Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fc␥ receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fc␥ receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.antibody-dependent cell-mediated cytotoxicity ͉ Fc␥R ͉ protein engineering ͉ cancer

Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-␤ (TGF-␤) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of ϳ620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/␣ 2 -macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.

ABSTRACTmRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 animino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using singlestranded RNA probes prepared with SP6 polymerase.Many hormones exert their effects on cellular metabolism and gene expression by regulating the intracellular level of cAMP, which in turn regulates the activity of cAMPdependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) (1-3). The holoenzyme, consisting of two identical regulatory (R) and two catalytic (C) subunits, dissociates when each of the R subunits binds two cAMP molecules (3). The C subunit phosphorylates serine and threonine residues in substrate proteins involved in mediating the biological response to hormonal stimulation. Although many of the effects of cAMP-dependent protein kinase on cellular metabolism involve phosphorylation of substrate proteins by the C subunit (3), there is evidence that the free R subunits also may play a direct role in the regulation of cellular functions (4, 5).There are at least three types of cAMP-dependent protein kinase: type I, which is the predominant form in bovine skeletal muscle; type II, which is the major form in bovine heart; and a neural type II, which is found in bovine brain (2,6). The difference between the various types ofprotein kinase lies in the type of R subunit associated with C subunit. Although three forms of the C subunit have been characterized by isoelectric focusing, C subunits from both types of cAMP-dependent protein kinase have otherwise similar physical and enzymatic properties (1, 7). In addition, the structure of the R and C subunits appears to have been conserved over the course of evolution, since a functional enzyme can be reconstituted using yeast C subunit and bovine R subunit (8).The C subunit of cAMP-dependent protein kinase has been studied extensively. The complete amino acid sequence of the bovine heart C subunit has been published (9), and some of the amino acid residues that form the C site have been identified by photoaffinity labeling (10, 11). These active-site residues include a lysine at position 72, which oc...