed 20. Melgar-Lesmes , P. , Perramon , M. & Jiménez , W . Roles of the Hepatic Endocannabinoid and Apelin Systems in the Pathogenesis of Liver Fibrosis . Cells 8 , 1311 ( 2019 ). OpenUrl 21. Tan , S. , Liu , H. , Ke , B. , Jiang , J. & Wu , B . The peripheral CB receptor antagonist JD5037 attenuates liver fibrosis via a CB receptor/β-arrestin1/Akt pathway . Br. J. Pharmacol . 177 , 2830 – 2847 ( 2020 ). OpenUrl PubMed 22. ↵ Eddin , L. B. , Meeran , M. F. N. , Subramanya , S. B. , Jha , N. K. & Ojha , S . Therapeutic potential of agents targeting cannabinoid type 2 receptors in organ fibrosis . Pharmacol. Res. Perspect . 12 , e1219 ( 2024 ). OpenUrl 23. ↵ Louvet , A. et al. Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice . Hepatology 54 , 1217 – 1226 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 24. Denaës , T. et al. The Cannabinoid Receptor 2 Protects Against Alcoholic Liver Disease Via a Macrophage Autophagy-Dependent Pathway . Sci. Rep . 6 , ( 2016 ). 25. ↵ Tortora , C. et al. Effects of CB2 Receptor Modulation on Macrophage Polarization in Pediatric Celiac Disease . Biomedicines 10 , 874 ( 2022 ). 26. ↵ Wynn , T. & Barron , L . Macrophages: Master Regulators of Inflammation and Fibrosis . Semin. Liver Dis . 30 , 245 – 257 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 27. ↵ Hanuš , L. et al. HU-308: A specific agonist for CB2, a peripheral cannabinoid receptor . Proc. Natl. Acad. Sci . 96 , 14228 – 14233 ( 1999 ). OpenUrl Abstract / FREE Full Text 28. ↵ An , D. , Peigneur , S. , Hendrickx , L. A. & Tytgat , J . Targeting Cannabinoid Receptors: Current Status and Prospects of Natural Products . Int. J. Mol. Sci . 21 , 5064 ( 2020 ). OpenUrl CrossRef PubMed 29. ↵ Malfitano , A. M. , Basu , S. , Maresz , K. , Bifulco , M. & Dittel , B. N . What we know and do not know about the cannabinoid receptor 2 (CB2) . Semin. Immunol . 26 , 369 – 379 ( 2014 ). OpenUrl CrossRef PubMed 30. ↵ Dhawan , J. et al. Evaluation of the in vivo receptor occupancy for the behavioral effects of cannabinoids using a radiolabeled cannabinoid receptor agonist, R-[ 125/131 I]AM2233 . Synapse 60 , 93 – 101 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 31. ↵ Mackie , K . Mechanisms of CB1 receptor signaling: endocannabinoid modulation of synaptic strength . Int. J. Obes . 30 , S19 – S23 ( 2006 ). OpenUrl 32. ↵ Mallat , A. , Teixeira-Clerc , F. , Deveaux , V. , Manin , S. & Lotersztajn , S . The endocannabinoid system as a key mediator during liver diseases: new insights and therapeutic openings: The endocannabinoid system and liver diseases . Br. J. Pharmacol . 163 , 1432 – 1440 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 33. ↵ Tam , J. et al. Endocannabinoids in liver disease . Hepatology 53 , 346 – 355 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 34. ↵ Smith , A. J . New Horizons in Therapeutic Antibody Discovery: Opportunities and Challenges versus Small-Molecule Therapeutics . J. Biomol. Screen . 20 , 437 – 453 ( 2015 ). OpenUrl CrossRef PubMed 35. ↵ Han , K. H. et al. CB1 and CB2 cannabinoid receptors differentially regulate the production of reactive oxygen species by macrophages . Cardiovasc. Res . 84 , 378 – 386 ( 2009 ). OpenUrl CrossRef PubMed 36. ↵ Rinaldi-Carmona , M. et al. SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor . J. Pharmacol. Exp. Ther . 284 , 644 – 650 ( 1998 ). OpenUrl Abstract / FREE Full Text 37. ↵ Portier , M. et al. SR 144528, an antagonist for the peripheral cannabinoid receptor that behaves as an inverse agonist . J. Pharmacol. Exp. Ther . 288 , 582 – 589 ( 1999 ). OpenUrl Abstract / FREE Full Text 38. ↵ Rinaldi-Carmona , M. et al. SR141716A, a potent and selective antagonist of the brain cannabinoid receptor . FEBS Lett . 350 , 240 – 244 ( 1994 ). OpenUrl CrossRef PubMed Web of Science 39. ↵ Chen , X. et al. Involvement of β-arrestin-2 and Clathrin in Agonist-Mediated Internalization of the Human Cannabinoid CB2 Receptor . Curr. Mol. Pharmacol . 7 , 67 – 80 ( 2014 ). OpenUrl CrossRef PubMed 40. ↵ Saroz , Y. , Kho , D. T. , Glass , M. , Graham , E. S. & Grimsey , N. L . Cannabinoid Receptor 2 (CB2) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes . ACS Pharmacol. Transl. Sci . 2 , 414 – 428 ( 2019 ). OpenUrl PubMed 41. ↵ Gerlo , S. et al. Cyclic AMP: a selective modulator of NF-κB action . Cell. Mol. Life Sci. CMLS 68 , 3823 – 3841 ( 2011 ). OpenUrl PubMed 42. ↵ Rakotoarivelo , V. , Mayer , T. Z. , Simard , M. , Flamand , N. & Di Marzo , V . The Impact of the CB2 Cannabinoid Receptor in Inflammatory Diseases: An Update . Molecules 29 , 3381 ( 2024 ). OpenUrl CrossRef PubMed 43. ↵ Chen , S. et al. Macrophages in immunoregulation and therapeutics . Signal Transduct. Target. Ther . 8 , 1 – 35 ( 2023 ). OpenUrl PubMed 44. ↵ Wang , S. et al. TGF-β/Smad3 signalling regulates the transition of bone marrowderived macrophages into myofibroblasts during tissue fibrosis . Oncotarget 7 , 8809 – 8822 ( 2015 ). OpenUrl 45. Jiang , Y. et al. Macrophages in organ fibrosis: from pathogenesis to therapeutic targets . Cell Death Discov . 10 , 1 – 11 ( 2024 ). OpenUrl PubMed 46. Zhu , L. , Fu , X. , Chen , X. , Han , X. & Dong , P . M2 macrophages induce EMT through the TGF-β/Smad2 signaling pathway . Cell Biol. Int . 41 , 960 – 968 ( 2017 ). OpenUrl PubMed 47. ↵ Parola , M. & Pinzani , M . Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues . Mol. Aspects Med . 65 , 37 – 55 ( 2019 ). OpenUrl CrossRef PubMed 48. Rockey , D. C . Current and Future Anti-Fibrotic Therapies for Chronic Liver Disease . Clin. Liver Dis . 12 , 939 – 962 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 49. Popovic , P. J. , Zeh , H. J. & Ochoa , J. B. Arginine and Immunity . J. Nutr . 137 , 1681S – 1686S ( 2007 ). OpenUrl Abstract / FREE Full Text 50. ↵ Koyama , Y. & Brenner , D. A. Liver inflammation and fibrosis . J. Clin. Invest . 127 , 55 – 64 ( 2017 ). OpenUrl CrossRef PubMed 51. Carpino , G. et al. Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation . Dig. Liver Dis . 37 , 349 – 356 ( 2005 ). OpenUrl CrossRef PubMed 52. Tone , K. , Stappers , M. H. T. , Willment , J. A. & Brown , G. D . C-type lectin receptors of the Dectin-1 cluster: Physiological roles and involvement in disease . Eur. J. Immunol . 49 , 2127 – 2133 ( 2019 ). OpenUrl CrossRef PubMed 53. Karsdal , M. A. et al. Collagen biology and non-invasive biomarkers of liver fibrosis . Liver Int. Off. J. Int. Assoc. Study Liver 40 , 736 – 750 ( 2020 ). OpenUrl 54. Ouyang , W. & O’Garra , A . IL-10 Family Cytokines IL-10 and IL-22: from Basic Science to Clinical Translation . Immunity 50 , 871 – 891 ( 2019 ). OpenUrl CrossRef PubMed 55. Zhang , W.-J. , Chen , S.-J. , Zhou , S.-C. , Wu , S.-Z. & Wang , H. Inflammasomes and Fibrosis . Front. Immunol . 12 , ( 2021 ). 56. Schmidt-Arras , D. & Rose-John , S . IL-6 pathway in the liver: From physiopathology to therapy . J. Hepatol . 64 , 1403 – 1415 ( 2016 ). OpenUrl CrossRef PubMed 57. Jondle , C. N. et al. Macrophage galactose-type lectin-1 (MGL1) deficiency is associated with increased neutrophilia and hyper inflammation in Gram-Negative pneumonia . J. Immunol. Baltim. Md 1950 196 , 3088 – 3096 ( 2016 ). OpenUrl 58. Szczykutowicz , J . Ligand Recognition by the Macrophage Galactose-Type C-Type Lectin: Self or Non-Self?—A Way to Trick the Host’s Immune System . Int. J. Mol. Sci . 24 , 17078 ( 2023 ). 59. Madsen , D. H. et al. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway . J. Cell Biol . 202 , 951 – 966 ( 2013 ). OpenUrl Abstract / FREE Full Text 60. Parameswaran , N. & Patial , S . Tumor Necrosis Factor-α Signaling in Macrophages . Crit. Rev. Eukaryot. Gene Expr . 20 , 87 – 103 ( 2010 ). OpenUrl CrossRef PubMed 61. ↵ Teixeira-Clerc , F. et al. Beneficial paracrine effects of cannabinoid receptor 2 on liver injury and regeneration . Hepatology 52 , 1046 – 1059 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 62. ↵ Balistreri , E. et al. The cannabinoid WIN55, 212-2 abrogates dermal fibrosis in scleroderma bleomycin model . Ann. Rheum. Dis . 70 , 695 – 699 ( 2011 ). OpenUrl Abstract / FREE Full Text 63. ↵ Paish , H. L. et al. A Bioreactor Technology for Modeling Fibrosis in Human and Rodent Precision-Cut Liver Slices . Hepatology ( 2019 ) doi: 10.1002/hep.30651 . OpenUrl CrossRef PubMed 64. ↵ Bhattacharya , D. et al. Repositioning of a novel GABA-B receptor agonist, AZD3355 (Lesogaberan), for the treatment of non-alcoholic steatohepatitis . Sci. Rep . 11 , 20827 ( 2021 ). OpenUrl PubMed 65. ↵ Ullmer , C. et al. Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7 . Br. J. Pharmacol . 167 , 1448 – 1466 ( 2012 ). OpenUrl CrossRef PubMed 66. Ohta , S. et al. Agonistic Antibodies Reveal the Function of GPR56 in Human Glioma U87-MG Cells . Biol. Pharm. Bull . 38 , 594 – 600 ( 2015 ). OpenUrl CrossRef PubMed 67. Ma , Y. et al. Structure-guided discovery of a single-domain antibody agonist against human apelin receptor . Sci. Adv . 6 , eaax7379 ( 2020 ). OpenUrl FREE Full Text 68. Ren , H. et al. Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors. Commun . Biol . 3 , 146 ( 2020 ). 69. Haubrich , J. , et al. A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers . Proc. Natl. Acad. Sci . 118 , e2105848118 ( 2021 ). OpenUrl Abstract / FREE Full Text 70. Trilleaud , C. et al. Agonist anti-ChemR23 mAb reduces tissue neutrophil accumulation and triggers chronic inflammation resolution . Sci. Adv . 7 , eabd1453 ( 2021 ). OpenUrl FREE Full Text 71. Fontaine , T. et al. Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist . Nat. Commun . 15 , 7029 ( 2024 ). OpenUrl CrossRef PubMed 72. ↵ Biswas , S. De novo design of hundreds of functional GPCR-targeting antibodies enabled by scaling test-time compute . 2025.05.28.656709 Preprint at doi: 10.1101/2025.05.28.656709 ( 2025 ). OpenUrl Abstract / FREE Full Text 73. ↵ Strand , V. et al. Immunogenicity of Biologics in Chronic Inflammatory Diseases: A Systematic Review . BioDrugs 31 , 299 – 316 ( 2017 ). OpenUrl CrossRef PubMed 74. ↵ Manglik , A. , Kobilka , B. K. & Steyaert , J . Nanobodies to Study G Protein–Coupled Receptor Structure and Function . Annu. Rev. Pharmacol. Toxicol . 57 , 19 – 37 ( 2017 ). OpenUrl CrossRef PubMed 75. ↵ Morales , P. , Goya , P. & Jagerovic , N . Emerging strategies targeting CB2 cannabinoid receptor: Biased agonism and allosterism . Biochem. Pharmacol . 157 , 8 – 17 ( 2018 ). OpenUrl PubMed 76. ↵ Chen , Q. & Tesmer , J. J. G . G protein–coupled receptor interactions with arrestins and GPCR kinases: The unresolved issue of signal bias . J. Biol. Chem . 298 , ( 2022 ). 77. ↵ Wen , A. Y. , Sakamoto , K. M. & Miller , L. S . The Role of the Transcription Factor CREB in Immune Function . J. Immunol. Baltim. Md 1950 185 , 6413 – 6419 ( 2010 ). OpenUrl 78. ↵ Correa , F. , Mestre , L. , Docagne , F. & Guaza , C . Activation of cannabinoid CB2 receptor negatively regulates IL-12p40 production in murine macrophages: role of IL-10 and ERK1/2 kinase signaling . Br. J. Pharmacol . 145 , 441 – 448 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 79. ↵ Correa , F. et al. Anandamide enhances IL-10 production in activated microglia by targeting CB(2) receptors: roles of ERK1/2, JNK, and NF-kappaB . Glia 58 , 135 – 147 ( 2010 ). OpenUrl CrossRef PubMed 80. ↵ Ma , X. , Qiu , J. , Zou , S. , Tan , L. & Miao , T . The role of macrophages in liver fibrosis: composition, heterogeneity, and therapeutic strategies . Front. Immunol . 15 , 1494250 ( 2024 ). OpenUrl PubMed 81. ↵ Pratim Das , P. & Medhi , S. Role of inflammasomes and cytokines in immune dysfunction of liver cirrhosis . Cytokine 170 , 156347 ( 2023 ). 82. ↵ Roth , K. et al. Interleukin-10 disrupts liver repair in acetaminophen-induced acute liver failure . Front. Immunol . 14 , ( 2023 ). 83. ↵ Soppert , J. et al. Blood Endotoxin Levels as Biomarker of Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-analysis . Clin. Gastroenterol. Hepatol. Off. Clin. Pract. J. Am. Gastroenterol. Assoc . 21 , 2746 – 2758 ( 2023 ). OpenUrl 84. ↵ Dixon , L. J. , Barnes , M. , Tang , H. , Pritchard , M. T. & Nagy , L. E . Kupffer cells in the liver . Compr. Physiol . 3 , 785 – 797 ( 2013 ). OpenUrl PubMed 85. ↵ Zipfel , A. , Schenk , M. , Metzdorf , B. , Bode , C. & Viebahn , R . Release of TNF-alpha from lipopolysaccharide (LPS)-stimulated Kupffer cells in serum- and nutrient-free medium . Inflammation 25 , 287 – 292 ( 2001 ). OpenUrl CrossRef PubMed 86. ↵ Osawa , Y. et al. Tumor necrosis factor-α promotes cholestasis-induced liver fibrosis in the mouse through tissue inhibitor of metalloproteinase-1 production in hepatic stellate cells . PloS One 8 , e65251 ( 2013 ). OpenUrl PubMed 87. ↵ Hirano , T . IL-6 in inflammation, autoimmunity and cancer . Int. Immunol . 33 , 127 – 148 ( 2021 ). OpenUrl CrossRef PubMed 88. ↵ Geiling , R. , Wallace , K. & Han , Y.-P . Interleukin-1 participates in the progression from liver injury to fibrosis . Am. J. Physiol. Gastrointest. Liver Physiol . 296 , ( 2009 ). 89. ↵ Baeza-Raja , B. et al. Pharmacological inhibition of P2RX7 ameliorates liver injury by reducing inflammation and fibrosis . PloS One 15 , ( 2020 ). 90. ↵ Menten , P. , Wuyts , A. & Van Damme , J . Macrophage inflammatory protein-1 . Cytokine Growth Factor Rev . 13 , ( 2002 ). 91. ↵ Seki , E. et al. CCR1 and CCR5 promote hepatic fibrosis in mice . J. Clin. Invest . 119 , ( 2009 ). 92. ↵ Wang , Z. , Du , K. , Jin , N. , Tang , B. & Zhang , W . Macrophage in liver Fibrosis: Identities and mechanisms . Int. Immunopharmacol . 120 , 110357 ( 2023 ). 93. ↵ Bagher , A. M . The Endocannabinoid System as a Therapeutic Target in Diabetic Peripheral Neuropathic Pain: A Review . J. Microsc. Ultrastruct . 10 , 47 – 54 ( 2021 ). OpenUrl PubMed 94. Lin , X. et al. A peripheral CB2 cannabinoid receptor mechanism suppresses chemotherapy-induced peripheral neuropathy: evidence from a CB2 reporter mouse . PAIN 163 , 834 ( 2022 ). 95. ↵ Carey , L. M. et al. Peripheral sensory neuron CB2 cannabinoid receptors are necessary for both CB2-mediated antinociceptive efficacy and sparing of morphine tolerance in a mouse model of anti-retroviral toxic neuropathy . Pharmacol. Res . 187 , 106560 ( 2023 ). 96. ↵ Baxter , E. W. et al. Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes . J. Immunol. Methods 478 , 112721 ( 2020 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted August 15, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Schwimmer bioRxiv 2025.07.11.664477; doi: https://doi.org/10.1101/2025.07.11.664477 Share This Article: Copy Citation Tools Cannabinoid Receptor 2 Activating Antibodies: A Promising Therapeutic Strategy for Macrophage-Driven Fibro-Inflammatory Diseases Raghavender Reddy Gopireddy , Dipankar Bhattacharya , Swastik Sen , Monica A. Schwartz , Scott L. Friedman , Richard C. Yu , Toshi Takeuchi , Lauren J. 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