West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.

Monoclonal antibodies (mAb) are widely used in the treatment of non-Hodgkin's lymphoma and autoimmune diseases. Although the mechanism of action in vivo is not always known, the therapeutic activity of several approved mAbs depends on the binding of the Fc; regions to low-affinity Fc; receptors (Fc;R) expressed on effector cells. We did functional genetic screens to identify IgG1 Fc domains with improved binding to the low-affinity activating Fc receptor CD16A (Fc;RIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (Fc;RIIB). Identification of new amino acid residues important for Fc;R binding guided the construction of an Fc domain that showed a dramatically enhanced CD16A binding and greater than a 100-fold improvement in antibody-dependent cell-mediated cytotoxicity. In a xenograft murine model of B-cell malignancy, the greatest enhancement of an Fc-optimized anti-human B-cell mAb was accounted for by improved binding to Fc;RIV, a unique mouse activating Fc;R that is expressed by monocytes and macrophages but not natural killer (NK) cells, consistent with experimental and clinical data suggesting that mononuclear phagocytes, effector cells expressing both activating and inhibitory Fc;R, are critical mediators of B-cell depletion in vivo. By using mice transgenic for human CD16A, enhanced survival was observed due to expression of CD16A-158 phe on monocytes and macrophages as well as on NK cells in these mice. The design of new generations of improved antibodies for immunotherapy should aim at Fc optimization to increase the engagement of activating Fc;R present on the surface of tumor-infiltrating effector cell populations. [Cancer Res 2007;67(18):8882-90]

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The neurofibromatosis 2 tumor suppressor protein schwannomin/merlin is commonly mutated in schwannomas and meningiomas. Schwannomin, a member of the 4.1 family of proteins, which are known to link the cytoskeleton to the plasma membrane, has little known function other than its ability to suppress tumor growth. Using yeast two-hybrid interaction cloning, we identified the HGF-regulated tyrosine kinase substrate (HRS) as a schwannomin interactor. We verified the interaction by both immunoprecipitation of endogenous HRS with endogenous schwannomin in vivo as well as by using bacterially purified HRS and schwannomin in vitro. We narrowed the regions of interaction to include schwannomin residues 256-579 and HRS residues from 480 to the end of either of two HRS isoforms. Schwannomin molecules with a L46R, L360P, L535P or Q538P missense mutation demonstrated reduced affinity for HRS binding. As HRS is associated with early endosomes and may mediate receptor translocation to the lysosome, we demonstrated that schwannomin and HRS co-localize at endosomes using the early endosome antigen 1 in STS26T Schwann cells by indirect immunofluorescence. The identification of schwannomin as a HRS interactor implicates schwannomin in HRS-mediated cell signaling.

Objective. To exploit the physiologic Fc␥ receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications.Methods. DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the ␤-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. Conclusion. This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.Activation-inhibition coupling, the pairing of a positive signal with an inhibitory loop, controls the magnitude and duration of many biologic processes (1-3). In B lymphocytes, recognition of an antigen by the clonotypic B cell receptor (BCR) induces a signal that can direct clonal expansion, differentiation, the release of cytokines, and, ultimately, Ig production. Uncontrolled activation is prevented by exhaustion of the activating stimulus as well as by the triggering of a negative feedback loop that involves the engagement of an inhibitory Fc␥ receptor (Fc␥R), Fc␥RIIb (CD32B) (4). The latter mechanism is triggered when the BCR recognizes immune-complexed antigen, resulting in the concomitant engagement of CD32B by the Fc domain of the complex-bound IgG, thus preventing the expansion of B cell clones that share the same specificity as that recognized by the soluble IgG. Attesting to its critical role in immune regulation, CD32B-knockout mice on the Th1-prone C57BL/6 background develop a lupuslike glomerulonephritis (5,6). We have focused on CD32B inhibitory signaling in B lymphocytes as a model system for the development of an alternative class of biologics that exploit activation-inhibition coupling for the control of immune activation. In addition to Ig

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TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.