Cost-effective manufacture of an allogeneic GM-CSF-secreting breast tumor vaccine in an academic cGMP facility.

BACKGROUND: GM-CSF-secreting, allogeneic cell-based cancer vaccines have shown promise for the treatment of a variety of solid tumors. We have now applied this approach to breast cancer. The aim of these studies was to optimize expansion parameters, qualify the manufacturing process, and establish expected outcomes for cGMP-compliant manufacturing of two GM-CSF-secreting breast tumor cell lines. METHODS: The variables affecting the efficiency of expanding and formulating two allogeneic GM-CSF-secreting cell lines, 2T47D-V and 3SKBR3-7, were systematically evaluated. Production criteria investigated included alternative cell culture vessels (flasks vs. cell factories), centrifugation time and speed variables for large volume cell concentration, cell seeding density, the minimal concentration of FBS required for maximal cell expansion, and the dose and timing of irradiation in relation to cryopreservation. RESULTS: These studies demonstrate that, in comparison with standard 150-cm2 tissue culture flasks, Nunc 10-Stack Cell Factories are a more efficient and practical cell culture vessel for vaccine cell line manufacture. Centrifugation optimization studies using the COBE 2991 Cell Processor established that a speed of 2000 r.p.m. (450 g) for 2 min reliably concentrated the cells while maintaining acceptable viability and bioactivity. Radiation studies established that lethal irradiation prior to cryopreservation does not compromise the quality of the product, as measured by post-thaw cell viability and GM-CSF cell line-specific secretion levels. Finally, studies aimed at optimizing the production of one vaccine cell line, 3SKBR3-7, demonstrated that seeding the cells at a higher density and maintaining them in half the initial concentration of FBS maximized the yield of bioactive cells, resulting in significant cost savings. DISCUSSION: A manufacturing process that simultaneously maximizes cell yield, minimizes cell manipulation and maintains vaccine cell potency is critical for producing cell-based cancer vaccines in an academic setting. These studies define a feasible, reproducible and cost-effective methodology for production of a GM-CSF-secreting breast cancer vaccine that is cGMP compliant.

Mesenchymal stem cells as vehicles for gene delivery.

Mesenchymal stem cells contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and marrow stroma. Transduction of mesenchymal stem cells from species other than humans is required for the development of disease models in which mesenchymal stem cells-based gene delivery is evaluated. Attempts to transduce mesenchymal stem cells from some species with amphotropic retroviral vectors were unsuccessful, leading to comparative mesenchymal stem cells transductions with xenotropic and gibbon-ape leukemia virus envelope-pseudotyped retroviral vectors. Human, baboon, canine, and rat mesenchymal stem cells were transduced optimally with amphotropic vector supernatants. In contrast, sheep, goat, and pig mesenchymal stem cells showed highest transduction levels with xenotropic retroviral vector supernatant, and rabbit mesenchymal stem cells were transduced optimally with gibbon-ape-enveloped vectors. Using a myeloablative canine transplantation model and gene-marked canine mesenchymal stem cells, the biodistribution of infused and ex vivo expanded mesenchymal stem cells were examined. The majority of transduced canine mesenchymal stem cells were found in the bone marrow samples. The current study shows the use of mesenchymal stem cells as a delivery vehicle for gene transfer studies, and validates the feasibility of delivering mesenchymal stem cells to the marrow compartment for stromal regeneration after cancer-associated cytotoxic therapies.