T-Lymphocytes Transduced With Sfcmm-3 Vector In A Closed System

Safety, yield and scalability represent critical issues in production of gene-modified cells (GMC) for clinical applications. Currently open systems, based on culture flasks, are used for production of TK-transduced T cells. Such systems may be used, without significant risk of contamination, only if limited numbers of cells (up to 108-109) are required. Thus a method for cell manipulation and culturing in a closed system has been developed.Lymphocytes are cultured in bags, activated with OKT3, then transduced with the vector SFCMM-3, carrying the HSV-TK and Delta LNGFR genes. After a few days, lymphocytes are collected and the percentage of transduced cells is evaluated by FACS analysis. Transduced cells are sorted by antibody conjugated magnetic beads and then expanded in culture. The full process lasts 10 days. The following instruments are used: CytoMate (TM) Cell Processing System (Nexell Therapeutic Inc.), for cell thawing, washing and harvest, and lsolex (TM) 300 Magnetic Cell Separation System (Baxter), for selection of the transduced cells.The closed-system is very efficient and the cell yield is higher than that obtained in the open-system (final cell number/initial cell number: 0.64 versus 0.39). A detailed analysis of the process indicates that the steps in which the closed system shows major advantages are transduction (23% versus 17%) and selection (recovery of the cells 67% versus 36%). The purity of the final product is very similar and shows the homogeneity of the final population of GMC (94% and 95% LNGFR positive cells, respectively). Only the proliferation rate is lower in the closed than in the open system, especially post-transduction (2.5 versus 4.7) and post-selection (3.6 versus 5.6). However, the development of cell culturing using different bags or different culture media could improve cell growth.These results demonstrate the feasibility of a method for transduction and selection of GMC in a closed system that is safe and efficient.