Receptor FcgammaRIIb Research Articles

Exercise Inhibits Doxorubicin-Induced Cardiotoxicity via Regulating B Cells.

Doxorubicin is an effective chemotherapeutic agent, but its use is limited by acute and chronic cardiotoxicity. Exercise training has been shown to protect against doxorubicin-induced cardiotoxicity, but the involvement of immune cells remains unclear. This study aimed to investigate the role of exercise-derived B cells in protecting against doxorubicin-induced cardiotoxicity and to further determine whether B cell activation and antibody secretion play a role in this protection. Mice that were administered with doxorubicin (5 mg/kg per week, 20 mg/kg cumulative dose) received treadmill running exercise. The adoptive transfer of exercise-derived splenic B cells to μMT-/- (B cell-deficient) mice was performed to elucidate the mechanism of B cell regulation that mediated the effect of exercise. Doxorubicin-administered mice that had undergone exercise training showed improved cardiac function, and low levels of cardiac apoptosis, atrophy, and fibrosis, and had reduced cardiac antibody deposition and proinflammatory responses. Similarly, B cell pharmacological and genetic depletion alleviated doxorubicin-induced cardiotoxicity, which phenocopied the protection of exercise. In vitro performed coculture experiments confirmed that exercise-derived B cells reduced cardiomyocyte apoptosis and fibroblast activation compared with control B cells. Importantly, the protective effect of exercise on B cells was confirmed by the adoptive transfer of splenic B cells from exercised donor mice to μMT-/- recipient mice. However, blockage of Fc gamma receptor IIB function using B cell transplants from exercised Fc gamma receptor IIB-/- mice abolished the protection of exercise-derived B cells against doxorubicin-induced cardiotoxicity. Mechanistically, we found that Fc gamma receptor IIB, an important B cell inhibitory receptor, responded to exercise and increased B cell activation threshold, which participated in exercise-induced protection against doxorubicin-induced cardiotoxicity. Our results demonstrate that exercise training protects against doxorubicin-induced cardiotoxicity by upregulating Fc gamma receptor IIB expression in B cells, which plays an important anti-inflammatory role and participates in the protective effect of exercise against doxorubicin-induced cardiotoxicity.

Lupus exacerbation in ovalbumin-induced asthma in Fc gamma receptor IIb deficient mice, partly due to hyperfunction of dendritic cells.

Although allergy might be another factor that exacerbates lupus as demonstrated by several epidemiologic studies, the direct correlation between lupus activities and allergy is still in question. To explore the correlation between allergic reaction and lupus activities. The allergic asthma model using ovalbumin (OVA) administration in wildtype (WT) and Fc gamma receptor IIb deficient (FcgRIIb-/-) mice (a lupus-prone model) together with in vitro experiments on bone marrow-derived dendritic cells (DCs) were performed. At 2-weeks-post OVA, both WT and FcgRIIb-/- mice demonstrated similar allergic reaction as indicated by an elevation of IgE and IL-4 in serum with asthma-liked lung histology (lung weight, inflammatory score, and bronchial thickness) with increased spleen weight. Apoptosis in the lungs and spleens (activated caspase 3 immunohistochemistry) was detected only in OVA-administered FcgRIIb-/- mice. Surprisingly, OVA-administered FcgRIIb-/- mice, demonstrated active lupus nephritis, as indicated by anti-dsDNA, proteinuria, and renal immune complex deposition (immunohistochemistry analysis) implying an impact of allergy on lupus activities. Meanwhile, serum creatinine and gut permeability defect (FitC-dextran assay and endotoxemia) were not different between the FcgRIIb-/- mice with OVA versus with control. In parallel, FcgRIIb-/- DCs were more susceptible to activations by OVA and lipopolysaccharide (LPS) than WT DCs as demonstrated by CD80 with major histocompatibility complex II (MHC II) using flow cytometry analysis. OVA-induced allergy in FcgRIIb-/- mice exacerbated lupus activity, possibly due to hyper-responsiveness of FcgRIIb-/- DCs over WT from the loss of inhibitory FcgRIIb. The proper control of allergy might be beneficial for lupus.

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis.

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.

Inhibitory Fc-Gamma IIb Receptor Signaling Induced by Multivalent IgG-Fc Is Dependent on Sialylation.

Immunoglobulin (IgG) Fc glycosylation has been shown to be important for the biological activity of antibodies. Fc sialylation is important for the anti-inflammatory activity of IgGs. However, evaluating the structure-activity relationship (SAR) of antibody Fc glycosylation has been hindered using simplified in vitro models in which antibodies are often displayed in monomeric forms. Presenting antibodies in monomeric forms may not accurately replicate the natural environment of the antibodies when binding their antigen in vivo. To address these limitations, we used different Fc-containing molecules, displaying their Fc domains in monovalent and multivalent fashion. Given the inhibitory role of Fc gamma receptor IIb (FcγRIIb) in autoimmune and inflammatory diseases, we focused on evaluating the impact of Fc sialylation on the activation of FcγRIIb. We report for the first time that in human cellular systems, sialic acid mediates the induction of FcγRIIb phosphorylation by IgG-Fc when the IgG-Fc is displayed in a multivalent fashion. This effect was observed with different types of therapeutic agents such as sialylated anti-TNFα antibodies, sialylated IVIg and sialylated recombinant multivalent Fc products. These studies represent the first report of the specific effects of Fc sialylation on FcγRIIb signaling on human immune cells and may help in the characterization of the anti-inflammatory activity of Fc-containing therapeutic candidates.

Fc gamma receptor IIb in tumor-associated macrophages and dendritic cells drives poor prognosis of recurrent glioblastoma through immune-associated signaling pathways.

Background: Among central nervous system tumors, glioblastoma (GBM) is considered to be the most destructive malignancy. Recurrence is one of the most fatal aspects of GBM. However, the driver molecules that trigger GBM recurrence are currently unclear. Methods: The mRNA expression data and clinical information of GBM and normal tissues were collected from the Chinese Glioma Genome Atlas The Cancer Genome Atlas (TCGA), and REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) cohorts. The DESeq2 R package was used to identify the differentially expressed genes between primary and recurrent GBM. ClueGO, Kyoto Encyclopedia of Genes and Genomes (KEGG), Biological Process in Gene ontology (GO-BP), and the Protein ANalysis THrough Evolutionary Relationships (PANTHER) pathway analyses were performed to explore the enriched signaling pathways in upregulated DEGs in recurrent GBM. A gene list that contained potential oncogenes that showed a significant negative correlation with patient survival from The Cancer Genome Atlas was used to further screen driver candidates for recurrent GBM. Univariate Cox proportional hazards regression analyses were used to investigate the risk score for the mRNA expression of the candidates. Single-cell RNA sequencing (scRNA-Seq) analyses were used to determine the cell type-specific distribution of Fc gamma receptor II b (FcγRIIb) in GBM. Immunohistochemistry (IHC) was used to confirm the FcγRIIb-positive cell populations in primary and paired recurrent GBM. Results: Through DEG analysis and overlap analysis, a total of 10 genes that are upregulated in recurrent GBM were screened. Using validation databases, FcγRIIb was identified from the 10 candidates that may serve as a driver for recurrent GBM. FCGR2B expression, not mutation, further showed a highly negative correlation with the poor prognosis of patients with recurrent GBM. Furthermore, scRNA-Seq analyses revealed that tumor-associated macrophage- and dendritic cell-specific FCGR2B was expressed. Moreover, FcγRIIb also showed a strong positive correlation coefficient with major immune-associated signaling pathways. In clinical specimens, FcγRIIb-positive cell populations were higher in recurrent GBM than in primary GBM. Conclusion: This study provides novel insights into the role of FcγRIIb in recurrent GBM and a promising strategy for treatment as an immune therapeutic target.

Obesity Exacerbates Lupus Activity in Fc Gamma Receptor IIb Deficient Lupus Mice Partly through Saturated Fatty Acid-Induced Gut Barrier Defect and Systemic Inflammation

The prevalence of obesity is increasing, and the coexistence of obesity and systemic lupus erythematosus (lupus) is possible. A high-fat diet (HFD) was orally administered for 6 months in female 8-week-old Fc gamma receptor IIb deficient (FcgRIIb−/−) lupus or age and gender-matched wild-type (WT) mice. Lupus nephritis (anti-dsDNA, proteinuria, and increased creatinine), gut barrier defect (fluorescein isothiocyanate dextran), serum lipopolysaccharide (LPS), serum interleukin (IL)-6, liver injury (alanine transaminase), organ fibrosis (liver and kidney pathology), spleen apoptosis (activated caspase 3), and aorta thickness (but not weight gain and lipid profiles) were more prominent in HFD-administered FcgRIIb−/− mice than the obese WT, without injury in regular diet-administered mice (both FcgRIIb−/− and WT). In parallel, combined palmitic acid (PA; a saturated fatty acid) with LPS (PA + LPS) induced higher tumor necrotic factor-α, IL-6, and IL-10 in the supernatant, inflammatory genes (inducible nitric oxide synthase and IL-1β), reactive oxygen species (dihydroethidium), and glycolysis with reduced mitochondrial activity (extracellular flux analysis) when compared with the activation by each molecule alone in both FcgRIIb−/− and WT macrophages. However, the alterations of these parameters were more prominent in PA + LPS-administered FcgRIIb−/− than in the WT cells. In conclusion, obesity accelerated inflammation in FcgRIIb−/− mice, partly due to the more potent responses from the loss of inhibitory FcgRIIb against PA + LPS with obesity-induced gut barrier defect.

Impaired B Cell Expression of the Inhibitory Fcγ Receptor IIB in Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease in which pathogenic immunoglobulin G antibodies bind to acetylcholine receptors (or to functionally related molecules at the neuromuscular junction). B cell expression of the inhibitory immunoglobulin G receptor, Fc-gamma receptor (FcγR)IIB, maintains peripheral immune tolerance, and its absence renders B cells hyperresponsive to autoantigen. Here, we report that FcγRIIB expression levels are substantially reduced in B lineage cells derived from immunotherapy-naïve patients with acetylcholine receptor antibody-positive early-onset MG. In contrast, genetic variants associated with impaired FcγRIIB expression are not enriched in MG, indicating post-transcriptional dysregulation. FcγR-targeted therapies could have therapeutic benefits in MG. ANN NEUROL 2022;92:1046-1051.

Gut Barrier Damage and Gut Translocation of Pathogen Molecules in Lupus, an Impact of Innate Immunity (Macrophages and Neutrophils) in Autoimmune Disease.

The gut barrier is a single cell layer that separates gut micro-organisms from the host, and gut permeability defects result in the translocation of microbial molecules from the gut into the blood. Despite the silent clinical manifestation, gut translocation of microbial molecules can induce systemic inflammation that might be an endogenous exacerbating factor of systemic lupus erythematosus. In contrast, circulatory immune-complex deposition and the effect of medications on the gut, an organ with an extremely large surface area, of patients with active lupus might cause gut translocation of microbial molecules, which worsens lupus severity. Likewise, the imbalance of gut microbiota may initiate lupus and/or interfere with gut integrity which results in microbial translocation and lupus exacerbation. Moreover, immune hyper-responsiveness of innate immune cells (macrophages and neutrophils) is demonstrated in a lupus model from the loss of inhibitory Fc gamma receptor IIb (FcgRIIb), which induces prominent responses through the cross-link between activating-FcgRs and innate immune receptors. The immune hyper-responsiveness can cause cell death, especially apoptosis and neutrophil extracellular traps (NETosis), which possibly exacerbates lupus, partly through the enhanced exposure of the self-antigens. Leaky gut monitoring and treatments (such as probiotics) might be beneficial in lupus. Here, we discuss the current information on leaky gut in lupus.

Abstract 3423: A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity with anti-HER2 therapy

Abstract Therapeutic antibodies have improved survival of both hematologic and solid cancer patients, inducing long-lasting responses and even cures. Clinically successful antibodies exert antitumor activity by targeting tumor cells directly (e.g. anti-HER2 mAb in HER2+ breast cancer, anti-CD20 mAb in B cell cancer) or by targeting and activating immune cells that seek and destroy cancer cells in the tumor microenvironment (immune checkpoint blocking antibodies; ICB). Still, many patients fail to respond or acquire resistance to these therapies. Understanding mechanisms and overcoming resistance to distinct classes of antibody drugs, is therefore clearly warranted and has the potential to further improve cancer outcomes. The inhibitory Fc gamma receptor (FcγR) IIB imbues resistance to cancer immunotherapy by several mechanisms, acting both on tumor and immune effector cells. We previously developed an FcγRIIB blocking antibody (BI-1206) which is currently being explored in clinical trials (NCT03571568, NCT04219254, and NCT02933320). BI-1206 was found to have cytolytic activity against malignant B cells and the ability to block rituximab internalization from tumor cells, as well as enhance rituximab therapeutic activity in mice humanized for CD20 and FcγRIIB or bearing relapsed/refractory CLL in vivo. Emerging data demonstrate that FcγRs modulate the therapeutic activity of many therapeutic antibodies among them HER2 targeting antibodies such as Trastuzumab. Trastuzumab alone or in combination with chemotherapy significantly improves overall survival of HER2+ breast cancer patients. However, many patients remain uncured and develop Trastuzumab resistance resulting in relapse of the disease. Means of improving anti-HER2 therapy and overcoming resistance are therefore highly desirable. We report the generation of a fully human FcγRIIB-blocking antibody (BI-1607) engineered to eliminate Fc-mediated FcγR binding and function (Fc-null anti-FcγRIIB). Using a mechanism-of-action-matched surrogate antibody, we demonstrate that Fc-null anti-FcγRIIB enhances therapeutic efficacy, both in tumor growth and overall survival of anti-HER2 in the syngeneic immune competent TUBO mouse model. This enhanced therapeutic efficacy is associated with increased tumor influx of myeloid and NK-cells. Collectively our results provide proof-of-concept for Fc-null anti-FcγRIIB to enhance anti-HER2 in the treatment of cancer. Citation Format: Linda Mårtensson, Robert Oldham, Marie Borggren, Mathilda Kovacek, Therese Blidberg, Ulla-Carin Tornberg, Ingrid Karlsson, Steve Beers, Mark Cragg, Ingrid Teige, Björn Frendeus. A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity with anti-HER2 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3423.

Preclinical Characterization of ASP2713, a Novel Igβ and FcγRIIB Cross-Linking Antibody, for Prediction of Human Pharmacokinetics and Clinically Effective Dose

Previously, we reported the fundamental pharmacological characteristics of a novel Igβ and Fc gamma receptor IIB cross-linking antibody, ASP2713, as a new treatment option for systemic lupus erythematosus. The aims of the present study were to investigate ASP2713’s characteristics with regard to pharmacological effect, pharmacokinetics (PK), and receptor occupancy, and to predict its human PK and clinically effective dose. The relationship between the concentration and receptor occupancy of ASP2713 for Igβ of B cell receptors was examined using whole blood B cells. Calculated EC50 values in cynomolgus monkeys and healthy volunteers were 0.35 and 0.058 μg/mL, respectively. Dose-dependent inhibition of anti-tetanus toxoid (TTx) antibody production, PK, and receptor occupancy of ASP2713 in TTx-sensitized cynomolgus monkeys suggested a minimally effective dose of 1 mg/kg by single intravenous (IV) administration. Scaling-up of monkey PK parameters to humans by allometric scaling predicted a clinically effective dose of 0.4 mg/kg IV administration at 4-week intervals to maintain a trough concentration in humans which achieved the same receptor occupancy expected at the effective dose in monkeys. This study aids in understanding the characteristics of ASP2713 and can be used as a basis for clinical dose setting.

Cellular Binding and Internalization Assay for an Anti-FcγRIIB Antibody Using Human Liver Non-parenchymal Cells.

A cellular assay for evaluating the binding and internalization of biologics using primary human liver sinusoidal endothelial cells (LSEC) is not readily available, since human LSEC generally lose their receptor expression and internalization activity during the purifying processes and cell culturing. Here, we propose a novel cell-based assay using human liver non-parenchymal cells (NPC) as an alternative method using LSEC. To identify the LSEC population, NPC were stained with CD31 and CD45, and analyzed by flow cytometry. The expression of Fc gamma receptor IIB (FcγRIIB), one of the LSEC markers was detected in the CD31-positive and the CD45-negative fractions. The concentration-dependent binding and internalization of the anti-FcγRIIB antibody was also quantified in the LSEC fraction in human NPC. Saturated binding and internalization curves were obtained for the anti-FcγRIIB antibody. In the case of the negative control antibody, however, binding and internalization were negligible. The findings reported here indicate that cell-based assays using fresh human liver NPC will be useful for evaluating the binding and internalization of biologics as well as for determining pharmacokinetic parameters.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.

A Comparison Between 1 Day versus 7 Days of Sepsis in Mice with the Experiments on LPS-Activated Macrophages Support the Use of Intravenous Immunoglobulin for Sepsis Attenuation.

BackgroundBecause survival and death after sepsis are partly due to a proper immune adaptation and immune dysregulation, respectively, survivors and moribund mice after cecal ligation and puncture (CLP) sepsis surgery and in vitro macrophage experiments were explored.MethodsCharacteristics of mice at 1-day and 7-days post-CLP, the representative of moribund mice (an innate immune hyper-responsiveness) and survivors (a successful control on innate immunity), respectively. In parallel, soluble heat aggregated immunoglobulin (sHA-Ig), a representative of immune complex, was tested in lipopolysaccharide (LPS)-activated macrophages together with a test of intravenous immunoglobulin (IVIG), a molecule of adaptive immunity, on CLP sepsis mice.ResultsExcept for a slight increase in alanine transaminase (liver injury), IL-10, endotoxemia, and gut leakage (FITC-dextran assay), most of the parameters in survivors (7-days post-CLP) were normalized, with enhanced adaptive immunity, including serum immunoglobulin (using serum protein electrophoresis) and activated immune cells in spleens (flow cytometry analysis). The addition of sHA-Ig in LPS-activated macrophages reduced supernatant cytokines, cell energy (extracellular flux analysis), reactive oxygen species (ROS), several cell activities (proteomic analysis), and Fc gamma receptors (FcgRs) expression. The loss of anti-inflammatory effect of sHA-Ig in LPS-activated macrophages from mice with a deficiency on Fc gamma receptor IIb (FcgRIIb-/-), the only inhibitory signaling of FcgRs family, when compared with wild-type macrophages, implying the FcgRIIb-dependent mechanism. Moreover, IVIG attenuated sepsis severity in CLP mice as evaluated by serum creatinine, liver enzyme (alanine transaminase), serum cytokines, spleen apoptosis, and abundance of dendritic cells in the spleen (24-h post-CLP) and survival analysis.ConclusionImmunoglobulin attenuated LPS-activated macrophages, partly, through the reduced cell energy of macrophages and might play a role in sepsis immune hyper-responsiveness. Despite the debate over IVIG’s use in sepsis, IVIG might be beneficial in sepsis with certain conditions.

Discovery of a novel Igβ and FcγRIIB cross-linking antibody, ASP2713, and its potential application in the treatment of systemic lupus erythematosus

B cell-targeted therapies have evolved as established therapies for systemic lupus erythematosus (SLE); however, existing approaches still do not thoroughly satisfy clinical requirements due to limited efficacy against memory B cells, autoantibody-producing plasmablasts and disease heterogeneity. To provide a new treatment option for SLE, we created a novel anti-Igβ antibody with enhanced affinity for Fc gamma receptor (FcγR) IIB called ASP2713. ASP2713 cross-reacted with both human and cynomolgus monkey Igβ and showed increased binding affinity for human and monkey FcγRIIB compared to native human IgG1. This binding property allows dominant B cell binding and induction of intrinsic negative feedback signals. In human B cells, ASP2713 significantly and concentration-dependently induced FcγRIIB ITIM phosphorylation, while suppressing proliferation under B cell receptor stimulation. This pharmacological effect was also confirmed in in vitro B cell proliferation and antibody production assays using peripheral B cells isolated from patients with SLE. In a cynomolgus monkey tetanus toxoid-induced antibody production model, ASP2713 almost completely inhibited the increase in antigen-specific antibodies with superior efficacy to rituximab. Additionally, ASP2713 significantly suppressed recall antibody production in response to secondary tetanus toxoid immunization, indicating the memory B cell- and plasmablast-targeting potential of ASP2713. Our results suggest that ASP2713 may have therapeutic potential as a treatment for SLE, where B cells play a pathogenic role.

Abstract 1642: A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity

Abstract Therapeutic antibodies have improved survival of both hematologic and solid cancer patients, inducing long-lasting responses and even cures. Clinically successful antibodies exert antitumor activity by targeting tumor cells directly e.g. anti-CD20 mAb in B cell cancer, or by targeting and activating immune cells that seek and destroy cancer cells in the tumor microenvironment (immune checkpoint blocking antibodies;ICB). Still, many patients fail to respond or acquire resistance to these therapies. Understanding mechanisms and overcoming resistance to distinct classes of antibody drugs, is therefore clearly warranted and has the potential to further improve cancer outcomes. The inhibitory Fc gamma receptor (FcγR) IIB may precipitate resistance to cancer immunotherapy by several mechanisms, acting both on tumor and immune effector cells. We previously developed an FcγRIIB blocking antibody (BI-1206). Based on its cytolytic activity against B cell cancer cells and ability to block rituximab internalization from tumor cells, as well as enhance rituximab therapeutic activity in mice humanized for CD20 and FcγRIIB or bearing relapsed/refractory CLL in vivo, BI-1206 is being explored in clinical trials (NCT03571568; NCT04219254). Emerging data demonstrate that FcγRs can modulate the therapeutic activity of ICB antibodies (anti-PD-1/PD-L1 and anti-CTLA-4). While both anti-CTLA-4 and anti-PD-1/L1 mAb are effective and approved for cancer therapy, and combined checkpoint blockade improves therapeutic responses, anti-CTLA-4 therapies are associated with tolerability issues. Means of enhancing the anti-CTLA-4 therapeutic window are therefore desirable in order to deliver this therapy to more patients. Recent preclinical and retrospective clinical data indicate that anti-CTLA-4 antibodies may promote antitumor immunity by dual mechanisms; “releasing the brakes” on T effector cells and depleting immune suppressive intratumoral T regulatory (Treg) cells. Anti-CTLA-4 induced Treg depletion was shown to be FcγR-dependent. Here, we report the generation of a novel, fully human FcγRIIB-blocking antibody (BI-1607) engineered to eliminate Fc-mediated FcγR binding and function (Fc-null anti-FcγRIIB). Using a mechanism-of-action-matched surrogate antibody, we demonstrate that Fc-null anti-FcγRIIB enhances therapeutic efficacy of submaximally efficacious doses of anti-CTLA-4 in responsive (MC38) or resistant (CT26) syngeneic immune competent mouse models. Of relevance to the clinical setting where low dose anti-CTLA-4/anti-PD-1 combination therapies have been approved for different indications, we further demonstrate that triplet therapy significantly enhanced survival in the checkpoint blockade-poorly responsive B16 tumor model. Collectively our results provide proof-of-concept for Fc-null anti-FcγRIIB to enhance anti-CTLA-4 ICB in the treatment of cancer. Citation Format: Linda Mårtensson, Robert Oldham, Marie Borggren, Mathilda Kovacek, Therese Blidberg, Ali Roghanian, Ulla-Carin Thornberg, Ingrid Karlsson, Stephen A. Beers, Mark S. Cragg, Ingrid Teige, Bjorn L. Frendéus. A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1642.

Bcr-Abl1 Kinase-Independent Upregulation of FcγRIIb Mediates Malignant Signaling That Is Critical for Leukemogenesis

Chronic myeloid leukemia (CML) is provoked by the chromosomal translocation t(9;22) within the hematopoietic stem cell (HSC) compartment, and this mutation gives rise to the oncogenic tyrosine kinase Bcr-Abl1. Although tyrosine kinase inhibitor (TKI) therapy results in enormous clinical success, TKIs fail to eradicate the disease initiating leukemic stem cell population (LSC) in the majority of CML patients. In a transgenic CML mice (SCLtTA/Bcr-Abl), which we have previously demonstrated to serve as an excellent model to study Bcr-Abl1 independent stem cell persistence, we identified the ITIM carrying Fc gamma receptor IIb (FcγRIIb; CD32) to be 2.8-fold upregulated in Bcr-Abl1+ versus control bone-marrow (BM) derived LSK (lin-;Sca-1+;c-kit+) cells, using microarray and qRT-PCR. Aiming to evaluate the role of FcγRIIb function in Bcr-Abl1 mediated leukemogenesis, we previously retrovirally infected 5-FU treated SCLtTA/Bcr-Abl BM cells (CD45.1+) using FcγRIIb shRNA or scrambled control and transplanted these cells into FVB/N wildtype (WT) CD45.2+ recipients. Spleen weight in recipients receiving shRNA transduced stem and progenitor cells was significantly reduced (352 ± 59.1 mg), as compared to scrambled controls (568.1 ± 101.7 mg). Stem cell compartment analysis revealed decreased leukemic cells of shRNA versus scrambled control transplanted mice in the BM LSK compartment (lin-, c-kit+, Sca-1+, CD45.1+, GFP+, 1.38-fold, p≤0.05). In addition, these effects were even more pronounced upon total depletion of FcγRIIb using the retroviral Bcr-Abl1 model. In these mice, complete loss of FcγRIIb decreased malignant BM LSK cells by 3.5-fold (p<0.05) as compared to Bcr-Abl1 transduced FcγRIIb+/+recipients. Here, we have now studied the oncogenic mechanisms of FcγRIIb expression using the transgenic mice, a retroviral CML mouse model, as well as human cells.Ectopic Bcr-Abl1 expression in human TF-1 cells increased FcγRIIb levels by 4-fold (p<0.001). In lin- SCLtTA/Bcr-Abl BM cells, FcγRIIb was increased 1.4-fold (p<0.001), and receptor upregulation was thus higher in the LSK compartment (2.8-fold) as compared to lin- cells. We then studied the effect of TKI treatment on FcγRIIb expression in Bcr-Abl1 transduced lin- BM and lin-SCLtTA/Bcr-Abl cells using imatinib or dasatinib. However, both TKIs did not reduce FcγRIIb levels, suggesting that persistent receptor upregulation is regulated independently of the Bcr-Abl1 kinase. Next, we used HoxB8-immortalized progenitor cells from FcγRIIb-/- vs wt BM and virally introduced Bcr-Abl1 or ev. CFU (colony forming unit) potential in Bcr-Abl1 positive FcγRIIb -/- cells was significantly decreased 4-fold (p<0.01) as compared to Bcr-Abl1 transduced wt cells. Interestingly, re-introduction of FcγRIIb into Bcr-Abl1 positive FcγRIIb-/- cells significantly increased CFU capacity again. CFU reduction was also observed upon overexpression of an SH2-SHIP1 domain in leukemic cells, as SHIP1 is the predominant FcγRIIb binding partner upon activation. Furthermore, depletion of the receptor, using shRNAs, decreased the proliferation rate in CML cells (2.3-fold on day 4, p<0.01) and this was accompanied by an increase in G0/G1 phase and decrease in S and G2/M phase in CML cells lacking the receptor. Along the same line, expression of p16, p19 and p21 were significantly increased in FcγRIIb depleted CML cells. CFU potential as well as proliferation was likewise decreased (10-fold, p<0.01; 2.8-fold p<0.01) in shRNA-mediated FcγRIIb knock-down immortalized SCLtTA/Bcr-Abl CML cells. Western-Blot analysis showed that FcγRIIb is phosphorylated in leukemic cells, and analysis of downstream signal pathways revealed decreased levels of p-ERK and p-p38 among others in FcγRIIb-/-, compared to FcγRIIb+/+ Bcr-Abl1 transduced cells. Taken together, we demonstrate that the FcγRIIb upregulation on LSC from transgenic CML mice, progenitor cells and human cells critical for CML cell biology. Genetic depletion or knock-down of the receptor reduces CFU capacity and cell growth which is mediated due to impaired leukemic downstream signaling. As FcγRIIb inactivation significantly impairs CML development and LSC burden in vivo and is upregulated independent of the Bcr-Abl1 kinase this receptor represents an attractive novel target. DisclosuresBruemmendorf:Novartis: Research Funding. Koschmieder:Roche: Other: Clinical Trial participation; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding.

Lipopolysaccharide-Enhanced Responses against Aryl Hydrocarbon Receptor in FcgRIIb-Deficient Macrophages, a Profound Impact of an Environmental Toxin on a Lupus-Like Mouse Model.

Fc gamma receptor IIb (FcgRIIb) is the only inhibitory-FcgR in the FcgR family, and FcgRIIb-deficient (FcgRIIb−/−) mice develop a lupus-like condition with hyper-responsiveness against several stimulations. The activation of aryl hydrocarbon receptor (Ahr), a cellular environmental sensor, might aggravate activity of the lupus-like condition. As such, 1,4-chrysenequinone (1,4-CQ), an Ahr-activator, alone did not induce supernatant cytokines from macrophages, while the 24 h pre-treatment by lipopolysaccharide (LPS), a representative inflammatory activator, prior to 1,4-CQ activation (LPS/1,4-CQ) predominantly induced macrophage pro-inflammatory responses. Additionally, the responses from FcgRIIb−/− macrophages were more prominent than wild-type (WT) cells as determined by (i) supernatant cytokines (TNF-α, IL-6, and IL-10), (ii) expression of the inflammation associated genes (NF-κB, aryl hydrocarbon receptor, iNOS, IL-1β and activating-FcgRIV) and cell-surface CD-86 (a biomarker of M1 macrophage polarization), and (iii) cell apoptosis (Annexin V), with the lower inhibitory-FcgRIIb expression. Moreover, 8-week-administration of 1,4-CQ in 8 week old FcgRIIb−/− mice, a genetic-prone lupus-like model, enhanced lupus characteristics as indicated by anti-dsDNA, serum creatinine, proteinuria, endotoxemia, gut-leakage (FITC-dextran), and glomerular immunoglobulin deposition. In conclusion, an Ahr activation worsened the disease severity in FcgRIIb−/− mice possibly through the enhanced inflammatory responses. The deficiency of inhibitory-FcgRIIb in these mice, at least in part, prominently enhanced the pro-inflammatory responses. Our data suggest that patients with lupus might be more vulnerable to environmental pollutants.

Prominent Indomethacin-Induced Enteropathy in Fcgriib Defi-cient lupus Mice: An Impact of Macrophage Responses and Immune Deposition in Gut.

A high dose of NSAIDs, a common analgesic, might induce lupus activity through several NSAIDs adverse effects including gastrointestinal permeability defect (gut leakage) and endotoxemia. Indomethacin (25 mg/day) was orally administered for 7 days in 24-wk-old Fc gamma receptor IIb deficient (FcgRIIb-/-) mice, an asymptomatic lupus model (increased anti-dsDNA without lupus nephritis), and age-matched wild-type (WT) mice. Severity of indomethacin-induced enteropathy in FcgRIIb-/- mice was higher than WT mice as demonstrated by survival analysis, intestinal injury (histology, immune-deposition, and intestinal cytokines), gut leakage (FITC-dextran assay and endotoxemia), serum cytokines, and lupus characteristics (anti-dsDNA, renal injury, and proteinuria). Prominent responses of FcgRIIb-/- macrophages toward lipopolysaccharide (LPS) compared to WT cells due to the expression of only activating-FcgRs without inhibitory-FcgRIIb were demonstrated. Extracellular flux analysis indicated the greater mitochondria activity (increased respiratory capacity and respiratory reserve) in FcgRIIb-/- macrophages with a concordant decrease in glycolysis activity when compared to WT cells. In conclusion, gut leakage-induced endotoxemia is more severe in indomethacin-administered FcgRIIb-/- mice than WT, possibly due to the enhanced indomethacin toxicity from lupus-induced intestinal immune-deposition. Due to a lack of inhibitory-FcgRIIb expression, mitochondrial function, and cytokine production of FcgRIIb-/- macrophages were more prominent than WT cells. Hence, lupus disease-activation from NSAIDs-enteropathy-induced gut leakage is possible.

Abstract PO-26: Prognostic significance of Fc gamma receptor IIB expression in the response of previously untreated diffuse large B-cell lymphomas to anti-CD20 monoclonal antibodies: Differing impact of rituximab and obinutuzumab

Abstract Background: The anti-CD20 monoclonal antibody (mAb), obinutuzumab (G), has shown improved outcomes versus rituximab (R) in indolent lymphomas; however, no improvement was seen in diffuse large B-cell lymphoma (DLBCL), and the molecular basis is unclear. The inhibitory Fc gamma receptor IIB (Fc γRIIB), expressed on lymphoma cells, can impair the effects of direct targeting mAbs, such as R, by binding and internalizing them, reducing opsonization and limiting FcγR-mediated killing. We hypothesized that FcγRIIB expression on cellular effectors and/or the lymphoma confers treatment resistance in some patients (pts) and evaluated if outcomes differ for therapies involving a non-internalized mAb (G). Methods: We evaluated correlates between FCGR2B mRNA and/or FcγRIIB protein expression and pt outcomes in two discovery cohorts of de novo DLBCL treated with R-CHOP (Arthur et al. 2018, n=372; Schmitz et al. 2018, n=234), and the phase III GOYA trial (NCT01287741; n=552), which compared R-CHOP with G-CHOP in pts with previously untreated DLBCL. FCGR2B mRNA expression was assessed by RNA-Seq, and protein expression was assessed in evaluable cohorts by immunohistochemistry, using tissue microarrays with macrophages identified by CD68. FCGR2B expression was also measured by a NanoString assay (Arthur cohort). Cox regression analyzed the impact of FcγRIIB/FCGR2B expression on progression-free survival (PFS), with univariate and multivariate models adjusted for International Prognostic Index (IPI), cell of origin (COO), and BCL2 protein expression. Results: In the discovery cohorts, a higher FCGR2B expression was significantly associated with shorter PFS (Arthur: HR 1.09 [95% CI: 1.01–1.19], P=0.036; Schmitz: HR 1.13 [95% CI: 1.02–1.26], P=0.0243). Expression by NanoString strongly correlated with RNA-Seq, confirming the association with shorter PFS (HR 1.13 [95% CI: 1.04–1.23], P=0.0048). In GOYA, a significant association between PFS and FCGR2B was observed in the R arm (HR 1.26 [95% CI: 1.00–1.58], P=0.0455), with no prognostic effect observed for G (HR 0.91 [95% CI: 0.69–1.20], P=0.5). Pts with high FCGR2B expression appeared to benefit more from G than R (HR 0.67 [95% CI: 0.44–1.02], P=0.0622), in contrast to pts with low FCGR2B expression (HR 1.58 [95% CI: 1.00–2.50], P=0.0503). In both Arthur and GOYA cohorts, FCGR2B expression by RNA-Seq was associated with FcγRIIB on the tumor, which correlated with a shorter PFS for R (HR 2.17 [95% CI: 1.04–4.50], P=0.03), but not G (HR 1.37 [95% CI: 0.66–2.87], P=0.4). This prognostic effect on PFS was independent of established prognostic biomarkers, IPI, COO and BCL2. Conclusion: High FcγRIIB/FCGR2B expression in pts with DLBCL has prognostic value in those treated with R and may confer differential responsiveness to R or G. Citation Format: Laura K. Hilton, Malgorzata Nowicka, Margaret Ashton-Key, Chantal E. Hargreaves, Chern Lee, Russell Foxall, Matthew J. Carter, Stephen A. Beers, Kathleen N. Potter, Christopher R. Bolen, Christian Klein, Andrea Knapp, Farheen Mir, Matthew Rose-Zerilli, Cathy Burton, Wolfram Klapper, David W. Scott, Laurie H. Sehn, Umberto Vitolo, Maurizio Martelli, Marek Trneny, Graham W. Slack, Pedro Farinha, Jonathan C. Strefford, Mikkel Z. Oestergaard, Ryan D. Morin, Mark S. Cragg. Prognostic significance of Fc gamma receptor IIB expression in the response of previously untreated diffuse large B-cell lymphomas to anti-CD20 monoclonal antibodies: Differing impact of rituximab and obinutuzumab [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-26.

Pharmacokinetic prediction of an antibody in mice based on an in vitro cell-based approach using target receptor-expressing cells

In vivo pharmacokinetics (PK) studies using mice and monkeys are the main approaches for evaluating and predicting the PK of antibodies, and there is a strong demand for methods that do not require animal experiments. In this work, we focused on quantitatively predicting the nonlinear PK of an antibody based on cell-based assays. An anti-mouse Fc gamma receptor IIB antibody was used as a model antibody. To determine the PK parameters related to nonspecific elimination in vivo, the plasma concentration profile at 100 mg/kg, at which target-specific clearance is saturated, was analyzed by a 2-compartment model. To estimate the parameters related to target-specific elimination, the Michaelis–Menten constant (Km) and the maximum elimination rate (Vmax) were determined by an uptake assay using Chinese hamster ovary (CHO) cells expressing the target receptor. Finally, the integration of all of these parameters permitted the PK to be predicted at doses ranging from 1 to 100 mg/kg regardless of whether target-specific clearance was saturated or nonsaturated. The findings presented herein show that in vitro assays using target-expressing cells are useful tools for obtaining PK parameters and predicting PK profiles and, in some cases, eliminate the need for in vivo PK studies using experimental animals.