We explored the biology of IL-15 and showed that efficient production of IL-15 is possible only by co-expression in the same cell with the so-called IL-15 Receptor-alpha. We also showed that a second form of IL-15 (SSP IL-15) previously identified in humans and rodents as intracellular or nuclear IL-15 is also efficiently secreted from the cells when co-expressed with the IL-15 Receptor alpha. These results shed new light in the biology and regulation of IL-15 and provide methods for the efficient production and clinical application of this cytokine. Cell lines overproducing soluble bioactive IL-15/IL-15 Receptor alpha heterodimers have been constructed and were used for the production of the bioactive form of IL-15 found in the body. IL-15 purified from over-producing human cells was injected in mice and shown to be bioactive. IL-15 is of interest due to its ability to stimulate the growth, activation and survival of lymphocytes, including CD8 and NK cells. Thus, IL-15 has been considered for cancer immunotherapy and for support of the growth of cytotoxic cell clones after adoptive transfer. Other proposed uses of IL-15 are in lymphopenia, in supporting NK cell growth and activation after NK transfer, and as vaccine adjuvant. We have shown that IL-15 injection accelerates the recovery of lymphocytes in mice rendered lymphopenic after treatment with cytotoxic drugs. We also showed that hetIL-15 can replace the need for lymphodepletion in Adoptive Cell Transfer (ACT), since the injected cells can survive, proliferate and enter the tumors after hetIL-15 treatment. This may have important clinical implications for ACT protocols. We have used the previously developed technologies of RNA optimization to optimize expression of IL-15 cytokine, and have shown that we can over-produce bioactive cytokine after DNA delivery in mice and macaques. DNA delivery of vectors expressing heterodimeric IL-15 leads to systemically active levels of cytokine and the increased proliferation of NK and T cells. We have also used optimized vectors to express IL-12 cytokine in animals. Efficient expression results in bioactive levels, which increase immune response after DNA vaccination, thus becoming important molecular adjuvant for our vaccines. This work established methods to optimize expression of the IL-12 family of cytokines (IL-12, IL-23, IL-27, IL-35). Efficient expression of IL-27 after DNA delivery demonstrated synergy with IL-2 in the elimination of neuroblastoma metastases in mice. We have also developed methods for large-scale isolation and purification of exosomes. We have shown that hetIL-15 is incorporated in exosomes and have produced sufficient quantities for animal experiments. We propose that the versatility of exosomes can be used for the delivery of immunotherapy exosomes to disease sites (tumor or lymphoid tissue). We developed methods for the efficient delivery of exosomes to tumor sites.
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