This thesis presents my research work developing and then employing microbeads containing highly selected and then genetically engineered cells as a delivery platform of cytokines and chemokines, to achieve antitumor effects in a mouse model. To successfully deliver the cytokines and chemokines into the tumor microenvironment, known to be Hypoxic, Acidic and Nutritionally deprived (HAN), a human cell line - HEK293 cell, was selected under HAN conditions for survival and growth. The selected cell line, 293 HAN, proved to be an excellent vehicle due to its high expression rate of the relevant bio-molecules in vitro, and its resilience and viability, in vivo, under encapsulated conditions. It was shown that the encapsulated 293HAN could survive and proliferate for at least 28 and 14 days in subcutaneous and peritumoral subcutaneous tissues, respectively, while the wild type HEK293 cells died within 14 and 7 days, respectively. Afterwards, 293HAN cells were transfected with BMGneo vectors into which were inserted cDNAs of relevant cytokines and chemokines, namely mGM-CSF, mIFNγ, mIP1O, mCCL21, mCCL27 and hIL-15. The production of these bio-molecules by the transfected cells was confirmed to be high e.g. 3800, 2300, 890 and 910 ng/106cells/24 hours of mGM-CSF, mIFNγ, hIL-15 and mIP10, respectively, but with lower rates for mCCL21 and mCCL27. After that, the cell microencapsulation system was optimized by development of a new alginate purification procedure and a new polyanion-polycation complex, namely alginate-PDADMAC, for microencapsulation. The microbead production of relevant cytokines and chemokines from the encapsulated cells was stable for at least 2 weeks, in vitro. A method for the rapid and repeated, non-destructive assessment of the cell number inside microbeads and the measurement of the cytokines and chemokines secreted from these microbeads, was also standardized and proven to be useful.
Finally, the microbeads containing cells capable of secreting the cytokines and chemokines were used for anti-tumor therapy in a mouse (C57BL/6) model with B16 melanoma, EL4 or EG7 tumors. It was demonstrated that mGM-CSF is indeed very potent in stimulating the anti-tumor, cellular mediated, immune response (CMIR) m mice and the source of mGM-CSF could be provided not only by allogeneic tumor cells and xenogeneic non-tumor cells themselves, but also by microbeads containing engineered xenogeneic non-tumor cells (HEK293 and 293HAN) capable of secreting the cytokine. In a treatment format, the injected microbeads secreting mGM-CSF had to be mixed with a source of tumor antigen and supplied separately from the tumor site since the mGM-CSF actually stimulated tumor growth when delivered at the tumor site. It was demonstrated that mGM-CSF secreting microbeads plus irradiated B16 cells, was effective in protecting the mice from challenge with the same tumor, and was also an effective agent for antitumor treatment. In attempts to find a more effective regime for antitumor therapy, I also investigated the potential synergisms of various cytokines and chemokines known or likely to be able to moderate the CMIR. Among the test cytokines and chemokines, the combination groups of mGMCSF plus mIFNγ, mGMCSF plus hIL-15, mGM-CSF plus hIL-15 plus mIP10, hIL-15 plus mIP10, and mIFNγ plus mIP10 proved very promising in tumor immunotherapy. In the B16 tumor model, all these combinations could retard the growth of the DO (vaccine delivered on the same day as tumor "seed") and D-5/D-4 (treatment of pre-existing) tumor. These experiments also showed that mGM-CSF plus hIL-15 could cure 66.6% of mice when the initial B16 cell number was low - 5x10"* tumor cells in the DO model. The mechanism of the immunotherapy of these groups involved CD8+ T lymphocytes, NK cells and CD11c+ Dendritic Cells. In the groups containing mIP10, involvement of an anti-angiogenesis effect was mooted. By transfer of the combination groups for the immunotherapy of EG7 tumor, 83.3% mice had tumor regression, remaining tumor free for more than 3 weeks. The cure rate from mGM-CSF plus hIL-15 plus mIP10 microbeads was 100%. The results presented in the thesis prove the hypothesis that cytokines and chemokines delivered into the tumor environment can enhace host immunity against a pre-existing tumor. Early intervention is crucial for late stage patient responsiveness. It is concluded that the depot cytokines and chemokines, locally delivered into tumor sites by microbeads containing genetically engineered cyto/chemokine producing cells, is a promising regime for tumor immunotherapy.