Specification
METHOD FOR PRODUCING A POLYPEPTIDE
FIELD OF THE INVENTION
The invention relates generally to polypeptides and more specifically to cytokine antagonist polypeptides, and to methods of producing cytokine antagonist polypeptides.
BACKGROUND OF THE INVENTION
Cytokines are polypeptides secreted by cells of the immune system and exert regulatory effects on the cells of the immune system. They have been reported to play a major role in the pathogenesis of numerous diseases, including allergic rhinitis, atopic dermatitis, allergic asthma, some parasitic infections, and cancer.
The cellular responses to cytokines are mediated through receptors found on the surfaces . of responsive cells. The cytokine receptors may include intracellular, transmembrane, and extracellular components. The extracellular portion of some cytokine receptor polypeptides can be expressed in a soluble fo'rmTWhen added to a population of cells known to be responsive to the cognate cytokine, soluble cytokine receptor polypeptides can inhibit the function of the cytokine. For example, a polypeptide that includes the extracellular portion of the IL-13 receptor has been reported to inhibit the function of EL-13 function in vitro and in vivo.
The expression level of soluble cytokine antagonists, including inhibitors based on the extracellular portions of the IL-13 receptor polypeptide, in cell culture, however, is low. This can limit the commercial feasibility of manufacturing cytokine antagonist. Thus, there is aneed for an effective method of producing a high level of a soluble cytokine antagonist from cell culture.
SUMMARY OF THE INVENTION
The invention is based in part on the discovery of an improved method for producing an IL-13 antagonist polypeptide. The IL-13 antagonist polypeptide produced in the method is recovered in high yields and in a stable form. The method additionally results in production of a high proportion of the BL-13 antagonist polypeptide in a dimeric form, which is the most active form of the antagonist polypeptide.
The invention also provides for a pharmaceutical composition that includes the cytokine antagonist polypeptide of this method as well as a method of reducing the level of a cytokine.,. e.g., IL-13 in a patient that includes administering to the patient a therapeutically effective amount of this pharmaceutical composition.
Li one aspect the invention provides a method of producing an IL-13 antagonist polypeptide. In the method, a culture medium is provided that includes a host cell. The host cell expresses a nucleic acid encoding the IL-13 antagonist polypeptide and the host cell expresses a nucleic acid encoding a complexing polypeptide for the IL-13 antagonist polypeptide. The host cell is cultured under conditions allowing for expression of the IL-13 antagonist polypeptide and the complexing polypeptide. The IL-13 antagonist polypeptide is recovered from the culture medium, thereby producing the IL-13 antagonist polypeptide.
Examples of suitable complexing polypeptides include IL-13 (including an IL-13 polypeptide with the amino acid sequence of a human IL-13 polypeptide);'an' IL-13 receptor binding fragment of an IL-13 polypeptide, an antibody to an IL-13 receptor polypeptide, and EL-6 (including an IL-6 polypeptide with the amino acid sequence of a human IL-6 polypeptide).
In some embodiments, the nucleic acid encoding the IL-13 antagonist polypeptide is a nucleic acid endogenous with respect to the host cell.
In some embodiments, the nucleic acid encoding the complexing polypeptide is an exogenous nucleic acid.
The method optionally includes introducing the exogenous nucleic acid into the host cell.
In some embodiments, more antagonist polypeptide is recovered when the IL-13 antagonist polypeptide is co-expressed with the complexing polypeptide than when the IL-13 antagonist polypeptide is expressed in the absence of the complexing polypeptide.
In some embodiments, the host cell is cultured at a temperature of from about 29 °C to about 39 °C when expressing the nucleic acid encoding the IL-13 antagonist polypeptide and the complexing polypeptide. For example the temperature can be about, e.g., 30 °C, 32 °C3 34 °C, 36 °C, or 37°C, or 38°C.
The host cell can be, e.g., a stably transfected cell (such as a stably transfected>Chinese Hamster Ovary (CHO) cell). Alternatively, the host cell can be a transiently transfected cell (such as a transiently transfected COS cell).
In some embodiments, the IL-13 antagonist polypeptide includes an extracellular moiety of an EL-13 receptor polypeptide fused to at least a portion of an irnmunoglobulin polypeptide. Examples of an IL-13 receptor polypeptide include an IL-13Rαl, IL-13Rα2, or IL-4 receptor polypeptide chain.
In some embodiments, the IL-13 antagonist polypeptide includes an Fc region of an irnmunoglobulin γl polypeptide.
An example of an IL-13 antagonist polypeptide is IL-13 Rcc.2Fc. In some embodiments, aggregation of the expressed IL-13 antagonist polypeptide is reduced relative to aggregation of the DL-13 antagonist polypeptide expressed in a host cell not expressing the nucleic acid encoding the complexing polypeptide for the IL-13 polypeptide. For example, in various embodiments, aggregation is reduced at least about 10%, 30%, 50%, 70%, 80%, 90% or more relative to aggregation of the IL-13 antagonist polypeptide expressed in a host cell not expressing the nucleic acid encoding the complexing polypeptide for the IL-13 polypeptide.
In a further aspect, the invention provides a method of producing an IL-13 Rcc2.Fc polypeptide by providing a culture medium that includes a cell, wherein the cell expresses a nucleic acid encoding IL-13 Ro2.Fc polypeptide and a nucleic acid encoding a complexing polypeptide for the IL-13 Ra2.Fc polypeptide. The cell is cultured under conditions allowing for expression of the IL-13 Ra2.Fc polypeptide and the complexing polypeptide; and the IL-13
Ra2.Fc polypeptide is recovered from the culture medium, thereby producing the IL-13 Ra2.Fc polypeptide.
Also within the invention is a method of producing an IL-13 Ra2.Fc polypeptide by providing a culture medium comprising a cell that expresses a nucleic acid encoding the IL-13 Rcc2.Fc polypeptide and a nucleic acid encoding an IL-13 polypeptide. The cell is cultured under conditions allowing for expression of the IL-13 Ra2.Fc polypeptide and the IL-13 polypeptide. The IL-13 Ra2.Fc polypeptide is recovered from the culture medium, thereby producing" the IL-13 Ra2.Fc polypeptide.
In some embodiments, more IL-13 Ra2.Fc polypeptide is recovered when the IL-13 Ra2.FcF polypeptide is co-expressed with IL-13 than when the IL-13 Rcc2.Fc polypeptide is expressed in the absence of IL-13.
In a further aspect, the invention provides an IL-13 antagonist polypeptide (e.g., an IL-13 Rol.Fc polypeptide) produced by the methods described herein and a pharmaceutically acceptable carrier.
In a still further aspect, the invention provides a purified preparation of a soluble IL-13 antagonist polypeptide, wherein at least 40% of the polypeptide is present as a monomer or dimer following incubation for at least one week at 4 °C. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the polypeptide is present as a monomer or dimer.
Also within the invention is method of reducing the level of a cytokine in a patient comprising administering to the patient a therapeutically effective.amount of a composition that includes a cytokine polypeptide antagonist polypeptide (including an IL-13 antagonist polypeptide) described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification,
including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is an autoradiogram showing 35S-labeIed polypeptides from COS cell lines.
FIG IB is an autoradiogram showing 35S-labeled polypeptides from COS cell lines prepared by Protein A precipitation.
FIG. 2 is a schematic diagram depicting the circular map of IL-13 expression plasmid pTMNhIL13H6EK.
FIG. 3 is a graph showing the level of IL-13R
Documents
Application Documents
| # |
Name |
Date |
| 1 |
5757-delnp-2005-pct-311.pdf |
2011-08-21 |
| 1 |
5757-DELNP-2005_EXAMREPORT.pdf |
2016-06-30 |
| 2 |
5757-delnp-2005-abstract.pdf |
2011-08-21 |
| 2 |
5757-delnp-2005-pct-308.pdf |
2011-08-21 |
| 3 |
5757-delnp-2005-pct-304.pdf |
2011-08-21 |
| 3 |
5757-delnp-2005-assignment.pdf |
2011-08-21 |
| 4 |
5757-delnp-2005-pct-237.pdf |
2011-08-21 |
| 4 |
5757-delnp-2005-claims.pdf |
2011-08-21 |
| 5 |
5757-delnp-2005-pct-220.pdf |
2011-08-21 |
| 5 |
5757-delnp-2005-correspondence-others.pdf |
2011-08-21 |
| 6 |
5757-delnp-2005-pct-210.pdf |
2011-08-21 |
| 6 |
5757-delnp-2005-description (complete).pdf |
2011-08-21 |
| 7 |
5757-delnp-2005-gpa.pdf |
2011-08-21 |
| 7 |
5757-delnp-2005-drawings.pdf |
2011-08-21 |
| 8 |
5757-delnp-2005-form-5.pdf |
2011-08-21 |
| 8 |
5757-delnp-2005-form-1.pdf |
2011-08-21 |
| 9 |
5757-delnp-2005-form-18.pdf |
2011-08-21 |
| 9 |
5757-delnp-2005-form-3.pdf |
2011-08-21 |
| 10 |
5757-delnp-2005-form-2.pdf |
2011-08-21 |
| 11 |
5757-delnp-2005-form-18.pdf |
2011-08-21 |
| 11 |
5757-delnp-2005-form-3.pdf |
2011-08-21 |
| 12 |
5757-delnp-2005-form-1.pdf |
2011-08-21 |
| 12 |
5757-delnp-2005-form-5.pdf |
2011-08-21 |
| 13 |
5757-delnp-2005-drawings.pdf |
2011-08-21 |
| 13 |
5757-delnp-2005-gpa.pdf |
2011-08-21 |
| 14 |
5757-delnp-2005-description (complete).pdf |
2011-08-21 |
| 14 |
5757-delnp-2005-pct-210.pdf |
2011-08-21 |
| 15 |
5757-delnp-2005-correspondence-others.pdf |
2011-08-21 |
| 15 |
5757-delnp-2005-pct-220.pdf |
2011-08-21 |
| 16 |
5757-delnp-2005-claims.pdf |
2011-08-21 |
| 16 |
5757-delnp-2005-pct-237.pdf |
2011-08-21 |
| 17 |
5757-delnp-2005-assignment.pdf |
2011-08-21 |
| 17 |
5757-delnp-2005-pct-304.pdf |
2011-08-21 |
| 18 |
5757-delnp-2005-abstract.pdf |
2011-08-21 |
| 18 |
5757-delnp-2005-pct-308.pdf |
2011-08-21 |
| 19 |
5757-DELNP-2005_EXAMREPORT.pdf |
2016-06-30 |
| 19 |
5757-delnp-2005-pct-311.pdf |
2011-08-21 |