Delivery of IFNalpha and VEGF165b by microencapsulated cells: Preparation and in vitro analysis

The pivotal role of angiogenesis in the growth and spread of all solid tumors has driven the cancer research on applying antiangiogenic factors to suppress formation of new blood vessels in order to prevent or slow tumor growth. Glycoproteins, Interferon alpha (IFNalpha) and VEGF 165b are of particular interest in this study. IFNalpha is a multifunctional cytokine with many physiological effects including antiangiogenesis effects. VEGF165b is a competitive antagonist of the Vascular Endothelial Growth Factor (VEGF) receptor and has been reported to successfully inhibit angiogenesis.;The results suggest that one cell line, HEK293, can produce IFNalpha and VEGF165b simultaneously. This process has the advantage of ease of manipulation and low cost but is somewhat limited by the fact that production of IFNalpha and VEGF165b cannot be controlled. Microencapsulation of IFNalpha or VEGF165b producing cells demonstrates that encapsulated cells grow and remain viable within the microcapsules. The IFNalpha and VEGF165b released from microencapsulated HEK293 IFNalpha or HEK293 VEGF165b producing cells were similar to the control IFNalpha and VEGF165b produced by non-microencapsulated cells. Co-encapsulation and mixing of microencapsulated HEK293 IFNalpha producing cells and HEK293 VEGF165b producing cells offer an efficient system for simultaneous production of these two proteins. This process would have bioprocessing advantages and thus would be cost effective.;The results show that sialylation level can be increased using recombinant procedures in VEGF165b produced by HEK293 cells. No improvements after sialylation in protein VEGF165b in vivo half-life is observed. In another experiment the O-glycosylation site of IFNalpha is successfully abolished using site-directed mutagenesis to increase IFNalpha circulatory half-life. This procedure, however, failed to generate N-glycosylated IFNalpha suggesting that the presence of a consensus sequon is not sufficient to provide required glycosylation. A non-glycosylated IFNalpha was used to evaluate the impact of O-glycosylation on its in vivo half life and pharmacokinetic. Results show O-glycosylated IFNalpha offers no improvement in protein half-life, in experimental rats, compared to non-glycosylated IFNalpha mutants.;This study demonstrates that microencapsulated HEK293 cells can be a potent system for continuous and targeted delivery of IFNalpha or VEGF 165b in various biomedical applications. Specifically, this study may have implications in future bioprocessing for production of IFNalpha or VEGF165b and in cell based therapeutic applications. However, further studies are required before the complete potential of this process can be fully evaluated.;The thesis goal is to develop a system for simultaneous production of IFNalpha2b (IFNalpha) and VEGF165b and targeted delivery to enhance their antiangiogenic properties. For this purpose, HEK293 cells were developed to produce IFNalpha and VEGF165b simultaneously. The potential of a stable HEK293 cell line producing IFNalpha or VEGF165 b to continuously deliver IFNalpha or VEGF165b after encapsulation in alginate-poly-l-lysine-alginate (APA) microcapsules was evaluated. For a better delivery system, coencapsulation of HEK293 VEGF165b producing cells and HEK293 IFNalpha producing cells or a mixture of encapsulated HEK293 cells producing either IFNalpha or VEGF165b were studied. Attempts were also made to increase the bioactivity (pharmacodynamic) of IFNalpha by modifying its O-glycosylation to an N-glycosylation site and of VEGF 165b by increasing its sialylation level. The bioactivity was investigated in experimental rats.