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The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.

Jalah R, Rosati M, Ganneru B, Pilkington GR, Valentin A, Kulkarni V, Bergamaschi C, Chowdhury B, Zhang GM, Beach RK, Alicea C, Broderick KE, Sardesai NY, Pavlakis GN, Felber BK - J. Biol. Chem. (2013)

Bottom Line: We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit.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.Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, USA.

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.

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Murine IL-12 DNA treatment of B16 melanoma tumor.A, C57BL/6 mice (groups of 9–10 mice) were inoculated with 105 B16 melanoma cells via the intravenous route. Two days later, 5 or 50 ng of the wild type (plasmid WLV-105M) or optimized (plasmid AG250) murine IL-12 DNA were injected using hydrodynamic DNA delivery. Sham vector DNA (CMVkan; 50 ng) was injected into the control mice. Mice were bled at day 1 post-DNA injection, and the plasma levels of murine IL-12p70 were determined. B, after 3 weeks, the mice were sacrificed, and the lung nodules were counted (bottom). p values using the Mann-Whitney two-tailed t test are shown. C, correlation of plasma IL-12 levels (measured at day 1 post-DNA injection) and number of lung nodules (at week 3 post-tumor injection) from the data shown in Fig. 6A. Error bars, S.E.
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Figure 7: Murine IL-12 DNA treatment of B16 melanoma tumor.A, C57BL/6 mice (groups of 9–10 mice) were inoculated with 105 B16 melanoma cells via the intravenous route. Two days later, 5 or 50 ng of the wild type (plasmid WLV-105M) or optimized (plasmid AG250) murine IL-12 DNA were injected using hydrodynamic DNA delivery. Sham vector DNA (CMVkan; 50 ng) was injected into the control mice. Mice were bled at day 1 post-DNA injection, and the plasma levels of murine IL-12p70 were determined. B, after 3 weeks, the mice were sacrificed, and the lung nodules were counted (bottom). p values using the Mann-Whitney two-tailed t test are shown. C, correlation of plasma IL-12 levels (measured at day 1 post-DNA injection) and number of lung nodules (at week 3 post-tumor injection) from the data shown in Fig. 6A. Error bars, S.E.

Mentions: To examine the biological activity of our DNA plasmids, we also compared the anti-tumoral effects of the low and high murine IL-12p70-expressing plasmids in a B16 melanoma cancer model. C57BL/6 mice were inoculated with B16 melanoma cells, and 2 days later the animals received a single administration of IL-12 DNAs expressing low (plasmids WLV-105M, wild type) or high (plasmid AG250, optimized) levels of murine IL-12p70 or control DNA via hydrodynamic delivery. Two doses of each DNA (5 and 50 ng, respectively) were tested. The IL-12p70 plasma levels were determined at 1 day after DNA injection (Fig. 7A), and after 3 weeks, the mice were sacrificed and tumor nodules in lungs were counted (Fig. 7B).


The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.

Jalah R, Rosati M, Ganneru B, Pilkington GR, Valentin A, Kulkarni V, Bergamaschi C, Chowdhury B, Zhang GM, Beach RK, Alicea C, Broderick KE, Sardesai NY, Pavlakis GN, Felber BK - J. Biol. Chem. (2013)

Murine IL-12 DNA treatment of B16 melanoma tumor.A, C57BL/6 mice (groups of 9–10 mice) were inoculated with 105 B16 melanoma cells via the intravenous route. Two days later, 5 or 50 ng of the wild type (plasmid WLV-105M) or optimized (plasmid AG250) murine IL-12 DNA were injected using hydrodynamic DNA delivery. Sham vector DNA (CMVkan; 50 ng) was injected into the control mice. Mice were bled at day 1 post-DNA injection, and the plasma levels of murine IL-12p70 were determined. B, after 3 weeks, the mice were sacrificed, and the lung nodules were counted (bottom). p values using the Mann-Whitney two-tailed t test are shown. C, correlation of plasma IL-12 levels (measured at day 1 post-DNA injection) and number of lung nodules (at week 3 post-tumor injection) from the data shown in Fig. 6A. Error bars, S.E.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585113&req=5

Figure 7: Murine IL-12 DNA treatment of B16 melanoma tumor.A, C57BL/6 mice (groups of 9–10 mice) were inoculated with 105 B16 melanoma cells via the intravenous route. Two days later, 5 or 50 ng of the wild type (plasmid WLV-105M) or optimized (plasmid AG250) murine IL-12 DNA were injected using hydrodynamic DNA delivery. Sham vector DNA (CMVkan; 50 ng) was injected into the control mice. Mice were bled at day 1 post-DNA injection, and the plasma levels of murine IL-12p70 were determined. B, after 3 weeks, the mice were sacrificed, and the lung nodules were counted (bottom). p values using the Mann-Whitney two-tailed t test are shown. C, correlation of plasma IL-12 levels (measured at day 1 post-DNA injection) and number of lung nodules (at week 3 post-tumor injection) from the data shown in Fig. 6A. Error bars, S.E.
Mentions: To examine the biological activity of our DNA plasmids, we also compared the anti-tumoral effects of the low and high murine IL-12p70-expressing plasmids in a B16 melanoma cancer model. C57BL/6 mice were inoculated with B16 melanoma cells, and 2 days later the animals received a single administration of IL-12 DNAs expressing low (plasmids WLV-105M, wild type) or high (plasmid AG250, optimized) levels of murine IL-12p70 or control DNA via hydrodynamic delivery. Two doses of each DNA (5 and 50 ng, respectively) were tested. The IL-12p70 plasma levels were determined at 1 day after DNA injection (Fig. 7A), and after 3 weeks, the mice were sacrificed and tumor nodules in lungs were counted (Fig. 7B).

Bottom Line: We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit.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.Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, USA.

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.

Show MeSH
Related in: MedlinePlus