Development of antibodies against the notch ligand Delta-Like-1 by phage display with activity against breast cancer cells.

Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER+) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER+ breast cancer cells is reported. Human DLL1 proteins, containing the essential regions for binding to the Notch receptor and Notch signalling activation, were produced and used to select specific scFv antibody fragments by phage display. Fifteen unique scFvs were identified and reformatted into full IgGs. Characterization of these antibodies by ELISA, surface plasmon resonance and flow cytometry enabled selection of three specific anti-DLL1 IgGs, sharing identical VH regions, with nM affinities. Cellular assays on ER+ breast cancer MCF-7 cells showed that one of the IgGs (IgG-69) was able to partially impair DLL1-mediated activation of the Notch pathway, as determined by Notch reporter and RT-qPCR assays, and to attenuate cell growth. Treatment of MCF-7 cells with IgG-69 reduced mammosphere formation, suggesting that it decreases the breast cancer stem cell subpopulation. These results support the use of this strategy to develop and identify potential anti-DLL1 antibodies candidates against breast cancer.

On-line detection of microbial contaminations in animal cell reactor cultures using an electronic nose device.

An electronic nose (EN) device was used to detect microbial and viral contaminations in a variety of animal cell culture systems. The emission of volatile components from the cultures accumulated in the bioreactor headspace, was sampled and subsequently analysed by the EN device. The EN, which was equipped with an array of 17 chemical gas sensors of varying selectivity towards the sampled volatile molecules, generated response patterns of up to 85 computed signals. Each 15 or 20 min a new gas sample was taken generating a new response pattern. A software evaluation tool visualised the data mainly by using principal component analysis. The EN was first used to detect microbial contaminations in a Chinese hamster ovary (CHO) cell line producing a recombinant human macrophage colony stimulating factor (rhM-CSF). The CHO cell culture was contaminated by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida utilis which all were detected. The response patterns from the CHO cell culture were compared with monoculture references of the microorganisms. Second, contaminations were studied in an Sf-9 insect cell culture producing another recombinant protein (VP2 protein). Contaminants were detected from E. coli, a filamentous fungus and a baculovirus. Third, contamination of a human cell line, HEK-293, infected with E. coli exhibited comparable results. Fourth, bacterial contaminations could also be detected in cultures of a MLV vector producer cell line. Based on the overall experiences in this study it is concluded that the EN method has in a number of cases the potential to be developed into a useful on-line contamination alarm in order to support safety and economical operation for industrial cultivation.

Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiae.

The importance of controlling the expression of heterologous cutinase in a recombinant Saccharomyces cerevisiae SU50 strain was investigated. Maximum specific growth rate and the biomass yield increased 1.91 and 1.16 fold, respectively, when cutinase production was induced by galactose in a pre-fermentation step. However, only 19% of specific cell activity was obtained in comparison to other fermentations following a pre-fermentation step without induction of cutinase expression. Thus, the pre-fermentation step was performed using a selective medium not containing galactose, and the fermentation was performed with a cheaper and complex non-selective medium containing galactose. Under these conditions, and with the aim of maximising the specific cutinase activity, a pre-fermentation with low volume and high density of viable cells must be used. However, due to the low pre-fermentation volume, low yeast cell concentrations and low specific cell activities were obtained after 96 h of fermentation. Otherwise, when the aim was to maximise cutinase yield and productivity, a pre-fermentation volume of 10% (v/v) in relation to fermentation and in the exponential growth phase with a cell concentration between 1.1 and 1.8 g dcw/l should be used. A higher pre-fermentation volume, such as 20% (v/v), would still be economical in the case of a pre-fermentation with low cell density or low cell viability.