Formulation and production of a blood-free and chemically defined virus production media for VERO cells.

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.

Inhibition of tumor angiogenesis by the matrix metalloproteinase-activated anthrax lethal toxin in an orthotopic model of anaplastic thyroid carcinoma.

Patients with anaplastic thyroid carcinoma (ATC) typically succumb to their disease months after diagnosis despite aggressive therapy. A large percentage of ATCs have been shown to harbor the V600E B-Raf point mutation, leading to the constitutive activation of the mitogen-activated protein kinase pathway. ATC invasion, metastasis, and angiogenesis are in part dependent on the gelatinase class of matrix metalloproteinases (MMP). The explicit targeting of these two tumor markers may provide a novel therapeutic strategy for the treatment of ATC. The MMP-activated anthrax lethal toxin (LeTx), a novel recombinant protein toxin combination, shows potent mitogen-activated protein kinase pathway inhibition in gelatinase-expressing V600E B-Raf tumor cells in vitro. However, preliminary in vivo studies showed that the MMP-activated LeTx also exhibited dramatic antitumor activity against xenografts that did not show significant antiproliferative responses to the LeTx in vitro. Here, we show that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression in both toxin-sensitive and toxin-resistant ATC cells via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival that is comparable with that produced by the multikinase inhibitor sorafenib. Our results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced tumors with well-established vascular networks. Taken together, these results suggest that the MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo antitumor mechanism of this novel toxin. Therefore, the MMP-activated LeTx could be used not only in the clinical management of V600E B-Raf ATC but potentially in any solid tumor.