Redirecting adenoviruses to tumour cells using therapeutic antibodies: Generation of a versatile human bispecific adaptor.

Effective use of adenovirus-5 (Ad5) in cancer therapy is heavily dependent on the degree to which the virus's natural tropism can be subverted to one that favours tumour cells. This is normally achieved through either engineering of the viral fiber knob or the use of bispecific adaptors that display both adenovirus and tumour antigen receptors. One of the main limitations of these strategies is the need to tailor each engineering event to any given tumour antigen. Here, we explore bispecific adaptors that can utilise established anti-cancer therapeutic antibodies. Conjugates containing bacterially derived antibody binding motifs are efficient at retargeting virus to antibody targets. Here, we develop a humanized strategy whereby we synthesise a re-targeting adaptor based on a chimeric Ad5 ligand/antibody receptor construct. This adaptor acts as a molecular bridge analogous to therapeutic antibody mediated cross-linking of cytotoxic effector and tumour cells during immunotherapy. As a proof or principle, we demonstrate how this adaptor allows efficient viral recognition and entry into carcinoma cells through the therapeutic monoclonal antibodies Herceptin/trastuzumab and bavituximab. We show that targeting can be augmented by use of contemporary antibody enhancement strategies such as the selective elimination of competing serum IgG using "receptor refocusing" enzymes and we envisage that further improvements are achievable by enhancing the affinities between the adaptor and its ligands. Humanized bispecific adaptors offer the promise of a versatile retargeting technology that can exploit both clinically approved adenovirus and therapeutic antibodies.

Restricted processing of glycans by endomannosidase in mammalian cells.

Removal of α-glucose residues from nascent glycoproteins in the early secretory pathway is a requirement for further N-glycan maturation. Although deglucosylation is a stepwise process mediated by endoplasmic reticulum-associated glucosidases I and II for most glycoproteins, Golgi endo-α-mannosidase provides a backup mechanism for glycoprotein deglucosylation. Although conserved in mammals, in certain cell lines, endomannosidase activity in vitro appears to differ from its activity in cells following glucosidase inhibition. Here, we show that in bovine cells this is explained by restricted substrate specificity allowing processing of Glc(1)Man(7)GlcNAc(1/2) and Glc(1)Man(5)GlcNAc(1/2) but not fully glucosylated glycans that build up when glucosidases are inhibited. Our data further demonstrate that such specificity is determined genetically rather than post-translationally. We also demonstrate that the bovine endomannosidase is transcriptionally upregulated by comparison with glucosidase II in Madin-Darby bovine kidney cells and speculate that this is to compensate for the reduced catalytic activity as measured in the recombinant form of the enzyme.