Expression of IL-12 family heterodimers
Inventors
Felber, Barbara K. • Pavlakis, George N.
Assignees
US Department of Health and Human Services
Publication Number
US-9546202-B2
Publication Date
2017-01-17
Expiration Date
2029-05-11
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 for improved expression of IL-12 family cytokine heterodimeric proteins 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 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.
The present invention demonstrates that optimized ratios of the subunits result in greatly increased extracellular levels of the heterodimeric proteins. It identifies optimal ratios of subunits for several heterodimeric cytokines, including IL-12 family cytokines, such as IL-12 chains p35 and p40, IL-23 chains p19 and p40, IL-27 chains p28 and EBI3. Use of optimized expression strategies leads to improvement of cytokine expression and is a general approach for expressing any multimeric protein.
Claims Coverage
The patent includes multiple independent claims covering dual expression vectors, mammalian cells containing these vectors, compositions comprising these vectors, and methods for promoting stability and secretion of IL-12 family cytokines by expressing subunits at non-equimolar ratios.
Dual expression vector for expressing IL-12 family cytokine subunits at non-equimolar ratios
A dual expression vector comprising first and second expression cassettes for expressing the first and second subunits of an IL-12 family cytokine protein, wherein the subunits are expressed at a relative ratio other than 1:1, and the IL-12 family cytokine is IL-23 or IL-27.
Expression ratio range in dual expression vector
The first and second subunits are expressed at a ratio in the range of about 3:1 to about 15:1.
Promoter control of subunit expression in dual expression vectors
The first subunit is expressed under control of a human CMV promoter and the second subunit is expressed under control of a simian CMV promoter.
IL-23 dual expression vector with specified subunits
A dual expression vector wherein the IL-12 family cytokine is IL-23, the first subunit is IL-23 p40 and the second subunit is IL-23 p19.
Sequence identity in IL-23 dual expression vector
The p19 subunit is encoded by a nucleic acid sequence with at least 95% identity to SEQ ID NO:26 and the p40 subunit with at least 95% identity to SEQ ID NO:33.
IL-27 dual expression vector
A dual expression vector wherein the IL-12 family cytokine is IL-27.
Sequence identity in IL-27 dual expression vector
In the IL-27 dual expression vector, the p28 subunit is encoded by a nucleic acid sequence sharing at least 95% identity with SEQ ID NO:29, and the EBI3 subunit shares at least 95% identity with SEQ ID NO:30.
Mammalian cells comprising dual expression vectors
Mammalian cells containing the dual promoter expression vector expressing the IL-12 family cytokine subunits at non-equimolar ratios.
Compositions with dual expression vector
Compositions comprising the dual expression vector and a pharmaceutically acceptable excipient.
Method of promoting stability and secretion of IL-27 heterodimer
Method of promoting stability and secretion of an IL-27 heterodimer by expressing p28 and EBI3 subunits in a relative ratio that is not equimolar.
Sequence identity in method of promoting IL-27 heterodimer stability
In the method, p28 is expressed from a nucleic acid sequence sharing at least 95% identity with SEQ ID NO:29 and EBI3 from a sequence sharing at least 95% identity with SEQ ID NO:30.
Method of increasing expression level of IL-12 family cytokine by expressing subunits at determined ratio
A method comprising determining the ratio of alpha and beta subunits that produces increased expression level, and expressing the subunits at that ratio, with defined ranges depending on the cytokine: (i) IL-12 p35 and p40 expressed at 1:3 to 1:15 ratio, (ii) IL-23 p19 and p40 expressed at 1:3 to 1:15 ratio, (iii) IL-27 p28 and EBI3 expressed in non-equimolar amounts.
Specific subunit ratios in method for IL-12 expression
For IL-12, p35 and p40 are expressed at a ratio of about 1:3 to 1:15.
Specific subunit ratios in method for IL-23 expression
For IL-23, p19 and p40 are expressed at a ratio of about 1:3 to 1:15.
Specific subunit ratios in method for IL-27 expression
For IL-27, p28 and EBI3 are expressed in non-equimolar amounts.
Expression level increase achieved by optimized ratios
The level of IL-12 family cytokine protein expression is increased at least 3-fold compared to expressing the subunits at a 1:1 ratio.
The claims cover dual expression vectors and methods expressing IL-12 family cytokine subunits at non-equimolar ratios (typically about 3:1 to 15:1), particularly for IL-23 and IL-27. The claims also cover mammalian cells containing such vectors, compositions including them, and methods for increasing expression and stability of these cytokines by expressing subunits at determined non-equimolar ratios.
Stated Advantages
Increased levels and stability of expression of IL-12 family cytokine heterodimeric proteins compared to equimolar subunit expression.
Optimized subunit ratios result in greatly increased extracellular levels of heterodimeric proteins, boosting cytokine production and secretion efficiency.
Improved cytokine expression prevents negative effects due to excess production of single chains and allows efficacious concentrations in vivo.
Documented Applications
Use in improved expression of heterodimeric and multimeric cytokines and other proteins of mammalian origin in vitro and in vivo.
Use of improved IL-12 family cytokine expression constructs as adjuvants co-delivered with vaccine antigens in antimicrobial, anticancer, and mucosal immunity therapies.
Interested in licensing this patent?