Cross-linking Of Fc Receptors Research Articles

Interleukin-3-dependent potentiation of IgE responsiveness in mouse basophils.

Basophils produce interleukins (IL)-4 in response to various stimuli and may contribute to type 2 immune responses to various infections and allergens. We found that resting basophils freshly isolated from mice produce IL-4 in response to IL-3 but not to high-affinity Fc receptor (FcεRI) cross-linking (CL), yet both required the immunoreceptor tyrosine-based activation motif (ITAM) containing adaptor Fc receptor γ-chain (FcRγ), while basophils activated in vitro by IL-3 become responsive to FcεRI CL. Acquisition of responsiveness to FcεRI CL occurred upon infection with Trichinella spiralis or administration of superantigen. Because cultured basophils return to a quiescent state upon starvation with IL-3 with surface FcεRI levels unchanged, this acquisition is reversible and probably reflects intracellular events requiring protein synthesis. Interestingly, similar activation-associated acquisition was observed for responsiveness to other stimuli, including CD200R3 CL, which is known to signal via DAP-12, and the allergen protease papain. This acquisition of responsiveness to FcεRI CL was inhibited by Jak inhibitor. Thus, the IL-3 signal bifurcates downstream of Jak, into two distinct pathway, one leading to IL-4 production and the other to render basophils competent to respond to stimuli dependent on ITAM-containing adaptors DAP12 and FcRγ for IL-4 production.

IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype.

Simple SummaryWhen activated by tumour antigen-specific IgEs, monocytes may contribute to the restriction of cancer growth in animal models of cancer. In this study, we investigated the effects of IgE stimulation on the activation state of human monocytes from healthy subjects and from patients with cancer. Cross-linking of cognate Fc receptors by IgE on human monocytes potentiated: (a) upregulation of activatory and down regulation of regulatory monocyte cell surface markers; (b) phosphorylation of intracellular protein kinases in monocytes previously described to be downstream of the mast cell and basophil FcεRI signalling pathway; (c) ovarian cancer patient monocyte-mediated cytotoxic killing of tumour cells and release of pro-inflammatory mediators, potentially associated with favourable patient survival. IgE can therefore activate human monocytes to acquire a pro-inflammatory phenotype capable of mediating effector functions against tumour cells. This may contribute to the mechanism of cancer immunotherapy using IgE antibodies.IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNFα, MCP-1, IL-10, CXCL-10, IL-1β, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.

ICOS agonism by JTX-2011 (vopratelimab) requires initial T cell priming and Fc cross-linking for optimal T cell activation and anti-tumor immunity in preclinical models.

Immunotherapy checkpoint inhibitors, such as antibodies targeting PD-1 and CTLA-4, have demonstrated the potential of harnessing the immune system to treat cancer. However, despite encouraging results particularly with respect to survival, only a minority of patients benefit from these therapies. In clinical studies aimed at understanding changes in the immune system following immunotherapy treatment, ICOS (Inducible T cell CO-Stimulator) was shown to be significantly up-regulated on CD4+ T cells and this was associated with clinical activity, indicating that ICOS stimulatory activity may be beneficial in the treatment of solid tumors. In this report, we describe the generation of specific, species cross-reactive, agonist antibodies to ICOS, including the humanized clinical candidate, JTX-2011 (vopratelimab). Preclinical studies suggest that the ICOS stimulating antibodies require Fc receptor cross-linking for optimal agonistic activity. Notably, the ICOS antibodies do not exhibit superagonist properties but rather require T cell receptor (TCR)-mediated upregulation of ICOS for agonist activity. Treatment with the ICOS antibodies results in robust anti-tumor benefit and long-term protection in preclinical syngeneic mouse tumor models. Additional benefit is observed when the ICOS antibodies are administered in combination with anti-PD-1 and anti-CTLA-4 therapies. Based on the preclinical data, JTX-2011 is currently being developed in the clinical setting for the treatment of solid tumors.

CD40 Enhances Type I Interferon Responses Downstream of CD47 Blockade, Bridging Innate and Adaptive Immunity.

Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancers by potentiating antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing the phagocytosis of tumor cells by macrophages and enhancing the cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; both of these processes are potentiated by CD40 signaling. Here we generated a novel, two-sided fusion protein incorporating the extracellular domains of SIRPα and CD40L, adjoined by a central Fc domain, termed SIRPα-Fc-CD40L. SIRPα-Fc-CD40L bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor cross-linking. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Murine SIRPα-Fc-CD40L outperformed CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and synergized with immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated a type I interferon response in macrophages and potentiated the activity of ADCP-competent targeted antibodies both in vitro and in vivo These data illustrated that whereas CD47/SIRPα inhibition could potentiate tumor cell phagocytosis, CD40-mediated activation of a type I interferon response provided a bridge between macrophage- and T-cell-mediated immunity that significantly enhanced durable tumor control and rejection.

Pathogenic Antibodies in Heparin-Induced Thrombocytopenia Specifically Target an Immunodominant Region on Platelet Factor 4

Introduction: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that occurs when heparin binds to platelet factor 4 (PF4) forming immunogenic complexes. Anti-PF4/heparin IgG antibodies bind PF4/heparin complexes, leading to cross-linking of FcγRIIa receptors on platelets and FcγRI on monocytes, resulting in platelet activation, thrombocytopenia, and thrombosis. The current diagnostic challenge is that the majority of patients suspected of HIT yield false-positive results in immunoassays, since up to 50% of patients will make anti-PF4/heparin antibodies but will not develop HIT. The antibody response in HIT patients is polyclonal, making it difficult to identify a common pathogenic epitope. The disparities between anti-PF4/heparin antibodies that activate platelets (pathogenic HIT antibodies) and those that do not (non-pathogenic anti-PF4/heparin antibodies) present a significant challenge in diagnosing HIT. The objective of this study was to map and characterize the critical immunodominant region on PF4 for the binding of pathogenic antibodies in confirmed HIT patients. Methods: We used sera with anti-PF4/heparin antibodies from patients with confirmed HIT (n=10). Post-cardiopulmonary bypass patients (CPB; n=10) and healthy individuals (n=10) were used as controls. Confirmed HIT patients met clinical criteria (4Ts ≥ 4) and tested positive in both the anti-PF4 IgG/A/M immunoassay (OD > 0.4; range 2.33 - 3.90) and in the serotonin release assay (SRA release > 20%; range 88-100%). CPB patients all received heparin but did not develop HIT, tested positive in the anti-PF4 IgG/A/M immunoassay (OD > 0.4; range 0.42 - 2.73), and tested negative in the SRA (SRA release < 20%; range 0-18%). We previously used alanine scanning mutagenesis and identified 30 amino acids that were on the surface of PF4 and were likely a part of the region essential for the binding of pathogenic HIT antibodies. From those results, we used the panel of 30 PF4 mutants and tested their ability to bind to HIT, CPB, and healthy control sera. Loss of binding to PF4 mutants was applied to in-silico structural analysis to determine binding regions specific for pathogenic and non-pathogenic antibodies. We also determined binding affinities of pathogenic and non-pathogenic anti-PF4/heparin antibodies using biolayer interferometry (BLI). Results: When 30 PF4 mutants were used to test the effect of the amino acid changes on the binding of HIT and CPB patient sera, an average of 8 different PF4 mutants resulted in more than 35% loss of binding to confirmed HIT sera when compared to wild-type PF4. None of the 30 PF4 mutants resulted in more than 35% loss of binding to CPB sera. Structural analysis demonstrated that the amino acids of PF4 that significantly affected the binding of HIT sera, but not CPB sera, were clustered to a specific region on PF4, similar to the region of KKO, but with varying epitopes. Using BLI, anti-PF4/heparin antibodies of confirmed HIT patients had a stronger binding response to PF4 and PF4/heparin than that of CPB patients and healthy controls. Overall, we were able to show a significant difference between confirmed HIT sera and the false-positive antibodies of CPB patients that did not develop HIT (P < 0.01). Conclusion: This work shows that among the polyclonal response in HIT, pathogenic HIT antibodies must bind to the critical immunodominant region on PF4 with high affinity. This ensures the proper spatial configuration of the antibodies for Fc-receptor cross-linking, platelet activation, and subsequently HIT. This study has implications for the development of novel epitope-targeted diagnostic and therapeutic approaches for HIT. Disclosures Arnold: Novartis: Honoraria, Research Funding; Rigel: Consultancy, Research Funding; Principia: Consultancy; Bristol-Myers Squibb: Research Funding.

Monomer hapten and hapten-specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms.

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

Simultaneous blockade of immune checkpoint molecules and co-stimulation of the TNF receptor superfamily (TNFRSF) is predicted to improve overall survival in human cancer. TNFRSF co-stimulation depends upon coordinated antigen recognition through the T cell receptor followed by homotrimerization of the TNFRSF, and is most effective when these functions occur simultaneously. To address this mechanism, we developed a two-sided human fusion protein incorporating the extracellular domains (ECD) of PD-1 and OX40L, adjoined by a central Fc domain, termed PD1-Fc-OX40L. The PD-1 end of the fusion protein binds PD-L1 and PD-L2 with affinities of 2.08 and 1.76 nM, respectively, and the OX40L end binds OX40 with an affinity of 246 pM. High binding affinity on both sides of the construct translated to potent stimulation of OX40 signaling and PD1:PD-L1/L2 blockade, in multiple in vitro assays, including improved potency as compared to pembrolizumab, nivolumab, tavolixizumab and combinations of those antibodies. Furthermore, when activated human T cells were co-cultured with PD-L1 positive human tumor cells, PD1-Fc-OX40L was observed to concentrate to the immune synapse, which enhanced proliferation of T cells and production of IL-2, IFNγ and TNFα, and led to efficient killing of tumor cells. The therapeutic activity of PD1-Fc-OX40L in established murine tumors was significantly superior to either PD1 blocking, OX40 agonist, or combination antibody therapy; and required CD4+ T cells for maximum response. Importantly, all agonist functions of PD1-Fc-OX40L are independent of Fc receptor cross-linking. Collectively, these data demonstrate a highly potent fusion protein that is part of a platform, capable of providing checkpoint blockade and TNFRSF costimulation in a single molecule, which uniquely localizes TNFRSF costimulation to checkpoint ligand positive tumor cells.

Pentraxins and Fc Receptor-Mediated Immune Responses.

C-reactive protein (CRP) is a member of the pentraxin family of proteins. These proteins are highly conserved over the course of evolution being present as far back as 250 million years ago. Mammalian pentraxins are characterized by the presence of five identical non-covalently linked subunits. Each subunit has a structurally conserved site for calcium-dependent ligand binding. The biological activities of the pentraxins established over many years include the ability to mediate opsonization for phagocytosis and complement activation. Pentraxins have an important role in protection from infection from pathogenic bacteria, and regulation of the inflammatory response. It was recognized early on that some of these functions are mediated by activation of the classical complement pathway through C1q. However, experimental evidence suggested that cellular receptors for pentraxins also play a role in phagocytosis. More recent experimental evidence indicates a direct link between pentraxins and Fc receptors. The Fc receptors were first identified as the major receptors for immunoglobulins. The avidity of the interaction between IgG complexes and Fc receptors is greatly enhanced when multivalent ligands interact with the IgG binding sites and activation of signaling pathways requires Fc receptor crosslinking. Human pentraxins bind and activate human and mouse IgG receptors, FcγRI and FcγRII, and the human IgA receptor, FcαRI. The affinities of the interactions between Fc receptors and pentraxins in solution and on cell surfaces are similar to antibody binding to low affinity Fc receptors. Crystallographic and mutagenesis studies have defined the structural features of these interactions and determined the stoichiometry of binding as one-to-one. Pentraxin aggregation or binding to multivalent ligands increases the avidity of binding and results in activation of these receptors for phagocytosis and cytokine synthesis. This review will discuss the structural and functional characteristics of pentraxin Fc receptor interactions and their implications for host defense and inflammation.

Abstract 5559: Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy

Abstract Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely due to the fact that these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. OX40L-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the fact that the ECDs are in opposite orientation. Here we report the generation of a two-sided fusion protein incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain. Initial confirmation of the structural integrity of TIM3-Fc-OX40L was performed using functional ELISA assays, wherein simultaneous binding to GAL9 and OX40 was observed. A potential advantage of utilizing the intact ECD of TIM3 is the ability to bind all candidate TIM3 ligands, and the TIM3 end of the fusion protein binds both GAL9 and phosphatidylserine (PS) on the surface of human tumor cells. The OX40L end of the fusion protein binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in cells engineered to overexpress OX40 and an NFκB-luciferase reporter in the absence of Fc receptor cross-linking. Additionally, the TIM3-Fc-OX40L ARC added to primary human PBMCs along with the super-antigen Staphylococcal enterotoxin B, induced secretion of the cytokines IL2 and TNFα. In vitro human T cell/tumor cell co-culture studies indicated that TIM3-Fc-OX40L stimulated enhanced T cell mediated killing of GAL9 positive human tumor cells as compared to OX40L-Fc controls. In vivo, TIM3-Fc-OX40L stimulated significant expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells and vaccinated with Ova/Alum. Time-lapse high content live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L functionally tethering T cells to tumor cells and eliciting cytotoxic activity (tumor killing). Finally, the therapeutic activity of TIM3-Fc-OX40L in established murine MC38, B16.F10 and CT26 tumors was significantly superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. Importantly, a pharmacodynamic biomarker of tumor rejection was identified by the elevation in serum cytokines post-treatment. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is uniquely advantageous because the construct links those two signals in the same microenvironmental context, at the time in which T cells are engaging cognate tumor antigen. Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5559.

Abstract 5567: Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy

Abstract Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely because these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. SIRPα-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of SIRPα (CD172a) and the ECD of CD40L, adjoined by a central Fc domain. The SIRPα end of the ARC binds immobilized CD47 at 3.59 nM affinity and binds CD47 on the surface of human tumor cells both in vitro and in vivo, but does not bind human platelets or RBCs. Further, no hemolytic activity was observed with the human SIRPα-Fc-CD40L ARC in vitro as compared to CD47 mAbs. The CD40L end of the ARC binds immobilized CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates functional activity (independent of Fc receptor cross-linking) in NFκB-luciferase reporter cells (CD40 driven activation of NFκB). Addition of SIRPα-Fc-CD40L to an ex vivo super-antigen (SEB) cytokine release assay stimulated secretion of IL2 and TNFα from human PBMC. Furthermore, time-lapse high content live cell imaging has shown that when activated human macrophages were co-cultured with CD47 positive human tumor cells, SIRPα -Fc-CD40L was shown to enhance phagocytosis. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to either CD47 blocking antibody, CD40 agonist antibody or combination antibody therapy. Notably, the superior anti-tumor immunity observed with SIRPα-Fc-CD40L was not accompanied by significant mucosal inflammation and weight loss stimulated by murine CD40 agonist antibodies. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule. Signal replacement of CD47 by CD40L may uniquely poise macrophages in the tumor microenvironment for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis. Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5567.

Agonist Redirected Checkpoint (ARC), SIRPα-Fc-CD40L, for Cancer Immunotherapy

Abstract Combination immunotherapy with bispecific abs, linked scFv’s or T cell engagers have not shown that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule, likely due to a loss of target avidity. Fusion proteins incorporating the extracellular domain of type I membrane proteins (eg. Enbrel) or type II membrane proteins (eg. SIRPα-Fc), linked via an antibody Fc domain, are both functional despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of SIRPα (CD172a) and the ECD of CD40L, adjoined by a central Fc domain. The SIRPα end of the ARC binds CD47 at 3.59 nM affinity, but does not cause hemolysis, compared to CD47 mAbs. The CD40L end binds CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates NFκB-functional activity (independent of Fc receptor cross-linking) and IL2 and TNFα secretion in an ex vivo super-antigen (SEB) assay. Furthermore, live cell imaging shows that SIRPα-Fc-CD40L enhanced phagocytosis of tumor cells by primary macrophages. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to either CD47 blocking antibody, CD40 agonist antibody or their combination. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed. These data demonstrate feasibility of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which may uniquely poise macrophages in the TME for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis.

Agonist Redirected Checkpoint (ARC), TIM3-Fc-OX40L, for Cancer Immunotherapy

Abstract Combination immunotherapy with bispecific abs, linked scFv’s or T cell engagers have not shown that both checkpoint blockade and TNFR activation can be achieved with a single molecule, likely due to a loss of target avidity. Fusion proteins incorporating the extracellular domain of type I membrane proteins (eg. Enbrel) or type II membrane proteins (eg. OX40L-Fc), linked via an antibody Fc domain, are both functional despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain. The TIM3 end of the ARC binds GAL9 and phosphatidylserine (PS) in vitro and in vivo, and OX40L binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in the absence of Fc receptor cross-linking, stimulates IL2/TNFa secretion with the superantigen SEB, and increases the in vitro killing of GAL9+ tumor cells. In vivo, TIM3-Fc-OX40L stimulated expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells (+ova/alum). Live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L tethering T cells to tumor cells and eliciting cytotoxic activity (tumor killing). Finally, the therapeutic activity of TIM3-Fc-OX40L in established murine MC38, B16.F10 and CT26 tumors was superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. These data demonstrate feasibility of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is unique with this molecule compared to antibody approaches, since all TIM3 ligands can be targeted simultaneously.

Blocking dephosphorylation at Serine 120 residue in t-SNARE SNAP-23 leads to massive inhibition in exocytosis from mast cells

Mast cells (MCs) respond to allergen challenge by release of pre-stored inflammatory mediators from their secretory granules, on cross-linking of Fc(epsilon) receptor I (Fc(epsilon)RI) receptors. The target-SNARE (t-SNARE) SNAP-23 has been shown to play an important role in MC exocytosis and undergoes transient phosphorylation at Serine 95 (S95) and Serine 120 (S120), concomitant with mediator release. During current study we explored the importance of transient nature of phosphorylation at S120 in MC exocytosis. A phosphomimetic SNAP-23-S120D mutant of rodent SNAP-23 was cloned into EGFP vector and its effect on the exocytosis and the mechanisms involved was studied in RBL-2H3 MC line. Secretion reporter assay with SNAP-23-S120D transfected MCs revealed a very significant inhibition of exocytosis, and reduced ruffling in response to Fc(epsilon)RI cross-linking. Further, the effect of this mutation on localization of SNAP-23 in MCs was studied. Immunofluorescence microscopy studies and membrane-cytosol fractionation of green fluorescent protein-tagged SNAP- 23-S120D (GFP-SNAP-23-S120D) transfected MCs showed that a large proportion of GFP-SNAP-23-S120D was residing in cytosol unlike wild-type SNAP-23, in resting and activated MCs and even the membrane associated portion was on internal lysosomal membranes than plasma membrane. These studies imply that dephosphorylation of S120 is important for SNAP-23 membrane association dynamics and subsequently MC degranulation.

Antibody-Dependent Cellular Cytotoxicity Responses to Seasonal Influenza Vaccination in Older Adults.

Older adults are at high risk of influenza disease, but generally respond poorly to vaccination. Antibody-dependent cellular cytotoxicity (ADCC) may be an important component of protection against influenza infection. An improved understanding of the ADCC response to influenza vaccination in older adults is required. We studied sera samples from 3 groups of subjects aged ≥65 years (n = 16-17/group) receiving the 2008/2009 seasonal trivalent influenza vaccine (TIV). Subjects had minimal pre-existing hemagglutination inhibiting (HAI) antibodies and TIV induced either no, low, or high HAI responses. Serum ADCC activity was analyzed using Fc receptor cross-linking, NK cell activation, and influenza-infected cell killing. Most subjects from TIV nonresponder, low responder, and high responder groups had detectable ADCC antibodies prevaccination, but baseline ADCC was not predictive of HAI vaccine responsiveness. Interestingly, ADCC and HAI responses tracked closely across all groups, against all 3 TIV hemagglutinins, and in all ADCC assays tested. Older adults commonly have pre-existing ADCC antibodies in the absence of high HAI titers to circulating influenza strains. In older vaccinees, ADCC response mirrored HAI antibodies and was readily detectable despite high postvaccination HAI titers. Alternate measures of vaccine responsiveness and improved vaccinations in this at-risk group are needed.

Abstract 4693: Fc effector bioassays enable faster and quantitative measurement of ADCC and ADCP mechanisms of action

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are recognized as important mechanisms of action (MOA) of therapeutic antibodies. Primary peripheral blood mononuclear cells (PBMCs) are routinely used in traditional bioassays to measure potency, stability of antibody drugs in ADCC and ADCP. However these methods are labor intensive and highly variable. Here we report the development of a luciferase-based reporter assay that measures activation of effector cells via cross-linking of Fc receptors with target cell-bound antibodies. We will discuss functional cell-based Fc effector reporter bioassays developed to measure Human FcgRIIIa (V158 and F158 variants), Human FcgRIIa (H131 and R131 variants), Human FcgRI as well as Mouse FcgRIV and FcgRIII. Compared to primary cell-based assays these reporter bioassays are less variable, easier to use and provide more consistent analysis of the important MOAs that are critical to for drug development programs. In qualification studies, performed in accordance with ICH guidelines for antibody screening and characterization, we tested the bioassays for specificity, accuracy, precision and linearity. Citation Format: Zhi-Jie Jey Cheng, Rich Moravec, Aileen Paguio, Denise Garvin, Gopal B. Krishnan, Frank Fan, Mei Cong. Fc effector bioassays enable faster and quantitative measurement of ADCC and ADCP mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2017-4693

Selective activation of anti-CD73 mechanisms in control of primary tumors and metastases

ABSTRACTThe emerging role for CD73 in driving cancer growth and metastasis has presented opportunities to develop anti-CD73 monoclonal antibodies (mAbs) in the treatment of human cancers. Blockade of CD73 by antagonistic CD73 mAbs ameliorates tumor growth and metastasis via the inhibition of enzymatic and non-enzymatic CD73 pathways. In this study, we investigated whether Fc-receptor cross-linking represented a non-redundant mechanism by which anti-CD73 mAbs exert potent suppression of solid tumors and metastases. We engineered four anti-CD73 mAbs, each different in their ability to modulate CD73 enzymatic function and bind Fc receptors. mAbs recognizing a similar epitope of CD73 (CD73–04, TY/23 and 2C5) displayed the greatest antitumor activity. Importantly, we observed that the optimal control of metastasis by anti-CD73 mAbs involved primarily Fc receptor engagement, while suppression of solid tumors required both, enzyme inhibition and activation of Fc receptors. Engagement of Fc-receptors was also essential for optimal anti-metastatic effect in combination with either A2AR inhibitor or anti-PD-1 mAb treatment. The control of experimental metastases relied on the activation of host NK cells and IFNγ, while NK cells, CD8+ T cells and IFNγ were needed for effective antitumor effect in the spontaneous metastases model. These observations advance our understanding of the enzymatic and non-enzymatic functions of anti-CD73 mAbs in solid tumors and metastases. Altogether, these findings will greatly assist in the design of anti-CD73 mAbs to be used as either single agents or in combination with other immunotherapeutic molecules or targeted therapies.

Antibody Functional Assays as Measures of Fc Receptor-Mediated Immunity to HIV - New Technologies and their Impact on the HIV Vaccine Field.

Background:There is now intense interest in the role of HIV-specific antibodies and the engagement of FcγR functions in the control and prevention of HIV infection. The analyses of the RV144 vaccine trial, natural progression cohorts, and macaque models all point to a role for Fc-dependent effector functions, such as cytotoxicity (ADCC) or phagocytosis (ADCP), in the control of HIV. However, reliable assays that can be reproducibly used across different laboratories to measure Fc-dependent functions, such as antibody dependent cellular cytotoxicity (ADCC) are limited.Method:This brief review highlights the importance of Fc properties for immunity to HIV, particular-ly via FcγR diversity and function. We discuss assays used to study FcR mediated functions of HIV-specific Ab, including our recently developed novel cell-free ELISA using homo-dimeric FcγR ecto-domains to detect functionally relevant viral antigen-specific antibodies.Results:The binding of these dimeric FcγR ectodomains, to closely spaced pairs of IgG Fc, mimics the engagement and cross-linking of Fc receptors by IgG opsonized virions or infected cells as the es-sential prerequisite to the induction of Ab-dependent effector functions. The dimeric FcγR ELISA reli-ably correlates with ADCC in patient responses to influenza. The assay is amenable to high throughput and could be standardized across laboratories.Conclusion:We propose the assay has broader implications for the evaluation of the quality of anti-body responses in viral infections and for the rapid evaluation of responses in vaccine development campaigns for HIV and other viral infections.

Revisiting the Immunoglobulin Intramolecular Signaling Hypothesis

A generation ago, the immunoglobulin intramolecular signaling, or allosteric, hypothesis was abandoned in favor of the associative hypothesis, which posited that Fc receptor crosslinking produced the increased affinity of antigen-antibody complexes. This essay argues that there is sufficient evidence to resuscitate the allosteric hypothesis, at least for some antibodies.

Abstract 2668: RG7116, a novel humanized anti-HER3 antibody with superior preclinical in vitro and in vivo efficacy in combination with, everolimus and other anti-cancer agents

Abstract HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. HER3 is a kinase dead receptor, but by forming heterodimers with other HER family receptors, HER3 works as amplifier for PI3 kinase driven tumorigenesis. It has been reported that tumors treated with EGFR-, HER2-, cMET-or mTOR targeted therapies can escape via HER3 activation or upregulation. HER3 is expressed in a large variety of tumors for example in non-small cell lung cancer (NSCLC), head and neck, colorectal, gastric, pancreatic, breast, ovarian, thyroid and prostate cancer. Anti-HER3 antibodies can work via various mechanisms including: (1) blocking ligand (HRGs) binding to the receptor, (2) blocking heterodimerization with other HER family members (HER1, 2 and 4), (3) downregulation of the receptor from the cell surface, and (4) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). The first three mechanisms lead to inhibition of HER3 phosphorylation and downstream signaling thereby resulting in tumor cell growth inhibition, while ADCC is a mechanism of direct target cell killing triggered by cross-linking of Fc receptors on immune effector cells (e.g. NK cells, macrophages). RG7116 is a novel humanized and glycoengineered IgG1 antibody currently in clinical development, that binds to HER3 with high affinity. This antibody prevents ligand binding and receptor heterodimerization, thereby blocking receptor phosphorylation. In various tumor xenograft models monotherapy treatment with this antibody leads to substantial tumor growth inhibition. Only in a few cases monotherapy treatment resulted in complete tumor remission. Most of the models initially show convincing efficacy, but tumors regrow after a while. Combination therapy with other HER targeted therapies such as pertuzumab, cetuximab or erlotinib, lead to complete remission in preclinical models. The combination of RG7116 with downstream signaling inhibitors like: everolimus (mTOR) andand other anti-cancer agents leads to increased efficacy. Citation Format: Birgit Bossenmaier, Thomas Friess, Martin Weisser, Stefanie Lechner, Esther Abraham, Monika Hoch, Christian Mirschberger. RG7116, a novel humanized anti-HER3 antibody with superior preclinical in vitro and in vivo efficacy in combination with, everolimus and other anti-cancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2668. doi:10.1158/1538-7445.AM2014-2668

A combined experimental and computational study of Vam3, a derivative of resveratrol, and Syk interaction.

Spleen tyrosine kinase (Syk) plays an indispensable role through preliminary extracellular antigen-induced crosslinking of Fc receptor (FcR) in the pathogenesis of autoimmune disorders, such as rheumatoid arthritis. In this study, we identify Vam3, a dimeric derivative of resveratrol isolated from grapes, as an ATP-competitive inhibitor of Syk with an IC50 of 62.95 nM in an in vitro kinase assay. Moreover, docking and molecular dynamics simulation approaches were performed to get more detailed information about the binding mode of Vam3 and Syk. The results show that 11b-OH on ring-C and 4b-OH on ring-D could form two hydrogen bonds with Glu449 and Phe382 of Syk, respectively. In addition, arene-cation interaction between ring-D of Vam3 and Lys402 of Syk was also observed. These results indicate that ring-C and D play an essential role in Vam3–Syk interaction. Our studies may be helpful in the structural optimization of Vam3, and also aid the design of novel Syk inhibitors in the future.