WO2000015781A1

WO2000015781A1 - Antagonists of fibroblast growth factor

Info

Publication number: WO2000015781A1
Application number: PCT/US1999/020638
Authority: WO
Inventors: Brian T. Edmonds
Original assignee: Eli Lilly And Company
Priority date: 1998-09-11
Filing date: 1999-09-10
Publication date: 2000-03-23
Also published as: AU5914299A

Abstract

Polypeptides that are related structurally to the protein, SPROUTY-1, can be produced recombinantly and can be used to inhibit vascularization, for example, in the context of counteracting abnormal cell proliferation or undesired angiogenesis. By the same token, a polynucleotide coding for such a polypeptide, and a complement thereof, finds application in inhibiting, for example, prostate cell proliferation, adrenal tumor growth; limiting the range of FGF levels in vivo; inhibiting an adverse effect of FGF; inhibiting bFGF and FGFR-1-mediated signaling; conteracting FGF overexpression associated with certain types of tumors controlling the growth, development, and/or differentiation of any cell responsive to FGF signaling; treating various pathologies involving hypersensitive responses to FGF; treating conditions in which there is an underexpression of sprouty-1 and treating developmental anomalies associated with disregulated FGF signaling.

Description

ANTAGONISTS OF FIBROBLAST GROWTH FACTOR

FIELD OF THE INVENTION

The present invention relates to polypeptides that bear sequence similarities to any of several homologs of a human protein, SPROUTY, and to polynucleotides that encode such polypeptides. The invention further relates to vectors, host cells, and antibodies, and to methods for producing and using these different embodiments of the invention.

BACKGROUND OF THE INVENTION

SPROUTY is a cysteine-rich protein which blocks intercellular signaling by fibroblast growth factor (FGF). The sprouty polypeptide was identified initially, in

Drosophila, as an antagonist of an FGF signaling pathway which affected apical branching in airways of the insect. Hacohen, PhD thesis, Stanford University (1997);

Hacohen et al., Cell 92: 253 (1998). Mutations in the gene encoding SPROUTY that resulted in the removal or truncation of the cysteine-rich C-terminal domain of the SPROUTY protein produced a phenotype with excessive branching in Drosophila airways.

Hacohen et al, supra (1998) used a sprouty-encoding cDNA sequence, derived from Drosophila, to identify three human homologs. Three mouse homologs also have been identified. WO 98/20032. A 124-residue, cysteine-rich domain is highly conserved in these human and mouse homologs. Antagonists of growth factor signaling pathways play important role in developmental patterning by limiting the range of the cognate inducer. Hacohen (1998), supra.

The FGF family has at least ten members: acidic FGF (aFGF or FGF-1), basic

FGF (bFGF or FGF-2), FGF-3 (int02), FGF-4 (hst/kFGF), FGF-5, FGF-6, keratinocyte growth factor (KGF orFGF-7), androgen- induced growth factor (AIGF or

FGF-8), glia-activating factor (GAF or FGF-9), and FGF-10. Yamasaki et al , J. Biol.

Chem. 271: 15919 (1996). The signaling of the FGFs is mediated by a dual-receptor system, consisting of four high-affinity tyrosine kinase receptors, termed fibroblast growth factor receptors (FGFRs), and a number of low-affinity heparan sulfate proteoglycan receptors. Klagsburn et al, Cell 67: 229 (1991); Givol, FASEB J. 6: 3362 (1992); Johnson et al. , Adv. Cancer Res. 60: 1 (1993); Fernig et al, Prog. Growth Factor Res. 5: 353 (1994); Mason, Cell 78: 547 (1994); Burgess et al , "Proliferation in cancer regulatory mechanisms of neoplastic cell growth," in CELL 154, Oxford Univ. Press (1996).

FGFRs are glycoproteins, typically composed of three extracellular, immunoglobulin-like domains, a hydrophobic transmembrane region, and a cytoplasmic domain that contains a tyrosine kinase catalytic domain. Alternative mRNA splicing mechanisms result in receptor isoforms that display unique ligand binding properties. Ligand interaction with FGFRs also requires the presence of endogenous heparan sulfate proteoglycan or exogenous heparin and results in oligomerization, activation of the cytoplasmic receptor tyrosine kinase and receptor autophosphorylation. Subsequent signaling is mediated through a number of substrates for tyrosine phosphorylation, including mitogen-activated protein kinases (MAPKs) or ERK-1, ERK-2 and ERK-3. Mason, Cell 78: 547 (1994); Friesel et al , FASEB J. 9: 919 (1995).

Basic FGF (FGF-2) is nearly ubiquitous in its distribution, so highly specific mechanisms regulate its bioavailability . Baird, Molec. Reprod. Develop. 39:43 (1994). Various processes have been proposed to regulate FGF bioavailability, which include: (1) regulation of FGF activities by its low- and high-affinity receptors; (2) proteolytic regulation of FGF-2 activity; and (3) structural modification of FGF by post- translational modifivation. Id., citing Feige et al, Med. Sci 8:805 (1992), and Baird et al, Br. Med. J. 45:438 (1989).

In the nervous system, one mechanism of growth-factor action involves fibroblast growth factor-2 (FGF-2) and its receptor, which accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. Stachowiak, M.K. et al., Molec. Neurbio. 15: 257 (1997). Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. Id. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Id. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by transactivating factors interacting with an upstream regulatory promoter region. Id. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. Id. A similar regulation of nuclear FGF-2 and FGFRl is observed in neural crest-derived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Id. The regulation of the nuclear content of FGF-2 and FGFRl is thought to be one mechanism controlling growth and proliferation of glial and neuronal cells. Id.

Growth factors, such as FGF, have been found to be overexpressed in many tumors. FGF-1, FGF-2 and FGF-7 are overexpressed in human pancreatic cancer. Yamanaka et al. , Cancer Res. 53: 5289 (1993); Siddiqi et al. , Biochem. Biophys. Res. Commun. 215: 309 (1995) Pancreatic cancers also overexpress transforming growth factor betas (TGF-betas) that usually inhibit the growth of epithelial cells. Korc M., Surgical One. Clinics North Amer. 7:25 (1988) The tyrosine growth factor receptors include certain fibroblast growth factors (FGF) receptors (FGFR) and ligands. Id. Overexpression of FGF-2 correlates with shorter post-operative survival. Siddiqi et al supra. FGF-8 is expressed in high frequency in prostate cancers and breast tissues and may be involved in hormone-related tumorigenesis in these tissues. Tanaka et al , Cancer Res. 58: 2053 (1998). FGF-2 and its receptors are expressed in human colorectal cancer. Burns et al, Cell and Tumor Biol. 39: 206 (1998).

Antibodies that bind to a growth factor receptor have been shown to serve as a growth factor receptor antagonist. See U.S. Patent 5,646,036. Polypeptides can serve as an antagonist to a growth factor to inhibit an adverse effect of the growth factor. See U.S. Patent 5,444,151. One FGF antagonist is being developed to treat hormonally refractory prostate cancer. FDC Reports, 1998 WL 8441618 (1998). Antagonists of FGF can be used to inhibit endothelial cell proliferation in angiogenesis. Inhibition of bFGF and FGFR- 1 -mediated signaling, using bFGF and FGFR- 1 -specific antisense oligonucleotides, blocks angiogenesis in human melanomas, for example, Cancer Wkly Plus (September 1, 1997). There is a need, therefore to counteract the overexpressed FGF. Therefore, it would be useful to have an antagonist of FGF to oppose and couteract the effects of overexpressed or unchecked FGF activity.

SUMMARY OF THE INVENTION

It therefore is an object of the present invention to provide a class of therapeutic antagonists to counteract or block FGF activity. It is another object of the present invention to provide therapeutic antagonists of FGF when SPROUTY is underexpressed or fails to maintain the FGF-SPROUTY homeostasis.

Another object of the present invention is to provide an isolated polynucleotide, or the complement thereof, encoding an amino acid sequence comprising the sequence set forth in SEQ ID NO: 1, exclusive of SEQ ID NO: 11; SEQ ID NO : 12; SEQ ID NO: 13 ; SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16. It is a further object of the invention to provide an isolated polynucleotide, or the complement thereof, encoding an amino acid sequence comprising the sequence set forth in SEQ ID NO: 2, exclusive of SEQ ID NO: 11; SEQ ID NO : 12; SEQ ID NO: 13 ; SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO: 16. Yet another object of the present invention is a polynucleotide in which the amino acid sequence comprises the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 7, in which the sequence is devoid of the first 19 amino acids, which are FGSSACQVSTDCQNSRSLH (as shown in SEQ ID NO:24). Another object of the present invention is an isolated polypeptide encoded by a polynucleotide comprising the sequence set forth in SEQ ID NO: 1, or the complement thereof, exclusive of SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19 ; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO 22 and SEQ ID NO: 23; or an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 7 in which the SEQ ID No. 7 sequence is devoid of the first 19 amino acids, which are FGSSACQVSTDCQNSRSLH (shown in SEQ ID NO: 24). Another object of the present invention is an isolated polynucleotide encoding a polypeptide described in this or the previous paragraph. Yet another object of the present invention is to provide a method of inhibiting angiogenesis in a mammal by administering to a mammal a pharmacologically effective amount of such described SPROUTY polypeptides.

A further object of the present invention is an isolated nucleic acid that hybridizes under stringent conditions to the described polynucleotides encoding SPROUTY. Another object of the present invention is a composition having a carrier or diluent and such a described isolated nucleic acid.

Yet other objects of the present invention are isolated polypeptides having at least 20 contiguous amino acids of SEQ ID Nos. 3, 5, 7 or 9. Another object of the present invention is a composition of a carrier or diluent and an isolated polypeptide of the types described as the objects of the present invention.

Another object of the present invention includes vectors having the nucleic acid sequences described as objects of the invention and host cells containing such isolated nucleic acid sequences.

Other objects of the present invention include an antibody or at least one fragment thereof that binds an epitope specific to at least any one of the SPROUTY- 1 polypeptides described in the instant application. A further object of the present invention is a host cell that expresses at least one antibody or at least one fragment that binds an epitope specific to at least any one of the SPROUTY- 1 polypeptides described in the instant application. Yet another object of the instant invention provides a method for producing such antibodies by culturing such a host cell. Another object of the instant invention includes a transgenic or chimeric non-human animal having at least one of such host cells. Yet a further object of the invention includes a method for producing at least one SPROUTY- 1 polypeptide, by translating a nucleic acid of the instant invention under conditions so that the SPROUTY- 1 polypeptide is expressed in detectable or recoverable amounts. Another object of the instant invention includes a method for identifying compounds that bind at least one SPROUTY- 1 polypeptide, by admixing at least one isolated SPROUTY- 1 polypeptide of the instant invention, with at least one test compound or composition; and detecting at least one binding interaction between said at least one SPROUTY- 1 polypeptide and the test compound or composition.

Another object of the instant invention is to provide a method of limiting the range of FGF levels in vivo, in a mammal by administering to the mammal a pharmacologically effective amount of a polypeptide of the instant invention. Yet another object of the instant invention is to provide a method of limiting the range of FGF levels in vivo, in a mammal, by administering to the mammal a pharmacologically effective amount of a polypeptide of the instant invention. A futher object of the instant invention provides a method of inhibiting an adverse effect of FGF in a mammal, by administering to the mammal a pharmacologically effective amount of a polypeptide of the instant invention.

A further object of the instant invention provides a method of treating prostate cancer in a mammal by administering a pharmacologically effective amount of a polypeptide of the instant invention. Another object is to provide a method of inhibiting bFGF and FGFR- 1 -mediated signaling in a mammal by administering a pharmacologically effective amount of a polypeptide of the instant invention. A further object of the claimed invention provides a method of counteracting FGF overexpression associated with certain types of tumors in a mammal by administering a polypeptide of the instant invention.

A further object of the instant invention provides a method of controlling the growth, development or differentiation of any cell responsive to FGF signaling in a mammal by administering a polypeptide of the instant invention. Such differentiation includes metanephrogenesis, angiogenesis, tumorogenesis and neurogenesis.

Yet another object of the instant invention provides a method of controlling the growth, development, or differentiation of adrenal gland cells responsive to FGF signaling in a mammal by administering a polypeptide of the instant invention. Another object of the instant invention provides a method of treating a pathology involving hypersensitive responses to FGF in a mammal by administering a polypeptide of the instant invention. Yet a further object of the instant invention provides a method of treating a mammal having an adrenal gland pathology involving hypersensitive responses to FGF in a mammal by administering a polypeptide of the instant invention. Another object of the instant invention provides a method of treating developmental anomalies associated with disregulated FGF signaling, in a mammal, by administering a polypeptide of the instant invention. A further object of the instant invention provides a method of counteracting SPROUTY underexpression associated with certain types of tumors or pathology in a mammal by administering a polypeptide of the instant invention. Yet another object of the invention provides a method to counteract SPROUTY underexpression associated with certain types of tumors in a mammal by administering a pharmacologically effective amount of a polypeptide of the instant invention.

In several of these methods, the polypeptide is acting on glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, or colorectal carcinoma cells. Further, in some of the methods of the instant invention, the mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Certain types of FGF, such as basic FGF (FGF-2), are nearly ubiquitous in their distribution, requiring highly specific mechanisms to regulate their bioavailability and activity. Various processes have been described to regulate FGF bioavailability. Baird supra (1994). The instant claimed invention shows that novel SPROUTY polypeptides are also nearly ubiquitous in most human tissues, and are overabundant in the tissues of certain disease states. These novel SPROUTY polypeptides function as antagonists of FGF activity and inhibit FGF action. The SPROUTY polypeptides regulate a target cell's ability to activate FGF in its local milieu. In other words, the instant invention recognizes an important homeostasis between FGF and SPROUTY. When FGF increases in concentration or distribution, SPROUTY keeps FGF in check. When SPROUTY is underexpressed, FGF may become overexpressed. When FGF is overexpressed, SPROUTY can limit the FGF activity. SPROUTY also may be used as replacement therapy or when needed to augment or maintain the FGF-SPROUTY homeostasis.

Several homologs of SPROUTY have been discovered and, through insights related to their primary structure, a class of FGF antagonists is described. These FGF antagonists, sharing the aforementioned cysteine-rich domain, are candidates for therapeutic compositions suitable for treating pathological conditions characterized by a dysfunction in an FGF signaling pathway, as would occur, for example, with unchecked expression of FGF.

Polypeptides that are related structurally to the protein, SPROUTY- 1, can be produced recombinantly and can be used to inhibit vascularization, for example, in the context of counteracting abnormal cell proliferation or undesired angiogenesis. The instant claimed invention employs SPROUTY polypeptides to provide a new FGF antagonist to block such FGF-dependent activities as angiogenesis, tumor growth, reproduction, and diseases of cell proliferation. A polynucleotide coding for such a polypeptide, and a complement thereof, finds application in inhibiting, for example, prostate cell proliferation, adrenal tumor growth; limiting the range of FGF levels in vivo; inhibiting an adverse effect of FGF; inhibiting bFGF and FGFR- 1 -mediated signaling; counteracting FGF overexpression associated with certain types of tumors controlling the growth, development, and/or differentiation of any cell responsive to FGF signaling; treating various pathologies involving hypersensitive responses to FGF; treating conditions in which there is an underexpression of sprouty-1 and treating developmental anomalies associated with disregulated FGF signaling. A method of counteracting SPROUTY underexpression associated with certain types of mammalian tumors entails adminstering SPROUTY polypeptide. Also, SPROUTY can be used to act on such disregulated cells as glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells. Further, SPROUTY can be used to treat overexpressed FGF or disregulated FGF in such diseases as: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

Accordingly, the present invention comprehends nucleic acid molecules, isolated, recombinant or synthetic, that code for a SPROUTY homolog sequence, as discussed above, or a biologically active portion or variants thereof. In the present description these nucleic acid molecules, are designated collectively as "sprouty polynucleotides."

In addition to the therapeutic utility mentioned above, the present invention also encompasses isolated nucleic acid molecules of sufficient length and complementarity to a sprouty polynucleotide to be useful as probes or as amplification primers in the detection, quantitation, or isolation of gene transcripts. For example, isolated nucleic acids of the present invention can be used: (1) as probes to detect deficiencies in the level of mRNA; (2) in screenings for detecting mutations in the gene (e.g., substitutions, deletions, or additions), (3) for monitoring upregulation of expression or changes in biological activity as described herein in screening assays of compounds, and/or for detection of any number of allelic variants (polymorphisms) of the gene.

The nucleic acid molecules of the present invention also can be used to express recombinant SPROUTY polypeptides, which can be used as immunogens in the preparation and/or screening of antibodies. The isolated nucleic acids of the present invention also can be employed for use in sense or antisense suppression of one or more

SPROUTY-1 genes, nucleic acids or genes in a host cell, or tissue. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention also can be used to modulate transcription or translation of at least one nucleic acid. The SPROUTY polypeptides, SPROUTY polynucleotides and antibodies specific for SPROUTY polypeptides may be used as diagnostic reagents to identify when SPROUTY polypeptides levels in vivo are either elevated or lower than normal.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the shorter sprouty amino acid consensus sequence.

Figure 2 shows the longer sprouty amino acid consensus sequence.

Figure 3 shows the amino acid sequence of SEQ ID NO. 3. Figure 4 shows the nucleic acid sequence of SEQ ID NO. 4.

Figure 5 shows the amino acid sequence of SEQ ID NO. 5.

Figure 6 shows the nucleic acid sequence of SEQ ID NO. 6.

Figure 7 shows the amino acid sequence of SEQ ID NO. 7.

Figure 8 shows the nucleic acid sequence of SEQ ID NO. 8. Figure 9 shows the amino acid sequence of SEQ ID NO. 9.

Figure 10 shows the nucleic acid sequence of SEQ ID NO. 10.

Figure 11 shows the hspryl amino acid sequence.

Figure 12 shows the hspry2 amino acid sequence.

Figure 13 shows the hspry3 amino acid sequence. Figure 14 shows the mspryl amino acid sequence.

Figure 15 shows the mspry4 amino acid sequence.

Figure 16 shows the drosophila spry amino acid sequence.

Figure 17 shows the hspryl nucleic acid sequence. Figure 18 shows the hspry2 nucleic acid sequence. Figure 19 shows the hspry3 nucleic acid sequence. Figure 20 shows the mspryl nucleic acid sequence. Figure 21 shows the mspry2 nucleic acid sequence. Figure 22 shows the mspry4 nucleic acid sequence.

Figures 23a and 23b show the drosophila spry nucleic acid sequence. Figure 24 shows a comparison of amino acid sequences of SEQ ID Nos. 3, 5, 7, and 9. Figures 25 A and 25B are photographs of two RT-PCR Northern blots. Figure 26 shows the amino acid sequence of SEQ ID NO. 24. Figure 27 shows the nucleic acid sequence of SEQ ID NO. 25.

CITATIONS

All references to publications cited herein are entirely incorporated herein by reference, as they show the state of the art at the time of the present invention, to describe and enable the present invention. Publications refer to scientific, patent publication or any other information available in any media format, including all recorded, electronic or printed formats. The following citations are entirely incorporated by reference: Ausubel et al, ed., Current Protocols in Molecular Biology, Greene Publishing (1987-1988); Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan et al. , eds., Current Protocols in Immunology. Greene Publishing (1994-1988).

DEFINITIONS The following definitions of terms are intended to correspond to those as well- known in the art. The following terms are therefore not limited to the definitions given, but are used according to the state of the art, as demonstrated by cited and/or contemporary publications.

A "polynucleotide" comprises at least 5 - 10 nucleotides of a nucleic acid (RNA, DNA or combination thereof), provided by any means, such as synthetic, recombinant isolation or purification method steps.

The terms "complementary" or "complementarity" as used herein refer to the capacity of purine, pyrimidine, synthetic or modified nucleotides to associate by partial or complete complementarity through hydrogen or other bonding to form partial or complete double or triple stranded nucleic acid molecules. The following base pairs occur by complete complementarity: (i) guanine (G) and cytosine (C); (ii) adenine (A) and thymine (T); and adenine (A) and uracil (U). "Partial complementarity" refers to association of two or more bases by one or more hydrogen bonds or attraction that is less than the complete complementarity as described above. Partial or complete complementarity can occur between any two nucletides, including naturally occurring or modified bases, e.g., as listed in 37 CFR sec. 1.822. All such nucleotides are included in polynucleotides of the invention as described herein. "Fragment" refers to a fragment, piece, portion, or sub-region of a nucleic acid or polypeptide molecule as disclosed herein, such that the fragment comprises 4 or more amino acids, or 10 or more nucleotides, that are contiguous in the referenced polypeptide or nucleic acid molecule. A fragment thereof may or may not retain biological activity. For example, a fragment of a polypeptide disclosed herein could be used as an antigen to raise a specific antibody against the referenced polypeptide molecule.

The term "fusion protein" denotes a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked on a single polypeptide chain. The term "polypeptide" also includes such fusion proteins.

"Host cell" refers to any eucaryotic, procaryotic, or fusion or other cell or pseudo cell or membrane containing construct that is suitable for propagating and/or expressing an isolated nucleic acid that is introduced into the host cell by any suitable means known in the art (e.g. , but not limited to, transformation or transfection, or the like). The cell can be part of a tissue or organism, isolated in culture or in any other suitable form.

The term "hybridization" as used herein refers to a process in which a partially or completely single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. Hybridization can occur under conditions of low, moderate to high stringency, with high stringency preferred. The degree of hybridization depends upon, for example, the degree of homology, the stringency conditions, and the length of hybridizing strands. By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA, RNA, or both which has been removed from its native or naturally occurring environment. For example, recombinant nucleic acid molecules contained or generated in culture, a vector and/or a host cell are considered isolated for the purposes of the present invention. Further examples of isolated nucleic acid molecules include recombinant nucleic acid molecules maintained in heterologous host cells or purified (partially or substantially) nucleic acid molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the nucleic acid molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically, purified from or provided in cells containing such nucleic acids, where the nucleic acid in other than a naturally occurring form, quantitatively or qualitatively.

"Isolated" used in reference to at least one polypeptide of the invention describes a state of isolation such that the peptide or polypeptide is not in a naturally occurring form and/or has been purified to remove at least some portion of cellular or non-cellular molecules with which the protein is naturally associated. However, "isolated" may include the addition of other functional or structural polypeptides for a specific purpose, where the other peptide may occur naturally associated with at least one polypeptide of the present invention. A "nucleic acid probe," "oligonucleotide probe," or "probe" as used herein comprises at least one detectably labeled or unlabeled nucleic acid which hybridizes under specified hybridization conditions with at least one other nucleic acid. This term also refers to a single or partially double stranded nucleic acid, oligonucleotide or polynucleotide that will associate with a complementary or partially complementary target nucleic acid to form at least a partially double-stranded molecule. A nucleic acid probe may be an oligonucleotide or a nucleotide polymer. A probe can optionally contain a detectable moiety which may be attached to the end(s) of the probe or be internal to the sequence of the probe, termed a "detectable probe" or "detectable nucleic acid probe. " A "primer" is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule, e.g., using a amplification reaction, such as, but not limited to, a polymerase chain reaction (PCR), as known in the art. The term "promoter" refers to a nucleic acid sequence that directs the initiation of transcription, for example, of DNA to RNA. An inducible promoter is one that is regulatable by environmental signals, such as carbon source, heat, or metal ions, for example. The term "stringency" refers to hybridization conditions for nucleic acids in solution. High stringency conditions disfavor non-homologous base pairing. Low stringency conditions have much less of this effect. Stringency may be altered, for example, by temperature and salt concentration, or other conditions, as well known in the art. A non-limiting example of "high stringency" conditions includes, for example, (a) a temperature of about 42° C , a formamide concentration of about 20% , and a low salt (SSC) concentration; or, alternatively, a temperature of about 65° C, or less, and a low salt (SSPE) concentration; (b) hybridization in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C (See, e.g., Ausubel et al , ed., Current Protocols in Molecular Biology, 1987-1998, Wiley Interscience, New York, at §2.10.3). "SSC" comprises a hybridization and wash solution. A stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0. "SSPE" comprises a hybridization and wash solution. A IX SSPE solution contains 180 mM NaCl, 9mM Na2HPO4, 0.9 mM Na2HPO4 and 1 mM EDTA, pH 7.4. The term "vector" as used herein refers to a nucleic acid compound used for introducing exogenous or endogenous nucleic acid into host cells. A vector comprises a nucleotide sequence which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses, and bacteriophages, in a natural state or which have undergone recombinant engineering, are non-limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated nucleic acid molecule.

The various restriction enzymes disclosed and described herein are commercially and/or available and the manner of use of the enzymes including reaction conditions, cofactors, and other requirements for activity are well known to one of ordinary skill in the art (New England Biolabs, Boston; Life Technologies, Rockville, Md.). Reaction conditions for particular enzymes are preferably carried out according to the manufacturer's recommendation. NUCLEIC ACID MOLECULES

Unless otherwise indicated, all nucleotide sequences identified by sequencing a nucleic acid molecule herein were identified using an automated nucleic acid sequencer, and all amino acid sequences of polypeptides encoded by nucleic acid molecules identified herein were identified by codon correspondence or by translation of a nucleic acid sequence identified as described herein or as known in the art. Therefore, as is well known in the art that for any nucleic acid sequence identified by this automated approach, any nucleotide sequence identified herein may contain some errors which are reproducibly correctable by resequencing using well-known methods. Nucleotide sequences identified by automation are typically at least about 95 % to at least about 99.999% identical to the actual nucleotide sequence of the sequenced nucleic acid molecule. The actual sequence can be more precisely identified by other approaches including manual nucleic acid sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in an identified nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the identified amino acid sequence encoded by an identified nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced nucleic acid molecule, beginning at the point of such an insertion or deletion. Using the information provided herein, such as the nucleotide sequences encoding at least a 3-50 amino acid fragment of at least one of SEQ ID NOS: 1, 2, 3, 5, 7 and 9, or a deposited vector comprising at least one of these sequences, a nucleic acid molecule of the present invention encoding a SPROUTY- 1 polypeptide can be obtained using well-known cloning and/or screening procedures, such as those for cloning cDNAs using mRNA as starting material.

One of the identified nucleic acid sequences encoding a human sprouty- 1 protein, shown as SEQ ID NO: 4, encodes a polypeptide of 295 amino acid residues (SEQ ID NO: 3). SEQ ID NO: 4 was isolated from a library constructed using polyA RNA isolated from adrenal tumor tissue removed from a 57-year-old Caucasian female during a unilateral right adrenalectomy. The pathology of this tissue source indicated pheochromocytoma (chromaffin cell tumor), forming a nodular mass completely replacintg the medulla of the adrenal gland cDNA synthesis was initiated using an oligo(dT) primer. Double-stranded cDNA was blunted and cloned into the pINCY vector.

Another identified nucleic acid sequence, SEQ ID NO: 6, encodes a human sprouty- 1 polypeptide (SEQ ID NO: 5) that is identical to the amino acid sequence encoded by SEQ ID NO: 4, namely, SEQ ID No. 3, except that SEQ ID No. 3 contains a glutamic acid (E) residue toward the amino end. Both SEQ ID Nos. 3 and 5 contain a large consensus sequence shown in SEQ ID NOS: 1 and 2. These polynucleotides could be from different genes. SEQ ID NO: 6 has an expression pattern that suggests it is abundantly expressed in prostate, thyroid, parathyroid, adrenal tumor and rheumatoid arthritis.

A third identified nucleic acid sequence (SEQ ID NO: 8) encodes a human sprouty- 1 protein that differs from the SPROUTY- 1 polypeptides shown in SEQ ID NOS: 3 and 5, yet includes a large consensus sequence shown in SEQ ID NOS: 1 and 2. SEQ ID NO: 8 has an expression pattern that suggests it is ubiquitously expressed and abundantly expressed in abnormal prostate (benign prostatic hypertrophy with adenocarcinoma) and abnormal skin (Patau's syndrome). SEQ ID NO: 8 encodes a polypeptide of 339 amino acid residues (SEQ ID NO: 7). Notably, another emodiment of the claimed invention is directed to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7, yet the polypeptide sequence is devoid of the first 19 amino acids, which are FGSSACQVSTDCQNSRSLH, as shown in SEQ ID NO: 24. Another aspected of the claimed invention is directed to a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 7 without the first 19 amino acids, namely FGSSACQVSTDCQNSRSLH .

A fourth identified nucleic acid sequence encoding a human sprouty- 1 protein, shown as SEQ ID NO: 10, differs from all three clones shown in SEQ ID NOS: 4, 6 and 8, yet encodes the large amino acid consensus sequence shown in SEQ ID NOS: 1 and 2. SEQ ID NO: 10 has an expression pattern that suggests it is abundantly expressed in pancreatic tumor, gallbladder, heart tumor (myxoma), neuroganglion tumor (ganglioneuroma) and normal placenta. SEQ ID NO: 10 encodes a polypeptide of 167 amino acid residues (SEQ ID NO: 9).

As indicated, nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA can be triple-, double- stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the antisense strand. Nucleic acid molecules of the present invention can be in the form of hybrid or inverted hybrid

RNA-DNA oligonucleotides which may or may not contain modifications, such as those of the form described in U.S. Patent 5,652,355; U.S. Patent 5,652,356; U.S. Patent 5,149,797; U.S. Patent 5,663,153 and U.S. Patent 5,723,335.

Isolated nucleic acid molecules of the present invention include nucleic acid molecules comprising an open reading frame (ORF) shown in SEQ ID NOS: 4, 6, 8 and 10; nucleic acid molecules comprising the coding sequence for a SPROUTY- 1 polypeptide; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one SPROUTY- 1 polypeptide as described and enabled herein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific SPROUTY- 1 polypeptides of the present invention. See, e.g., Ausubel, et al.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a SPROUTY- 1 polypeptide having an amino acid sequence of any of SEQ ID

Nos. 1, 2, 3, 5, 7 or 9, or combinations thereof.

The invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NOS: 4, 6, 8 and 10 or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly nucleic acid molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting transcription, translation and/or expression of the SPROUTY-1 gene in human tissue, for instance, by Northern blot analysis for mRNA detection.

NUCLEIC ACID FRAGMENTS

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having at least 10 nucleotides of a nucleotide sequence of a deposited cDNA or a nucleotide sequence shown in SEQ ID NOS: 4, 6, 8 or 10, and is intended fragments at least about 10 nt, at least about 15 nt, at least about 30 nt, and at least about 40 nt in length, which are useful, inter alia as diagnostic probes and primers as described herein. Of course, larger fragments such as at least about 50, 100, 120, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, and/or 4000 nt in length, are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown at least one of SEQ ID NOS: 4, 6, 8 or 10. By a fragment at least 10 nt in length, for example, is intended fragments which include 10 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOS: 4, 6, 8 or 10, or consensus sequences thereof, as determined by methods known in the art.

Such nucleotide fragments are useful according to the present invention for screening DNA sequences that code for one or more fragments of a SPROUTY-1 polypeptide as described herein. As indicated, nucleic acid molecules of the present invention which comprise a nucleic acid encoding a SPROUTY-1 polypeptide can include, but are not limited to, those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA); an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding a polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.

Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of a SPROUTY-1 polypeptide.

OLIGONUCLEOTIDE AND POLYNUCLEOTIDE PROBES

In another aspect, the invention provides a polynucleotide (either DNA or RNA) that comprises at least about 10 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably at least about 30- 2000 nt of a nucleic acid molecule described herein. These are useful as diagnostic probes and primers as discussed above and in more detail below.

By a portion of a polynucleotide of "at least 10 nt in length," for example, is intended 10 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited nucleic acid or the nucleotide sequence as shown in SEQ ID NOS: 4, 6, 8 or 10.

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly (A) of a SPROUTY-1 polypeptide cDNA shown in SEQ ID NOS: 4, 6, 8 or 10, or to a complementary stretch of T (or U) residues, would not be included in a probe of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

The present invention also provides subsequences of full-length nucleic acids. Any number of subsequences can be obtained by reference to SEQ ID NOS: 4, 6, 8 or 10, and using primers which selectively amplify, under stringent conditions to: at least two sites to the polynucleotides of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it. A variety of methods for obtaining 5' and/or 3' ends is well known in the art. See, e.g., RACE (Rapid Amplification of Complementary Ends) as described in Frohman, M. A., in PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc. , San Diego, 1990), pp. 28-38.); see also, U.S. Pat. No. 5,470,722, and Current Protocols in Molecular Biology, Unit 15.6, Ausubel, et al. , Eds., Greene Publishing and Wiley-Interscience, New York (1995). Thus, the present invention provides SPROUTY-1 polynucleotides having the sequence of the SPROUTY-1 gene, nuclear transcript, cDNA, or complementary sequences and/or subsequences thereof.

Primer sequences can be obtained by reference to a contiguous subsequence of a polynucleotide of the present invention. Primers are chosen to selectively hybridize, under PCR amplification conditions, to a polynucleotide of the present invention in an amplification mixture comprising a genomic and/or cDNA library from the same species. Generally, the primers are complementary to a subsequence of the amplicon they yield. In some embodiments, the primers will be constructed to anneal to the sequence toward the 5' terminal end of the codon of the polynucleotides, or the complements thereof, of the present invention. The primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50. Thus, the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length. A non- annealing sequence at the 5' end of the primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon.

The amplification primers may optionally be elongated in the 3' direction with additional contiguous nucleotides from the polynucleotide sequences, such as SEQ ID NOS: 4, 6, 8 or 10, from which they are derived. The number of nucleotides by which the primers can be elongated is selected from the group of integers consisting of from at least 1 to at least 25. Thus, for example, the primers can be elongated with an additional 1, 5, 10, or 15 nucleotides. Those of skill will recognize that a lengthened primer sequence can be employed to increase specificity of binding (i.e. , annealing) to a target sequence.

The amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra. The resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of the present invention. Methods for protein synthesis from PCR derived templates are known in the art and available commercially. See, e.g. , Amersham Life Sciences, Inc, Catalog '97, p.354.

POLYNUCLEOTIDES WHICH SELECTIVELY HYBRIDIZE TO A POLYNUCLEOTIDE AS DESCRIBED HEREIN

As indicated above, the present invention provides isolated nucleic acids comprising SPROUTY-1 polynucleotides, wherein the polynucleotides selectively hybridize, under selective hybridization conditions, to a polynucleotide as discussed, supra. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides. For example, polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a dicot or monocot nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA libraries can be normalized to increase the representation of rare sequences. Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.

Optionally, the polynucleotides of this embodiment will share an epitope with a polypeptide encoded by the polynucleotides described above. Thus, these polynucleotides encode a first polypeptide which elicits production of antisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide described above. The polynucleotides of this embodiment embrace nucleic acid sequences which can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.

Screening polypeptides for specific binding to antisera can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 15 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. Such methods are described in PCT patent publication Nos. WO91/17271, WO91/18980, WO91/19818, and WO93/08278. Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent publication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Patent Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, CA).

POLYNUCLEOTIDES COMPLEMENTARY TO THE POLYNUCLEOTIDES As indicated above, the present invention provides isolated nucleic acids comprising SPROUTY-1 polynucleotides, wherein the polynucleotides are complementary to the polynucleotides described herein, above. As those of skill in the art will recognize, complementary sequences base-pair throughout the entirety of their length with such polynucleotides (i.e., have 100% sequence identity over their entire length). Complementary bases associate through hydrogen bonding in double stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.

Antisense Inhibitors Given the disclosed nucleic acid sequence encoding the hspry polypeptide, namely SEQ ID Nos.4, 6, 8, and 10, certain spry-specific inhibitors of expression may be rationally designed. Most commonly, these inhibitors will be relatively small RNA or DNA molecules because they can be designed to be highly specific. In general, so- called "antisense" molecules will have a sequence which is complementary to a portion of the spry mRNA, preferably the pre-mRNA, i.e., the pre-splicing version. More preferred antisense molecules will be specific for the polynucleotides encoding the spry consensus sequence shown in SEQ ID Nos. 1 or 2. One particularly preferred class of antisense molecules is directed to the control elements for splicing and/or translation. The "splicing control elements" include the splice junctions. As indicated, the antisense molecules can have a variety of chemical constitutions, so long as they retain the ability specifically to bind at the indicated control elements. Thus, especially preferred molecules are oligo-DNA, RNA, DNA- RNA hybrid, chimerics or mixed backbone oligonucleotides and protein nucleic acids (PNAs). The oligonucleotides of the present invention can be based, for example, upon ribonucleotide or deoxyribonucleotide monomers linked by phosphodiester bonds, or by analogues linked by methyl phosphonate, phosphorothioate, other bonds or combinations thereof. These can be engineered using standard synthetic techniques to very specifically bind the targeted control region(s). While these molecules may also be large, they are preferably relatively small, i.e., corresponding to less than about 50 nucleotides. Such oligonucleotides may be prepared by methods well-known in the art, for instance using commercially available machines and reagents available from Perkin- Elmer/ Applied Biosystems (Foster City, CA). Phosphodiester-linked oligonucleotides are particularly susceptible to the action of nucleases in serum or inside cells, and therefore in a preferred embodiment the oligonucleotides of the present invention contain at least one phosphorothioate or methyl phosphonate-linked analogues, which have been shown to be nuclease-resistant. See Stein et al , PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDE ANALOGUES in "Oligodeoxynucleotides - Antisense Inhibitors of Gene Expression" Cohen, Ed. McMillan Press, London (1988). Persons knowledgeable of this field will be able to select other linkages or combinations thereof for use in the present invention.

To select the preferred length for an antisense oligonucleotide, a balance must be struck to gain the most favorable characteristics. Shorter oligonucleotides 10-15 bases in length readily enter cells, but have lower gene specificity. In contrast, longer oligonucleotides of 20-30 bases offer superior gene specificity, but show decreased kinetics of uptake into cells. See Stein et al. , Science 261: 1004 (1993). In a preferred embodiment this invention contemplates using oligonucleotides approximately 14 to 25 nucleotides long. Antisense molecules can be delivered in a variety of ways. They may be synthesized and delivered as a typical pharmaceutical, usually parenterally. They may be formulated as detailed below, but one preferred formulation involved encapsulation association with cationic liposomes. Alternatively, antisense molecules may be delivered using gene therapy methods, detailed below. Using gene therapy vectors, single, or multiple tandem copies of antisense molecules can be used.

Administration of an antisense oligonucleotide to a subject can be effected orally or by subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Other modes of administration are comtemplated such as intravitreol injection, transplacental delivery or inhalation can be used if appropriate for the condition being treated. Pharmaceutical compositions of the present invention, however, are advantageously administered in the form of injectable compositions. A typical composition for such purpose comprises a pharmaceutically acceptable solvent or diluent and other suitable, physiologic compounds. For instance, the composition may contain oligonucleotide and about 10 mg of human serum albumin per milliliter of a phosphate buffer containing NaCl. As much as 700 milligrams of antisense oligodeoxynucleotide has been administered intravenously to a patient over a course of 10 days (i.e., 0.05 mg/kg/hour) without signs of toxicity. Sterling, "Systemic Antisense Treatment Reported," Genetic Engineering News 12: 1, 28 (1992).

CONSTRUCTION OF NUCLEIC ACIDS

The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well known in the art.

The nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention. The nucleic acid of the present invention - excluding the polynucleotide sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Typically, the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.

RECOMBINANT METHODS FOR CONSTRUCTING NUCLEIC ACIDS

The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. While isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art.

NUCLEIC ACID SCREENING AND ISOLATION METHODS

The cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.

For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0% to 50% . The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100 percent; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and can be used according to the present invention without undue experimentation, based on the teaching and guidance presented herein. According to the present invention, the use of nucleic acids encoding portions of SPROUTY polypeptide according to the present invention, as amplification primers, allows for advantages over known amplification primers, due to the increase in sensitivity, selectivity and/or rate of amplification. Known methods of DNA or RNA amplification include, but are not limited to polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. patent Nos. 4,683,195, 4,683,202, 4,800, 159, 4,965, 188, to Mullis et al; 4,795,699 and 4,921,794 to Tabor et al; 5,142,033 to Innis; 5,122,464 to Wilson et al ; 5,091,310 to Innis; 5,066,584 to Gyllensten et al; 4,889,818 to Gelfand et al; 4,994,370 to Silver et al; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediated amplification which uses antisense RNA to the target sequence as a template for double stranded DNA synthesis (U.S. patent No. 5,130,238 to Malek et al, with the tradeneame NASBA), the entire contents of which patents are herein entirely incorporated by reference.

For instance, PCR technology can be used to amplify the sequences of polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. , U.S. Patent No. 4,683,202 (1987); and, PCR Protocols A Guide to Methods and Applications, Innis et al. , Eds., Academic Press Inc., San Diego, CA (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.

SYNTHETIC METHODS FOR CONSTRUCTING NUCLEIC ACIDS

The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al. , Meth. Enzymol. 68: 90 (1979); the phosphodiester method of Brown et al. , Meth. Enzymol. 68: 109 (1979); the diethylphosphoramidite method of Beaucage et al. , Tetra. Lett. 22: 1859 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22(20): 1859 (1981), e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter et al. , Nucleic Acids Res., 12: 6159 (1984); and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

RECOMBINANT EXPRESSION CASSETTES The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a full-length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell.

Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter SPROUTY-1 content and/or composition in a desired tissue.

In some embodiments, isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution.

A polynucleotide of the present invention can be expressed in either sense or antisense orientation as desired. It will be appreciated that control of gene expression in either sense or antisense orientation can have a direct impact on the observable characteristics.

Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. A variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov et al , Nucleic Acids Res 14: 4065 (1986), describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of similar work by the same group is that by Knorre et al. , Biochimie 67: 785 (1985). Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J Am Chem Soc 109: 1241 (1987)). Meyer et al , J Am Chem Soc 111: 8517 (1989), effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee et al, Biochemistry 27: 3197 (1988). Use of crosslinking in triple-helix forming probes was also disclosed by Home et al. , J Am Chem Soc 112: 2435 (1990). Use of N4, N4-eιhanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, J Am Chem Soc 108: 2764 (1986); Nucleic Acids Res 14: 7661(1986); Feteritz et al, J. Am. Chem. Soc. 113: 4000 (1991). Various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Patent Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and, 5,681941.

VECTORS AND HOST CELLS

The present invention also relates to vectors which include isolated nucleic acid molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of SPROUTY-1 polypeptides or fragments thereof by recombinant techniques, as well known in the art. See, eg., Sambrook, et al , 1989; Ausubel, et al , 1987-1989, each entirely incorporated herein by reference. The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include, e.g., dihydrofolate reductase or neomycin resistance for eucaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art. Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHlόa, pNHlδA, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eucaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16. The polypeptide can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a polypeptide to facilitate purification. Such regions can be removed prior to final preparation of a polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

EXPRESSION OF PROTEINS IN HOST CELLS

Using nucleic acids of the present invention, one may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made. In brief summary, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/ translation terminator. One of skill would recognize that modifications can be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences. EXPRESSION IN PROKARYOTES

Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al, Nature 198: 1056 (1977)), the tryptophan (tip) promoter system (Goeddel et al. , Nucleic Acids Res. 8: 4057 (1980)) and the lambda derived PL promoter and N- gene ribosome binding site (Shimatake et al, Nature 292: 128 (1981)). The inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al. , Gene 22: 229 (1983); Mosbach, et al , Nature 302: 543 (1983)).

EXPRESSION IN EUKARYOTES

A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a SPROUTY-1 polypeptide of the present invention can be expressed in these eukaryotic systems.

Synthesis of heterologous proteins in yeast is well known. Sherman et al. , Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to produce the protein in yeast. Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3 -phosphogly cerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al , Immunol. Rev. 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992).

Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line. See Schneider, J. Embryol. Exp. Morphol. 27: 353 (1987).

As with yeast, when higher animal or plant host cells are employed, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al. , J. Virol. 45: 773 (1983)). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M. , Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning Vol. II a Practical Approach, D.M. Glover, Ed., IRL Press, Arlington, Virginia pp. 213-238 (1985).

EXPRESSED PROTEIN PURIFICATION

A SPROUTY-1 polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxy lapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eucaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention can be glycosylated or can be non-glycosylated. In addition, polypeptides of the invention can also include an initial modified methionine residue, in some cases as a result of host- mediated processes. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.

SPROUTY-1 POLYPEPTIDES AND FRAGMENTS AND VARIANTS

The invention further provides an isolated SPROUTY-1 polypeptides having fragments or specified variants of the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in SEQ ID NOS: 1, 2, 3, 5, 7 or 9.

The isolated proteins of the present invention comprise a polypeptide having at least 5 - 10 amino acids encoded by any one of the polynucleotides of the present invention as discussed more fully, supra, or polypeptides which are conservatively modified variants thereof.

Amino acid sequence variants of a polypeptide sequence can be substitutional. Substitutional variant typically contain the exchange of one amino acid for another at one or more sites within the protein. Substititions preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include such changes as: leucine to isoleucine and threonine to serine.

Exemplary polypeptide sequences are provided in SEQ ID NOS: 1, 2, 3, 5, 7 or 9. The proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 10 to the number of residues in the SPROUTY-1 polypeptides disclosed. Optionally, this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acids in length, often at least 50, 60, 70, 80, or 90 amino acids in length. Further, the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes biologically active polypeptides of the present invention (e.g., enzymes). Biologically active polypeptides have a specific activity at least 20%, 30%, or 40%, and preferably at least 50% , 60% , or 70% , and most preferably at least 80% , 90%, or 95% - 100% of that of the native (non-synthetic), endogenous polypeptide. Further, the substrate specificity (kcat/Km) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide. Typically, the Km will be at least 30%, 40%, or 50%, that of the native (non-synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or 90 % . Methods of assaying and quantifying measures of enzymatic activity and substrate specificity (kcat/Km), are well known to those of skill in the art.

Generally, the polypeptides of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention encoded by a polynucleotide of the present invention as described, supra. Exemplary polypeptides include those disclosed, such as those disclosed in SEQ ID NO: 1, 2, 3, 5, 7 or 9. Further, the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide. Immunoassays for determining binding are well known to those of skill in the art. A preferred immunoassay is a competitive immunoassay as discussed, infra. Thus, the proteins of the present invention can be employed as immunogens for constructing antibodies immunoreactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.

A SPROUTY-1 polypeptide of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.

Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions for any given SPROUTY-1 polypeptide will not be more than 20, 10, 5, or 3, such as 1-20 or any range or value therein, as specified herein.

Amino acids in a SPROUTY-1 polypeptide of the present invention that are essential for function can be identified by methods known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as protein binding or apoptosis and/or transforming activity. Sites that are critical for ligand-protein binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al. , J. Moi Biol. 224: 899(1992) and de Vos et al, Science 255: 306 (1992)). SPROUTY-1 polypeptides of the present invention can include but are not limited to, at least one selected from:

(a) SEQ ID NO: 3;

(b) SEQ ID NO: 5;

(c) SEQ ID NO: 7; (a) SEQ ID NO: 9;

A SPROUTY-1 polypeptide can further comprise a polypeptide encoded by the contiguous amino acids of SEQ ID NOS: 3, 5, 7 or 9.

A SPROUTY-1 polypeptide further includes an amino acid sequence selected from one or more of SEQ ID NOS: 1, 2, 3, 5, 7 or 9. Non-limiting mutants that can enhance or maintain at least one of the listed activities include, but are not limited to, any of the above polypeptides, further comprising at least one mutation in the amino acid sequence of the SPROUT- 1 polypeptide sequences identified in SEQ ID NOS 3, 5, 7, or 9, as long as the polypeptide maintains its function as an antagonist of FGF. Preferably, the identified FGF antagonistic SPROUTY polypeptides have at least one mutation at the position(s) discussed below. The amino acid position numbers identified correspond to the sequence shown between amino acids 156 and 294 of Figure 24. The amino acid position numbers within Figure 24 correspond to Figure 3 at amino acids 156 through 294; Figure 5 at amino acids 157 through 295; Figure 7 at amino acids 200 through 338; Figure 9 at amino acids 200 through 338; Figure 1 at amino acids 18 through 156 and Figure 2 at amino acids 150 through 287. The particular amino acids are referred to by their conventional one letter codes, as they are presented in the particular SEQ ID numbers identified. Specific examples of the preferred mutations include at least one of the following, including any combination thereof in which the SPROUTY polypeptide maintains its function as an antagonist of FGF: the Q at amino acid position 158 is changed to a D; the G at amino acid 165 is changed to a K; the A at amino acid 168 is changed to a Y; the T at amino acid 172 is changed to a P; the C at amino acid 176 is changed to a D; the L at amino acid 177 is changed to a W; the A at amino acid 178 is changed to an I; the N at amino acid 180 is changed to a D; the R at amino acid 181 is changed to a K; the E at amino acid 188 is changed to a Q; the S at amino acid 189 is changed to an N; the M at amino acid 190 is changed to a V; the V at amino acid 191 is changed to an I; the E at amino acid 192 is changed to a D; the M at amino acid 197 is changed to a V; the L at amino acid 199 is changed to a C; the I at amino acid 203 is changed to an L; the G at amino acid 213 is removed so that the E at 212 is contiguous with the D at 214; the S at amino acid 215 is changed to an N; the Y at amino acid 216 is changed to a C; the S at amino acid 217 is changed to an A; the S at amino acid 230 is changed to a T; the Y at amino acid 232 is changed to a W; the L at amino acid 233 is changed to an S; the C at amino acid 234 is changed to an A; the A at amino acid 237 is changed to a V; the L at amino acid 246 is changed to a W; the P at amino acid 249 is changed to an L; the R at amino acid 259 is changed to a W; the R at amino acid 260 is changed to a C; the W at amino acid 264 is changed to an R; the I at amino acid 265 is changed to a V; the H at amino acid 266 is changed to an N; the Y at amino acid 279 is changed to a C; the L at amino acid 282 is changed to a V; the E at amino acid 283 is changed to a P; the S at amino acid 284 is changed to a T; the C at amino acid 285 is changed to a V; the S at amino acid 287 is changed to a P; the G at amino acid 289 is changed to an N; the Q at amino acid 290 is changed to an F; the G at amino acid 291 is changed to an E; and the S at amino acid 294 is changed to a T.

ANTIGENIC/EPITOPE COMPRISING SPROUTY-1 PEPTIDE AND POLYPEPTIDES

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention according to methods well known in the art. See, e.g., Colligan, et al,. ed., Current Protocols in Immunology, Greene Publishing, N.Y. (1993-1998), Ausubel, supra, entirely incorporated herein by reference.

The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein. An "immunogenic epitope" can be defined as a part of a polypeptide that elicits an antibody response when the whole polypeptide is the immunogen. On the other hand, a region of a polypeptide molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a polypeptide generally is less than the number of antigenic epitopes. See, for instance, Geysen et al. , Proc. Natl. Acad. Sci. USA 81: 3998 (1983).

As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a polypeptide molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a polypeptide sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked polypeptide. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.A. Antibodies that react with preidentified sites on polypeptides. Science 219: 660 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a polypeptide, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact polypeptides (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson, et al. , Cell 37: 767(1984) at 777. Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.

The epitope-bearing peptides and polypeptides of the invention can be produced by any conventional means. Houghten, R. A., General method for the rapid solid- phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82: 5131 (1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al (1986). As one of skill in the art will appreciate, SPROUTY-1 polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al. , Nature 331 : 84 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric SPROUTY-1 polypeptide or polypeptide fragment alone (Fountoulakis et al. , J. Biochem 270: 3958 (1995)).

PRODUCTION OF ANTIBODIES The polypeptides of this invention and fragments thereof may be used in the production of antibodies. The term "antibody" as used herein describes antibodies, fragments of antibodies (such as, but not limited, to Fab, Fab', Fab2', and Fv fragments), and modified versions thereof, as well known in the art (e.g., chimeric, humanized, recombinant, veneered, resurfaced or CDR-grafted) such antibodies are capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived. The instant invention also encompasses single chain polypeptide binding molecules.

The production of antibodies, both monoclonal and polyclonal, in animals is well known in the art. See, e.g., Colligan, supra, entirely incorporated herein by reference.

Single chain antibodies and libraries thereof are yet another variety of genetically engineered antibody technology that is well known in the art. (See, e.g. Bird et al , Science 242: 423(1988); PCT Publication Nos. WO 88/01649, WO 90/14430, and WO 91/10737. Single chain antibody technology involves covalently joining the binding regions of heavy and light chains to generate a single polypeptide chain. The binding specificity of the intact antibody molecule is thereby reproduced on a single polypeptide chain.

Antibodies included in this invention are useful in diagnostics, therapeutics or in diagnostic/therapeutic combinations . The polypeptides of this invention or suitable fragments thereof can be used to generate polyclonal or monoclonal antibodies, and various inter-species hybrids, or humanized antibodies, or antibody fragments, or single-chain antibodies. The techniques for producing antibodies are well known to skilled artisans. (See, e.g., Colligan supra ; Monoclonal Antibodies: Principles & Applications Ed. J.R.Birch & E.S. Lennox, Wiley-Liss, 1995.

A polypeptide used as an immunogen may be modified or administered in an adjuvant, by subcutaneous or intraperitoneal injection into, for example, a mouse or a rabbit. For the production of monoclonal antibodies, spleen cells from immunized animals are removed, fused with myeloma or other suitable known cells, and allowed to become monoclonal antibody producing hybridoma cells in the manner known to the skilled artisan. Hybridomas that secrete a desired antibody molecule can be screened by a variety of well known methods, for example ELISA assay, Western blot analysis, or radioimmunoassay (Lutz et al , Exp. Cell Res. 175, 109 (1988); Monoclonal

Antibodies: Principles & Applications Ed. J.R.Birch & E.S. Lennox, Wiley-Liss, 1995; Colligan, supra).

For some applications labeled antibodies are desirable. Procedures for labeling antibody molecules are widely known, including for example, the use of radioisotopes, affinity labels, such as biotin or avidin, enzymatic labels, for example horseradish peroxidase, and fluorescent labels, such as FITC or rhodamine (See, e.g., Colligan, supra).

Labeled antibodies are useful for a variety of diagnostic applications. In one embodiment the present invention relates to the use of labeled antibodies to detect the presence of a SPROUTY-1 polypeptide. Alternatively, the antibodies could be used in a screen to identify potential modulators of a SPROUTY-1 polypeptide. For example, in a competitive displacement assay, the antibody or compound to be tested is labeled by any suitable method. Competitive displacement of an antibody from an antibody- antigen complex by a test compound such that a test compound-antigen complex is formed provides a method for identifying compounds that bind HPLFP.

TRANSGENICS AND CHIMERIC NON-HUMAN MAMMALS

The present invention is also directed to a transgenic non-human eukaryotic animal (preferably a rodent, such as a mouse) the germ cells and somatic cells of which contain genomic DNA according to the present invention which codes for a SPROUTY- 1 polypeptide. At least one SPROUTY-1 nucleic acid can be introduced into the animal to be made transgenic, or an ancestor of the animal, at an embryonic stage, preferably the 1-1000 cell or oocyte, stage, and preferably not later than about the 64-cell stage. The term "transgene," as used herein, means a gene which is incorporated into the genome of the animal and is expressed in the animal, resulting in the presence of at least one SPROUTY-1 polypeptide in the transgenic animal.

There are several means by which such a sprouty-1 nucleic acid can be introduced into a cell or genome of the animal embryo so as to be chromosomally incorporated and expressed according to known methods.

Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain the a SPROUTY-1 polypeptide nucleic acid of the present invention, such as animals produced when fewer than all of the cells of the morula are transfected in the process of producing the transgenic animal, are also intended to be within the scope of the present invention.

Chimeric non-human mammals having human cells or tissue engrafted therein are also encompassed by the present invention, which may be used for testing expression of at least one SPROUTY-1 polypeptide in human tissue and/or for testing the effectiveness of therapeutic and/or diagnostic agents associated with delivery vectors which preferentially bind to a SPROUTY-1 polypeptide of the present invention. Methods for providing chimeric non-human mammals are provided, for example, in U.S. Patents 5,633,076; 5,639,940; 5,709,843; 5,663,481; 5,652,373 and 5,589,604, which are entirely incorporated herein by reference, for their description of how to engraft human cells or tissue into non-human mammals.

The techniques described in Leder, U.S. Patent 4,736,866 (hereby entirely incorporated by reference) for producing transgenic non-human mammals may be used for the production of a transgenic non-human mammal of the present invention. The various techniques described in Palmiter et al , Ann. Rev. Genet. 20: 465 (1986), the entire contents of which are hereby incorporated by reference, may also be used.

The animals carrying at least one SPROUTY-1 polypeptide nucleic acid can be used to test compounds or other treatment modalities which may prevent, suppress or cure a pathology using the SPROUTY-1 polypeptide or sprouty-1 nucleic acid of the present invention. Such transgenic animals will also serve as a model for testing of diagnostic methods for the same diseases. Transgenic animals according to the present invention can also be used as a source of cells for cell culture. Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLE 1: EXPRESSION AND PURIFICATION OF A SPROUTY-1 POLYPEPTIDE IN E. Coli

The bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc. , Chatsworth, CA). pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., and suitable single restriction enzyme cleavage sites. These elements are arranged such that a DNA fragment encoding a polypeptide can be inserted in such as way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide. However, a polypeptide coding sequence can optionally be inserted such that translation of the six His codons is prevented and, therefore, a polypeptide is produced with no 6 X His tag.

The nucleic acid sequence encoding the desired portion of a SPROUTY-1 polypeptide lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers (based on the sequences presented, (e.g., as presented in SEQ ID NO: 4), which anneal to the amino terminal sequences of the desired portion of a SPROUTY-1 polypeptide and to sequences in the deposited construct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.

For cloning a SPROUTY-1 polypeptide, the 5' and 3' primers have nucleotides corresponding or complementary to a portion of the coding sequence of a sprouty-1 , e.g., as presented in SEQ ID NOS: 4, 6, 8 or 10, according to known method steps. One of ordinary skill in the art would appreciate, of course, that the point in a polypeptide coding sequence where the 5' primer begins can be varied to amplify a desired portion of the complete polypeptide shorter or longer than the mature form. The amplified SPROUTY-1 nucleic acid fragments and the vector pQE60 are digested with appropriate restriction enzymes and the digested DNAs are then ligated together. Insertion of the SPROUTY-1 DNA into the restricted pQE60 vector places a SPROUTY-1 polypeptide coding region including its associated stop codon downstream from the IPTG-inducible promoter and in-frame with an initiating AUG. The associated stop codon prevents translation of the six histidine codons downstream of the insertion point.

The ligation mixture is transformed into competent E. coli cells using standard procedures such as those described in Sambrook, et al. ,1989; Ausubel, 1987-1998. E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described herein. This strain, which is only one of many that are suitable for expressing SPROUTY-1 polypeptide, is available commercially from QIAGEN, Inc. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 mg/ml) and kanamycin (25 mg/ml). The O/N culture is used to inoculate a large culture, at a dilution of approximately 1: 25 to 1 : 250. The cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.

The cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCl, pH8. The cell debris is removed by centrifugation, and the supernatant containing the SPROUTY- 1 is dialyzed against 50 mM Na-acetate buffer pH6, supplemented with 200 mM NaCl. Alternatively, a polypeptide can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH 7.4, containing protease inhibitors. After renaturation the polypeptide is purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, an affinity chromatography step such as an antibody column is used to obtain pure SPROUTY-1 polypeptide. The purified polypeptide is stored at 4°C or frozen at -80°C.

EXAMPLE 2: CLONING AND EXPRESSION OF A SPROUTY-1 POLYPEPTIDE IN A BACULOVIRUS EXPRESSION SYSTEM

In this illustrative example, the plasmid shuttle vector pA2 GP is used to insert the cloned DNA encoding the mature polypeptide into a baculovirus to express a SPROUTY-1 polypeptide, using a baculovirus leader and standard methods as described in Summers et al. , A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 polypeptide and convenient restriction sites such as BamHI, Xbal and Asp718. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide.

Other baculovirus vectors are used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al , Virology 170: 31.

The cDNA sequence encoding the mature SPROUTY-1 polypeptide in the deposited or other clone, lacking the AUG initiation codon and the naturally associated nucleotide binding site, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. Non-limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of a sprouty-1 , e.g., as presented in SEQ ID NOS: 4, 6, 8 or 10, according to known method steps. The amplified fragment is isolated from a 1 % agarose gel using a commercially available kit (e.g., "Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is then digested with the appropriate restriction enzyme and again is purified on a 1 % agarose gel. This fragment is designated herein "FI. "

The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1 % agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "VI."

Fragment FI and the dephosphorylated plasmid VI are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates. Bacteria are identified that contain the plasmid with the human sprouty- 1 gene using the PCR method, in which one of the primers that is used to amplify the gene and the second primer is from well within the vector so that only those bacterial colonies containing the SPROUTY-1 gene fragment will show amplification of the DNA. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBac SPROUTY-1.

Five mg of the plasmid pBacSPROUTY-1 is co-transfected with 1.0 mg of a commercially available linearized baculovirus DNA ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al, Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 mg of

BaculoGold™ virus DNA and 5 mg of the plasmid pBacSPROUTY-1 are mixed in a sterile well of a microtiter plate containing 50 ml of serum-free Grace's medium (Life Technologies Inc., Rockville, MD). Afterwards, 10 ml Lipofectin plus 90 ml Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27 °C for four days. After four days the supernatant is collected and a plaque assay is performed, according to known methods. An agarose gel with "Blue Gal" (Life Technologies Inc., Rockville, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Rockville, MD, page 9-10). After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ml of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C. The recombinant virus is called V-SPROUTY-1. To verify the expression of the SPROUTY-1 DNA, Sf9 cells are grown in

Grace's medium supplemented with 10% heat inactivated FBS. The cells are infected with the recombinant baculovirus V-SPROUTY-1 at a multiplicity of infection ("MOI") of about 2. Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, MD). If radiolabeled polypeptides are desired, 42 hours later, 5 mCi of 35S-methionine and 5 mCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation. The polypeptides in the supernatant as well as the intracellular polypeptides are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled). Microsequencing of the amino acid sequence of the amino terminus of purified polypeptide can be used to determine the amino terminal sequence of the mature polypeptide and thus the cleavage point and length of the secretory signal peptide.

EXAMPLE 3: CLONING AND EXPRESSION OF SPROUTY-1 IN MAMMALIAN CELLS

A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded polypeptide. The DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al , Biochem J. 227: 277 (1991); Bebbington et al , Bio /Technology 10: 169 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of polypeptides.

The expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al. , Molec. Cell. Biol. 5: 438 (1985)) plus a fragment of the CMV-enhancer (Boshart et al, Cell 41: 521 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.

EXAMPLE 3(A): CLONING AND EXPRESSION IN COS CELLS The expression plasmid, pSPROUTY-1 HA, is made by cloning a cDNA encoding SPROUTY-1 into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.). The expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eucaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker. The HA tag corresponds to an epitope derived from the influenza hemagglutinin polypeptide described by Wilson et al. , Cell 37: 767-778 (1984). The fusion of the HA tag to the target polypeptide allows easy detection and recovery of the recombinant polypeptide with an antibody that recognizes the HA epitope. pcDNAIII contains, in addition, the selectable neomycin marker. A DNA fragment encoding the SPROUTY-1 is cloned into the polylinker region of the vector so that recombinant polypeptide expression is directed by the CMV promoter. The plasmid construction strategy is as follows. The SPROUTY-1 cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of SPROUTY-1 in E. coli. Non-limiting examples of suitable primers include those based on the coding sequences presented in SEQ ID NOS: 4, 6, 8 or 10, as they encode SPROUTY-1 polypeptides as described herein.

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with suitable restriction enzyme(s) and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems,

11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the SPROUTY-1 -encoding fragment.

For expression of recombinant SPROUTY-1 , COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al , Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of SPROUTY-1 by the vector.

Expression of the SPROUTY-1 -HA fusion polypeptide is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow et al. , Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 0.5 % DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated polypeptides then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls. The vector pC4 is used for the expression of SPROUTY-1 polypeptide. Plasmid pC4 is a derivative of the plasmid ρSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented. For example, see Alt et al. , J Biol. Chem. 253: 1357 (1978); Hamlin, J. L. and Ma, C, Biochem. et Biophys. Acta, 1097: 107 (1990); and Page, M. J. and Sydenham, M.A., Biotechnology 9: 64 (1991). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained which contain the amplified gene integrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al. , Molec. Cell. Biol. 5: 438 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al , Cell 41: 521 (1985)). Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene. Other high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the SPROUTY-1 in a regulated way in mammalian cells (Gossen, M. , & Bujard, H., Proc. Natl. Acad. Sci. USA 89: 5547 (1992)). For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1 % agarose gel.

The DNA sequence encoding the complete SPROUTY-1 polypeptide including its nucleotide binding site is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. Non-limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of a sprouty-1, for example, as presented in SEQ ID NOS: 4, 6, 8 or 10, according to known method steps. The amplified fragment is digested with suitable endonucleases and then purified again on a 1 % agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis. Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection. 5 mg of the expression plasmid pC4 is cotransfected with 0.5 mg of the plasmid pSV2-neo using lipofectin. The plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 mM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.

EXAMPLE 4: TISSUE DISTRIBUTION OF SPROUTY-1 mRNA EXPRESSION AND OVEREXPRESSION

Northern blot analysis was carried out to examine SPROUTY-1 gene expression in both normal and diseased human tissues, using methods described by, among others, Sambrook et al, cited above. Typically, a cDNA probe containing the nucleotide sequence encoding of SEQ ID NO: 4, encoding a SPROUTY-1 polypeptide, is labeled with 32P using the rediprime(TM) DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT 1200-1. The purified labeled probe is then used to examine various human tissues, both normal and pathologic, for SPROUTY-1 mRNA. Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) are obtained from Clontech and are examined with the labeled probe using ExpressHyb hybridization solution (Clontech) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at -70 °C overnight, and films developed according to standard procedures.

Two additional types of Northern blots were performed: a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Northern and an Electronic Northern. Electronic Northerns identify the nucleic acid libraries in which a given gene is expressed, and indicate the relative abundance of its expression in a particular tissue. Some of the libraries tested in the Electronic Northern protocol included Pharmaceutical's LifeSeq^® database of human genomic and cDNA sequences. See www.incyte.com. The transcription profile for the Sprouty homologs was determined using the following RT-PCR method. Two different pairs of PCR probes were designed. One pair encompassed the highly conserved carboxy-terminal domain. The second pair of probes was designed to be specific for SEQ ID No. 8. Each homologs was amplified by PCR simultaneously while two internal control genes are also being amplified. Standard PCR reactions were performed using the Advantage cDNA polymerase

(Clontech) enzyme. The PCR cycling conditions were as follows: (Step 1) 94°C for 5 min; (Step 2) 94°C for 45 sec ; (Step 3) 60°C for 45 sec; (Step 4) 72°C for 2 min.; (Step 5) Repeat steps 2-4, 29 more times; (Step 6) 72°C for 7 min.; (Step 7) held at 4 C. The templates for the reactions were twenty different first-strand cDNA panels (Clontech) isolated from different human tissues. The human tissues used were: brain, heart, kidney, liver, lung, pancreas, placenta, skeletal muscle, colon, ovary, peripheral blood lymphocytes, prostate, small intestine, spleen, testis, thymus, bone marrow, fetal liver, lymph, and tonsil and are identified in the figure. The control markers run on the 2% agarose, IX TBE gel were the Low Mass Ladder (Gibco-BRL), in which the sizes of the bands are 2000 bp, 1200 bp, 800 bp, 400 bp, 200 bp, and 100 bp.

The first set of sprouty gene PCR primers were designed to amplify the sprouty homolog sequence of SEQ ID No. 8 specifically. PCR of the SEQ ID No. 8 employed two primers from this sequence, specifically sptlhlUl l with sptlhlL233, and the sprouty PCR product size was 289 bp. PCR of the sprouty homolog consensus sequence, which is common to SEQ ID Nos. 4, 6, 8 and 10, employed two primers common to these four sequences, sptlh3U289 with sptlh3L624, and the sprouty PCR product size was 289 bp.

The identified primers have the following sequences: sptlhlUll 5'-AGTGCATGCCAGGTTTCCACTGATT-3' [in SEQ ID No. 8] sptlhlL233 5'-CCGAGGAGCAGGTCTTTTCACCAC-3' [in SEQ ID No. 8] sptlh3U289 5'-TTCACAATCACACTGCTGCTCTAGATACCT

[in SEQ ID Nos. 4, 6, 8 and 10] sptlh3L624 5'-AAATCCATGAGTTAGACCTTGGCAACAG

[in SEQ ID Nos.4, 6, 8 and 10].

The internal control genes used for these experiments are human β-actin and the human transferrin receptor. The human β-actin gene control employed a β-actin 5' primer (bactin5pri) with a β-actin 3' primer (bactin3pri) and the PCR product size was 838 bp. The human transferrin receptor gene control employed a human transferrin receptor 5' primer (hutfr5prim) with a human transferrin receptor 3' primer (hutfr3prim) and the PCR product size was 1347 bp. The following internal control primers were purchased from Clontech: bactin5pri 5 '-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3 ' bactin3pri 5 '-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3 ' hutfr5prim 5'-CCACCATCTCGGTCATCAGGATTGCCT-3' hutfr3prim 5 '-TTCTCATGGAAGCTATGGGTATCACAT-3 ' .

The resulting RT-PCR Northern blots are shown in Figures 25 A and 25B. They indicate that the SPROUTY homolog sequence of SEQ ID No. 8 is expressed in nearly all tissues tested, including brain, heart, kidney, liver, lung, pancreas, placenta, skeletal muscle, colon, ovary, prostate, small intestine, spleen, testis, thymus, bone marrow, fetal liver, lymph, tonsil, but not in peripheral blood lymphocytes. A consensus sequence common to each of the homolog sequences of SEQ ID Nos. 4, 6, 8 and 10, is expressed ubiquitously as well, with the same exception of peripheral blood lymphocytes. Since the consensus sequence is common to each of the homolog sequences, its ubiquitous expression may reflect that of just SEQ ID No. 8, or the other homologs (SEQ ID No. 4, 6, and/or 8) may be expressed ubiquitously as well. The

PCR product size from both the SEQ ID No. 8-specific primers and the primers for the consensus sequence was the same 296bp. Although each of the SPROUTY homologs may be ubiquitously expressed, the individual homologs are upregulated in certain disease states identified in the Electronic Northerns, discussed below. Each of these homologs may be allelic variants of the same gene.

Electronic Northern results showed a SPROUTY-1 mRNA expressed abundantly in adrenal tumor. A second cDNA probe (shown as SEQ ID NO: 6) containing the nucleotide sequence encoding another SPROUTY-1 polypeptide (SEQ ID NO: 5) is also expressed abundantly in prostate, thyroid, parathyroid, adrenal tumor and rheumatoid arthritis.

A third cDNA probe, shown as SEQ ID NO: 8, is expressed abundantly in abnormal prostate (benign prostatic hypertrophy with adenocarcinoma) and abnormal skin (Patau's syndrome). SEQ ID NO: 8 encodes a polypeptide shown in SEQ ID NO: 7. Similarly, SEQ ID NO: 25 encodes a polypeptide shown in SEQ ID NO: 24.

A fourth cDNA probe, shown as SEQ ID NO: 10, has an expression pattern that suggests it is expressed abundantly in pancreatic tumor, gallbladder, heart tumor (myxoma), neuroganglion tumor (ganglioneuroma) and normal placenta. SEQ ID NO: 10 encodes the polypeptide shown in SEQ ID NO: 9.

It will be clear that the invention can be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

Claims

WE CLAIM:

1. An isolated polynucleotide, or the complement thereof, encoding an amino acid sequence comprising the sequence set forth in SEQ ID NO: 24.

2. An isolated SPROUTY-1 polypeptide encoded by a polynucleotide comprising the sequence set forth in SEQ ID NO: 25 or a portion thereof, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 24.

3. An isolated polynucleotide encoding a polypeptide according to claim 2.

4. A method of inhibiting angiogenesis in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to claim 2.

5. An isolated nucleic acid, comprising a polynucleotide that hybridizes under stringent conditions to a polynucleotide according to claim 1.

6. A composition, comprising a carrier or diluent and an isolated polynucleotide, according to any one of claims 1, 3 or 5.

7. An isolated SPROUTY-1 polypeptide, comprising at least 20 contiguous amino acids of SEQ ID NO: 24.

8. A composition, comprising a carrier or diluent and an isolated polypeptide according to either claim 2 or claim 7.

9. A vector, comprising a polynucleotide according to any one of claims 1, 3 or 5.

10. A host cell comprising an isolated polynucleotide according to any one of claims 1 , 3 or 5.

11. An antibody or at least one fragment thereof that binds an epitope specific to the SPROUTY-1 polypeptide according to either claim 2 or claim 7.

12. A host cell, expressing at least one antibody or at least one fragment thereof according to claim 11.

13. A method for producing at least one antibody, comprising culturing a host cell according to claim 12.

14. A method for producing the SPROUTY-1 polypeptide, comprising translating a polynucleotide according to any of claims 1 , 3 or 5 under conditions that the SPROUTY-1 polypeptide is expressed in detectable or recoverable amounts.

15. A transgenic or chimeric non-human animal, comprising at least one host cell according to claim 10.

16. A method for identifying compounds that bind at least one SPROUTY-1 polypeptide, comprising a) admixing at least one isolated SPROUTY-1 polypeptide according to either claim 2 or claim 7 with at least one test compound or composition; and b ) detecting at least one binding interaction between said at least one SPROUTY-1 polypeptide and the test compound or composition.

17. A method of limiting the range of FGF levels in vivo, in a mammal, comprising administering to said mammal a pharmacologically effective amount of the polypeptide according to either claim 2 or claim 7.

18. A method of inhibiting an adverse effect of FGF in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

19. A method of treating prostate cancer in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

20. A method of inhibiting bFGF and FGFR- 1 -mediated signaling in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

21. A method of counteracting FGF overexpression associated with certain types of tumors in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

22. A method of controlling the growth, development or differentiation of any cell responsive to FGF signaling in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

23. A method of controlling the growth, development, or differentiation of adrenal gland cells responsive to FGF signaling in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

24. A method of treating a pathology involving hypersensitive responses to FGF in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

25. A method of treating a mammal having an adrenal gland pathology involving hypersensitive responses to FGF in a mammal, said method comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

26. A method of treating developmental anomalies associated with disregulated FGF signaling, in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

27. A method of counteracting SPROUTY-1 underexpression associated with certain types of tumors or pathology in a mammal, comprising administering to said mammal a pharmacologically effective amount of a polypeptide according to either claim 2 or claim 7.

28. The method of claim 17, wherein said polypeptide is acting on cells selected from the group consisting of: glioma mmor cells, astrocytic mmor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

29. The method of claim 18, wherein said polypeptide is acting on cells selected from the group consisting of: glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

30. The method of claim 20, wherein said polypeptide is acting on cells selected from the group consisting of: glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

31. The method of claim 21 , wherein said polypeptide is acting on cells selected from the group consisting of: glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

32. The method of claim 22, wherein said polypeptide is acting on cells selected from the group consisting of: glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

33. The method of claim 24, wherein said polypeptide is acting on cells selected from the group consisting of: glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

34. The method of claim 26, wherein said polypeptide is acting on cells selected from the group consisting of: glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

35. The method of claim 27, wherein said polypeptide is acting on cells selected from the group consisting of: glioma tumor cells, astrocytic tumor cells, cerebellar neurons, adrenal medullary cells, neuronal cells, astrocytoma cells, glioblastoma cells, glial cells, pancreatic cancer cells, prostatic epithelial cells, prostatic stromal cells, myfibroblasts, kupffer cells, hepatocytes, endometrial cells, and colorectal carcinoma cells.

36. The method of claim 17, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

37. The method of claim 18, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

38. The method of claim 20, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

39. The method of claim 21, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal mmor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

40. The method of claim 22, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

41. The method of claim 24, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

42. The method of claim 26, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hyperplasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

43. The method of claim 27, wherein said mammal has a disease selected from the group consisting of: polycystic kidney disease, nephropathy, melanoma, lung fibrosis, hepatic fibrogenesis, sarcoma, squamous lung cancer, scleroderma, Kaposi's sarcoma, vascular tumor, glioma tumor, astrocytic tumor, cerebellar neuron tumor, adrenal tumor, neuronal tumor, astrocytoma, glioblastoma, glial tumor, pancreatic cancer, prostatic hype╧ålasia, prostatic tumor, endometrial insufficiency, and colorectal carcinoma.

44. The method of claim 22, wherein said differentiation is selected from the group consisting of metanephrogenesis, angiogenesis, mmorogenesis and neurogenesis.