Inhibitory Fc Research Articles

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.

KMT1E-mediated chromatin modifications at the FcγRIIb promoter regulate thymocyte development.

This work examines the role the lysine methyltransferase KMT1E (Setdb1) in thymocyte development. We have developed and described a T cell-specific conditional knockout of Setdb1. A partial block was seen at the double-positive to single-positive transition, causing reduced numbers of single-positive T cells in the thymus and periphery. Knockout thymocytes had reduced numbers of CD69(+) and T-cell receptor TCRβ(+) cells and increased numbers of apoptotic cells in the double-positive compartment, suggesting an alteration in the selection process. Transcriptional profiling of thymocytes revealed that Setdb1 deletion derepresses expression of FcγRIIb, the inhibitory Fc receptor. We demonstrate that a KMT1E-containing complex directly interacts with the FcγRIIb promoter and that histone H3 at lysine 9 tri-methylation at this promoter is dependent on Setdb1 expression. Derepression of FcγRIIb causes exacerbated signaling through the TCR complex, with specifically increased phosphorylation of ZAP70, affecting selection. This work identifies KMT1E as a novel repressor of FcγRIIb and identifies an underappreciated role of FcγRIIb in fine tuning thymocyte development.

CD44 antibody-mediated amelioration of murine immune thrombocytopenia (ITP): mouse background determines the effect of FcγRIIb genetic disruption.

Several monoclonal antibodies to CD44 can successfully ameliorate murine immune thrombocytopenia (ITP). As these antibodies may be a potential replacement for intravenous immune globulin (IVIG) in the treatment of ITP and other autoimmune diseases, an understanding of their mechanisms of action is important. The role of the inhibitory Fc receptor (FcγRIIb) in the mechanism of action of IVIG and therapeutic CD44 antibodies remains uncertain. To assess if FcγRIIb in splenic macrophages plays a critical role in the action of these two therapeutics, splenectomized mice and mice genetically deficient in FcγRIIb on different backgrounds were evaluated. Thrombocytopenia was induced in FcγRIIb-deficient mice on B6;129S, C57BL/6, and BALB/C backgrounds, as well as splenectomized mice and control mice by platelet (PLT) antibody. PLT counts were enumerated before and after treatment with anti-CD44, red blood cell antibodies, or IVIG. Anti-CD44 is ineffective at inhibiting thrombocytopenia in B6;129S FcγRIIb-deficient mice but, like IVIG, is effective in splenectomized mice and FcγRIIb-deficient mice on the BALB/C and C57BL/6 background. These data suggest that 1) the B6;129S background itself is unlikely to be the sole reason for anti-CD44's inability to function in B6;129S FcγRIIb-deficient mice, 2) the simple loss of macrophage FcγRIIb expression alone is insufficient to explain anti-CD44 ameliorative function, and 3) a combination of mouse background genes in addition to FcγRIIb genetic disruption may affect the ability of anti-CD44 to function therapeutically. Similarities between IVIG and anti-CD44 mechanisms suggest that patients responsive to IVIG may also potentially respond to anti-CD44 treatment.

Gammaglobulins, autoantibodies, therapeutic antibodies and anti-drug antibodies in rheumatoid arthritis.

Gammaglobulins, autoantibodies, therapeutic antibodies and anti-drug antibodies in rheumatoid arthritis.

Dichotomy in FcγRIIB deficiency and autoimmune-prone SLAM haplotype clarifies the roles of the Fc receptor in development of autoantibodies and glomerulonephritis.

BackgroundThe significance of a unique inhibitory Fc receptor for IgG, FcγRIIB (RIIB), in the prevention of spontaneous production of autoantibodies remains controversial, due mainly to the fact that the RIIB locus is adjacent to the autoimmune-related SLAM locus harboring the genes coding for signaling lymphocyte activation molecules, making it difficult to isolate the effect of RIIB deletion from that of SLAM in gene-targeted mice. Our objective was to determine the influence of RIIB deletion on the spontaneous development of autoimmune diseases and to compare it with that of potentially pathogenic SLAM.ResultsWe established two congenic C57BL/6 (B6) strains, one with the RIIB deletion and the other with SLAM, by backcrossing 129/SvJ-based RIIB-deficient mice into the B6 genetic background extensively. The RIIB deficiency indeed led to the production and/or accumulation of a small amount of anti-nuclear autoantibodies (ANAs) and to weak IgG immune-complex deposition in glomeruli without any obvious manifestation of lupus nephritis. In contrast, pathogenic SLAM in the B6 genetic background induced ANAs but no IgG immune-complex deposition in the kidneys. Naïve SLAM mice but not RIIB-deficient mice exhibited hyperplasia of splenic germinal centers.ConclusionThe present results clarify the roles of RIIB in preventing production and/or accumulation of a small amount of ANAs, and development of glomerulonephritis. The combined effects of RIIB deletion and pathogenic SLAM can lead to severe lupus nephritis in the B6 genetic background.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0047-y) contains supplementary material, which is available to authorized users.

Fc gamma receptor IIb on GM-CSF macrophages controls immune complex mediated inhibition of inflammatory signals.

BackgroundIn rheumatoid arthritis (RA) macrophages play a major role in amplifying synovial inflammation. Important activating signals are those induced by Toll-like receptor (TLR) ligands and by activated T cells. The balance between activating and inhibitory Fc gamma receptors (FcγRs) on macrophages might be crucial in modulating these inflammatory responses. The purpose of this study was to determine FcγR expression on pro- and anti-inflammatory macrophages (gmMφ and mMφ, respectively) and identify functional consequences on immune complex uptake and macrophage activation.MethodsHuman monocytes were isolated and differentiated into gmMφ and mMφ. A full FcγR characterization of both macrophage subtypes was performed and uptake of fluorescent immune complexes (ICs) was determined. FcγRIIb isoforms were determined by qPCR. Macrophages were stimulated via different TLRs or cytokine activated T cells in the presence or absence of ICs and cytokine production was determined. Blocking studies were performed to look into the pathways involved.ResultsmMφ expressed high levels of the activating FcγRIIa and FcγRIII and low levels of the inhibitory FcγRIIb, while the FcγR balance on gmMφ was shifted towards the inhibitory FcγRIIb. This was accompanied by a clear increase in FcγRIIb1 mRNA expression in gmMφ. This resulted in higher IC uptake by mMφ compared to gmMφ. Furthermore, FcγR-mediated stimulation of gmMφ inhibited TLR2, 3, 4 and 7/8 mediated cytokine production via FcγRIIb and PI3K signaling. In addition, gmMφ but not mMφ produced TNFα upon co-culture with cytokine activated T cells, which was reduced by IC binding to FcγRIIb. The latter was dependent on PI3K signaling and COX2.ConclusionsFcγR expression patterns on gmMφ and mMφ are significantly different, which translates in clear functional differences further substantiating FcγRIIb as an interesting target for inflammation control in RA and other autoimmune/inflammatory diseases.

IgE and IL-33-mediated triggering of human basophils inhibits TLR4-induced monocyte activation.

Basophils are circulating granulocytes, best known as effector cells in allergic reactions. Recent studies in mice suggest that they might also participate in the suppression of chronic inflammation. The aim of this study was to assess the ability of purified human basophils to modulate monocyte responses upon IL-33 and IgE triggering. Activation of human basophils with IL-33 induced the production of IL-4 and the release of histamine, and enhanced their IgE-mediated activation. In addition, basophils triggered with IL-33 and anti-IgE significantly suppressed the LPS-induced production of the proinflammatory cytokine TNF-α and the upregulation of the costimulatory molecule CD80 by monocytes. These effects were mainly explained by the release of histamine, as they could be inhibited by the histamine receptor 2 antagonist ranitidine, with a smaller contribution of IL-4. In contrast, basophil-derived IL-4 and histamine had opposing effects on the expression of the inhibitory Fc γ receptor IIb and the production of IL-10 by monocytes. Our data show that basophils can influence monocyte activation and suggest a previously unrecognized role for human basophils in the modulation of monocyte-mediated immune responses, through the balanced secretion of histamine and IL-4.

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.

FcγRIIB limits protection against F. tularensis LVS challenge induced by inactivated F. tularensis (VAC7P.964)

Abstract We have previously demonstrated that intranasal (i.n.) immunization with inactivated Francisella tularensis (Ft) live vaccine strain (LVS) targeted to Fcγ receptors (FcγR) can induce partial protection against a lethal mucosal challenge with Ft SchuS4. This protection is antibody (Ab) and interferon gamma (IFNγ) dependent. FcγRIIB is the only inhibitory Fc receptor (FcR) in the FcγR family, and has been reported to regulate IgG production, which plays a significant role in protection against Ft LVS and Ft SchuS4 challenge. Thus, we sought to determine the impact of FcγRIIB on immune protection against Ft infection. We utilized inactivated Ft (iFt) as an immunogen. Naïve or iFt-immunized FcγRIIB knockout (KO) versus wildtype (WT) C57BL/6 mice were challenged with varying doses of Ft LVS. We observed no significant difference in survival between naïve KO versus WT mice or in basal levels of Ft-specific IgG or IgM. However, KO mice immunized with iFt showed significantly increased survival as compared to iFt-immunized WT mice. Furthermore, Ft-specific IgG and IgA production were elevated in the serum from iFt-immunized KO versus WT mice. IFNγ levels were also enhanced in iFt-immunized KO versus WT mice. In summary, our studies demonstrate that FcγRIIB influences the level of protection against Ft challenge generated by iFt immunization by limiting the production of Ft-specific Ab and IFNγ, which have been shown to play significant roles in protection against Ft infection.

Asymmetrical Fc Engineering Greatly Enhances Antibody-dependent Cellular Cytotoxicity (ADCC) Effector Function and Stability of the Modified Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

Immunotherapy Targeting Inhibitory Fcγ Receptor IIB (CD32b) in the Mouse Is Limited by Monoclonal Antibody Consumption and Receptor Internalization

Genetic deficiency of the inhibitory Fc receptor, FcγRIIB (CD32b), has been shown to augment the activity of activatory FcγR and promote mAb immunotherapy. To investigate whether mAbs capable of blocking FcγRIIB have similar capacity, we recently generated a panel of specific anti-mouse FcγRIIB mAbs that do not cross-react with other FcRs, allowing us to study the potential of FcγRIIB as a therapeutic target. Previous work revealed a number of these mAbs capable of eliciting programmed cell death of targets, and in the present study we demonstrated their ability to promote target cell phagocytosis. However, in a variety of murine tumor models, anti-FcγRIIB mAbs demonstrated limited therapeutic activity despite optimized treatment regimens. Unexpectedly, we observed that the anti-FcγRIIB mAbs are rapidly and extensively consumed in vivo, both by the tumor and host cells, including B cells, leading to a precipitous loss from the circulation. Closer analysis revealed that the anti-FcγRIIB mAbs become extensively internalized from the cell surface within 24 h in vivo, likely explaining their suboptimal efficacy. Subsequent studies revealed that anti-FcγRIIB mAb immunotherapy was effective when used against FcγRIIB(+) tumors in FcγRIIB(-/-) recipients, indicating that consumption of the mAb by nontumor cells is the primary limitation of these reagents. Importantly, similar rates of internalization were not seen on human target cells, at least in vitro. These studies further highlight the need to determine the propensity of mAb therapeutics to internalize target receptors and also identify potential key differences between human and mouse cells in this respect.

Molecular Basis for Downregulation of C5a-Mediated Inflammation by IgG1 Immune Complexes in Allergy and Asthma

Allergy and asthma are triggered primarily by the binding of allergen-specific immunoglobulin E (IgE)-allergen complexes to their receptors, recognition of the allergens by antigen-presenting cells, and allergen presentation to the T cells. These events lead to mucus secretions, runny nose, itchy eyes, sneezing, airway hyperresponsiveness, and nasal congestion. Complement 5a (C5a) has emerged as a central molecule that mediates these allergic reactions. Many allergens and allergen-specific IgG immune complexes (IgG-ICs) cause complement activation and C5a generation. C5a interaction with its receptor (C5aR) leads to the infiltration and activation of several immunologic cell types and the secretion of pathogenic inflammatory and proinflammatory mediators. However, IgG1-IC binding to the IgG inhibitory Fc gamma receptor (FcγRIIB) suppresses C5aR-mediated inflammatory signaling and, hence, may reduce the inflammatory immune responses through this FcγRIIB-mediated pathway. Reviews of the IgG1-IC interactions with C5a-mediated inflammatory immune responses suggest that IgG1-IC-C5a inhibitory therapy may reduce inflammation in allergic diseases.

Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

The utility of Plasmodium berghei as a rodent model for anti-merozoite malaria vaccine assessment.

Rodent malaria species Plasmodium yoelii and P. chabaudi have been widely used to validate vaccine approaches targeting blood-stage merozoite antigens. However, increasing data suggest the P. berghei rodent malaria may be able to circumvent vaccine-induced anti-merozoite responses. Here we confirm a failure to protect against P. berghei, despite successful antibody induction against leading merozoite antigens using protein-in-adjuvant or viral vectored vaccine delivery. No subunit vaccine approach showed efficacy in mice following immunization and challenge with the wild-type P. berghei strains ANKA or NK65, or against a chimeric parasite line encoding a merozoite antigen from P. falciparum. Protection was not improved in knockout mice lacking the inhibitory Fc receptor CD32b, nor against a Δsmac P. berghei parasite line with a non-sequestering phenotype. An improved understanding of the mechanisms responsible for protection, or failure of protection, against P. berghei merozoites could guide the development of an efficacious vaccine against P. falciparum.

Abstract 1250: Fibronectin domains engineered for specificity to individual murine Fc gamma receptors modulate tumoricidal activity of immune cells.

Abstract Activating and inhibitory Fc gamma receptors (FcγRs) can play a large role in the efficacy of certain antibody therapeutics. The Fc regions of the antibody bind to the FcγRs expressed on immune cells and modulate responses against antibody-coated tumor cells. Previous studies with FcγR knock-out mice have shown that antibody-mediated tumor clearance is improved when activating FcγRs are bound preferentially over inhibitory FcγRs. The ectodomains of FcγRs are highly homologous, making it challenging to engineer Fc regions to specifically bind individual FcγRs with high affinity and lack binding to the other FcγRs. To address this challenge, we engineered the human tenth type III fibronectin (Fn3) domain scaffold to bind individual murine FcγRs at epitopes that are distinct from the Fc binding region for testing in a mouse model. The most established pre-clinical models of cancer are mouse models; therefore studying the effect of modulating individual murine FcγRs should provide insight into engaging specific FcγRs as an anti-cancer therapeutic for humans. Using directed evolution with yeast surface display, we isolated Fn3 clones specific for murine FcγRI, FcγRII, FcγRIII, and FcγRIV through a combination of depletion, enrichment, and mutagenesis steps. Cell titrations measured with flow cytometry confirmed the binding specificity of the Fn3s. Binding epitopes were determined by fluorescence activated cell sorting of a single-point mutant yeast library of FcγR and competition studies. To confirm the biological activity of the Fn3 clones, a tumor-targeting single-chain variable fragment (scFv) was fused to each Fn3 clone. Each scFv-Fn3 construct was antigen specific and bound specifically to the FcγR that it was designed to target. The murine colon carcinoma cell line MC38 stably transfected with carcinoembryonic antigen (CEA) was used for our studies. Tumor cells were pre-incubated with scFv-Fn3 constructs targeting CEA and then combined with thioglycollate- induced peritoneal macrophages pre-treated with interferon gamma. The phagocytic activity of the macrophages was measured using flow cytometry. Pre-incubation with scFv-Fn3 constructs specific for FcγRI or FcγRIV significantly increased phagocytosis compared with pre-incubations with the control scFv-Fn3 construct. Pre-incubation with a combination of both the FcγRI- and FcγRIV-specific constructs resulted in a phagocytosis level close to that of murine IgG2a, which can interact with all of the FcγRs. These Fn3 tools will allow us to conduct future studies on the immune response that is generated by triggering individual FcγRs in other in vitro assays and in vivo models of cancer. This work was supported by a Sanofi Aventis Biomedical Innovation Award and the NIH/NIGMS Biotechnology Training Program. Citation Format: Tiffany F. Chen, Kevin Li, K. Dane Wittrup. Fibronectin domains engineered for specificity to individual murine Fc gamma receptors modulate tumoricidal activity of immune cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1250. doi:10.1158/1538-7445.AM2013-1250

Expression of inhibitory Fc receptor (FcγRIIB) is a marker of poor response to rituximab monotherapy in follicular lymphoma

BackgroundFcγRIIB promotes rituximab internalisation on various B-cell targets, including in follicular lymphoma, which may lead to reduced efficacy. We analysed diagnostic tumour samples from the SAKK 35/98 trial, which has follow-up data of nearly 10 years to determine the relation of FcγRIIB expression with responses and clinical outcomes after rituximab monotherapy in follicular lymphoma. MethodsAvailable archived tissue samples were stained with an anti-human FcγRIIB antibody. Positive samples were graded into negative/low intensity staining (n=116) or medium/high staining (n=13) by a histopathologist masked to clinical outcomes. Failure-free survival (FFS) was defined as time from first rituximab infusion until failure to achieve complete/partial response at week 12, progression, relapse, a second cancer, or death from any cause. Objective response rate (ORR) was associated with intensity staining levels with Fisher's exact test. All time-to-event endpoints were evaluated with the Kaplan-Meier method; groups were compared with the log-rank test. Hazard ratio (HR) was assessed with Cox proportional hazards models. FindingsPatients expressing medium/high levels of FcγRIIB were less likely to respond to rituximab than were those with negative/low levels (ORR 23·1% [95% CI 7·5–50·9] vs 58·6% [49·5–67·2], p=0·02). FFS was higher in the negative/low staining group than in the medium/high staining group (median 8·3 months [95% CI 2·8–13·4, IQR 2·76–28·5] vs 2·8 [not calculable, 2·76–2·76], p=0·002; HR 0·43 [95% CI 0·23–0·78]). There was a non-significant trend towards better overall survival in the low/negative group compared with the medium/high group (median 140·0 months vs 50·0, p=0·13; HR 0·56 [95% CI 0·26–1·20]). InterpretationElevated FcγRIIB expression level is associated with poor response to rituximab in patients with follicular lymphoma. This group may show better results with non-internalising type II antibodies, a hypothesis for validation in future prospective clinical trials. FundingCancer Research UK.

Expression of Inhibitory Fc Receptor (FcγRIIB) Is a Marker of Poor Response to Rituximab Monotherapy in Follicular Lymphoma (FL).

AbstractAbstract 2747 Background:Single-agent immunotherapy with rituximab is a viable treatment option for low risk FL, with limited toxicity and a long duration of response in some patient subsets. We have previously shown that high expression of FcγRIIB promotes rituximab internalisation on various B cell targets, including FL (Blood 2011 118:2530–2540), something not seen with type II anti-CD20 antibodies. The SAKK 35/98 trial examined rituximab monotherapy in FL and now has long-term follow-up data of almost 10 years (JCO 2010 28:4480–4484). We analysed diagnostic tumour samples from this trial to determine the relationship of FcγRIIB expression to responses and clinical outcomes after rituximab treatment in FL. Methods:202 patients (pts) with newly diagnosed or relapsed FL received induction treatment with rituximab 375 mg/m2 weekly for 4 weeks. Pts with stable or responding disease at week 12 were randomized into 2 groups: no further treatment or prolonged treatment with single infusions of rituximab 375 mg/m2 at weeks 12, 20, 28 and 36. Archived tissue samples from 135 evaluable pts were stained using an anti-human FcγRIIB antibody (clone EP888Y, Abcam) at a dilution of 1:3000 on a Dako autostainer. The samples were pretreated with the Dako EnVisionFLEX target retrieval solution high pH and detection using the Dako AS-Link 48 with Dako EnVision flex plus detection kit. Positive samples were graded into negative/low intensity staining (n=120) versus medium/high (n=13) by an expert lymphoma histopathologist blinded to the clinical outcomes. Data from 2 slides and response at week 12 data for 4 pts were unavailable (1 of whom also has missing slide data), resulting in 130 pts available for analysis. Failure-free survival (FFS) was defined as time from registration until failure to achieve complete/partial response at week 12, progression, relapse, a second cancer or death from any cause. Objective response rate (ORR) was associated with intensity staining levels using Fisher’s exact test. All time-to-event endpoints were evaluated using the Kaplan-Meier method; groups were compared using the log-rank test. The hazard ratio (HR) was assessed using Cox proportional hazards models. Results:Registered and randomised pts had very similar baseline characteristics; previously untreated pts had slightly more favourable characteristics but were balanced between the 2 treatment arms. Pts expressing medium/high levels of FcγRIIB were less likely to respond to rituximab by week 12 (ORR 58.1% vs 23.1%, Fisher’s exact test, p=0. 02), a finding independent of prior therapy. For FFS, there was a statistically significant difference (p=0.001; HR=0.42; 95% confidence interval (C.I.): 0.23–0.77) between the negative/low staining group (median: 21.4 months; 95% C.I.: 7.0–34.2) and the medium/high staining group (median: 7.0 months; 95% C.I.: Not calculable). The interaction between staining levels and randomised treatment groups for FFS was not statistically significant. There was a non-significant trend towards better overall survival in the low/negative group (median: 140.0 vs 50.0 months; p=0.15; HR=0.57; 95% C.I.: 0.27–1.23); however the event rate was lower (36.8% vs 61.5%). Conclusion:Elevated FcγRIIB expression level is associated with poor response to rituximab in pts with FL. This group may show better results with non-internalising type II antibodies, a hypothesis for validation in future prospective clinical trials. Disclosures:Ghielmini:Roche: Honoraria, Speakers Bureau. Johnson:Roche: Honoraria.

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.

157P - Rituximab Induced Internalisation of B-Cells CD20 Receptor is Independent of the Inhibitory FC Receptor (CD32B) Intracellular Cell Signalling

ABSTRACT The anti-CD20 monoclonal antibody, rituximab, has improved treatment outcomes in B-cell malignancies. However, several B-cell lymphomas either do not respond to rituximab or develop resistance. Internalisation of rituximab may be partly responsible for this resistance. We recently showed that the level of the inhibitory Fc receptor (CD32B) at the cell surface controls the rate of rituximab internalisation. However, the precise mechanism involved has not been elucidated. Different isoforms of CD32B exist (B1 and B2), only one of which (B2) has previously been associated with an ability to internalise after engagement of immune complexes. The B1 form in contrast contains an additional intracellular region that makes it more resistant to internalisation. We therefore investigated the role of the two different CD32B isoforms (B1 and B2) and the associated intracellular tail on rituximab internalisation. CD32B1 and CD32B2 isoforms were stably transfected into CD32B-ve mouse IIA1.6 and human Ramos lymphoma cells, and flow cytometry was then used to determine the relative rates of CD32B and CD20 internalisation. Additionally, we generated mutant versions of the CD32B receptors, including those lacking the entire cytoplasmic domain to assess the importance of intracellular signalling. In contrast to expectations both B1 and B2 CD32B isoforms were downregulated upon engagement with CD32B mAb as a surrogate for immune complexes, although in agreement with earlier reports the CD32B2 isoform internalised more extensively. However, the rate of rituximab internalisation occurred equally with both isoforms and was dependent on relative expression of CD32B and the CD20:CD32B ratio at the cell surface rather than any specific activity imparted by the CD32 intracellular domain. These studies suggest that the intracellular part of CD32B is redundant for rituximab-induced CD20 internalisation and imply that internalisation is augmented by CD32B through its physical ability to bind the Fc region of rituximab at the cell surface. Disclosure All authors have declared no conflicts of interest.

Development and characterisation of monoclonal antibodies specific for the murine inhibitory FcγRIIB (CD32B)

Fc receptors (FcRs) play a key role in regulating and coordinating responses from both innate and adaptive arms of the immune system. The inhibitory Fc gamma receptor II (FcγRIIB; CD32) is central to this regulation with FcγRIIB(-/-) mice demonstrating augmented responses to mAb immunotherapy, elevated incidence and severity of auto-immunity, and increased response to mAb-mediated cancer therapy. To date, these observations have remained unexploited therapeutically, partly through a lack of specific mAb reagents capable of exclusively binding mouse FcγRIIB. Thus almost all of the FcγRIIB-binding mAb currently available, such as 2.4G2, also bind FcγRIII (CD16), and polyclonal reagents have limited availability and are of unproven specificity and avidity, making in vivo manipulation of FcγRIIB impossible. Following an extensive immunisation protocol using FcγRIIB(-/-) mice, we recently produced three unique mAb that are suitable for this purpose. Here we characterise these novel reagents and demonstrate that they fall into two distinct categories; those which cause phosphorylation and subsequent activation of FcγRIIB (agonistic) and those that block receptor phosphorylation (antagonistic). These two types of mAb exhibit different characteristics in a range of biochemical, cellular, and functional assays relevant to FcγRIIB activity and mAb therapy.