Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcgamma receptors.

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 Fcgamma regions to low-affinity Fcgamma receptors (FcgammaR) 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 (FcgammaRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcgammaRIIB). Identification of new amino acid residues important for FcgammaR 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 FcgammaRIV, a unique mouse activating FcgammaR 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 FcgammaR, 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 FcgammaR present on the surface of tumor-infiltrating effector cell populations.

Transcription regulation of human chemokine receptor CCR3: evidence for a rare TATA-less promoter structure conserved between drosophila and humans.

The chemokine receptor CCR3 has a critical function in the pathogenesis of eosinophilic diseases and is an entry co-receptor for HIV-1. We describe here the genomic organization and general transcriptional control mechanism for the human gene CCR3. We identified six cDNA transcripts formed by alternative splicing of eight exons and seven introns. CCR3 contains a 37-bp core promoter domain (-3 to +34 relative to the transcription start point) lacking a TATA box but inclusive of an initiator sequence, a G at +24, and a downstream promoter element (DPE) at +28 to +33 common for Drosophila melanogaster but heretofore described for only two other human genes. Mutation of these elements significantly attenuates CCR3 transcription, as predicted by a model of RNA pol II engagement with DPE-containing Drosophila promoters. These results provide evidence for the functional conservation of a DPE-dependent, general transcription control mechanism between Drosophila and human genes.