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
1998-06-16
2004-04-27
Caputa, Anthony C. (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...
C435S320100, C536S023100, C536S023500, C536S024310
Reexamination Certificate
active
06727077
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel human gene encoding a polypeptide which is a novel member of the heregulin family. More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named heregulin-like factor, hereinafter referred to as “HLF”. HLF polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to primary cancers, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying agonists and antagonists of HLF activity.
BACKGROUND OF THE INVENTION
The proto-oncogene termed erbB2 (or HER2) encodes a 185 kDa transmembrane tyrosine kinase molecule designated p185erbB2. The overexpression of this receptor molecule correlates strongly with a poor prognosis in a number of human cancers including, among others, breast, ovarian, endometrium, fallopian tube, cervix, and colon (Nowak, F., et al.,
Exp. Cell Res.
231:251-259, 1997; Cirisano, F. D. and Karlan, B. Y.,
J. Soc. Gynecol. Investig.
3(3):99-105; 1996). Variously spliced transcripts of the heregulin (HRG) gene have been found to indirectly stimulate p185erbB2 through transphosphorylation or receptor heterodimerization with erbB3 and p180erbB4. A 45 kDa protein, designated HRG alpha specifically induces tyrosine phosphorylation of p185erbB2 and has been purified from the conditioned medium of a human breast tumor cell line (Holmes, W. E., et al.,
Science
256:1205-1210; 1992). A second, related HRG molecule of 52 kDa, which may be the product of a novel gene, rather than a novel HRG gene splice product, has been identified which exhibits similar characteristics including induction of transient membrane ruffling, lamellipodia formation, cell motility and proliferation of human breast cancer cells (Kung, W., et al.,
Biochem. Biophys. Res. Commun.
202(3):1357-1365; 1994). In addition, more recent studies have reported that heregulins can induce tyrosine phosphorylation not only of p185erbB2, but of several additional EGFR-related family members including erbB3 and p180erbB4 (Tzahar, E., et al.,
J. Biol. Chem.
269:25226-25223; 1994; Plowman, G. D., et al.,
Nature
366:473-475; 1993).
Lewis and colleagues (
Cancer Res.
56:1457-1465; 1996) recently performed an extensive analysis of the effects of the heregulin family of proteins on a panel of breast and ovarian tumor cell lines. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody 4D5. In nearly all cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. In addition to the effects of the heregulin family of proteins on breast and ovarian cells, similar effects have also been recently observed on human Schwann cells (Levi, A. D., et al.,
J. Neurosci.
15(2):1329-1340; 1995; Morrissey, T. K., et al.,
Proc. Natl. Acad. Sci. USA
92(5):1431-1435; 1995) suggesting that the heregulin family of proteins play a key role in the genesis of a number of cancers.
The heregulin family of proteins consists at least of a number of splice variants of heregulin, the Neu differentiating factor, the glial growth factors-I, -II, and -III, and a protein that stimulates muscle acetylcholine receptor synthsis (ARIA). In addition to the obvious role such polypeptides may play in oncogenic events, these proteins have also been exploited as Pseudomonas exotoxin A fusion proteins to inhibit the growth of several mammary carcinoma cell lines as well as to cause growth retardation of transplanted human breast tumor cells in mice (Jeschke, M., et al.,
Int. J. Cancer
60(5):730-739; 1995).
Thus, there is a need for polypeptides that function as regulators of oncogenic events and existing tumors. Therefore, there is a need for identification and characterization of such human polypeptides which can play a role in detecting, preventing, ameliorating or correcting such disorders.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the HLF polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNA clone was deposited as DNA plasmid with the American Type Culture Collection (“ATCC”) on Jun. 19, 1997, and assigned ATCC Deposit Number 209123. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209. The nucleotide sequence determined by sequencing the deposited HLF clone, which is shown in
FIGS. 1A and 1B
(SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 157 amino acid residues, beginning in frame with a serine residue at the amino-terminal end of the polypeptide corresponding to nucleotide positions 2-4, and a predicted molecular weight of about 17.7 kDa. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence shown in SEQ ID NO:2, or the complete amino acid sequence encoded by the cDNA clone in ATCC Deposit Number 209123, which molecules also can encode additional amino acids fused to the N-terminus of the HLF amino acid sequence.
The HLF protein of the present invention shares sequence homology with the translation product of the human mRNA for heregulin (
FIG. 2
; SEQ ID NO:3), including the following conserved domains: (a) the predicted extracellular domain of about 101 amino acids; (b) the predicted transmembrane domain of about 19 amino acids, and (c) the predicted intracellular domain of about 35 amino acids. Heregulin is thought to be important in oncogenesis. The homology between heregulin and HLF indicates that HLF may also be involved in oncogenesis.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the HLF polypeptide having the complete amino acid sequence in SEQ ID NO:2 (i.e., positions 1 to 157 of SEQ ID NO:2) or the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209123; (b) a nucleotide sequence encoding the predicted extracellular domain of the HLF polypeptide having the amino acid sequence in SEQ ID NO:2 (i.e., positions 1 to 101 of SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 209123; (c) a nucleotide sequence encoding the predicted transmembrane domain of the HLF polypeptide having the amino acid sequence in SEQ ID NO:2 (i.e., positions 102 to 121 of SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 209123; (d) a nucleotide sequence encoding the predicted intracellular domain of the HLF polypeptide having the amino acid sequence in SEQ ID NO:2 (i.e., positions 122 to 157 of SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 209123; (e) a nucleotide sequence encoding a soluble HLF polypeptide having the extracellular and intracellular domains but lacking the transmembrane domain; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a) through (e) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (or as stated in another way, a nucleotide sequence at most 10% different, and more preferably 5%, 4%, 3%, 2% or 1% different from), any of the nucleotide sequences in (a) through (f) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a) through (f) above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the inventio
Hijazi Mai
King C. Richter
Ruben Steven M.
Young Paul E.
Canella Karen A.
Caputa Anthony C.
Human Genome Sciences Inc.
Human Genome Sciences Inc.
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