Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc␥RI, Fc␥RIIA, Fc␥RIIB, Fc␥RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc␥R; Fc␥RII and Fc␥RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc␥RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc␥RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

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Purpose: The purpose of the present study was to evaluate granulocyte macrophage colonystimulating factor (GM-CSF)^secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. Experimental Design: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a solubleVEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory Tcells were quantified by their CD4 + CD25hi and CD4 + FoxP3 + phenotype. Results: The present study established that GM-CSF^secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4 + and CD8 + tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory Tcells in the tumor, suggesting a novel mechanism ofVEGF-mediated immune suppression at the tumor site. Conclusion: GM-CSF^secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colonystimulating factor (GM-CSF)^secreting tumor cell immunotherapy, which is known to stimulate potent and long-lasting antigen-specific immune responses, in combination with PD-1blockade, which has been shown to augment cellular immune responses. Experimental Design: Survival studies were done in the B16 melanoma and CT26 colon carcinoma tumor models. Immune monitoring studies were done in the B16 model. GM-CSF^secret-ing tumor cell immunotherapy was administered s.c. and the anti^PD-1 antibody was administered i.p. Results:The studies reported here show that combining PD-1blockade with GM-CSF^secret-ing tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with strong antigen-specific T-cell responses detected by tetramer staining and an in vivo CTL assay, higher secretion levels of proinflammatory cytokines by splenocytes, and the persistence of functional CD8 + T cells in the tumor microenvironment. Furthermore, in the biweekly multiple treatment setting, repeated antigen-specificT-cell expansion was only observed following administration of the cellular immunotherapy with the PD-1 blockade and not when the cellular immunotherapy or PD-1 blockade was used as monotherapy. Conclusion:The combination of PD-1 blockade with GM-CSF^secreting tumor cell immunotherapy leads to significantly improved antitumor responses by augmenting the tumor-reactive T-cell responses induced by the cellular immunotherapy. Readministration of the cellular immunotherapy with the anti^PD-1antibody in subsequent immunotherapy cycles was required to reactivate theseT-cell responses.