Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-07-31
2002-07-02
Huff, Sheela (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S320100, C435S325000, C435S069100
Reexamination Certificate
active
06414130
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention provides a HER-2 binding antagonist. Specifically, intron retention has generated a novel HER-2 antagonist polypeptide that binds to the HER-2 receptor.
BACKGROUND OF THE INVENTION
The HER-2
eu (erbB-2) oncogene encodes a receptor-like tyrosine kinase (RTK) that has been extensively investigated because of its role in several human carcinomas (Hynes and Stern,
Biochim. et Biophy. Acta
1198:165-184, 1994; and Dougall et al.,
Oncogene
9:2109-2123, 1994) and in mammalian development (Lee et al.,
Nature
378:394-398, 1995). The sequence of the HER-2 protein was determined from a cDNA that was cloned by homology to the epidermal growth factor receptor (EGFR) mRNA from placenta (Coussens et al.,
Science
230:1132-1139, 1985) and from a gastric carcinoma cell line (Yamamoto et al.,
Nature
319:230-234, 1986). The HER-2 mRNA was shown to be about 4.5 kb (Coussens et al.,
Science
230:1132-1139, 1985; and Yamamoto et al.,
Nature
319:230-234, 1986) and encodes a transmembrane glycoprotein of 185 kDa in normal and malignant human tissues (p185HER-2) (Hynes and Steen,
Biochim. et Biophys. Acta
1198:165-184, 1994; and Dougall et al.,
Oncogene
9:2109-2123, 1994). The function of the HER-2 gene has been examined mainly by expressing the cDNA corresponding to the 4.5 kb transcript in transfected cells and from the structure and biochemical properties of the 185 kDa protein product. P185HER-2 consists of a large extracellular domain, a transmembrane segment, and an intracellular domain with tyrosine kinase activity (Hynes and Stern,
Biochim. et Biophys. Acta
1198:165-184, 1994; and Dougall et al.,
Oncogene
9:2109-2123, 1994). Overexpression of p185HER-2 causes phenotypic transformation of cultured cells (DiFiore et al.,
Science
237:178-182, 1987; and Hudziak et al.,
Proc. Natl. Acad. Sci. USA
84:7159-7163, 1987) and has been associated with aggressive clinical progression of breast and ovarian cancer (Slamon et al.,
Science
235:177-182, 1987; and Slamon et al.,
Science
244:707-712, 1989). p185HER-2 is highly homologous to the EGFR. However, a ligand that directly binds with high affinity to p185HER-2 has not yet been identified. Moreover, the signaling activity of HER-2 may be mediated through heterodimerization with other ligand-binding members of the EGFR family (Carraway and Cantley,
Cell
78:5-8, 1994; Earp et al.,
Breast Cancer Res. Treat.
35:115-132, 1995; and Qian et al.,
Oncogene
10:211-219, 1995).
Divergent proteins, containing regions of the extracellular domains of HER family RTKs, are generated through proteolytic processing of full length receptors (Lin and Clinton,
Oncogene
6:639-643, 1991; Zabrecky et al.,
J. Biol. Chem.
266:1716-1720, 1991; Pupa et al.,
Oncogene
8:2917-2923, 1993; Vecchi et al.,
J. Biol. Chem.
271:18989-18995, 1996; and Vecchi and Carpenter,
J. Cell Biol.
139:995-1003, 1997) and through alternative RNA processing (Petch et al.,
Mol. Cell. Biol.
10:2973-2982, 1990; Scott et al.,
Mol. Cell. Biol.
13:2247-2257, 1993; and Lee and Maihle,
Oncogene
16:3243-3252, 1998). The extracellular domain of p185HER-2 is proteolytically shed from breast carcinoma cells in culture (Petch et al.,
Mol. Cell. Biol.
10:2973-2982, 1990; Scott et al.,
Mol. Cell. Biol.
13:2247-2257, 1993; and Lee and Maihle,
Oncogene
16:3243-3252, 1998), and is found in the serum of some cancer patients (Leitzel et al.,
J. Clin. Oncol.
10:1436-1443, 1992) where it is may be a serum marker of metastatic breast cancer (Leitzel et al.,
J. Clin. Oncol.
10:1436-1443, 1992) and may allow escape of HER-2-rich tumors from immunological control (Baselga et al.,
J. Clin. Oncol.
14:737-744, 1966; and Brodowicz et al.,
Int. J. Cancer
73:875-879, 1997).
A truncated extracellular domain of HER-2 is also the product of a 2.3 kb alternative transcript generated by use of a polyadenylation signal within an intron (Scott et al.,
Mol. Cell. Biol.
13:2247-2257, 1993). The alternative transcript was first identified in the gastric carcinoma cell line, MKN7 (Yamamoto et al.,
Nature
319:230-234, 1986; and Scott et al.,
Mol. Cell. Biol.
13:2247-2257, 1993) and the truncated receptor was located within the perinuclear cytoplasm rather than secreted from these tumor cells (Scott et al.,
Mol. Cell. Biol.
13:2247-2257, 1993). However, no particular therapeutic, diagnostic or research utility has been ascribed to this truncated extracellular domain polypeptide. A truncated extracellular domain of the EGFR, generated by alternative splicing (Petch et al.,
Mol. Cell. Biol.
10:2973-2982, 1990) is secreted, exhibits ligand-binding, and dimerization properties (Basu et al.,
Mol. Cell. Biol.
9:671-677, 1989), and may have a dominant negative effect on receptor function (Basu et al.,
Mol. Cell. Biol.
9:671-677, 1989; and Flickinger et al.,
Mol. Cell. Biol.
12:883-893, 1992).
Therefore, there is a need in the art to find molecules that bind to cellular HER-2 and particularly molecules that bind to different sites than humanized antibodies to HER-2 (e.g., HERCEPTIN®. Such molecules would be useful therapeutic agents for various cancers that overexpress HER-2.
SUMMARY OF THE INVENTION
The present invention provides an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 at an affinity of at least 10
8
. Preferably, the isolated polypeptide is from about 69 to 79 amino acids in length. Preferably, the isolated polypeptide binds to a site on the ECD of HER-2 that is different from the site of binding of HERCEPTIN® (a marketed humanized monoclonal antibody that is used for the treatment of cancer and that binds to the ECD of HER-2).
The present invention further provides an isolated DNA sequence that codes on expression for a polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 at an affinity of at least 10
8
. Preferably, the isolated polypeptide is from about 69 to 79 amino acids in length. Preferably, the isolated polypeptide binds to a site on the ECD of HER-2 that is different from the site of binding of HERCEPTIN® (a marketed humanized monoclonal antibody that is used for the treatment of cancer and that binds to the ECD of HER-2). The present invention further provides a transfected cell comprising an expression vector having a DNA sequence that codes on expression for a polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 at an affinity of at least 10
8
.
The present invention further provides an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO. 2, wherein the C terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present. Preferably, the isolated polypeptide is from about 350 to 419 amino acids in length and four N-linked glycosylation sites are present. Preferably, the isolated polypeptide binds to a site on the ECD of HER-2 that is different from the site of binding of HERCEPTIN® (a marketed humanized monoclonal antibody that is used for the treatment of cancer and that binds to the ECD or HER-2).
The present invention further provides an isolated DNA sequence that codes on expression for a polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO. 3, wherein the C terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present. Preferably, the isolated polypeptide is from about 350 to 419 amino acids in length and four N-linked glycosylation are present. The present invention further provides a transfected cell comprising an expression vector having a DNA sequence that codes on expression for a polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO. 3, wher
Clinton Gail M.
Doherty Joni Kristin
Davis , Wright, Tremaine, LLP
Davison Barry L.
Huff Sheela
Hunt Jennifer
Oregon Health & Science University
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