Expression of IL-12 family heterodimers
Inventors
Felber, Barbara K. • Pavlakis, George N.
Assignees
US Department of Health and Human Services
Publication Number
US-8715964-B2
Publication Date
2014-05-06
Expiration Date
2029-05-11
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Abstract
The present invention provides methods of improving the levels and stability of expression of interleukin-12 family cytokine polypeptides by expressing the alpha and beta subunits of the polypeptides at their determined relative molar ratios that increase the levels and stability of expression of the heterodimer, e.g., in comparison to heterodimer expressed at an equimolar ratio.
Core Innovation
The present invention provides methods of improving the levels and stability of expression of interleukin-12 family cytokine polypeptides by expressing the alpha and beta subunits of the polypeptides at their determined relative molar ratios that increase the levels and stability of expression of the heterodimer, for example, in comparison to heterodimer expressed at an equimolar ratio.
Many proteins are multimeric, composed of multiple and different subunits. Expression of the respective subunits provides a critical step in the production of a functional protein. To obtain maximal production of such proteins it is important to optimize the expression levels of individual subunits. The invention is based, in part, on the discovery that production levels and secretion of several multimeric cytokines depends not only on the absolute levels of expression, but also on the relative levels of expression of individual subunits. Optimized ratios of the subunits resulted in greatly increased extracellular levels of the heterodimeric proteins. Optimal ratios of subunits have been identified for several heterodimeric cytokines including IL-12 family cytokines such as IL-12 chains p35 and p40, IL-23 chains p19 and p40, and IL-27 chains p28 and EBI3. The use of optimized expression strategies leads to improvement of cytokine expression. This strategy is of general application for the expression of any multimeric protein.
Claims Coverage
The patent document contains 9 main inventive features derived from the independent patent claims.
Determining an expression ratio for increased stability and secretion of IL-12 family heterodimers
A method of promoting the stability and secretion of an IL-12 family cytokine heterodimer that includes expressing the first subunit and IL-12 p40 subunit in a cell at a ratio in the range of about 1:3 to about 1:15.
Expression of IL-12 heterodimer at a defined ratio
Expressing IL-12 p35 subunit and p40 subunit at a relative ratio in the range of about 1:3 to about 1:15 to promote heterodimer stability and secretion.
Sequence identity of p35 and p40 subunits
Using p35 and p40 subunits that share at least 95% nucleic acid sequence identity with SEQ ID NO:34 (p35) and SEQ ID NO:33 (p40), respectively.
Co-transfection for controlled ratio expression
Expressing the p35 and p40 subunits at the determined ratio by cotransfecting a cell with nucleic acids encoding the subunits at a ratio in the range of 1:3 to 1:15.
Dual promoter plasmid for differential expression
Transfecting a cell with a single plasmid comprising a first nucleic acid encoding the p35 subunit under control of a first promoter and a second nucleic acid encoding the p40 subunit under control of a second promoter, where promoter strengths allow expression at a ratio in the range of 1:3 to 1:15.
Using simian and human CMV promoters for ratio control
Using the simian CMV promoter as the weaker promoter for the p35 subunit and the human CMV promoter as the stronger promoter for the p40 subunit to achieve the desired expression ratio.
Bicistronic nucleic acid expression
Expressing p35 and p40 subunits at the determined ratio by transfecting a cell with a bicistronic nucleic acid including an internal ribosomal entry site separating the two coding sequences.
Expression of IL-23 heterodimer at defined ratio
Expressing IL-23 p19 subunit and p40 subunit in a cell at a ratio in the range of about 1:3 to about 1:15 to promote stability and secretion.
Sequence identity of p19 and p40 subunits
Using p19 and p40 subunits that share at least 95% nucleic acid sequence identity with SEQ ID NO:26 (p19) and SEQ ID NO:33 (p40), respectively.
The inventive features involve methods to improve stability and secretion of IL-12 family cytokine heterodimers by expressing the alpha and beta subunits at defined molar ratios, particularly involving p35 and p40 subunits of IL-12 and p19 and p40 subunits of IL-23, using co-transfection, dual promoter vectors with differential promoter strengths, and bicistronic nucleic acids. Specific promoter usage and subunit sequence identity thresholds are part of the invention.
Stated Advantages
Increased levels and stability of heterodimer expression compared to equimolar expression of subunits.
Improved extracellular production of IL-12 family cytokines by optimizing relative expression ratios of subunits.
Achieving 3-fold to 30-fold or more increase in expression as measured in vitro or in vivo compared to equimolar ratios.
Prevention of negative effects due to excess production of single chains by expressing subunits at optimized ratios.
Documented Applications
Use of improved nucleic acid sequences encoding IL-12 family cytokines as adjuvants co-delivered with vaccine antigens for antimicrobial therapy, anticancer therapy, and stimulating mucosal immunity.
Production of enhanced levels of heterodimeric IL-12 family cytokines in mammalian host cells for in vitro or in vivo applications.
Gene transfer methods for delivery of vectors encoding IL-12 family cytokines for therapeutic purposes in mammalian hosts including humans, domestic, and agricultural animals.
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