Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.endoglycosidase | Fc glycosylation | glycoengineered antibodies | homogeneous antibodies | sugar oxazoline

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We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.

In recent years, baculovirus has emerged as a tool for high-efficiency gene transfer into mammalian cells. However, the level of gene expression is often limited by the strength of the mammalian promoter used. Here, we show that the baculovirus RING protein IE2 is a strong, promiscuous trans-activator in mammalian cells, dramatically upregulating the cytomegalovirus (CMV) promoter in both Vero E6 and U-2OS cells. Further study of the cellular mechanism for the activation led to the discovery of a novel IE2 nuclear body structure which contains a high concentration of G-actin and closely associates with RNA polymerase II, PML, and SUMO1. IE2 mutagenesis studies indicated that the RING and coiled-coil domains of IE2 were necessary for nuclear body formation, as well as for strong activation of the CMV promoter in mammalian cells. Overall, this study shows that the IE2 trans-activator could significantly advance the use of baculovirus in mammalian gene transfer and protein production.

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