Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-02-03
2001-02-13
Fitzgerald, David L. (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023400, C530S350000, C514S002600, C424S085100
Reexamination Certificate
active
06187564
ABSTRACT:
BACKGROUND OF THE INVENTION
A problem encountered in the practice of medicine when using proteins as injectable pharmaceuticals is the frequency at which those injections must be made in order to maintain a therapeutic level of the protein in the circulation. For example, erythropoietin has a relatively short plasma half-life (Spivak, J. L., and Hogans, B. B.,
Blood
, 73:90, 1989; McMahon, F. G., et al.,
Blood
, 76:1718, 1990). Therefore, therapeutic plasma levels are rapidly decreased, and repeated intravenous administrations must be made. An alternative route of administration is subcutaneous injection. This route offers slower absorption from the site of administration, thus causing a sustained release effect. However, significantly lower plasma levels are achieved and, thus, a similar frequency of injection, as is required with intravenous administration, must be used to produce a comparable therapeutic effect.
Modification of naturally occurring proteins which have therapeutic value is often attempted in an effort to increase the protein's biological activity. Several methods have been employed to increase the biological activity of therapeutic proteins. These methods often focus on increasing the size of the therapeutic agents. For example, the size of a protein can be increased through chemical conjugation with a reagent such as polyethylene glycol (PEG) (Knusli, C., et al.,
Brit. J. Haematol
. 82:654-663, 1992). This procedure, also known as “PEGylation”, has been reported with several protein agents, first as a means to reduce antigenicity, but also as a way to increase biological activity.
Another method of increasing a protein's size is through chemical cross-linking with another protein. For example, to increase the antigenicity of a protein, chemical cross-linking agents are used to conjugate the immunogenic protein to a carrier molecule such as immunoglobulin or serum albumin.
However, the conjugation of chemical compounds or inert molecules to a protein often results in a significant decrease of the overall biological activity, and of selected biological activity of the protein. (Knusli, C., et al.,
Brit. J. Haematol
., 82:654-663, 1992). These conjugations must be designed such that the resulting modified protein remains therapeutically efficacious and retains the desired biological properties of the unmodified, wild type (i.e., naturally-occurring) protein (Satake, R., et al.,
Biochem. Biophys. Acta
. 1038:125-129, 1990). Thus, it would be advantageous to be able to modify therapeutically active proteins to increase their biological activity which would result in less frequent injections or smaller doses of protein.
SUMMARY OF THE INVENTION
The present invention relates to modified proteins or polypeptides with increased biological activity, and methods of producing and using these modified proteins and polypeptides.
Increased biological activity results from the production of fusion proteins that result in protein multimers, e.g., dimers and trimers. Protein multimers are produced by expressing tandemly linked nucleic acids encoding the proteins of the present invention, or biologically active fragments, analogs, variants, mutants or derivatives of the proteins. The nucleic acids that encode the proteins are fused, as described herein. The proteins of the present invention can be fused directly to another protein, or can be fused via a linker, e.g., a peptide linker. The tandemly fused nucleic acid sequence is then inserted into an expression vector and introduced into a competent cell, either prokaryotic or eukaryotic, resulting in the production of a fusion protein multimer with increased biological activity.
Increased biological activity is defined herein as a prolonged plasma half-life (that is, a longer circulating half-life relative to the naturally occurring protein), and/or higher potency (i.e., requiring a smaller quantity relative to the naturally occurring protein to achieve a specified level of biological activity). Increased biological activity can also encompass a combination of the above-described activities, for example, a modified protein with higher potency that also exhibits a prolonged circulating half-life. Because the proteins of the present invention have increased biological activity, the frequency with which they must be administered is reduced, or the amount administered to achieve an effective dose is reduced. Additional advantages can also result from the modifications described herein. For example, new unpredictable activities can result, such as increased affinity for receptors or binding ligands, which can result in increased stimulation of signal generated by such binding. A reduced quantity of modified protein would then be necessary over the course of treatment as compared to the quantity necessary if unmodified protein were used.
Proteins encompassed by the present invention include any protein with therapeutic activity. Specifically encompassed by the present invention are cytokines, growth factors, and hormones which include, for example, the following: Interferon-&agr;, Interferon-&bgr;, Interferon-&ggr;, Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9, Interleukin-10, Interleukin-11, Interleukin-12, Interleukin-13, Interleukin-14, Interleukin-15, Interleukin-16, Erythropoietin, Colony-Stimulating Factor-1, Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, Leukemia Inhibitory Factor, Tumor Necrosis Factor, Lymphotoxin, Platelet-Derived Growth Factor, Fibroblast Growth Factors, Vascular Endothelial Cell Growth Factor, Epidermal Growth Factor, Transforming Growth Factor-&bgr;, Transforming Growth Factor-&agr;, Thrombopoietin, Stem Cell Factor, Oncostatin M, Amphiregulin, Mullerian-Inhibiting Substance, B-Cell Growth Factor, Macrophage Migration Inhibiting Factor, Endostatin, and Angiostatin. Descriptions of these proteins can be found in “Human Cytokines:
Handbook for Basic and Clinical Research
”, Aggarwal, B. B., and Gutterman, J. U., Eds., Blackwell Scientific Publications, Boston, Mass., (1992), which is herein incorporated by reference in its entirety.
More specifically, the present invention relates to modified erythropoietin with increased biological activity, as defined herein. The modified erythropoietin with increased biological activity of the present invention is a fusion protein comprising two or more erythropoietin molecules covalently fused, resulting in an erythropoietin multimer.
Also encompassed by the present invention are methods of making and using the fusion protein multimers described herein and methods of using them.
REFERENCES:
patent: 4816567 (1989-03-01), Cabilly et al.
patent: 4874813 (1989-10-01), O'Shannessy
patent: 5073627 (1991-12-01), Curtis et al.
patent: 5091513 (1992-02-01), Huston et al.
patent: 5114711 (1992-05-01), Bell et al.
patent: 5116964 (1992-05-01), Capon et al.
patent: 5225538 (1993-07-01), Capon et al.
patent: 5349053 (1994-09-01), Landolfi
patent: 5428130 (1995-06-01), Capon et al.
patent: 5478925 (1995-12-01), Wallach et al.
patent: 5547852 (1996-08-01), Seiler et al.
patent: 5580853 (1996-12-01), Sytkowski
patent: 5614184 (1997-03-01), Sytkowski et al.
patent: 5684136 (1997-11-01), Godowski
patent: 5688679 (1997-11-01), Powell
patent: 5883230 (1999-03-01), Schendel
patent: A-79357 (1992-01-01), None
patent: 0255231B1 (1988-02-01), None
patent: 0 283 244 B1 (1988-03-01), None
patent: 0267678A1 (1988-05-01), None
patent: 0306943A3 (1988-09-01), None
patent: 0 622 459 A1 (1994-04-01), None
patent: 0618227A1 (1994-05-01), None
patent: 0 816 510 A1 (1996-12-01), None
patent: WO 91/19739 (1991-12-01), None
patent: WO 92/04455 (1992-03-01), None
patent: 92/06117 (1992-04-01), None
patent: WO 92/06116 (1992-04-01), None
patent: WO 94/02611 (1994-03-01), None
patent: 94/09817 (1994-05-01), None
patent: 94/13806 (1994-06-01), None
patent: WO 95/25746 (1995-09-01), None
patent: WO 95/33057 (1995-12-01), None
patent: WO 96/19573 (1996-06-0
Beth Israel Deaconess Medical Center
Fitzgerald David L.
Hamilton Brook Smith & Reynolds P.C.
LandOfFree
DNA encoding erythropoietin multimers having modified... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with DNA encoding erythropoietin multimers having modified..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA encoding erythropoietin multimers having modified... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2609978