Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-03-06
2002-12-03
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S389600, C536S023500, C424S085100
Reexamination Certificate
active
06489293
ABSTRACT:
BACKGROUND
The glycoprotein hormone erythropoietin regulates the growth and differentiation of red blood cell (erythrocyte) progenitors. The hormone is produced in the fetal liver and adult kidney. Erythropoietin induces proliferation and differentiation of red blood cell progenitors through interaction with receptors on the surface of erythroid precursor cells.
Several approaches have been employed to identify those features of the protein that are relevant to its structure and function. Examination of the homologies among the amino acid sequences of erythropoietin proteins of various species has demonstrated several highly conserved regions (McDonald, J. D., et al.,
Mol. Cell. Biol
., 6:842-848 (1986)).
Oligonucleotide-directed mutagenesis has been used to prepare structural mutants of erythropoietin, lacking specific sites for glycosylation. Studies indicate that N-linked carbohydrates are important for proper biosynthesis and/or secretion of erythropoietin. These studies also show that glycosylation is important for in vivo, but not in vitro, biological activity (Dube, S., et al.,
J. Biol. Chem
., 263:17516-17521 (1988); Yamaguchi, K., et al.,
J. Biol. Chem
., 266:20434-20439 (1991); Higuchi, M., et al.,
J. Biol. Chem
., 267:7703-7709 (1992)).
Studies with monoclonal anti-peptide antibodies have shown that the amino terminus and the carboxy-terminal region (amino acids 152-166) of erythropoietin may be involved with biological activity. It has also been demonstrated that antibodies to amino acids 99-119 and 111-129 block the hormone's biological activity, apparently by binding to two distinct non-overlapping domains (99-110 and 120-129) (Sytkowski, A. J. and Donahue, K. A.,
J. Biol. Chem
., 262:1161-1165 (1987)). Thus, it was hypothesized that amino acids 99-129 were important in the formation of a functional region involved in receptor recognition, either through forming a necessary component of the protein's tertiary structure or through direct participation in receptor binding, or both. Preliminary experiments suggested that alterations in localized secondary structure within the 99-129 region resulted in inactivation of erythropoietin. Therefore, a possible structural role for amino acids 99-129 has been postulated. Recently, a series of experiments indicated that amino acids 99-110 (Domain 1) play a critical role in establishing the biologically active conformation of human erythropoietin (Chern, Y., et al.,
Eur. J. Biochem
., 202:225-229 (1991)).
These Domain 1 mutants, in which a group of three amino acids was deleted and replaced by two different amino acids, were found to be biologically inactive. Furthermore, these mutations in Domain 1 inhibited the secretion of the mutant erythropoietin into cell culture medium (Chern, Y., et al.,
Eur. J. Biochem
., 202:225-229 (1991)). Inhibition of secretion in mammalian cells is consistent with a profound structural change of the polypeptide hormone. Profound structural changes could significantly affect the ability of the hormone to interact with its cognate receptor. Thus, these mutant erythropoietin polypeptides are not suitable for elucidating the structure/function relationship that exists between erythropoietin and its cellular receptor. Nor are these mutants suitable erythropoietin antagonists for use, for example, in therapeutic treatment of polycythemias, or over production of erythropoietin. Thus, it would be beneficial to precisely determine which amino acids are critical to the erythropoietin polypeptide to maintain a stable, biologically active conformation which retains its secretable properties and its ability to bind to the erythropoietin receptor.
Moreover, the precise determination of critical amino acid residues would be useful to alter the biological activity of erythropoietin, either decreasing or increasing one or more biological properties of the protein.
SUMMARY OF THE INVENTION
The present invention relates to isolated DNA encoding mutated erythropoietin proteins which have altered biological activity, yet retain their secretable properties (i.e., secretable erythropoictin proteins). That is, the present invention relates to isolated DNA encoding secretable erythropoietin proteins which have at least one amino acid residue in Domain 1 which differs from the amino acid residue present in the corresponding position of wildtype erythropoietin and which have altered ability to regulate the growth and differentiation of red blood cell progenitors. Domain 1 of the mutants described herein refers to the amino acids which correspond to amino acids 99-110 (SEQ ID NO: 1) of the wildtype recombinant erythropoietin. Altered ability is defined as ability different from that of the wildtype recombinant erythropoietin ability to regulate the growth and differentiation of red blood cell progenitors. As used herein, altered ability to regulate the growth and differentiation of red blood cell progenitor cells refers to biological activity different from wildtype recombinant erythropoietin activity (i.e., altered biological activity relative to wildtype recombinant erythropoietin activity). The mutated erythropoietin proteins of the present invention can be secreted in homologous and heterologous expression systems. For example, the mutated erythropoietin proteins of the present invention can be secreted in mammalian, bacterial or yeast expression systems.
The present invention also relates to the modified secretable mutant erythropoietin proteins encoded by the isolated DNA described above. These modified secretable erythropoietin proteins have altered biological activities. For example, the modified secretable mutant erythropoietin may have decreased ability relative to wildtype erythropoictin protein to regulate growth and differentiation of red blood cell progenitor cells. As used herein, decreased ability to regulate growth and differentiation of red blood cell progenitor cells is also referred to as decreased biological activity relative to wildtype erythropoietin activity. Wildtype erythropoietin activity is also referred to herein as biological activity of wildtype erythropoietin. Alternately, a modified secretable mutant erythropoietin protein described herein may exhibit increased heat stability relative to wildtype erythropoietin protein.
The modified erythropoietin proteins described herein comprise an amino acid sequence with at least one amino acid residue different from the amino acid residue present at the corresponding position in Domain 1 in the wildtype erythropoietin. These erythropoietin proteins are referred to as modified secretable human recombinant erythropoietin proteins having altered ability (i.e., decreasing or enhancing ability) relative to wildtype erythropoietin protein to regulate the growth and differentiation of red blood cell progenitors.
The term modified, as used herein, includes substitution of a different amino acid residue, or residues, as well as deletion or addition of an amino acid residue, or residues.
Until the present invention, mutations within the erythropoietin sequence which result in the alteration of biological activity have also frequently resulted in a concurrent loss of secretability of the protein from transfected cells. This loss of secretability is consistent with a loss of structural integrity (Boissel, J-P. and Bunn, H. F., “The Biology of Hematopoiesis”, pp. 227-232, John Wiley and Sons, New York (1989)). Now, the sites critical to the maintenance of a stable, biologically active conformation have been identified by means of oligonucleotide-directed mutagenesis and have been found to occur in Domain 1 (amino acids 99-110) (SEQ ID NO: 1) of human recombinant erythropoietin. Modifications of the wildtype erythropoietin have been made and the encoded erythropoietin proteins have been expressed. The resulting mutant erythropoietin proteins described herein have altered erythropoietin regulating activity, as demonstrated in the art-recognized bioassay of Krystal, G.,
Exp. Hematol
., 11:649-660 (1983). Activity of the resulting erythropoietin pr
Grodberg Jennifer
Sytkowski Arthur J.
Beth Israel Deaconess Medical Center
DeBerry Regina M.
Hamilton Brook Smith & Reynolds P.C.
Kunz Gary L.
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