An optimized IL-12-Fc expands its therapeutic window, achieving strong activity against mouse tumors at tolerable drug doses.

Background: Interleukin-12 (IL-12) has emerged as one of the most potent cytokines for tumor immunotherapy due to its ability to induce interferon γ (IFNγ) and polarize Th1 responses. Clinical use of IL-12 has been limited by a short half-life and narrow therapeutic index.

Methods: We generated a monovalent, half-life-extended IL-12-Fc fusion protein, mDF6006, engineered to retain the high potency of native IL-12 while significantly expanding its therapeutic window.

Consistency of quality attributes for the glycosylated monoclonal antibody Humira® (adalimumab).

Humira® (adalimumab) is a recombinant human IgG1 monoclonal antibody (mAb) glycoprotein consisting of 1330 amino acids that is specific for human tumor necrosis factor (TNF). The biological activity and clinical profile of mAb therapeutics, including adalimumab, is influenced by their protein structure and glycosylation patterns, which can be affected by the expression system, cell culture conditions and purification process methodology. While clinical outcome cannot yet be attributed to many of the individual structural features that constitute a mAb, it is evident that detailed structural attribute analysis is necessary if structural contributions to function are to be comprehensively defined.

Paclitaxel uptake and transport in Taxus cell suspension cultures.

The transport of paclitaxel in Taxus canadensis suspension cultures was studied with a fluorescence analogue of paclitaxel (Flutax-2(®)) in combination with flow cytometry detection. Experiments were carried out using both isolated protoplasts and aggregated suspension cell cultures. Flutax-2(®) was shown to be greater than 90% stable in Taxus suspension cultures over the required incubation time (24 hours).

Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.

Flow cytometric identification of Paclitaxel-accumulating subpopulations.

An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC.

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity.

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures.

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells.

Preparation of single cells from aggregated Taxus suspension cultures for population analysis.

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures.