The Fc (crystallizable fragment) region of therapeutic antibodies can have an important role in their safety and efficacy. Although much is known about the structure-activity relationship of antibodies and the factors that influence Fc effector functions, a process has not yet been defined to clearly delineate how Fc functionality should be assessed and controlled during antibody development and manufacturing. In this article, we summarize the current knowledge of antibody Fc functionality, provide a strategy for assessing the effector functions of different classes of therapeutic antibodies (including Fc fusion proteins) and propose a path for routine testing and controls for manufacturers of antibody products.

Signaling through the erythropoietin receptor (EPO-R) is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. EPO induces homodimerization of the EPO-R, triggering activation of the receptor-associated kinase JAK2 and activation of STAT5. By mutating the eight tyrosine residues in the cytosolic domain of the EPO-R, we show that either Y343 or Y41 is sufficient to mediate maximal activation of STAT5; tyrosine residues Y429 and Y431 can partially activate STAT5. Comparison of the sequences surrounding these tyrosines reveals YXXL as the probable motif specifying recruitment of STAT5 to the EPO-R. Expression of a mutant EPO-R lacking all eight tyrosine residues in the cytosolic domain supported a low but detectable level of EPO-induced STAT5 activation, indicating the existence of an alternative pathway for STAT5 activation independent of any tyrosine in the EPO-R. The kinetics of STAT5 activation and inactivation were the same, regardless ofwhich tyrosine residue in the EPO-R mediated its activation or whether the alternative pathway was used. The ability of mutant EPO-Rs to activate STAT5 did not directly correlate with their mitogenic potential.Erythropoietin (EPO), a hormone synthesized by the kidney, is the primary regulator of mammalian erythropoiesis. Hormone binding to its cognate receptor is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. The erythropoietin receptor (EPO-R) (1) belongs to the large cytokine receptor superfamily, which is characterized by a WSXWS motif in the extracellular domain and by the absence of enzymatic activity in the cytoplasmic domain. To transmit a signal these receptors couple to protein tyrosine kinases and other signal transduction proteins. Ligand induced homodimerization of the EPO-R (2) triggers the activation of the receptor-bound protein tyrosine kinase JAK2 (3), resulting in a transient increase in tyrosine phosphorylation of several cellular proteins including the EPO-R. Since phosphotyrosine residues are specifically recognized by Src homology 2 (SH2) domains, this event enables the EPO-R to recruit a number of signal transduction proteins to the receptor, including SH-PTP1 (4), SH-PTP-2 (5), and P13 kinase (6).Little is known about how signals from the activated receptor complex assembled at the plasma membrane are transmitted to the nucleus and converted into gene activation. One system involves activation of the JAK-STAT pathway (for reviews see refs. 7 and 8). At least six STAT proteins, STAT1-STAT6, have been cloned (for review see ref. 9); all have an SH2 domain that allows it to bind to a phosphorylated tyrosine on a receptor or other protein. Following tyrosine phosphorylation by a Janus kinase or other kinase(s), STAT proteins dissociate from the receptor and form homo-or heterodimers via their SH2 domains. They then move to the nucleus and bind to and presumably activate specific genes. STAT proteins enable a signal to be transduced within minutes from the cell surface to the ...

Transforming growth factor-␤1 (TGF-␤1

An electrophile-responsive element (EpRE) in the 5' flanking region of the mouse glutathione S-transferase Ya subunit gene was recently found to be responsible for the induction of gene expression by xenobiotics that contain or acquire by metabolism an electrophilic center. We now find that this EpRE is composed of two adjacent 9-base-pair motifs related in sequence to the AP-1 binding site, a transcriptional enhancer originally identified as the phorbol 12-myristate 13-acetate (PMA) response element and known to be regulated by the binding of protein products of c-jun and c-fos genes. Synthetic oligonucleotides representing each of the AP-1-like binding sites of the EpRE and the AP-1 site consensus sequence were prepared and assayed for their enhancer activity and inducibility by tert-butylhydroquinone, ,B-naphthoflavone, and PMA. Single AP-1-like sequences showed a lower enhancer activity than an AP-1 consensus sequence and no inducibility.Two adjacent AP-1-like sites were found to act synergistically and to confer inducibility beyond that observed for a single AP-1 consensus sequence. Examination of the PMA-responsive region of a number of genes shows the presence of adjacent AP-1-like sites and indicates that the structure of the EpRE found in the Ya gene may occur more generally and may be important in regulating the magnitude of the electrophilic response. The present study demonstrates the binding and transactivation of the EpRE by Jun and Fos and indicates that the AP-1 site is part of the EpRE. The induction by PMA or tert-butylhydroquinone appears to be independent of protein kinase C activity since it is not affected by inhibitors of this enzyme.Induction of the electrophile-metabolizing enzymes glutathione transferases, glucuronosyltransferases, and NAD-(P)H:quinone reductase is considered a major mechanism of protection against mutagens, carcinogens, and other toxic compounds (1-3). Compounds that induce these enzymes include the diphenols, isothiocyanates, thiocarbamates, unsaturated dicarboxylic acids, etc., which contain or acquire by metabolism electrophilic centers (2). Many of these inducers are Michael reaction acceptors characterized by olefinic or acetylenic bonds rendered electrophilic by conjugation with electron-withdrawing substituents and share common structural features with glutathione transferase substrates (4). As a consequence the potency of inducers was found to parallel their efficiency as substrates for glutathione transferases (5).In a recent study of a mouse glutathione S-transferase (GST) Ya subunit gene, we have demonstrated (6) that a single cis-regulatory element in the 5' flanking region, between nucleotides (nt) -754 and -714 from the start of transcription, is responsible for Ya gene induction by compounds that are electrophilic (e.g., trans-4-phenyl-3-buten-2-one, dimethyl fumarate) or by compounds that are easily oxidized into electrophilic (tert-butylhydroquinone) and planar aromatic (e.g., 3-methylcholanthrene, P-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p...

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