Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization.

Rapidly producing drug-like antibody therapeutics for lead molecule discovery and candidate optimization is typically accomplished by large-scale transient gene expression technologies (TGE) with cultivated mammalian cells. The TGE methodologies have been extensively developed over the past three decades, yet produce significantly lower yields than the stable cell line approach, facing the technical challenge of achieving universal high expression titers for a broad range of antibodies and therapeutics modalities. In this study, we explored various parameters for antibody production in the TGE cell host Expi293FTM and ExpiCHO-STM with the transfection reagents ExpiFectamineTM and polyethylenimine. We discovered that there are significant differences between Expi293FTM and ExpiCHO-STM cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization.

Melanocortin 5 Receptor Expression and Recovery of Ocular Immune Privilege after Uveitis.

PURPOSE: The therapeutic use of the RPE-neuropeptide α-MSH suppresses experimental autoimmune uveitis (EAU). This suppression is partially through the α-MSH melanocortin 5 receptor (MC5r). Therefore, we examined the possible role of MC5r-expression in the recovery of RPE suppression of phagolysosome-activation in macrophages following α-MSH-treatment of EAU. METHODS: The conditioned media of cultured in situ RPE-eyecup from α-MSH-treated EAU wild-type and MC5r(-/-) mice were used to treat macrophages to assay for phagolysosome activation. RESULTS: MC5r(-/-) mice treated with α-MSH recovered from EAU, but with greater retinal damage, and the RPE suppressed phagolysosome activation in wild type but not in MC5r(-/-) macrophages. In addition, α-MSH did not suppress phagolysosome activation in MC5r(-/-) macrophages, and resting-MC5r(-/-) macrophages had augmented phagocytic activity. CONCLUSION: α-MSH treatment of EAU mediates a MC5r-dependent recovery of RPE suppression of phagolysosome activation in macrophages possibly altering antigen processing and presentation. Also, MC5r-expression helps protect the retina from inflammatory damage. In addition, MC5r-expression is important in the homeostatic maintenance of phagosome-maturation within macrophages.