Antinsense modulation of Her-1 expression

Details Antinsense modulation of Her-1 expression Antinsense modulation of Her-1 expression

2000-09-29

2002-09-03

LeGuyader, John L. (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S091100, C435S325000, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500

Reexamination Certificate

active

06444465

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods of modulating the expression of Her-1. Many human tumors have been found to overexpress this receptor and such enhanced expression has been shown to be correlated with poor prognosis. In particular, this invention relates to oligonucleotides specifically hybridizable with nucleic acids encoding human Her-1. These oligonucleotides have been found to inhibit the expression of Her-1.
BACKGROUND OF THE INVENTION
Her-1, also known as epidermal growth factor receptor (EGFR), is a specific receptor for epidermal growth factor (EGF) and transforming growth factor-&agr; (TGF-&agr;). When these mitogenic polypeptides bind to Her-1, tyrosine kinase activity of the receptor is induced, and this in turn triggers a series of events which regulate cell growth. A number of malignant and non-malignant disease conditions are now believed to be associated with Her-1, particularly aberrant expression of Her-1. Aberrant expression includes both increased expression of normal Her-1 and expression of mutant Her-1. Overexpression of Her-1 is found in many human tumors including most glioblastomas and breast, lung, ovarian, colorectal, bladder, pancreatic, squamous cell and renal carcinomas. Elevated Her-1 levels correlate with poor prognosis in human tumors. Her-1 is also implicated in nonmalignant diseases, such as psoriasis. The sequence of the mRNA encoding human Her-1 is known. Ullrich et al.,
Nature
, 1984, 309, 418; GenBank Accession Number X00588; and Kraus et al.,
Proc. Natl. Acad. Sci. USA
, 1989, 86, 9193; GenBank Accession Number M29366. The gene encoding Her-1 is also known as ERBB3 or c-erb-B1. Two Her-1 transcripts typically appear on Northern blots, one measuring 10 kb and one measuring 5.6 kb.
A number of inhibitors of Her-1 have been shown to be effective in inhibiting the growth of human tumor cells. Monoclonal antibodies to Her-1 and drugs which inhibit Her-1 tyrosine kinase activity can inhibit the growth of human cancer cell xenografts in nude mice. Normanno et al.,
Clin. Cancer Res
., 1996, 2, 601. The drug PD153035, which inhibits Her-1 tyrosine kinase activity, can inhibit the growth of A431 cells in nude mice, and tyrphostins, which inhibit the activity of Her-1 as well as other tyrosine kinases, have been shown to inhibit the growth of squamous carcinoma in nude mice. Kunkel et al.,
Invest. New Drugs
, 1996, 13, 295 and Yoneda et al.,
Cancer Res
., 1991, 51, 4430.
Vectors expressing Her-1 nucleic acid sequences in an orientation complementary to mRNA have been used to study the effects of Her-1 on proliferation of cultured cancer cells. Transfectants of the human epidermoid carcinoma KB cell line expressing Her-1 cDNA or RNA sequences in an orientation complementary to mRNA exhibited restored serum-dependent growth and impaired colony formation and growth in agar. Moroni et al.,
J. Biol. Chem
., 1992, 267, 2714. Human pancreatic carcinoma cells of the PC-7 cell line transfected with vectors expressing Her-1 cDNA sequences in an orientation complimentary to mRNA showed inhibited cell growth, colony formation and tumorigenicity in nude mice. Liu et al.,
Chinese Medical Journal
., 1995, 108, 653. Transfection of human colon cancer cell lines with Her-1 RNA expression vectors producing an oligonucleotide complementary to mRNA caused a reduction in cell proliferation and ability to grow on soft agar. Rajagopal et al.,
Int. J. Cancer
, 1995, 62, 661. Human rhabdomyosarcoma cells transfected with a plasmid expressing Her-1 cDNA in an orientation complementary to mRNA had greatly impaired proliferation. De Giovanni et al.,
Cancer Res
., 1996, 56, 3898.
Considerable research is being directed to the application of oligonucleotides complementary to mRNA and other oligomers for therapeutic purposes. Oligonucleotides complementary to mRNA have already been employed as therapeutic moieties in the treatment of disease states in animals and man, and compositions comprising oligomers complementary to mRNA have been shown to be capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Further, oligonucleotides complementary to mRNA have been safely administered to humans and clinical trials of approximately a dozen oligonucleotide drugs targeted to viral and cellular gene products are underway.
Oligodeoxyribonucleotides complementary to mRNA targeted to Her-1 have been encapsulated into liposomes linked to folate via a polyethylene glycol linker and delivered into cultured human epidermoid carcinoma KB cells. The oligonucleotides were a phosphodiester (P═O) 15-mer complementary to the Her-1 gene stop codon, or the same sequence with three phosphorothioate (P═S) linkages at each end. Both of these oligonucleotides reduced KB cell proliferation by greater than 90% after treatment with 3 &mgr;M oligonucleotide in folate-PEG-liposomes. In contrast, free P═O oligonucleotide caused almost no growth inhibition, and free P═S-capped oligonucleotide caused only a 15% growth inhibition, even at this high dosage level. Her-1 expression, measured by indirect immunofluorescence, was virtually abolished in cells treated with either of the folate-PEG-liposome-encapsulated oligonucleotides but Her-1 expression was qualitatively similar to untreated cells after treatment with free oligonucleotide. Wang et al.,
Proc. Natl. Acad. Sci. USA
, 1995, 92, 3318.
A 15-mer phosphorothioate oligonucleotide complementary to the translation initiation region of Her-1 mRNA was found to inhibit cell proliferation by over 25% in A431 cells, derived from a vulval carcinoma. This activity, though dose-dependent from 1-25 &mgr;M, was not mediated by an antisense mechanism, as demonstrated by a lack of reduction in either Her-1 protein or mRNA after oligonucleotide treatment. In addition, an 18-mer oligonucleotide complementary to mRNA targeted to the same region had no effect even at the highest (25 &mgr;M) dose, and neither oligonucleotide had any effect in the two other tumor-derived cell lines tested. Coulson et al.,
Mol. Pharm
., 1996, 50, 314.
The suppression of growth of pancreatic carcinoma cell lines by undisclosed oligonucleotides complementary to mRNA inhibiting the expression of TGF-&agr; and/or the Her-1 has been reported. Hall et al., unpublished data, reported in Hall and Lemoine, Models of Pancreatic Cancer, in
Cancer Surveys
, Volume 16: The Molecular Pathology of Cancer, 1993, p. 135-155.
Rubenstein et al. have reported treatment of established human-derived prostate tumor xenografts in nude mice by intralesional injection of oligonucleotides complementary to mRNA directed against mRNAs encoding TGF-&agr; and Her-1. The oligonucleotides included 39-mers complementary to 18 bases located 5′ and 3′ from the AUG mRNA translation initiation codon of either TGF-&agr; or Her-1 sequence. The oligonucleotides were phosphorothioated at each of three terminal bases at both the 5′ and 3′ ends. The oligonucleotides were administered either alone or in combination, with the combination treatment proving most effective.
J. Surg. Oncol
., 1996, 62, 194. In U.S. Pat. No. 5,610,288, Rubenstein et al. disclose polynucleotides of about 20 to 50 nucleic acid bases, most preferably about 40 nucleic acid bases in length, which preferentially hybridize to the start codon of the mRNA encoding Her-1. A preferred embodiment is a 39-mer including 18 bases complementary to the 5′ side of the translation initiation codon. This oligonucleotide inhibited PC-3 cell growth when administered in combination with an oligonucleotide complementary to mRNA targeted to TGF-&agr;. Alone, the Her-1 oligonucleotide gave inhibition of cell growth equivalent to that achieved with an inverted (5′ to 3′) version of the same sequence.
Disclosed in U.S. Pat. No. 5,914,269 (Bennett, et al.) are oligonucleotides targeting Her-1 and methods of modulating the expression of Her-1 using said oligonucleotides. Three of these oligonucleotides and their analogs have also been used

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