Inhibitory Fc Receptor FcγRIIB Research Articles

Antibody-Targeted TNFRSF Activation for Cancer Immunotherapy: The Role of FcγRIIB Cross-Linking.

Co-stimulation signaling in various types of immune cells modulates immune responses in physiology and disease. Tumor necrosis factor receptor superfamily (TNFRSF) members such as CD40, OX40 and CD137/4-1BB are expressed on myeloid cells and/or lymphocytes, and they regulate antigen presentation and adaptive immune activities. TNFRSF agonistic antibodies have been evaluated extensively in preclinical models, and the robust antitumor immune responses and efficacy have encouraged continued clinical investigations for the last two decades. However, balancing the toxicities and efficacy of TNFRSF agonistic antibodies remains a major challenge in the clinical development. Insights into the co-stimulation signaling biology, antibody structural roles and their functionality in immuno-oncology are guiding new advancement of this field. Leveraging the interactions between antibodies and the inhibitory Fc receptor FcγRIIB to optimize co-stimulation agonistic activities dependent on FcγRIIB cross-linking selectively in tumor microenvironment represents the current frontier, which also includes cross-linking through tumor antigen binding with bispecific antibodies. In this review, we will summarize the immunological roles of TNFRSF members and current clinical studies of TNFRSF agonistic antibodies. We will also cover the contribution of different IgG structure domains to these agonistic activities, with a focus on the role of FcγRIIB in TNFRSF cross-linking and clustering bridged by agonistic antibodies. We will review and discuss several Fc-engineering approaches to optimize Fc binding ability to FcγRIIB in the context of proper Fab and the epitope, including a cross-linking antibody (xLinkAb) model and its application in developing TNFRSF agonistic antibodies with improved efficacy and safety for cancer immunotherapy.

Memory T cell-mediated rejection is mitigated by FcγRIIB expression on CD8+ T cells.

Donor-reactive memory T cells generated via heterologous immunity represent a potent barrier to long-term graft survival following transplantation because of their increased precursor frequency, rapid effector function, altered trafficking patterns, and reduced reliance on costimulation signals for activation. Thus, the identification of pathways that control memory T cell survival and secondary recall potential may provide new opportunities for therapeutic intervention. Here, we discovered that donor-specific effector/memory CD8+ T cell populations generated via exposure to acute vs latent vs chronic infections contain differential frequencies of CD8+ T cells expressing the inhibitory Fc receptor FcγRIIB. Results indicated that frequencies of FcγRIIB-expressing CD8+ donor-reactive memory T cells inversely correlated with allograft rejection. Furthermore, adoptive T cell transfer of Fcgr2b-/- CD8+ T cells resulted in an accumulation of donor-specific CD8+ memory T cells and enhanced recall responses, indicating that FcγRIIB functions intrinsically to limit T cell CD8+ survival in vivo. Lastly, we show that deletion of FcγRIIB on donor-specific CD8+ memory T cells precipitated costimulation blockade-resistant rejection. These data therefore identify a novel cell-intrinsic inhibitory pathway that functions to limit the risk of memory T cell-mediated rejection following transplantation and suggest that therapeutic manipulation of this pathway could improve outcomes in sensitized patients.

Keeping T-he Killers at Bay via FcγRIIb

Understanding how cytotoxic Tcells are regulated is key for enhancing or suppressing their activity in tumor or transplantation settings. A study by Morris etal. (2020) in this issue of Immunity shows that crosslinking of inhibitory Fc receptor FcγRIIb by the suppressive cytokine Fgl2 limits cytotoxic CD8+ Tcell responses by inducing Tcell apoptosis.

Signaling through the Inhibitory Fc Receptor FcγRIIB Induces CD8+ T Cell Apoptosis to Limit T Cell Immunity

Effector CD8+ Tcells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of effector CD8+ Tcells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ Tcell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector Tcell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ Tcell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ Tcells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b-/-, CD8+ Tcells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ Tcell immunity.

Testing the role of the FcγRIIB immunoreceptor tyrosine-based inhibitory motif in regulation of the B cell immune response.

In vitro studies have demonstrated that the immunoreceptor tyrosine-based inhibitory motif (ITIM) of the inhibitory Fc receptor FcγRIIB is critical for mediating attenuation of signaling via immunoreceptor tyrosine-based activation motif (ITAM) containing receptors, such as the B cell antigen receptor (BCR), when FcγRIIB is co-cross-linked to these activation receptors. To test the role of the FcγRIIB ITIM motif in regulation of the B cell immune response in vivo, we constructed lines of transgenic mice expressing a form of FcγRIIB with an inactivating tyrosine (Y) to phenylalanine (F) mutation in the ITIM motif. Detailed studies of one of these lines, in which the mutant FcγRIIB was expressed on B cells and other cell types that normally express this receptor, were performed. No quantitative differences in germinal center (GC) B cell responses were observed between the mutant FcγRIIB transgenic line and control mice. However, serum antibody and antibody forming cell responses were often observed to be elevated in the ITIM mutant FcγRIIB transgenic mice as compared to controls, though not to the same extent as mice deficient in expression of FcγRIIB. Moreover, primary B cells from the ITIM mutant FcγRIIB line did not display the same level of augmented BCR signaling as primary FcγRIIB deficient B cells under conditions inducing co-cross-linking of FcγRIIB and the BCR. In total, these data suggest that a functional ITIM motif is not required for all in vivo inhibitory activity of this receptor. However, we also found that the transgenic ITIM mutant FcγRIIB receptor was expressed at abnormal levels in several hematopoietic lineages. Thus, confirmation of our findings will require the generation and analysis of mice in which an ITIM mutant form of FcγRIIB is expressed in vivo as is the endogenous receptor.

Synergistic effects of the inhibitory Fc receptor FcγRIIB deficiency and lupus-associated SLAM family genes on breaking germinal center tolerance checkpoint (BA2P.126)

Abstract The inhibitory Fc receptor FcγRIIB deficiency and lupus-associated signaling lymphocyte activation molecules (SLAMs) are suggested to contribute to autoimmunity in FcγRIIB deficient mice generated using 129Sv/J ES cells and backcrossed to B6 mice (B6-129.RIIBKO). To study the individual contribution of FcγRIIB deficiency and 129-derived SLAM genes to loss of germinal center (GC) tolerance we compared B6 mice deficient in FcγRIIB (B6.RIIBKO) and B6 mice congenic for 129-derived SLAM locus (B6.129-SLAM) with B6-129.RIIBKO and B6 controls. B6-129.RIIBKO mice developed numerous spontaneous germinal centers (Spt-GCs) with increased size producing high titers of autoantibodies against nuclear (ANA) and other self-antigens, alongside a hyperactive T cell phenotype. We also observed elevated Spt-GC and T-cell responses in B6.RIIBKO and B6.129-SLAM mice than B6 mice although much lower when compared to B6-129.RIIBKO mice. B6.RIIBKO and B6.129-SLAM mice also failed to produce high titers of ANAs in comparison to B6-129.RIIBKO. Ex vivo assays revealed that differentially expressed 129-SLAM genes promoted higher expression of activation molecules on B cells, T cells and DCs while FcγRIIB deficiency was sufficient to drive proliferation of B cells and T cells. Our results suggest that the co-operative effects of 129-derived SLAMs on activation and FcγRIIB deficiency on the proliferation of B cells and T cells are essential for breaking the GC tolerance checkpoint in B6-129.RIIBKO mice.

Analysis of a wild mouse promoter variant reveals a novel role for FcγRIIb in the control of the germinal center and autoimmunity

Genetic variants of the inhibitory Fc receptor FcγRIIb have been associated with systemic lupus erythematosus in humans and mice. The mechanism by which Fcgr2b variants contribute to the development of autoimmunity is unknown and was investigated by knocking in the most commonly conserved wild mouse Fcgr2b promoter haplotype, also associated with autoimmune-prone mouse strains, into the C57BL/6 background. We found that in the absence of an AP-1-binding site in its promoter, FcγRIIb failed to be up-regulated on activated and germinal center (GC) B cells. This resulted in enhanced GC responses, increased affinity maturation, and autoantibody production. Accordingly, in the absence of FcγRIIb activation-induced up-regulation, mice developed more severe collagen-induced arthritis and spontaneous glomerular immune complex deposition. Our data highlight how natural variation in Fcgr2b drives the development of autoimmune disease. They also show how the study of such variants using a knockin approach can provide insight into immune mechanisms not possible using conventional genetic manipulation, in this case demonstrating an unexpected critical role for the activation-induced up-regulation of FcγRIIb in controlling affinity maturation, autoantibody production, and autoimmunity.

Intravenous immunoglobulin does not increase FcγRIIB expression levels on monocytes in children with immune thrombocytopenia

Intravenous immunoglobulin (IVIG) produces a rapid and prolonged increase in the platelet counts of children with immune thrombocytopenia (ITP). The mechanism of IVIG efficacy in a murine model of ITP has been reported to operate through an IVIG-mediated increase in the expression of the inhibitory Fc receptor FcγRIIB(CD32B) on splenic macrophages. This investigation examined whether IVIG administration results in a similar increase in FcγRIIB expression on peripheral blood CD14(+) monocytes in 20 children with ITP. FcγRIIB expression on peripheral blood monocytes was measured by flow cytometry in ITP patients, before and after IVIG therapy, as well as in control subjects. Peripheral blood monocytes were labelled with fluorescent-specific antibodies. There were no significant differences in the absolute number of [corrected] CD14(+) CD32B(+) monocytes, and [corrected] the percentages of CD14(+) CD32B(+) cells in mononuclear cells or monocytes. [corrected]. We suggest that IVIG does not increase FcγRIIB expression in peripheral blood monocytes in children with ITP.

Immunity against a therapeutic xenoprotein/Fc construct delivered by gene transfer is reduced through binding to the inhibitory receptor FcγRIIb

Therapeutic xenoproteins are immunogenic and can induce neutralizing antibodies. When delivered by intramuscular injection of a plasmid vector, this mimics classical DNA vaccination. To demonstrate this, we chose Exendin-4 (Ex4), which is a glucagon-like peptide-1 mimetic xenoprotein in clinical use for treating type 2 diabetes. We constructed an Ex4 and mouse immunoglobulin (Ig)G1-Fc fusion fragment (Ex4/Fc), and hypothesized that it would have minimal immunogenicity as a result of its capacity to bind the inhibitory Fc receptor FcγRIIb expressed by B lymphocytes. Plasmid vectors encoding Ex4/Fc constructs, with wild-type or mutant Fc, were injected intramuscularly into mice, and local electroporation was applied to enhance gene transfer. Gene transfer was performed in both wild-type and FcγRIIb knockout mice. Antibody production was detected in serum by an enzyme-linked immunosorbent assay. Recombinant Ex4/Fc bound only to B cells expressing FcγRIIb. This binding was dependent on a motif in the Fc region, which we mutated to abolish binding (Ex4/Fcmut). Ex4 antibody was detected in mice treated with Ex4, as well as Ex4/Fcmut, but not in those treated with Ex4/Fc. Thus, wild-type Fc was associated with reduced immunogenicity. To confirm this was related to the presence of inhibitory Fc receptors, we also performed experiments in FcγRIIb-null mice. Mice lacking this receptor produced antibodies against all Ex4 constructs, including the wild-type Fc (Ex4/Fc). The present study shows that inhibitory FcγRIIb receptors interacting with the wild-type IgG1-Fc reduce immunity against Ex4/Fc, suggesting an approach for reducing the immunogenicity of therapeutic proteins in the context of gene therapy.

Intravenous gammaglobulin suppresses inflammation through a novel TH2 pathway

High-dose intravenous immunoglobulin is a widely used therapeutic preparation of highly purified immunoglobulin G (IgG) antibodies. It is administered at high doses (1-2 grams per kilogram) for the suppression of autoantibody-triggered inflammation in a variety of clinical settings. This anti-inflammatory activity of intravenous immunoglobulin is triggered by a minor population of IgG crystallizable fragments (Fcs), with glycans terminating in α2,6 sialic acids (sFc) that target myeloid regulatory cells expressing the lectin dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN; also known as CD209). Here, to characterize this response in detail, we generated humanized DC-SIGN mice (hDC-SIGN), and demonstrate that the anti-inflammatory activity of intravenous immunoglobulin can be recapitulated by the transfer of bone-marrow-derived sFc-treated hDC-SIGN(+) macrophages or dendritic cells into naive recipients. Furthermore, sFc administration results in the production of IL-33, which, in turn, induces expansion of IL-4-producing basophils that promote increased expression of the inhibitory Fc receptor FcγRIIB on effector macrophages. Systemic administration of the T(H)2 cytokines IL-33 or IL-4 upregulates FcγRIIB on macrophages, and suppresses serum-induced arthritis. Consistent with these results, transfer of IL-33-treated basophils suppressed induced arthritic inflammation. This novel DC-SIGN-T(H)2 pathway initiated by an endogenous ligand, sFc, provides an intrinsic mechanism for maintaining immune homeostasis that could be manipulated to provide therapeutic benefit in autoimmune diseases.

Lack of Binding of Monoclonal Protein Produced by Multiple Myeloma (MM) Cells to FcγRIIb Reduces Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Signaling and Provides the Rationale for a Novel Therapeutic Approach to MM

AbstractAbstract 455Functional B cell and plasma cell immune responses are dependent upon an exquisitely controlled process integrating signals from activating and inhibitory receptors present on the surface of these cells. These activating and inhibitory signaling pathways regulate both the quality and quantity of immunoglobulin (Ig) production. These signaling motifs, termed immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposed signaling modules that duel for control of plasma cellular activation within the immune system. The inhibitory Fc receptor FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow and their ability to produce most serum Ig. Activation of FcγRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing protein tyrosine phosphatase-1 (SHP-1) SHP-2 and the SH2-containing inositol 5-phosphatase (SHIP) in plasma cells. FcγRIIb-mediated SHIP phosphorylation results in enhanced degradation of PtdIns (3,4,5)P, which is required for B-cell antigen receptor (BCR)-induced recruitment and activation of downstream ITAM signaling molecules. The inhibitory IgG Fc receptor FcγRIIB was the first discovered and remains the best studied example of an ITIM-containing receptor. In this study, we first investigated the IgG-binding ability of 18 MM patients and 10 normal donors to FcγRIIb using flow cytometric analysis. Each serum sample was incubated with MHC1 cells that only express FcγRIIb but do not express FcγRI and FcγRIIa. After washing three times with 1 × PBS, anti-human IgG antibody conjugated with FITC was added to the cells for another 30 minutes. The results showed MM patients' serum IgG have much lower FcγRIIb-binding ability than normal human IgG (P<0.05). Immunohistochemical staining also demonstrated MM patients' sera showed obviously less binding to the FcγRIIb on MHC1 cells compared to serum samples from normal human donors. We further analyzed the FcγRIIb-SHIP signaling pathway in normal B-cells following exposure to MM and normal human sera. Fresh human B-cells were isolated using anti-CD20 antibodies with magnetic bead selection and the cells were washed in an acidic solution (pH 4) for 1 minute to remove any bound antigen from the cell surface. Raji B-cells were also acid-treated. First, these cells were exposed to MM patients' or normal human sera for 5, 15, 30, or 60 minutes to determine the maximum time point of SHIP phosphorylation. Using a standard Western blot protocol, phosphorylated SHIP and total SHIP protein expression were visualized using an enhanced chemiluminescence detection system. The maximum time point of SHIP phosphorylation was 15 minutes. The results showed that both normal human B-cells and Raji B-cells showed markedly lower SHIP phosphorylation/total SHIP following exposure to MM patient serum compared to normal human serum which is consistent with the lack of binding of human Ig to FcγRIIb. Our findings suggest that the monoclonal protein produced by MM patients has very low FcγRIIb-binding ability and is incapable of signaling through inhibitory ITIM pathway. Most importantly, FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow in addition to Ig production. Cross-linking of FcγRIIb induces apoptosis of plasma cells (Xiang Z et al Nat Immunol 8: 419-29, 2007). Thus, it is possible that this effect prevents the induction of apoptosis in MM cells. Previous studies have reported that normal Ig is capable of inducing B-cell apoptosis. Thus, it is possible that exposure of MM cells to normal immunoglobulin may be capable of inducing apoptosis of these cells and reducing their production of M-protein. We are currently evaluating the ITIM signal transduction pathway, M-protein production and apoptosis in MM and normal B-cells following exposure to normal and MM M-protein IgG. Disclosures:No relevant conflicts of interest to declare.

Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcγRIIb with Fc-engineered antibodies

The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcγRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcγRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to ∼430-fold greater affinity to FcγRIIb. Relative to native IgG1, the FcγRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcγRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcγRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcγRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.