Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-05-05
2002-05-14
Crouch, Deborah (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023500, C530S350000, C530S827000
Reexamination Certificate
active
06388062
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to p53 and its use in treatment of conditions associated with inappropriate levels of p53 tumor suppressor activity.
BACKGROUND OF THE INVENTION
Wild-type p53 is a sequence-specific transcription factor that induces cell cycle arrest or programmed cell death in response to DNA damage [W. Maltzman and L. Czyzyk,
Mol. Cell. Biol
., 4:1689-1694 (1984); M. B. Kastan et al.,
Cancer Res
., 51: 6304-6311 (1991); S. W. Lowe, et. al.,
Cell
, 74: 957-967 (1993); S. W. Lowe, et. al.,
Nature
, 362: 847-849 (1993); A. R. Clarke, et. al.,
Nature
. 362: 849-852 (1993); A. D. Leonardo, et. al.,
Genes Dev
., 8: 2540-2551 (1994)]. The N-terminus of p53 contains a transactivation domain [S. Fields and S. K. Jang,
Science
249: 1046-1049 (1990); T. Unger et al.,
EMBO J
. 11: 1383-1390 (1992)], the central region a sequence-specific DNA binding domain [J. Bargonetti, et. al.,
Genes Dev
., 7: 2565-2574 (1993); N. P. Pavletich, et. al.,
Genes Dev
, 7: 2556-2564 (1993); Y. Wang et. al.,
Genes Dev
, 7: 2575-2586 (1993); T. D. Halazonetis and A. N. Kandil, et. al.,
EMBO J
. 12: 5057-5064 (1993)], and the C-terminus a tetramerization domain [J. Milner, et. al.,
Mol. Cell. Biol
. 11: 12-19 (1991); H. W. Stürzbecher, et. al.,
Oncogene
, 7: 1513-1523 (1992); H. Sakamoto, et. al.,
Proc. Natl. Acad. Sci. U.S.A
. 91: 8974 (1994); P. Wang, et. al,
Mol. Cell. Biol
. 14: 5182-5191 (1994)]. The tetramerization domain mediates homo-oligomerization, which is required for high affinity sequence-specific DNA binding activity and tumor suppressor function [Pietenpol et. al.,
Proc. Natl. Acad. Sci. USA
, 91:1998-2002 (1994)].
In about half of all human tumors, the sequence-specific DNA binding domain of p53 is inactivated by point mutations [C. C. Harris,
Science
, 262: 1980-1981 (1993); S. Friend,
Science
, 265: 334-335 (1994); J. Bargonetti, et. al.,
Cell
, 65: 1083-1091 (1991); S. E. Kern et. al.,
Science
252: 1708-1711 (1991)]. The tumor-derived p53 mutants fail to suppress tumor growth [L. Diller, et. al.,
Mol. Cell. Biol
., 10: 5772-5781 (1990); S. J. Baker, et. al.,
Science
, 249: 912-915 (1990); D. Eliyahu, et. al,
Proc. Natl. Acad. Sci., USA
, 86: 8763-8767 (1989); W. E. Mercer, et. al.,
Proc. Natl. Acad. Sci. USA
, 87: 6166-6170 (1990); C. A. Finlay, et. al.,
Cell
, 57: 1083-1093 (1989)] and also transdominantly inhibit wild-type p53 [J. Martinez, et. al.,
Genes Dev
., 5: 151-159 (1991); J. Bargonetti, et. al,
Genes Dev
., 6: 1886-1898, (1992); S. E. Kern, et. al.,
Science
, 256: 827-830 (1992); J. Miner and E. A. Medcalf,
Cell
, 65: 765-774 (1991); D. Eliyahu, et. al,
Nature
312: 646-649 (1984); L. F. Parada, et. al.,
Nature
312: 649-651 (1984); J. R. Jenkins, et. al.,
Nature
312: 651-654 (1984)]. There is significant evidence that transdominant inhibition of wild-type p53 is mediated by sequestration of wild-type p53 into inactive mutant/wild-type heterotetramers. The ability of tumor-derived p53 mutants to transdominantly inhibit wild-type p53 requires an intact tetramerization domain. The isolated p53 tetramerization domain also inhibits wild-type p53 function [T. Unger, et. al.,
Mol. Cell. Biol
., 13: 5186-5194 (1993); E. Shaulian, et. al.,
Mol. Cell. Biol
., 12: 5581-5592 (1992); M. Reed, et. al.,
Gene Expression
3: 95-107 (1993)]. Chimeric p53 proteins that contain a heterologous tetramerization domain, instead of the native p53 tetramerization domain, are not transdominantly inhibited by tumor-derived p53 mutants.
Induction of wild-type p53 function in tumor cells leads to growth arrest or apoptosis (L. Diller, cited above; S. J. Baker, cited above; W. E. Mercer, cited above; E. Yonish-Rouach, et. al.,
Nature
, 352: 345-347 (1991); P. Shaw, et. al.,
Proc. Natl. Acad. Sci. USA
, 89: 4495-4499 (1992)]. Thus, introduction of wild-type p53 into tumor cells could in principle be utilized for therapy [T. Fujiwara, et. al.,
Cancer Res
., 53: 4129-4133 (1993); T. Fujiwara, et. al.,
Cancer Res
., 54: 2287-2291 (1994); T. Liu, et. al.,
Cancer Res
., 54: 3662-3667 (1994). One obstacle to the effectiveness of such therapy, however, is that about half of all human tumors express dominant negative p53 mutants [Harris et al, cited above, S. Friend et al, cited above, J. Bargonetti et al, cited above; S. E. Kern et. al., cited above.].
The design and analysis of a p53 chimeric protein in which the native p53 oligomerization domain is substituted with a modified leucine zipper that assembles as a tetramer has been reported [See, e.g., U. S. Pat. No. 5,573,925]. This engineered p53 protein is not transdominantly inhibited by tumor-derived p53 mutants, but is not as active as wild-type p53.
What are needed are constructs and methods for providing p53 tumor suppressor activity, particularly in the presence of insufficient activity due to the presence of tumor-derived p53 mutants or other factors.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a modified p53 protein containing a p53 DNA binding domain and a modified p53 tetramerization domain that does not hetero-oligomerize with the native p53 tetramerization domain. The modified tetramerization contains the sequence of amino acid residues 325 to 355 of a human p53 or a homologous p53 sequence which has been modified at one or more of the following residues (with reference to the numbering scheme of the human p53 in SEQ ID NO: 2): Leu330 substituted with Phe; Met340 substituted with Phe; Ala347 substituted with Ile; Leu348 substituted with Met; Ala353 substituted with Leu; Gln354 substituted with Leu; and/or Ala355 substituted with Asp.
In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier; and a modified p53 protein of the invention.
In still another aspect, the present invention provides a nucleic acid molecule encoding a modified p53 protein of the invention. Optionally, this molecule may further contain regulatory sequences which direct expression of said modified tetramerization domain or modified p53 protein.
In yet another aspect, the present invention provides a vector containing a nucleic acid sequence of the invention and sequences which direct expression thereof
In still a further aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier.
In yet a further aspect, the present invention provides a method of treating a condition associated with abnormal p53 activity comprising the step of administering a pharmaceutical composition as described above.
Other aspects and advantages of the present invention are described further in the detailed description of the preferred embodiments thereof
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides modified p53 proteins containing modified tetramerization domains having altered oligomerization specificity. The modified p53 proteins of the invention form homo-tetramers and have tumor suppressor function, but do not hetero-oligomerize with tumor-derived p53 mutants and resist transdominant inhibition.
The modified tetramerization domains of the invention contain at least one of the following substitutions within the hydrophobic core of the p53 tetramerization domain, which is located in the region of about amino acids 325 to about 355, with reference to human p53: Leu330 substituted with Phe; Met340 substituted with Phe; Ala347 substituted with Ile; Leu348 substituted with Met; Ala353 substituted with Leu; Gln354 substituted with Leu and/or Ala355 substituted with Asp. In a currently preferred embodiment, a modified p53 protein construct of the invention contains a modified human p53 tetramerization domain, with all seven of the amino acid substitutions identified herein.
The present inventors have found that the modified p53 tetramerization domains, when engineered into modified
Halazonetis Thanos D.
Stavridi Elena S.
Crouch Deborah
Howson and Howson
The Wistar Institute of Anatomy and Biology
Woitach Joseph T.
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