Clinton, Gail M. (Portland, OR, US)

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09/638834

07/08/2008

08/14/2000

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Oregon Health Sciences University (Portland, OR, US)

530/300

424/130.1, 514/2, 530/350

A61K38/17; A61K39/395; C07K14/705; C07K14/435

530/300, 424/130.1, 514/2, 530/350

4376110	Immunometric assays using monoclonal antibodies	March, 1983	David et al.
4933294	Method of detecting truncated epidermal growth factor receptors	June, 1990	Waterfield et al.
4946778	Single polypeptide chain binding molecules	August, 1990	Ladner et al.
5401638	Detection and quantification of neu related proteins in the biological fluids of humans	March, 1995	Carney et al.	435/7.23
5464751	Ligand for the neu gene product	November, 1995	Greene et al.
5514554	Methods and compositions for cancer therapy and for prognosticating responses to cancer therapy	May, 1996	Bacus
5571894	Recombinant antibodies specific for a growth factor receptor	November, 1996	Wels et al.
5578482	Ligand growth factors that bind to the erbB-2 receptor protein and induce cellular responses	November, 1996	Lippman et al.
5604107	Detection of neu p185 in cell lysates	February, 1997	Carney et al.	435/7.23
5677171	Monoclonal antibodies directed to the HER2 receptor	October, 1997	Hudziak et al.
5705157	Methods of treating cancerous cells with anti-receptor antibodies	January, 1998	Greene
5720937	In vivo tumor detection assay	February, 1998	Hudziak et al.	424/9.34
5747261	Protein related to but distinct from EGF receptor and antibodies reactive therewith	May, 1998	King et al.
5756456	Methods involving sensory and motor neuron derived factor (SMDF)	May, 1998	Ho et al.
5763213	Sensory and motor neuron derived factor (SMDF)	June, 1998	Ho et al.
5783186	Antibody-induced apoptosis	July, 1998	Arakawa et al.	424/143.1
5811098	Antibodies to HER4, human receptor tyrosine kinase	September, 1998	Plowman et al.
5837523	Compositions and methods of treating tumors	November, 1998	Greene et al.
5861301	Recombinant kinase insert domain containing receptor and gene encoding same	January, 1999	Terman et al.
5874528	Binding peptides which interact with ligand growth factors of the epidermal growth factor receptor and erbB-2 receptor	February, 1999	Lupu et al.
5876712	Immune reactivity to HER-2/neu protein for diagnosis and treatment of malignancies in which the HER-2/neu oncogene is associated	March, 1999	Cheever et al.
5910583	Antisense oligonucleotides against ERBB-2	June, 1999	Marks et al.
5919764	Compounds that bind to p185 and methods of using the same	July, 1999	Greene et al.
5985553	erbB-2 gene segments, probes, recombinant DNA and kits for detection	November, 1999	King et al.
6015567	HER2 extracellular domain	January, 2000	Hudziak et al.	424/277.1
6020306	Receptor-type tyrosine kinase and use thereof	February, 2000	Boyd et al.
6045797	Treatment or diagnosis of diseases or conditions associated with a BLM domain	April, 2000	Margolis et al.
6054561	Antigen-binding sites of antibody molecules specific for cancer antigens	April, 2000	Ring
6166082	Substituted 5-biarylpentanoic acids and derivatives as matrix metalloprotease inhibitors	December, 2000	Kluender et al.
6174889	Bicyclic heteroaromatic compounds as protein tyrosine kinase inhibitors	January, 2001	Cockerill et al.
6204011	Human receptor tyrosine kinase, KDR	March, 2001	Kendall et al.
6267958	Protein formulation	July, 2001	Andya et al.
6333169	HER2 extracellular domain	December, 2001	Hudziak et al.
6359115	Human receptor tyrosine kinase, KDR	March, 2002	Kendall et al.
6375929	Gene therapy for inhibition of angiogenesis	April, 2002	Thomas, Jr. et al.
6387371	Monoclonal antibodies directed to the HER2 receptor	May, 2002	Hudziak et al.
6399063	Monoclonal antibodies directed to the HER2 receptor	June, 2002	Hudziak et al.
6399743	Isolation and characterization of a rat epidermal growth factor related protein	June, 2002	Majumdar
6414130	HER-2 binding antagonists	July, 2002	Doherty et al.
6417168	Compositions and methods of treating tumors	July, 2002	Greene et al.
6441143	Methods and compositions for determining HER-2/neu expression	August, 2002	Koski
6541214	N-terminally truncated HER-2/neu protein as a cancer prognostic indicator	April, 2003	Clinton
20010014326	Protein formulation	August, 2001	Andya et al.
20010021505	ALK protein tyrosine kinase/receptor and ligands thereof	September, 2001	Morris et al.
20020045215	Isolation and characterization of a rat epidermal growth factor related protein	April, 2002	Majumdar
20020064785	Gene detection assay for improving the likelihood of an effective response to an ErbB antagonist cancer therapy	May, 2002	Mass
20020146420	Protein tyrosine kinase agonist antibodies	October, 2002	Bennett et al.
20020155527	C-erbB-2 exrernal domain: gp75	October, 2002	Stuart et al.
20020165193	Compositions and methods of treating tumors	November, 2002	Greene et al.
20020172984	Oligomerized receptors which affect pathways regulated by transmembrane ligands for elk-related receptor tyrosine kinases	November, 2002	Holland et al.
20020173458	Protein tyrosine kinase receptor polynucleotides, polypeptides, and antibodies	November, 2002	Ruben et al.
20030036179	Secreted and transmembrane polypeptides and nucleic acids encoding the same	February, 2003	Baker et al.
20030044842	Secreted and transmembrane polypeptides and nucleic acids encoding the same	March, 2003	Desnoyers et al.
20030044945	Secreted and transmembrane polypeptides and nucleic acids encoding the same	March, 2003	Baker et al.
20030055239	Human receptor tyrosine kinase, KDR	March, 2003	Kendall et al.
20030059863	N-terminally truncated HER-2/neu protein as a cancer prognostic indicator	March, 2003	Clinton
20030078222	Human receptor tyrosine kinase	April, 2003	Ghildyal et al.
20040022785	Expression of herstatin, an alternative HER-2/neu product, in cells that express either p185HER-2 or the EGF receptor inhibits receptor activity and cell growth	February, 2004	Clinton
20040052796	Expression of herstatin, an alternative to her-2/neu product, in cells that express either p85her-2 or the egf receptor inhibits receptor activity and cell growth	March, 2004	Clinton et al.
20050239088	Intron fusion proteins, and methods of identifying and using same	October, 2005	Shepard et al.

AUB-64135	March, 1991
CA2055441	November, 1990
CA2042064	February, 1991
CA2187781	September, 1995
CA2260061	January, 1998
CA2418083	February, 2002
EP0412116	April, 1989			DETECTION OF NEU GENE EXPRESSION AND PRODUCTS.
EP0494135	September, 1989			DETECTION AND QUANTIFICATION OF -i(NEU) RELATED PROTEINS IN THE BIOLOGICAL FLUIDS OF HUMANS.
EP0119528	May, 1992			Monoclonal antibodies to human renal cancer antigens and method.
EP0491675	June, 1992			Improvements relating to growth factors.
EP0171407	November, 1993			IMPROVEMENTS RELATING TO GROWTH FACTORS.
EP0474727	July, 1997			HER2 EXTRACELLULAR DOMAIN.
EP0600744	March, 1998			Tumor inhibiting tetrapeptides bearing modified phenethyl amides
EP1006194	June, 2000			C-erbB-2 external domain: GP75
EP1114863	July, 2001			Heregulins (HRGs), binding proteins of P185erb2
EP0444181	October, 2001			C-ERBB-2 EXTERNAL DOMAIN: GP75.
EP1304110	April, 2003			Bicyclic heteroaromatic compounds as protein tyrosine kinase inhibitors
EP1308455	May, 2003			A composition comprising anti-HER2 antibodies
WO/1985/003357	August, 1985			IMPROVEMENTS RELATING TO GROWTH FACTORS
WO/1989/010412	November, 1989			DETECTION OF NEU GENE EXPRESSION AND PRODUCTS
WO/1990/014357	November, 1990			HER2 EXTRACELLULAR DOMAIN
WO/1991/002062	February, 1991			C-ERBB-2 EXTERNAL DOMAIN: GP75
WO/1991/005264	April, 1991			DETECTION AND QUANTIFICATION OF NEU RELATED PROTEINS IN THE BIOLOGICAL FLUIDS OF HUMANS
WO/1991/011715	August, 1991			DETECTION OF A CHARACTERISTIC ANTIGEN FROM CARCINOMA IN BODY FLUIDS
WO/1992/014748	September, 1992			IDENTIFICATION OF A NOVEL HUMAN RECEPTOR TYROSINE KINASE GENE
WO/1992/020798	November, 1992			HEREGULINS (HRGs), BINDING PROTEINS OF P185?erb2�
WO/1993/014124	July, 1993			TIE, A NOVEL ENDOTHELIAL CELL RECEPTOR TYROSINE KINASE
WO/1995/025166	September, 1995			METHODS FOR TREATMENT OR DIAGNOSIS OF DISEASES OR CONDITIONS ASSOCIATED WITH ABNORMAL SIGNAL TRANSDUCTION
WO/1995/030331	November, 1995			COMPOSITIONS AND METHODS OF TREATING TUMORS
WO/1998/023782	June, 1996			A NEW ETS-RELATED GENE OVEREXPRESSED IN HUMAN BREAST AND EPITHELIAL CANCERS
WO/1998/002438	January, 1998			BICYCLIC HETEROAROMATIC COMPOUNDS AS PROTEIN TYROSINE KINASE INHIBITORS
WO/1999/019732	April, 1999			METHODS AND COMPOSITIONS FOR DETECTING HEPATITIS E VIRUS
WO/1999/039729	August, 1999			USE OF HEREGULIN AS AN EPITHELIAL CELL GROWTH FACTOR
WO/2000/027426	May, 2000			METHOD FOR ENHANCING PROLIFERATION OF INNER EAR HAIR CELLS USING LIGANDS FOR HER2 AND/OR HER3 RECEPTORS
WO/2000/029609	May, 2000			N-TERMINALLY TRUNCATED HER-2/NEU PROTEIN AS A CANCER PROGNOSTIC INDICATOR
WO/2000/044403	August, 2000			HER-2 BINDING ANTAGONISTS
WO/2001/001748	January, 2001			PEPTIDE COMPOUNDS THAT BIND HER2
WO/2001/026607	April, 2001			SMDF AND GGF NEUREGULIN SPLICE VARIANT ISOFORMS AND USES THEREOF
WO/2001/061356	August, 2001			HER-2 BINDING ANTAGONISTS
WO/2001/089566	November, 2001			GENE DETECTION ASSAY FOR IMPROVING THE LIKELIHOOD OF AN EFFECTIVE RESPONSE TO AN ERBB ANTAGONIST CANCER THERAPY
WO/2002/011677	February, 2002			TREATMENT OF HYPERPROLIFERATIVE DISEASES WITH EPIDERMAL GROWTH FACTOR RECEPTOR ANTAGONISTS
WO/2002/012335	February, 2002			A NEW EGF MOTIF REPEAT PROTEIN OBTAINED FROM A cDNA LIBRARY FROM HS-5 STROMAL CELL LINE
WO/2002/014470	February, 2002			EXPRESSION OF HERSTATIN, AN ALTERNATIVE HER-2/NEU PRODUCT, IN CELLS THAT EXPRESS EITHER P185HER-2 OR THE EGF RECEPTOR INHIBITS RECEPTOR ACTIVITY AND CELL GROWTH
WO/2002/090991	November, 2002			PROTEINS AND GENES FOR DIAGNOSIS AND TREATMENT OF ERBB2-RELATED CANCER
WO/2003/025141	March, 2003			GENETIC ANALYSIS FOR STRATIFICATION OF CANCER RISK
WO/2003/035843	May, 2003			SCREENING SYSTEM FOR MODULATORS OF HER2 MEDIATED TRANSCRIPTION AND HER2 MODULATORS IDENTIFED THEREBY
WO/2003/060071	July, 2003			ALBUMIN FUSION PROTEINS
WO/2005/016966	February, 2005			INTRON FUSION PROTEINS, AND METHODS OF IDENTIFYING AND USING SAME

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Harris, Alana M.

Holleran, Anne L.

Davison, Barry L.
Davis Wright Tremaine LLP

We claim:

1. A method for treating a solid tumor characterized by expression of EGF receptor, comprising administering, to a subject in need thereof determined to have a tumor expressing the EGF receptor, an agent that binds to the extracellular domain of EGF receptor, wherein the agent is selected from the group consisting of (a) an isolated polypeptide comprising the amino acid sequence of SEQ ID NOS:11, 15 or 17-26, or fragments thereof of about 50 to 79 contiguous residues in length, wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, (b) an isolated and glycosylated polypeptide comprising the amino acid sequence of SEQ ID NOS:12, 38 or 27-36, or fragments thereof of about 80 to 419 contiguous residues in length, wherein the C-terminal 79 contiguous amino acids are present, wherein at least one N-linked glycosylation site is present, and wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, (c) a monoclonal antibody that binds to the extracellular domain of EGF receptor, and (d) combinations thereof, with the proviso that where the composition comprises the monoclonal antibody it also comprises at least one of the agents of (a) or (b).

2. The method of claim 1, wherein the solid tumor that expresses EGF receptor is selected from the group consisting of squamous cell carcinoma, lung carcinoma, colon carcinoma, and glial cell tumors.

3. The method of claim 1, wherein the agent is the isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:11, 15 and 17-26, and fragments thereof of about 50 to 79 contiguous residues in length, wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹.

4. The method of claim 1, wherein the agent comprises a combination of the isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:11, 15 and 17-26, and fragments thereof of about 50 to 79 contiguous residues in length, wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, and the monoclonal antibody that binds to the extracellular domain of EGF receptor.

5. The method of claim 1, wherein the agent is the isolated and glycosylated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:12, 38 and 27-36, and fragments thereof of about 80 to 419 contiguous residues in length, wherein the C-terminal 79 contiguous amino acids are present, wherein at least one N-linked glycosylation site is present, and wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹.

6. The method of claim 1, wherein the agent is a combination of the isolated and glycosylated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:12, 38 and 27-36, and fragments thereof of about 80 to 419 contiguous residues in length, wherein the C-terminal 79 contiguous amino acids are present, wherein at least one N-linked glycosylation site is present, and wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, and the monoclonal antibody that binds to the extracellular domain of EGF receptor.

7. The method of claim 1, wherein the isolated polypeptide of (a) comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:11, 15 and 17-26.

8. The method of claim 1, wherein the isolated polypeptide of (b) comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:12, 38 and 27-36.

9. The method of claim 1, wherein the isolated polypeptide of (a) consists of an amino acid sequence selected from the group consisting of SEQ ID NOS:11, 15 and 17-26.

10. The method of claim 1, wherein the isolated polypeptide of (b) consists of an amino acid sequence selected from the group consisting of SEQ ID NOS:12, 38 and 27-36.

11. A method for treating a solid tumor characterized by expression of EGF receptor, comprising administering, to a subject in need thereof, an agent that binds to the extracellular domain of EGF receptor; wherein the agent comprises the amino acid sequence of SEQ ID NO:11.

12. A method for treating a solid tumor characterized by expression of EGF receptor, comprising administering, to a subject in need thereof, an agent that binds to the extracellular domain of EGF receptor, wherein the agent comprises the amino acid sequence of SEQ ID NO:12.

13. The method of claim 11, wherein the agent consists of the amino acid sequence of SEQ ID NO:11.

14. The method of claim 12, wherein the agent consists of the amino acid sequence of SEQ ID NO:12.

15. A method for treating a solid tumor characterized by expression of EGF receptor, comprising administering, to a subject in need thereof determined to have a tumor expressing the EGF receptor, an agent that binds to the extracellular domain of EGF receptor, wherein the agent is selected from the group consisting of (a) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1, or fragments thereof of about 50 to 79 contiguous residues in length, wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, (b) an isolated and glycosylated polypeptide comprising the amino acid sequence of SEQ ID NO:2, or fragments thereof of about 80 to 419 contiguous residues in length, wherein the C-terminal 79 contiguous amino acids are present, wherein at least one N-linked glycosylation site is present, and wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, (c) a monoclonal antibody that binds to the extracellular domain of EGF receptor, and (d) combinations thereof, with the proviso that where the composition comprises the monoclonal antibody it also comprises at least one of the agents of (a) or (b).

16. The method of claim 15, wherein the solid tumor that expresses EGF receptor is selected from the group consisting of squamous cell carcinoma, lung carcinoma, colon carcinoma, and glial cell tumors.

17. The method of claim 15, wherein the agent is the isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1, and fragments thereof of about 50 to 79 contiguous residues in length, wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹.

18. The method of claim 15, wherein the agent comprises a combination of the isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, and fragments thereof of about 50 to 79 contiguous residues in length, wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, and the monoclonal antibody that binds to the extracellular domain of EGF receptor.

19. The method of claim 15 wherein the agent is the isolated and glycosylated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, and fragments thereof of about 80 to 419 contiguous residues in length, wherein the C-terminal 79 contiguous amino acids are present, wherein at least one N-linked glycosylation site is present, and wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹.

20. The method of claim 15, wherein the agent is a combination of the isolated and glycosylated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, and fragments thereof of about 80 to 419 contiguous residues in length, wherein the C-terminal 79 contiguous amino acids are present, wherein at least one N-linked glycosylation site is present, and wherein the polypeptide binds to the extracellular domain of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, and the monoclonal antibody that binds to the extracellular domain of EGF receptor.

This work was supported by a grant from the Department of Defense (DOD) Breast Cancer Research Program. The United States Government has certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to signaling through ErbB family member receptors, and to novel methods and compositions for modulating HER-2, and EGF receptor-mediated signaling.

BACKGROUND OF THE INVENTION

A HER-2 binding antagonist is described and provided. Specifically, intron retention has generated a novel HER-2 antagonist polypeptide that binds to the HER-2 receptor.

The HER-2/neu (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 Stem, Biochim. et Biophys. 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 Stern, 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).

Group I receptor tyrosine kinases including the EGF-receptor (HER-1, erbB-1), HER-2 (erbB-2), HER-3 (erbB-3), and HER-4 (erbB-4) are widely expressed in epithelial, mesenchymal, and neuronal tissues and play fundamental roles in proliferation and differentiation. With the exception of p185HER-2, receptor tyrosine kinases are activated by binding to a variety of EGF-related growth factors. Ligand binding is coupled to receptor dimerization, tyrosine autophosphorylation, and signal activation. Independently of a specifically binding growth factor, p185HER-2 dimerizes with itself or is recruited as the preferred heterodimer partner where it transactivates receptor family members.

Enhanced amounts of group receptors at the cell membrane occurs frequently in human carcinomas. This elevation in number of receptors is likely to favor the formation of receptor oligomers resulting in amplified signaling. The EGF-receptor and p185 HER-2 have been most frequently and clearly associated with human malignancies. HER-2 is overexpressed in breast, ovarian, gastric, and endometrial carcinomas Elevated levels of p185HER-2 in 25-30% of breast and ovarian cancers predicts significantly lower survival rates and shorter time to relapse. Amplification and alteration of the EGF-receptor gene is often observed in squamous cell carcinoma of the lung (Pavelic et al., 1993) and in glial tumors (Libermann et al., 1985), particularly in glioblastoma, the most malignant glial tumor.

There have been extensive efforts directed toward defining the structure and function of the group I receptor extracellular domains in the interests of understanding the mechanism of receptor activation and in blocking receptor action at the cell surface. Receptor mutants consisting of the extracellular domain and a membrane anchor, in the absence of the cytoplasmic domain, are capable of dimerizing (Lemmon et al., 1997; Tzahar et al., 1997;Tanner and Kyte 1999) and forming kinase inactive complexes with cell surface receptors (Greene). The ectodomains of group I receptors have been divided into subdomains I, beginning at the N-terminus, through IV ending at the juxtamembrane position. Domains II and IV contain multiple cysteine residues that are conserved amongst the four group I receptors. Subdomains I and II appear to be a repeating unit of III and IV that may have arose by a gene duplication event (Ullrich et al., 1984). Deletion of subdomains I and II from the EGF receptor results in constitutive dimerization and oncogenic transformation in a ligand-independent fashion (Hayely et al., 1989; Carter and Kung 1994; Qian et al., 1994; Moscatello et al., 1996), and allows ligand independent heterodimer formation with the membrane anchored p185neu ectodomain mutant (Greene). While subdomain III contains the high affinity ligand binding site as shown for EGF binding to the EGF receptor (Wu et al., 1990 Woltjer et al., 1992 Lax et al., 1989; 1991), subdomain I has been suggested to serve as a low affinity site that is promiscuous in its ligand recognition (Lax et al., 1989; 1991 Tzahar et al., 1997). According to this model EGF-like ligands are bivalent with a high affinity site that binds to the direct receptor in subdomain III and a second, low affinity site with broad specificity for subdomain I that prefers interaction with p185HER-2, thereby explaining the status of p185HER-2 as the preferred dimer partner. Taken together these results suggest that subdomains I and II may exert a negative constraint on dimerization in the absence of ligand and could be important for recruitment of receptors into heterodimers.

Monoclonal antibodies against the ectodomains of p185HER-2 and the EGF-receptor have been shown to be effective in limiting growth of tumors. These antibodies bind to their receptor targets with high affinity and specificity and their toxicity is low. The mechanisms underlying the antitumorigenic effects of antibodies are unclear. The rhuMAb4D5 (HERCEPTIN®) antibody may act by downregulation of p185HER-2 at the cell surface (Hurwitz et al., 1995), which causes a reversible cytostatic effect on HER-2 mediated cell growth. Systemic administration of the monoclonal antibody rhuMAb4D5 (HERCEPTIN®) has been shown to have therapeutic efficacy, since it increases the time to recurrence in a subset of patients with metastatic breast cancer. High affinity humanized, monoclonal antibodies against the EGF receptor have also been used as antitumor agents. While the molecular mechanisms underlying the activity of EGF receptor antibodies remain elusive, those tested compete with growth factor binding. Antibody strategies that target p185HER-2 and the EGF-receptor, as well as heterodimers between these two receptors, have also been attempted. Preliminary evidence suggests that targeting both receptors may significantly augment antiproliferative effects.

Mutant receptors consisting of ectodomains have proved to be effective in inhibition of tumorigenesis. The membrane-anchored ectodomain of p185 neu, ectopically expressed in cells, functions as a dominant negative inhibitor based on its ability to dimerize with the ectodomains of group I receptors forming a kinase-inactive complex. P185neu ectodomain mutants are capable of specific inhibition of p185HER-2 homodimer signaling as well as trans-inhibition of EGF receptor signaling. Since p185HER-2 is the preferred heterodimer partner of group I RTKs, then the p185-ectodomain is capable of suppressing the activation of all group I receptors. However, membrane anchoring of ectodomain mutants is required to exert a dominant negative effect since interactions between soluble ectodomains and cell surface receptors are too weak to achieve complex formation.

SUMMARY OF THE INVENTION

The present invention is directed to a A naturally occurring inhibitor of the HER-2 receptor tyrosine kinase called herstatin which consists of the first 340 α-residues identical to N-terminal subdomains I and II of p185HER-2, followed by a novel C-terminus of 79 aa-residues and a stop codon specified by the inserted sequence. In contrast to soluble ectodomains, herstatin binds with high affinity (˜14 nM Kd) to cell surface p185HER-2. Although herstatin is secreted and complexes with p185HER-2 at the cell surface, it differs from other EGR-like ligands in its ability to inhibit the activity ofp185HER-2. In the current study we find that coexpression of herstatin with p185HER-2 causes a striking reduction in cell growth that corresponds with suppression of p185 autophosphorylation. Moreover the inhibitory activity of herstatin is extended to EGF activation of the EGF-receptor.

In another embodiment, a method for treating a solid tumor characterized by overexpression of EGF receptor is disclosed including administering an agent that binds to the extracellular domain (ECD) of EGF receptor. The agent is selected from the group consisting of (a) 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 EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, (b) 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, (c) a monoclonal antibody that binds to the ECD of EGF receptor, and (d) combinations thereof. The agent cannot be the monoclonal antibody alone.

In yet another embodiment, a pharmaceutical composition for treating solid tumors that overexpress EGF receptor is disclosed including an agent selected from the group consisting of (a) 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 EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹, (b) 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, (c) a monoclonal antibody that binds to the ECD of EGF receptor, and (d) combinations thereof. The agent cannot be the monoclonal antibody alone, and pharmaceutically acceptable carrier.

An even further embodiment, a method for targeting a therapeutic agent to solid tumor tissue is disclosed. The method includes targeting a therapeutic agent to a solid tumor wherein the solid tumor tissue is characterized by overexpression of EGF receptor. The method includes attaching the therapeutic agent to an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1. The polypeptide binds to the extracellular domain ECD of EGF receptor with an affinity binding constant of at least 10⁸ M⁻¹.

In yet another embodiment, a method for determining the prognosis of tumor treatment in a patient for a tumor that overexpresses EGF receptor is disclosed including: (a) obtaining a bodily fluid sample from a patient, wherein the bodily fluid is selected from the group consisting blood, serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilical cord tissue, amniotic fluid, chorionic villi tissue and combinations thereof; (b) measuring the amount of p68HER-2 receptor expressed using an anti-p68HER-2 receptor antibody-based assay, wherein the assay is selected from the group consisting of ELISA, immunoprecipitation, immunohistocytochemistry, and Western analysis; (c) measuring the amount of EGF receptor ECD in the bodily fluid; and (d) determining a ratio between the amount of p68HER-2 and EGF receptor, whereby the higher the p68HER-2 to EGF receptor ratio, the better the prognosis of the patient.

One advantage of herstatin is in the development of therapeutics, particularly against human cancers that involve group I receptor tyrosine kinases. Another advantage of herstatin is its ability to suppress the growth of p185HER-2 or EGF receptor overexpressing cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence and amino acid of the insert in the extracellular domain of HER-2. The HER-2 ECD coding sequence from exon 1-9 (primers A and B) was amplified by PCR from a cDNA library from SKOV-3 cells. A product of ˜1420 bp was found to be HER-2-specific by Southern blot analysis. This product was subcloned and the nucleotide sequence was determined. In panel A, a nucleotide sequence (287 bp: SEQ ID NO:13) is shown for the 275 bp insert (within the open-ended boxes) plus the immediately adjacent 5′ and 3′ sequences (framed by the open-ended boxes). The 275 bp insert sequence, using the numbering of Coussens et al. (Science 230:1132-1139, 1985), is located between nucleotide residues 1171 and 1172 and following amino acid residue 340 in p185HER-2. SEQ ID NO:14 (276 bp) shows the 275 bp insert sequence plus the immediately 5′ nucleotide (“G”). The consensus 5′ and 3′ splice sites at the arrows are shown in larger print. The inserted sequence is in-frame with 5′ HER-2 exon sequence and is deduced to encode a 79 amino acid extension (SEQ ID NO:15) following Arg 340 (R³⁴⁰). The novel 79 acid sequence (SEQ ID NO:15) encoded by the insert is proline-rich (19%) and has a consensus asparagine linked glycosylation site, which is underlined. A stop codon was found at nucleotides 236-238 within the inserted sequence. In panel B, the predicted product of the alternative transcript is a truncated secreted protein which contains subdomains I and II identical to p185 and is missing the transmembrane domain and cytoplasmic domain. If fully glycosylated, the expected size is 65-70 kDa. This polypeptide product is referred to as p68HER-2. Thus, the product will be a truncated secreted protein which is missing the transmembrane domain and cytoplasmic domain found in p185HER-2.

FIG. 2 shows the detection of alternative HER-2 transcripts containing the ECDIIIa sequence by Northern blot analysis. PolyA+ mRNA (2.5 μg) from different human fetal tissues (Clontech) or isolated from HEK-293 cells was resolved in a formalin agarose gel and transferred to a BrightStar® membrane (Ambion) in 10×SSC. The membrane was hybridized with a ³²P-labeled antisense RNA probe complimentary to the ECDIII sequence, stripped and reprobed with a ³²P-labeled cDNA probe specific for the 5′ HER-2 exon sequence. The membranes were washed under high stringency conditions and analyzed by phosphorimaging (Molecular Dynamics).

FIG. 3 shows a sequence-specific reactivity of anti-ECDIIIa with a protein of ˜68 kDa in a human embryonic kidney cell line (HEK293). Cell extract protein (20 μg) and 20 μl of media conditioned by HEK-293 cells were Western blotted and probed with anti-ECDIIIa diluted 1:10,000 (lanes 1 and 2) or with anti-ECDIIa diluted 1:10,000 containing 50 μg/ml purified His-tagged ECDIIIa peptide (lanes 3, 4).

FIG. 4 shows the expression of p185HER-2, relative to p68ECDIIIa expression, is markedly elevated in carcinoma cell lines in which the HER-2 gene is amplified. Cell extracts (15 μg of protein) from human embryonic kidney cell line (HEK293), nontumorigenic ovarian surface epithelial cell line (IOSEVAN), ovarian carcinoma cell line with HER-2 gene amplification (SKOV-3), nontumorigenic breast epithelial cell line (HBL100), and breast carcinoma cell lines with HER-2 gene amplification (BT474 and SKBR-3), were resolved by SDS-PAGE in 7.5% acrylamide gels and analyzed as a Western blot. The Western blot was probed with both antibodies specific for p68HER-2 (anti-ECDIIIa) and for p185HER-2 (anti-neu(C)).

FIG. 5 shows that p68ECDIIIa binds to p185HER-2. In panel A: Two mg of SKBR-3 cells extracted in nondenaturing buffer were immunoprecipitated with 511 anti-neu(N) specific for the N-terminal sequence of p68HER-2 and p185HER-2, or with 5 μl anti-neu(C) specific for the C-terminus of p185HER-2 and then probed as a Western blot with both anti-ECDIIIa specific for p68HER-2 and with anti-neu(C) specific for p185HER-2. In panel B: 100 μg of 17-3-1 cell extract were incubated in duplicate with 50 μl packed volume of NiNTA agarose (Qiagen) coupled to 20 μg of His-tagged ECDIIIa or to 20 μg His-tagged CREB fragment in 200 μl of wash buffer (20 mM Tris pH 8.0, 300 mM NaCl) at room temperature for 1 hr with shaking. The resin was then washed 4 times with 500 μl of wash buffer and proteins were eluted by incubation with 50 μl SDS-sample buffer at 100° C. for 2 min. Eluted proteins were analyzed by Western blot analysis using antibodies against the C-terminus of p185HER-2, anti-neu(C). In panel C: Monolayers of ˜10⁵ 3T3 cells or HER-2 transfected 17-3-1 cells in 12 well plates were washed twice with PBS and then incubated with 0.5 ml of serum-free media with 1% BSA and 39, 75, 150, and 300 nM of purified recombinant His-tagged ECDIIIa for 2 hrs at 4° C. Cells were washed 1 time in PBS containing 1% BSA and twice in PBS and then were extracted in denaturing buffer. Equal aliquots (20 μg protein) were analyzed by western blotting with antibodies specific for ECDIIIa (anti-ECDIIIa) or, in the upper panel, with antibodies specific for p185HER-2 (anti-neu(C)).

FIG. 6 shows that neither p68-rich conditioned media nor the ECDIIIa peptide stimulate tyrosine phosphorylation of p185HER-2. Monolayer cultures of 5 HER-2 transfected 17-3-1 cells were washed twice with PBS, incubated in serum-free media at 37° C. for 24 hrs, and then treated for 10 minutes with 75 or 150 μM His-tagged ECDIIIa or with 50×CM from HEK-293 cells that secrete high levels of p68 or 50×CM from SKOV-3 cells that have no detectable p68HER-2. The treated cells were extracted with denaturing buffer containing the phosphotyrosine phosphatase inhibitor vanadate (2 mM) and 20 μg/ml of cell extract protein from each sample were analyzed by Western blot analysis with monoclonal antibodies against phosphotyrosine (Sigma). The blot was stripped by incubation at 55° C. for 30 min in 62.5 mM Tris pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol and then reprobed with anti-neu(C) specific for p185HER-2.

FIG. 7 shows that p68HER-2 inhibited anchorage independent growth of tumorigenic cells. SKOV-3 ovarian cancinoma cells and HER-2 transfected 17-3-1 cells were suspended in media with 10% fetal bovine serum containing 0.3% agar (control conditions) to which was added 50× concentrated media conditioned by SKOV-3 cells (which contains no detectable p68HER-2 (−p68 CM)), or 50× concentrated media conditioned by HEK-293 cells (which contains 20 nM p68HER-2 (+p68 CM)). Five times 10³ cells were plated in triplicate for each experimental condition onto a 0.5 ml layer of media containing 0.5% agarose in 12 well plates. The results shown are plotted as the mean and standard deviation of the number of colonies with more than 50 cells in triplicate wells counted at 21 days of incubation. Similar results were observed in three separate experiments.

FIG. 8 shows the nucleotide (SEQ ID NO:16) and deduced amino acid sequence (SEQ ID NO:11) of HER-2 Intron 8. Human genomic DNA was subjected to PCR using primers that flank intron 8. PCR parameters were 30 cycles of 94° C. for 1 min, 62° C. for 1 min, 72° C. for 30 s, followed by 1 cycle of 72° C. for 7 min. A 410 bp product was gel purified and sequenced in the forward and reverse directions. The sequence shown is the most common sequence found within intron 8 from about 15 different individuals. Positions of sequence variation resulting in amino acid substitutions as disclosed herein are marked by Xs below the sequence.

FIGS. 9 and 10 show the Expression of herstatin in combination with p185HER-2 in transfected Cos-7 cells. Cos-7 cells were plated at 2×10⁵ cell/well into 6 well plates and transfected using Lipofectamine (BioRrad) as described in Materials and Methods. The cells were transfected with 1.5 ug of herstatin expression vector plus 1.5 ug of HER-2 expression vector, or with 1.5 ug of HER-2 expression vectors (all pcDNA3.1 from Invitrogen). In each case the total amount of DNA was adjusted to 3 ug with control empty vector. In FIG. 9, upper panel, the transfected cells were analyzed at 48 hrs as a Western blot and reacted with antibodies against p185HER-2, anti-neu (C), or in the lower panel with antibodies against the intron 8-encoded C-terminal sequence of herstatin (anti-Hst). In FIG. 10, the transfections were conducted in triplicate using the indicated plasmids with the inclusion of 0.5 ug of β-galactosidase galactosidase expression plasmid driven by a CMV promoter. At 48 hrs, the cells were extracted, cell protein was quantitated by BioRad protein dye kit, and the β-galactosidase activity was measured as described. The β-galactosidase activity was normalized to protein amounts and the mean results with the standard deviations are plotted. A similar result was obtained when β-galactosidase activity was normalized to number of cells originally plated into each well.

FIGS. 11 and 12 show the effects of herstatin on the growth of transfected cell colonies. CHO cells were seeded at 2×10⁵ cell/well in 6 well plates and triplicate wells were transfected as described in Materials and Methods with 3 μg of control empty plasmid (pcDNA 3.1; Invitrogen), 1.5 mg of p185HER-2 expression plasmid plus 1.5 μg mg of control DNA; 1.5 μg of p185HER-2 plus 1.5 μg of herstatin; or 1.5 μg of herstatin plus 1.5 μg of control DNA. At 48 hours after addition of DNA, the cells were trypsinized and diluted 1:10 into 6 well plates in the presence of 0.6 mg/ml of G418. The media was changed every two days. At 14 days, the plates were stained with crystal violet and washed. In the upper panel, the crystal violet stained plates were extracted by shaking at room temperature for 30 minutes with 1 ml of 0.1 M NaPhosphate pH 4.5 in 50% ethanol. The extracted crystal violet, diluted 10 fold, was quantitated by the absorbance at 515 nM. Dilutions of 10 fold gave readings of 0.1 to 1.0 which was in the linear range of absorbance versus cell number determined in pilot studies (not shown).

FIGS. 13, 14 and 15 show Herstatin expression inhibits p185HER-2 tyrosine phosphorylation in transfected cells. Cos-7 cells were plated into 6 well plates and transfected as in FIGS. 9 and 10. In FIG. 13, Panel a, cells in duplicate wells were transfected with 0.25, 0.5, 1.0, and 3 μg of fluorescent green protein (FGP) expression plasmid. Empty vector was added to make the total amount of DNA equal to 3 μg in each well. At 48 hrs, the fluorescent signal was quantitated at a wavelength of 520 nM for emission and 490 mM for excitation for emission and for excitation using a fluorescent plate reader. In FIGS. 14 and 15, cells were transfected with 0.5 μg of FGP plasmid with or without 1.5 μg of HER-2 plasmid and with the indicated amounts of herstatin expression plasmid. Empty vector was added to make the total amount of DNA at 5 μg in each well. At 48 hrs, the cells were washed twice with PBS, extracted in 100 μg of modified RIPA containing 1 mM phenylmethylsulfonylfluoride and 2 mM orthovanadate. The protein concentration in the clarified extracts was determined using the BioRad protein dye kit. The extracted protein, 20 μg, was resolved in 7.5% polyacrylamide SDS gels and analyzed as a Western blot as described in Materials and Methods first using 1 μg/ml of anti-phosphotyrosine antibody (anti-PTyr) (Panel C). The blot was stripped and then was reacted with antibodies against p185HER-2 (anti-neu(C)). The blots were developed using chemiluminescent reagent (Pierce) and exposed to Kodak film.

FIGS. 16, 17, and 18 show Herstatin expression inhibits EGF activation of the EGF receptor in transfected cells. CHO cells at 2×10⁵ cell/well were plated into 6 well plates and 24 hrs later duplicate wells were transfected with 0.5 μg of FGP marker plasmid with or without 1.5 μg of EGF receptor expression plasmid with the indicated amounts of herstatin expression plasmid. At 24 hours, the cells were washed twice with PBS and cultured in serum free media for an additional 24 hrs. In FIGS. 16 and 17 the cultures were incubated in the absence or presence of 100 ng/ml of EGF for 20 minutes and then extracted in modified RIPA as in FIGS. 13-15. 20 μg of protein from each well were resolved in 7% polyacrylamide SDS-gels and analyzed as a Western blot, first with 1 μg/ml of anti-phosphotyrosine antibody (FIG. 17). The blot was then stripped and probed with anti-EGF receptor antibody. In FIG. 18 the cultures were incubated in the absence or presence of 100 ng/ml of EGF for 24 hrs. The cell extracts were then analyzed as a Western blot using anti-EGF receptor antibody.

FIG. 19 shows the effects of the EGF receptor alone and in combination with herstatin on colony growth. CHO cells were plated in 6 well plates and triplicate wells were transfected with 1.5 ÿg of empty vector, with 1.5 μg of the EGF receptor expression plasmid, with 1.5 μg of the EGF receptor plus 1.5 μg of herstatin plasmid, or with 1.5 μg of herstatin plasmid. Empty vector was added to bring the total amount of DNA to 3 μg in each well. At 48 hrs after DNA was added, the cells in each well were collected by trypsinization and diluted 1:10 into 6 well plates in media containing 600 μg/ml of G418. The media was changed every two days and at 14 days, the cultures were stained with crystal violet. The dye from the stained cultures was extracted as in FIG. 1 and quantitated by absorbance at 415 nM. The mean value of triplicate wells was determine as the % of the control transfected wells and the standard deviation are plotted. A similar result was obtained in three separate experiments.

FIG. 20 shows immobilized intron 8-encoded peptide or herstatin pulls-down the EGF receptor and p185HER-2. About 100 μl of a 50% suspension of S-protein agarose (Novagen) was incubated with no peptide, with 50 μg of TBpex14 peptide (provided by Dr. B. Ullman, OHSU), 50 μg of intron 8-encoded peptide, or 50 μg full length recombinant herstatin at room temperature for 1 hr. Each of these peptides contained an S-protein tag encoded by the pET 30 expression plasmid (Novagen). The agarose samples were then washed twice with PBS and incubated at room temperature for 1 hr with 100 μg of A431 cell extract, for the EGF receptor, or 17-3-1 extract, for p185HER-2, solubilized in PBS containing 1% nonidet-p40 (PBSNP-40). After incubation with the cell extracts, the agarose samples were washed twice with 500 μl of PBS-NP40 and the proteins associated with the resin were eluted at 92° C. for 2 min in 40 μl of SDS-sample buffer. To ensure that equal amounts of the original peptides were complexed to the agarose, an aliquot extracted in SDS-sample buffer was analyzed by SDS-PAGE and Coomassie staining of a 17% polyacrylamide gel for TBpex14 and intron 8-encoded peptide, and a 10% polyacrylamide gel for p50 herstatin. To analyze receptor binding, an aliquot eluted from the agarose was analyzed as a Western blot using anti-EGFR or anti-p185HER-2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the initial discovery of an alternative HER-2 mRNA of 4.8 kb with a 274 bp insert identified as intron 8. The retained intron is in-frame and encodes 79 amino acids (SEQ ID NO:1) followed by a stop codon at nucleotide 236. The alternative mRNA predicts a truncated HER-2 protein that lacks the transmembrane and intracellular domains and contains 419 amino acids (SEQ ID NO:2); 340 residues that are identical to the N-terminus of p185HER-2 (SEQ ID NO:13) and 79 unique residues at the C-terminus (SEQ ID NO:1). Using specific antibodies against either the novel 79 amino acid residue C-terminal sequence [SEQ ID NO:1] or the N-terminus of p185HER-2, a 68 kDa protein product was identified (SEQ ID NO:2). This 68 kDa protein is the product of an alternative HER-2 transcript, and is found in cell extracts and in extracellular media from several cell lines. Expression of the alternative transcript was highest in a nontransfected human embryonic kidney cell line.

The results presented here show expression of alternative HER-2 mRNA, which contains an additional 274 nucleotides, probably intron 8. Consistent with this finding, an alternative transcript of ˜4.8 kb was detected in human fetal kidney tissue and in the human embryonic kidney cell line, HEK 293. Moreover, a transcript of 2.6 kb, which is the size expected if the sequence is retained in the 2.3 kb truncated HER-2 mRNA (Yamamoto et al., Nature 319:230-234, 1986; and Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993), was detected in human fetal liver tissue by Northern blot analysis using a probe specific for the inserted sequence or for the HER-2 ECD coding sequence (FIG. 2). The inserted sequence introduces a termination codon and predicts a novel 79 amino acid extension designated ECDIIIa at residue 340 of the p185HER-2 protein. The predicted protein therefore lacks the transmembrane and intracellular domains, but contains subdomains I and II of the extracellular domain of p185HER-2. As predicted, a secreted protein that contains N-terminal sequence of p185HER-2 and the C-terminal extension provided by the inclusion of the novel sequence was detected (FIGS. 3 and 5). The ECDIIIa protein was found to be 68 kDa which is the approximate size expected of the protein encoded by the alternative transcript if the five N-linked glycosylation sites found in subdomains I and II of p185HER-2 are glycosylated (Stern et al., Mol. Cell. Biol. 6:1729-1740, 1986).

The data presented herein demonstrate that p68HER-2 specifically binds to p185HER-2. The association with p185HER-2 may be conferred by the novel proline rich ECDIIIa domain rather than the N-terminal subdomains I and II of p68HER-2. While the HER-2 ECD, generated by in vitro deletion mutagenesis, also contains subdomains I and II, it does not associate with the extracellular domain of p185HER-2 unless engineered to enhance their proximity (Tzahar et al., EMBO J. 16:4938-4950, 1997; O'Rourke et al., Proc. Natl. Acad. Sci. USA 94:3250-3255, 1997; and Fitzpatrick et al., FEBS Letters 431:102-106, 1998). However, the unique ECDIIIa peptide binds with high affinity (nM concentrations) to p185HER-2 and to transfected 17-3-1 cells that overexpress p185HER-2 (FIG. 5). Preferential binding of the ECDIIIa domain peptide to 17-3-1 cells indicates that secreted p68HER-2 interacts with the extracellular region of p185HER-2 at the cell surface. Therefore, p68HER-2 and fragments thereof appear to be a naturally occurring HER-2 binding protein, encoded by the HER-2 gene. In contrast to EGFR family ligands (Groenen et al., Growth Factors 11:235-257, 1994), p68HER-2 lacks an EGF homology domain and contains the first 340 amino acids of the receptor itself, p185HER.

Previously described putative HER-2 ligands were found to associate indirectly with p185HER-2 only in a heterodimer with an EGFR family member (Heldin and Ostman, Cytokine Growth Factor Rev. 7:33-40, 1996). Although it is possible that ECDIIIa binds indirectly to p185HER-2 through a coreceptor, this seems unlikely since detergent solubilized p185HER-2 was specifically and efficiently “pulled down” by immobilized ECDIIIa peptide (FIG. 5B).

For all naturally occurring or engineered ligands for mammalian EGFR family members, binding is tightly coupled to stimulation of receptor dimerization and tyrosine phosphorylation (Hynes and Stern, Biochim. et Biophys. Acta 1198:165-184, 1994; Dougall et al., Oncogene 9:2109-2123, 1994; and Groenen et al., Growth Factors 11:235-257, 1994). Although they bind, neither p68HER-2 nor the ECDIIIa peptide was found to activate p185HER-2. Activation was assessed in two different cell lines that differ in the extent of p185HER-2 tyrosine phosphorylation, transfected 17-3-1 cells as well as SKOV-3 ovarian carcinoma cells. Furthermore in vitro self-phosphorylation activity, which is enhanced in dimeric forms of p185HER-2 (Dougall et al., Oncogene 9:2109-2123, 1994; and Lin et al., J. Cell. Biochem. 49, 290-295, 1992), was not stimulated by p68HER-2 or ECDIIIa. Similarly, the Argos protein, which is an extracellular inhibitor of the Drosophila EGF receptor and the only known antagonist of class I RTKs, did not simulate tyrosine phosphorylation of the receptor (Schweitzer et al., Nature 376:699-702, 1995). Likewise, Angiopoietin-2, a natural antagonist for the Tie 2 RTK, bound the endothelial receptor but failed to activate it (Maisonpierre et al., Science 277:55-60, 1997).

Without being bound by theory, since p68HER-2 occupies but does not activate, it could block dimerization of p185HR-2. By analogy, HER-2 ECD, when engineered to enhance its binding to RTKs, prevented the formation of productive dimers required for transphosphorylation and receptor activation thereby having a dominant negative effect (O'Rourke et al., Proc. Natl. Acad. Sci. USA 94:3250-3255, 1997). In contrast to the HER-2 ECD, soluble p68HER-2 exhibited strong binding to p185HER-2, yet also contains subdomain I and II of the ECD. Since subdomain I may be the low affinity, promiscuous ligand binding site required for recruitment of p185HER-2 into heteromeric complexes (Tzahar et al., EMBO J. 16:4938-4950, 1997), p68HER-2 could block this site and thereby obstruct recruitment of p185HER-2 into dimers. Alternatively, p68HER-2 could compete with an uncharacterized ligand for binding to p185HER-2. The tissue-specific expression of p68HER-2 in human fetal liver and kidney may function to modulate the extent to which p185HER-2 is occupied during development of these organs. Moreover, the overexpression of p185HER-2, relative to p68HER-2 in tumor cells with HER-2 gene amplification (FIG. 3), could occur though a selective pressure based on overcoming the effects of a binding protein such as p68HER-2. Therefore, p68HER-2 is the first example of a naturally occurring p185HER-2 binding protein that may prevent activation of p185HER-2.

Pharmaceutical Composition

The present invention further provides a pharmaceutical composition for treating solid tumors that overexpress HER-2, comprising an agent selected from the group consisting of (a) 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 with an affinity binding constant of at least 10⁸ M⁻¹ at an affinity of at least 110, (b) an isolated and glycosylated polypeptide having from about 80 to 419. or about 300 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, (c) a monoclonal antibody that binds to the ECD of HER-2, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone, and pharmaceutically acceptable carrier. Preferably, the agent is the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1. Most preferably, the agent is a combination of the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1 and the monoclonal antibody that binds to the ECD of HER-2.

The inventive pharmaceutical composition, comprising either or both of the inventive polypeptides and/or monoclonal antibody, can be administered to a patient either by itself (complex or combination) or in pharmaceutical compositions where it is mixed with suitable carriers and excipients. Inventive polypeptide can be administered parenterally, such as by intravenous injection or infusion, intraperitoneal injection, subcutaneous injection, or intramuscular injection. Inventive polypeptide can be administered orally or rectally through appropriate formulation with carriers and excipients to form tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like. Inventive polypeptide can be administered topically, such as by skin patch, to achieve consistent systemic levels of active agent. Inventive polypeptide is formulated into topical creams, skin or mucosal patch, liquids or gels suitable to topical application to skin or mucosal membrane surfaces. Inventive polypeptide can be administered by inhaler to the respiratory tract for local or systemic treatment of cancers characterized by overexpressing HER-2.

The dosage of inventive polypeptide suitable for use with the present invention can be determined by those skilled in the art from this disclosure. Inventive polypeptide will contain an effective dosage (depending upon the route of administration and pharmacokinetics of the active agent) of inventive polypeptide and suitable pharmaceutical carriers and excipients, which are suitable for the particular route of administration of the formulation (i.e., oral, parenteral, topical or by inhalation). The active inventive polypeptide is mixed into the pharmaceutical formulation by means of mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical formulations for parenteral administration include aqueous solutions of the inventive polypeptide in water-soluble form. Additionally, suspensions of the inventive polypeptide may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may optionally contain stabilizers or agents to increase the solubility of the complex or combination to allow for more concentrated solutions.

Pharmaceutical formulations for oral administration can be obtained by combining the active compound with solid excipients, such as sugars (e.g., lactose, sucrose, mannitol or sorbitol), cellulose preparations (e.g., starch, methyl cellulose, hydroxypropylmethyl cellulose, and sodium carboxymethyl cellulose), gelaten, gums, or polyvinylpyrrolidone. In addition, a desintegrating agent may be added, and a stabilizer may be added.

Processes for Synthesizing p68 and 79 aa C Terminal Region

Polypeptide synthesis is done by a group of standard procedures for polypeptide synthesis by sequential amino acids building through peptide synthesis equipment, following manufacturer's instructions for synthesizing peptides. Preferably, shorter polypeptides, of less than 100 amino acids, are best suited for the method of synthesis through sequential amino acid building of polypeptides. In addition, heterologous polypeptides can be expressed by transformed cells using standard recombinant DNA techniques to transform either prokaryotic or eukaryotic cells, provide appropriate growth media for their expression, and then purify the inventive polypeptide either from the media or from intracellular contents depending upon the type of cell used and its expression characteristics.

Methods for Treating Cancer with p68, 79 aa C Terminal Region, and Combinations

The present invention provides a method for treating a solid tumor characterized by overexpression of HER-2, or HER-2 variants (see Example 8) comprising administering an agent that binds to the extracellular domain (ECD) of HER-2, wherein the agent is selected from the group consisting of (a) 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 with an affinity binding constant of at least 10⁸ M⁻¹, (b) an isolated and glycosylated polypeptide having from about 80 to 419. or about 300 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, (c) a monoclonal antibody that binds to the ECD of HER-2, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone. Preferably, the solid tumor that overexpresses HER-2 is selected from the group consisting of breast cancer, small cell lung carcinoma, ovarian cancer, prostate cancer, gastric carcinoma, cervical cancer, esophageal carcinoma, and colon cancer. Preferably, the agent is the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1. Most preferably, the agent is a combination of the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1 and the monoclonal antibody that binds to the ECD of HER-2.

The p68HER-2 polypeptide described herein was found to bind to HER-2 and prevent signal transduction through the kinase domain. Without being bound by theory, the unique ECDIIIa domain mediates specific binding to p185HER-2 and the resulting interaction with p68ECDIIIa prevents p185HER-2 dimerization and subsequent signal transduction. Therefore, p68HER-2 functions as a HER-2 antagonist to prevent signal transduction by preventing dimerization as a necessary prerequisite for signal transduction. Thus, the mechanism of p68HER-2 as a HER-2 antagonist is different from the mechanism of binding agents, such as the 79 amino acid polypeptide described herein or a monoclonal antibody that binds to the EDC of HER-2. The inventive method provides that p68HER-2 inhibits tumor cell growth in tumors that overexpress HER-2 by providing a selective pressure for such tumor cells. Similarly, the HER-2 antagonists that are binding agents also inhibit tumor cell growth in tumors that overexpress HER-2 by providing selective pressure to such cells to prevent ligand binding to the ECD of HER-2 and prevent signal transduction even before potential dimerization.

Use of 79 aa C Terminal Region as a Targeting Molecule

The present invention further provides a method for targeting a therapeutic agent to solid tumor tissue, wherein the solid tumor tissue is characterized by overexpression of HER-2, comprising attaching the therapeutic agent to 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 with an affinity binding constant of at least 10⁸ M⁻¹. 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 or HER-2). It was discovered that the 79 amino acid polypeptide [SEQ ID NO. 1] exhibited surprising high affinity binding properties to the ECD of HER-2. Moreover, the site of such binding is different and unaffected by the site of binding of a marketed humanized monoclonal antibody (HERCEPTIN®). Therefore, the high binding affinity enables the 79 amino acid polypeptide to function as a targeting molecule to tumor cells expressing HER-2.

Anti-p68 Antibody as a Diagnostic/Prognostic Agent

The p68HER-2 ECDIIIa variant 3 (see TABLE 1, below) glycosylated polypeptide was expressed and used as an antigen for antibody production. Specifically, antibody specific for p68HER-2 was prepared by injecting rabbits with purified polyhistidine-tagged ECDIIIa variant 3 peptide, which is the same as the intron encoded novel C-terminus or p68HER-2, the domain that binds with high affinity to p185HER-2. The isolated polyclonal antibody detected pM quantities of ECDIIIa peptide or of p68HER-2 with high specificity (see FIGS. 3 and 5). Thus, an antibody specific for p68HER-2 is useful as a diagnostic agent for detecting p68HER-2 in bodily fluids and tumor tissues using diagnostic techniques, such as ELISA, immunoprecipitations, immunohistochemistry or Western analysis.

Antibodies that specifically recognize one or more epitopes of ECDIIIa, or epitopes of p68HER-2, or peptide fragments, and thus distinguish among ECDIIIa variants (see TABLE 1, below) are also encompassed by the invention. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The antibodies of the invention may be used, for example, in the detection of a particular p68HER-2 ECDIIIa variant in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients or tissue samples may be tested for the presence of particular variants, or for abnormal amounts particular variants.

Such antibodies may also be utilized in conjunction with, for example, compound screening schemes for the evaluation of the effect of test compounds on expression and/or activity of particular p69HER-2 variants. Additionally, such antibodies can be used in conjunction with the cancer treatment methods described herein.

For the production of antibodies, various host animals may be immunized by injection with e.g., polyhistidine-tagged ECDIIIa variant polypeptides, truncated ECDIIIa variant polypeptides, functional equivalents of the ECDIIIa variants or mutants of the ECDIIIa region. Such host animals may include but are not limited to rabbits, mice, hamsters and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (Nature 256:495-497, 1975; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026-2030, 1983), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Hybridomas producing mAb may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

Additionally, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Neuberger et al., Nature, 312:604-608, 1984; Takeda et al., Nature, 314: 452-454, 1985) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region (humanized).

Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Ward et al., Nature 334:544-546, 1989) can be adapted to produce single-chain antibodies against ECDIIIa variant gene products. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., Science, 246:1275-1281, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Antibodies to particular ECDIIIa variants can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” the ECDIIIa variant, using techniques well known to those skilled in the art. (Greenspan & Bona, FASEB J 7 (5):437-444, 1993; and Nissinoff, J. Immunol. 147:2429-2438, 1991). For example antibodies which bind to an ECDIIIa variant and competitively inhibit the binding of p68HER-2 to HER-2 receptor can be used to generate anti-idiotypes that “mimic” the ECDIIIa variant and, therefore, bind and neutralize HER-2 receptor. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in cancer therapeutic regimens.

Alternatively, antibodies to particular ECDIIIa variants that can act as agonists or antagonists of the ECDIIIa variant activity can be generated. Such antibodies will bind to the ECDIIIa variant and modulate the activity of p68HER-2 vis-à-vis p185HER-2 receptor-mediated signal transduction. Such antibodies may be particularly useful for treating particular cancers and/or modulating tumor differentiation. Accordingly, the present invention further provides a method for determining the prognosis of tumor treatment for a tumor that overexpresses HER-2, comprising: (a) obtaining a bodily fluid, wherein the bodily fluid is selected from the group consisting of blood, serum, urine, lymph, saliva, tumor tissue, and combinations thereof; and (b) measuring the amount of p68HER-2 expressed using an anti-p68HER-2 antibody-based assay, wherein the assay is selected from the group consisting of ELISA, immunoprecipitation, immunohistocytochemistry, and Western analysis. Preferably, the method for determining the prognosis of tumor treatment further comprises measuring the amount of p185HER-2 ECD in the bodily fluid, and determining a ratio between the amount of p68HER-2 and p185HER-2. The higher the ratio of p68HER-2:p185HER-2, the better the treatment prognosis.

ECDIIIa Region Variants as Diagnostic/Prognostic Agents

Example 11 (below) shows that the human sequence of intron 8 is both proline-rich and polymorphic. Sequencing of genomic DNA from fifteen different individuals resulted in the identification of 10 variable sequence regions within Her-2 Intron 8. See SEQ ID NO:10; FIG. 8, and Table 1. FIG. 8 shows the most common nucleotide and corresponding polypeptide sequences of intron 8. This region contains 10 different polymorphisms (marked by the letters W (2×), Y (3×), R, N, M (2×), and S (1×) in SEQ ID NO:10; or marked by an “X” in FIG. 8) that result in nonconservative amino acid substitutions (see legend to TABLE 1). For example, the polymorphism (C→G) at nucleotide position 161 (FIG. 8; TABLE 1) would result in a substitution of Arginine (R) for Proline (P) at amino acid residue #54 of SEQ ID NO:1, or residue #394 of SEQ ID NO:2. The N-terminal Glycine (G), designated as position 1 in FIG. 8 or SEQ ID NO:10, corresponds to amino acid residue 341 in the “herstatin” sequence (Doherty et al., Proc. Natl. Acad. Sci. USA 96:10,869-10,874, 1999). The nucleotide sequence shown in FIG. 1(A) (Doherty et al., Proc. Natl. Acad. Sci. USA 96:10,869-10,874, 1999), is a polymorphic form that differs at amino acid residues #6 and #73 from the most commonly detected sequence shown here in FIG. 8.

This result demonstrates that in the human population there are several variations in the intron-8 encoded domain that could lead to altered biochemical and biological properties among ECDIIIa-containing protein variants. An individual may, inter alia, be genetically heterozygous for two variants, homozygous for a given variant, or homozygous for a double variant. Both tumor progression and optimal treatment may vary depending upon the particular variants represented in a given individual.

This variability has both prognostic and diagnostic utility. The present invention shows that ECDIIIa-containing polypeptides can bind tightly to, and thus antagonize the HER-2 receptor. Such a specific, high-affinity interaction is dependent upon particular primary, secondary and tertiary structure of the ECDIIIa-containing polypeptide. The ECDIIIa region is proline-rich, and it is well known in the art that nonconservative substitution of proline residues, or other residues within a proline-rich sequence, in a given protein can have profound effects on its secondary and tertiary structure. Thus, the polymorphisms of the present invention are likely to embody significant structural, biochemical and biological differences relative to the most common polypeptide structure (shown in FIG. 8). Structural differences among ECDIIIa variant proteins may include for example, differences in size, electronegativity, or antigenicity. Differences in biological properties among ECDIIIa variants might be seen e.g., in the relative degree of cellular secretion, the nature and/or extent of modulation of the HER-2 receptor, pharmacokinetics (e.g., serum half-life, elimination profile), resistance to proteolysis, N-linked glycosylation patterns, etc. These biological differences, in turn, would be expected to alter tumor progression and thus optimal treatment protocols. Thus, the knowledge that an individual contains a particular ECDIIIa variant or variants (e.g., in individuals heterozygous for a given variant, or individuals with compound variants like variant 11 of Table 1), may, in itself, be prognostic of particular cancer susceptibility.

The apparent genetic heterogeneity of ECDIIIa region means that the nature of the particular ECDIIIa variation carried by an individual may have to be ascertained using sequence identity assays prior to attempting genetic diagnosis of the patient. The analysis can be carried out on any genomic DNA derived from bodily fluids of the patient, typically a blood sample from an adult or child, but alternatively may be serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilical cord tissue, amniotic fluid, and chorionic villi samples. It is expected that standard genetic diagnostic methods, such as hybridization or amplification assays, can be used. Either DNA or RNA, may, for example, be used in hybridization or amplification assays of biological samples to detect particular ECDIIIa variant sequences. Such sequence identity assays may include, but are not limited to, Southern or Northern analyses, single-stranded conformational polymorphism analysis, in situ hybridization assays, and polymerase chain reaction (“PCR”) analyses. Such analyses may reveal both quantitative and qualitative aspects of ECDIIIa variant sequence expression. Such aspects may include, for example, point mutations, and/or activation or inactivation of gene expression. Standard in situ hybridization techniques may be used to provide information regarding which cells within a given tissue express a particular ECDIIIa variant sequence.

Preferably, diagnostic methods for the detection of ECDIIIa variant nucleic acid molecules involve contacting and incubating nucleic acids, derived from cell types or tissues being analyzed, with one or more labeled nucleic acid reagents, or probes, specific for particular ECDIIIa variants. More preferably, PCR, or reverse transcription PCR, can be utilized to identify nucleotide variation within the ECDIIIa domain. PCR reaction conditions should be chosen which optimize amplified product yield and specificity, and, additionally, produce amplified products of lengths that may be resolved utilizing standard gel electrophoresis techniques. Such reaction conditions are well known to those of skill in the art, and important reaction parameters include, for example, length and nucleotide sequence of oligonucleotide primers, and annealing and elongation step temperatures and reaction times. Following the PCR reaction, the PCR products can be analyzed by methods such as heteroduplex detection, cleavage of RNA-DNA hybrids using Rnase A, single-stranded conformational polymorphisms, and denaturing gradient gel electrophoresis.

Additionally, if the particular ECDIIIA sequence variant is known to add or remove a restriction site, or to have significantly altered the size of a particular restriction fragment, a protocol based upon restriction fragment length polymorphism (“RFLP”) analysis may be appropriate.

ECDIIIa variants can also be analyzed at the expression level using sequence identity assays with bodily fluids derived from the patient, typically a blood sample from an adult or child, but may include serum, urine, lymph, saliva, tumor tissue, placental or umbilical cord cells, amniotic fluid, and chorionic villi samples. Well-known sequence identity assays for analyzing expression include, but are not limited to, mRNA-based methods, such as Northern blots and in situ hybridization (using a nucleic acid probe derived from the relevant cDNA), and quantitative PCR (as described by St-Jacques et al., Endocrinology 134:2645-2657, 1994).

Polypeptide-based methods (e.g., including but not limited to western blot analysis) including the use of antibodies specific for the ECDIIIa variant of interest, as discussed above, could also be used. These techniques permit quantitation of the amount of expression of a given ECDIIIa variant, at least relative to positive and negative controls. Preferably, a battery of monoclonal antibodies, specific for different ECDIIIa eptitopes or variants, could be used for rapidly screening cells or tissue samples to detect those expressing particular ECDIIIa variants, or for quantifying the level of ECDIIIa variant polypeptides. Preferred diagnostic methods for the quantitative or qualitative detection of ECDIIIa variant peptide molecules may involve, for example, immunoassays wherein particular ECDIIIa-containing peptides are detected by their interaction with anti-ECDIIIa variant specific antibodies. This can be accomplished for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorometric detection. The antibodies (or fragments thereof) useful in the present invention may, additionally, be em