High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system.

Therapeutic recombinant monoclonal antibodies (mAbs) are commonly produced by high-expressing, clonal, mammalian cells. Creation of these clones for manufacturing remains heavily reliant on stringent selection and gene amplification, which in turn can lead to genetic instability, variable expression, product heterogeneity and prolonged development timelines. Inclusion of cis-acting ubiquitous chromatin opening elements (UCOE™) in mammalian expression vectors has been shown to improve productivity and facilitate high-level gene expression irrespective of the chromosomal integration site without lengthy gene amplification protocols. In this study we have used high-throughput robotic clone selection in combination with UCOE™ containing expression vectors to develop a rapid, streamlined approach for early-stage cell line development and isolation of high-expressing clones for mAb production using Chinese hamster ovary (CHO) cells. Our results demonstrate that it is possible to go from transfection to stable clones in only 4 weeks, while achieving specific productivities exceeding 20 pg/cell/day. Furthermore, we have used this approach to quickly screen several process-crucial parameters including IgG subtype, enhancer-promoter combination and UCOE™ length. The use of UCOE™-containing vectors in combination with automated robotic selection provides a rapid method for the selection of stable, high-expressing clones.

A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies.

Recombinant monoclonal antibodies currently dominate the protein biologics marketplace. The path from target antigen discovery and screening, to a recombinant therapeutic antibody can be time-consuming and laborious. We describe a set of expression vectors, termed mAbXpress, that enable rapid and sequence-independent insertion of antibody variable regions into human constant region backbones. This method takes advantage of the In Fusion cloning system from Clontech, which allows ligation-free, high-efficiency insertion of the variable region cassette without the addition of extraneous amino acids. These modular vectors simplify the antibody reformatting process during the preliminary evaluation of therapeutic or diagnostic candidates. The resulting constructs can be used directly for transient or amplifiable, stable expression in mammalian cells. The effectiveness of this method was demonstrated by the creation of a functional, fully human anti-human CD83 monoclonal antibody.

A lipase-like gene from Heliothis virescens ascovirus (HvAV-3e) is essential for virus replication and cell cleavage.

A unique feature of ascovirus infection is cleavage of host cells into virus containing vesicles. It has been suggested that the virus induces apoptosis, either by expression of a caspase or other means, which is then diverted toward vesicle formation. There is little known about the mechanism of vesicle formation. Recent genome sequences of three ascoviruses indicated the presence of several putative open reading frames coding for proteins that could be involved in lipid metabolism. These proteins may play a role in rearrangement of membranes in infected host cells leading to formation of vesicles. Here, we analyzed a lipase-like gene (ORF19) from Heliothis virescens ascovirus (HvAV-3e) expressed from 8 h after infection and essential for virus replication and cell cleavage. In addition, ORF19 knock down by RNA interference inhibited virus replication indicating that the gene is indispensable for HvAV-3e replication. However, under enzymatic assays tested, we did not detect any lipase or esterase activity from ORF19.

Effects of Heliothis virescens ascovirus (HvAV-3e) on a novel host, Crocidolomia pavonana (Lepidoptera: Crambidae).

Ascoviruses are double-stranded DNA viruses which cause fatal disease in lepidopteran host larvae. They induce a unique pathology, causing cleavage of host cells into virion-containing vesicles. With the single exception of Diadromus pulchellus ascovirus, all ascoviruses have been exclusively reported from the Noctuidae. To investigate whether Heliothis virescens AV (HvAV-3e) has a broader host range at the family level, larvae of Crocidolomia pavonana F. (Lepidoptera: Crambidae), a major pest of brassica crops in tropical and sub-tropical regions of the Old World and Australasia, were inoculated with HvAV-3e. Larvae were readily infected by the ascovirus and feeding, growth and survival were significantly affected. However, the milky white discolouration of the haemolymph which is characteristic of ascovirus infection in noctuid hosts was not apparent. In further contrast to infected noctuid host larvae that do not develop to the pupal stage, a significant proportion of infected C. pavonana larvae pupated but all were killed at this stage. Thus, C. pavonana appears to be a semi-permissive host of the ascovirus, the presence of such hosts in the field might be an explanation for the conundrum for the ascovirus-noctuid-wasp relationship, helping explain the persistence of the ascovirus.