title: The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.
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.
authors.affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, USA.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Jalah
authors.forename: Margherita
authors.initials: M
authors.lastname: Rosati
authors.forename: Brunda
authors.initials: B
authors.lastname: Ganneru
authors.forename: Guy R
authors.initials: GR
authors.lastname: Pilkington
authors.forename: Antonio
authors.initials: A
authors.lastname: Valentin
authors.forename: Viraj
authors.initials: V
authors.lastname: Kulkarni
authors.forename: Cristina
authors.initials: C
authors.lastname: Bergamaschi
authors.forename: Bhabadeb
authors.initials: B
authors.lastname: Chowdhury
authors.forename: Gen-Mu
authors.initials: GM
authors.lastname: Zhang
authors.forename: Rachel Kelly
authors.initials: RK
authors.lastname: Beach
authors.forename: Candido
authors.initials: C
authors.lastname: Alicea
authors.forename: Kate E
authors.initials: KE
authors.lastname: Broderick
authors.forename: Niranjan Y
authors.initials: NY
authors.lastname: Sardesai
authors.forename: George N
authors.initials: GN
authors.lastname: Pavlakis
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Felber
mesh_terms: D000818:Animals; D006801:Humans; D007167:Immunotherapy; D053765:Interleukin-12 Subunit p35 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D053762:Interleukin-12 Subunit p40 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008253:Macaca mulatta; D008545:Melanoma / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000628:therapy; D051379:Mice; D009374:Neoplasms, Experimental / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000628:therapy; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
chemical_list: C508858:IL12A protein, human; C500879:IL12B protein, human; C508859:Il12a protein, mouse; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D011994:Recombinant Proteins