Abstract
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.
Highlights
The biosynthesis of IL-12p70 depends on the intracellular interaction of its p35 and p40 subunits
We investigated the interaction of the IL-12p35 and p40 subunits and found that p40 plays a critical role at the posttranslational level by stabilizing and promoting the transport of p35, which results in secretion of the heterodimeric IL-12p70
The IL-12p40 Subunit Regulates the Trafficking and Export of the p35 Subunit—To dissect steps in the biosynthesis of the IL-12p70 heterodimer, its p35 and p40 subunits were expressed independently from the CMV promoter using the mammalian expression vector pCMVkan, bypassing their natural transcriptional control. In addition to their AU-rich 3Ј-UTRs [12,13,14], we noted that the native p35 and p40 coding sequences have a relatively low AU content of ϳ50% and contain AU-rich segments, including AUUUA or AAUAA elements, which have been associated with low expression when present within the coding sequence, as we previously reported for HIV and SIV gag and env genes [25,26,27, 37], for IL-15 [30], and for the IL-15R␣ [18, 19]
Summary
The biosynthesis of IL-12p70 depends on the intracellular interaction of its p35 and p40 subunits. This posttranslational regulation mediated by the p40 subunit is conserved in mammals Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ϳ1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ϳ1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences Such optimized DNA plasmids produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. We investigated the interaction of the IL-12p35 and p40 subunits and found that p40 plays a critical role at the posttranslational level by stabilizing and promoting the transport of p35, which results in secretion of the heterodimeric IL-12p70. Our studies on the basic mechanism governing the biosynthesis of IL-12 led to the development of improved plasmids for practical in vivo applications
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