AU1247800A - Fibroblast growth factor 15 - Google Patents

Description

P:\OPER\MRO\27674-95.DIV -19/1/00 1A- FIBROBLAST GROWTH FACTOR This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention have been putatively identified as fibroblast growth factor/heparin binding growth factor, hereinafter referred to as "FGF-15". The invention also relates to inhibiting the action of such polypeptides.

10 Fibroblast growth factors are a family of proteins characteristic of binding to heparin and are, therefore, also called heparin binding growth factors (HBGF). Expression of different members of these proteins are found in various tissues and are under particular temporal and spatial control. These proteins are potent mitogens for a variety of cells of mesodermal, ectodermal, and endodermal origin, including fibroblasts, corneal and vascular endothelial cells, granulocytes, adrenal cortical cells, chondrocytes, myoblasts, vascular smooth muscle cells, lens epithelial cells, melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells.

Each member has functions overlapping with others and also has its unique spectrum 20 of functions. In addition to the ability to stimulate proliferation of vascular endothelial cells, both FGF-1 and 2 are chemotactic for endothelial cells and FGF-2 has been shown to enable endothelial cells to penetrate the basement membrane. Consistent with these properties, both FGF-1 and 2 have the capacity to stimulate angiogenesis. Another important feature of these growth factors is their ability to promote wound healing. Many other members of the FGF family share similar activities with FGF-1 and 2 such as promoting angiogenesis and wound healing. Several members of the FGF family have been shown to induce mesoderm formation and to modulate differentiation of neuronal cells, adipocytes and skeletal muscle cells.

Other than these biological activities in normal tissues, FGF proteins have been implicated in promoting tumorigenesis in carcinomas and sarcomas by promoting tumor vascularization and as transforming proteins when their expression is deregulated.

P:\OPERMRO\27674-95.DIV 19/1/ -2- The FGF family presently consists of eight structurally related polypeptides: basic FGF, acidic FGF, int 2, hst 1/k-FGF, FGF-5, FGF-6, keratinocyte growth factor, AIGF (FGF-8) and recently a glia-activating factor has been shown to be a novel heparin-binding growth factor which was purified from the culture supernatant of a human glioma cell line (Miyamoto, M. et al., Mol. and Cell. Biol., 13(7):4251-4259 (1993). The genes for each have been cloned and sequenced. Two of the members, FGF-1 and FGF-2, have been characterized under many names, but most often as acidic and basic fibroblast growth factor, respectively. The normal gene products influence the general proliferation capacity of the majority of mesoderm and neuroectoderm-derived cells. They are capable of inducing **oo 10 angiogenesis in vivo and may play important roles in early development (Burgess, W.H. and :Maciag, Annu. Rev. Biochem., 58:575-606, (1989)).

Many of the above-identified members of the FGF family also bind to the same receptors and elicit a second message through binding to these receptors.

A eukaryotic expression vector encoding a secreted form of FGF-1 has been 'introduced by gene transfer into porcine arteries. This model defines gene function in the arterial wall in vivo. FGF-1 expression induced intimal thickening in porcine arteries 21 days after gene transfer (Nabel, et al., Nature, 362:844-6 (1993)). It has further been demonstrated that basic fibroblast growth factor may regulate glioma growth and progression independent of its role in tumor angiogenesis and that basic fibroblast growth factor release or secretion may be required for these actions (Morrison, et al., J. Neurosci. Res., 34:502-9 (1993)).

Fibroblast growth factors, such as basic FGF, have further been implicated in the growth of Kaposi's sarcoma cells in vitro (Huang, et al., J. Clin. Invest., 91:1191-7 (1993)). Also, the cDNA sequence encoding human basic fibroblast growth factor has been cloned downstream of a transcription promoter recognized by the bacteriophage T7 RNA polymerase. Basic fibroblast growth factors so obtained have been shown to have biological activity indistinguishable from human placental fibroblast growth factor in mitogenicity, synthesis of plasminogen activator and angiogenesis assays (Squires, et al., J. Biol.

P:\OPER\MRO\27674-95. DIV -19/1/00 -3- Chem., 263:16297-302 (1988)).

U.S. Patent No. 5,155,214 discloses substantially pure mammalian basic fibroblast growth factors and their production. The amino acid sequences of bovine and human basic fibroblast growth factor re disclosed, as well as the DNA sequence encoding the polypeptide of the bovine species.

Newly discovered FGF-9 has around 30% sequence similarity to other members of the FGF family. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic and basic FGF. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence FGF (Miyamoto, M. et al., Mol. and Cell. Biol., 13(7):4251-4259 (1993). Further, FGF-9 was found to stimulate the cell growth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3, and PC-12 cells but not that of human umbilical vein endothelial cells (Naruo, et al., J. Biol. Chem., 268:2857-2864 (1993).

Basic FGF and acidic FGF are potent modulators of cell proliferation, cell motility, differentiation, and survival and act on cell types from ectoderm, mesoderm and endoderm.

These two FGFs, along with KGF and AIGF, were identified by protein purification.

However, the other four members were isolated as oncogenes., expression of which was restricted to embryogenesis and certain types of cancers. FGF-9 was demonstrated to be a mitogen against glial cells. Members of the FGF family are reported to have oncogenic potency. FGF-9 has shown transforming potency when transformed into BALB/c3T3 cells (Miyamoto, et al., Mol. Cell. Biol., 13(7):4251-4259 (1993).

Androgen induced growth factor (AIGF), also known as FGF-8, was purified from a conditioned medium of mouse mammary carcinoma cells (SC-3) simulated with testosterone.

AIGF is a distinctive FGF-like growth factor, having a putative signal peptide and sharing 30-40% homology with known members of the FGF family. Mammalian cells transformed with AIGF shows a remarkable stimulatory effect on the growth of SC-3 cells in the absence P:OPER\MRO\27674-95.DIV -19/1100 -4of androgen. Therefore, AIGF mediates androgen-induced growth of SC-3 cells, and perhaps other cells, since it is secreted by the tumor cells themselves.

The polypeptide of the present invention has been putatively identified as a member of the FGF family as a result of amino acid sequence homology with other members of the FGF family.

In accordance with one aspect of the present invention, there are provided novel mature polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with still another aspect of the present invention, there are provided processes for producing such polypeptides by recombinant techniques through the use of recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides of the present invention, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide of the present invention.

In accordance with a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for screening for agonists and antagonists thereto and for therapeutic purposes, for example, promoting wound healing for example as a result of burns and ulcers, to prevent neuronal damage due to neuronal disorders and promote neuronal growth, and to prevent skin aging and hair loss, to stimulate angiogenesis, mesodermal induction in early embryos and limb regeneration.

P:\OPER\MRO\27674-95.DIV 19/1/00 In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, there are provided antagonists against such polypeptides and processes for their use to inhibit the action of such polypeptides, for example, in the treatment of cellular transformation, for example, tumors, to reduce scarring and treat hyper-vascular diseases.

In accordance with another aspect of the present invention, there are provided nucleic 10 acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a polynucleotide encoding a polypeptide of the present invention.

o In accordance with yet another aspect of the present invention, there are provided diagnostic assays for detecting diseases or susceptibility to diseases related to mutations in a nucleic acid sequence of the present invention and for detecting over-expression of the polypeptides encoded by such sequences.

In accordance with another aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are meant only as illustrations of specific embodiments of the present invention and are not meant as limitations in any manner.

Figure 1 depicts the cDNA sequence and corresponding deduced amino acid sequence of Figure 2 illustrates the amino acid sequence homology between FGF-15 and the other P:\OPERMRO\27674-95.DIV -19/1/00 -6- FGF family members. Conserved amino acids are readily ascertainable.

In accordance with one aspect of the present invention, there are provided isolated nucleic acids molecules (polynucleotides) which encode for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NOS: 2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 97146 on May 12, 1995.

For the purposes of nomenclature, the nucleotide sequence set forth in Figure 1 (SEQ ID NO: 1) relates to the nucleotide sequence obtained by sequencing the cDNA contained in ATCC Deposit No. 97146.

The polynucleotide encoding FGF-15 of this invention was discovered initially in a cDNA library derived from human adrenal tumor tissue. It is structurally related to all members of the fibroblast growth factor family and contains an open reading frame encoding a polypeptide of 252 amino acids. Among the top matches are: 1) 41 identity and 66 sequence similarity to human FGF-9 over a stretch of 129 amino acids; and 2) 37 identity *and 59 similarity to human KGF over a region of 88 amino acids.

The FGF/HBGF family signature, GXLX(S,T,A,G)X6 (D,E)CXFXE is conserved in the polypeptide of the present invention, (X means any amino acid residue; means either D or E residue; X6 means any 6 amino acid residues).

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be doublestranded or single-stranded. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO: 1) or that of the deposited clone or may be a different coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1, (SEQ ID NO:1) or the deposited cDNA.

The polynucleotides which encodes for the mature polypeptide of Figure 1 (SEQ ID P:\OPER\MRO\27674-95.DIV 19/1/00 -7- NO:2) or for the mature polypeptides encoded by the deposited cDNA(s) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which 10 includes additional coding and/or non-coding sequence.

.i.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides .o having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptides encoded by the cDNA(s) of the deposited clone(s). The variants of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

SThus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO: 2) or the same mature polypeptides encoded by the cDNA(s) of the deposited clone(s) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or the polypeptides encoded by the cDNA(s) of the deposited clone(s). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID NO: 1) or of the coding sequence of the deposited clone(s). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptides.

P:%OPER\MRO\27674-95. DIV 19/1/00 -8- The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secret ion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.

The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein 10 remains.

4* Thus, for example, the polynucleotides of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a oo prosequence and a presequence (leader sequence).

4 The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

Fragments of the full length FGF-15 gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also P:\OPER\MRO\27674-95.DIV 19/I/00 -9be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete FGF-15 gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the FGF-15 gene by using the known DNA sequence to synthesize an oligonucleotide probe.

Labelled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridize to the 10 hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO: 1) or the deposited cDNA(s).

9 Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having at least a identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.

P:\OPER\MRO\27674-95.DIV 19/1/00 The deposit(s) referred to herein will be maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. These deposits are provided merely as a convenience and are not an admission that a deposit is required under 35 U.S.C. 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

10 The present invention further relates to an FGF polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or which has the amino acid sequence encoded by the deposited cDNA(s), as well as fragments, analogs and derivatives of such polypeptides.

15 The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or those encoded by the deposited cDNA(s), means polypeptides which retains essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.

The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the deposited cDNA(s) may be one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature P:\OPER\MRO\27674-95.DIV 19/1/00 -11 polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment 10 the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least a 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably 20 at least a 90% similarity (more preferably at least a 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least a 95% similarity (still more preferably a identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the P:\OPER\MRO\27674-95.DIV 191100 -12fragments may be employed as intermediates for producing the full-length polypeptides.

Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells may be genetically engineered (transduced or transformed or transfected) 10 with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the FGF genes. The culture conditions, such as temperature, pH and the like, are those previously 15 used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotide of the present invention may be employed for producing a polypeptide by recombinant techniques. Thus, for example, the polynucleotide sequence may 20 be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector or plasmid may be used as long as they are replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

P:\OPER\MRO\27674-95.DIV 19/1100 -13- The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli.

lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

S. In addition, the expression vectors preferably contain a gene to provide a phenotypic 10 trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E.

coli.

The vector containing the appropriate DNA sequence as herein above described, as 15 well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Salmonella typhimurium, Streptomyces; fungal cells, such as yeast; insect cells, such as Drosophilia S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant 20 cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTRC99A, P:\OPERWMRO\2767495.DIV -1911/0 -14pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transf erase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include LM'V immediate early, HSV S. thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.

10 Selection of the appropriate vector and promoter is well within the level of ordinary skill in :i the art.

In a further embodiment, the present invention relates to host cells containing the Sabove-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian 15 cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by o o calcium phosphate transfection, DEAE Dextran mediated transfection, or electroporation (Davis, Dibner, Battey, Basic Methods in Molecular Biology, 1986)).

20 The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, 1989), the disclosure of which is hereby incorporated by reference.

P:\OPER\MRO\27674-95.DIV 19/1/00 Transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, the ampicillin resistance 10 gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation, initiation and termination C' 15 sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation, initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from P:\OPER\MRO\27674-95.DIV 19/1/00 -16commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vect: s include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient 15 method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey 20 kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The polypeptide of the present invention may be recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose P:\OPER\MRO\27674-95.DIV 19/1/00 -17chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production 10 procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The polypeptide of the present invention, as a result of the ability to stimulate vascular 15 endothelial cell growth, may be employed in treatment for stimulating revascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration.

20 The polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. FGF-15 has the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.

P:\OPER\MRO\27674-95.DIV 9/1/00 -18- The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

The FGF-15 polypeptide may also be employed for preventing hair loss, since FGF family members activate hairforming cells and promotes melanocyte growth. Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

10 The FGF-15 polypeptide may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA, manufacture of DNA vectors and for the purpose of providing diagnostics and therapeutics for the treatment of 20 human disease.

This invention provides a method for identification of the receptors for the polypeptides of the present invention. The genes encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., Chapter 5, (1991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labelled. The polypeptides P:\OPER\MRO\27674-95.DIV 1911100 -19can be labeled by a variety of means including iodination or inclusion of a recognition site for a sitespecific protein kinase.

Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be 10 photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide 15 probes to screen a cDNA library to identify the genes encoding the putative receptors.

This invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, 20 the compound to be screened and thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3 thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance P:\OPER\MRO\27674-95.DIV 19/l/W or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

Examples of antagonist compounds include antibodies, or in some cases, oligonucleotides, which bind to the receptor for the polypeptide of the present invention but 10 elicit no second messenger response or bind to the FGF-15 polypeptide itself. Alternatively, a potential antagonist may be a mutant form of the polypeptide which binds to the receptors, however, no second messenger response is elicited and, therefore, the action of the polypeptide is effectively blocked.

15 Another antagonist compound to the FGF-15 gene and gene product is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of 20 the present invention, is used to design an antisense RNA oligonucleotide of from about to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of the polypeptides of the present invention. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide (Antisense Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the polypeptide.

P:\OPER\MRO\27674-95.DIV 19/1/00 -21 Potential antagonist compounds also include small molecules which bind to and occupy the binding site of the receptors thereby making e .e receptor inaccessible to its polypeptide such that normal biological activity is prevented. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules.

Antagonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e.

stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

The antagonists may also be employed to prevent hypervascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

The antagonists may also be employed to prevent the growth of scar tissue during wound healing.

The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, as hereinafter described.

The polypeptides, agonists and antagonists of the present invention may be employed in combination with a suitable pharmaceutical carrier to comprise a pharmaceutical composition for parenteral administration. Such compositions comprise a therapeutically effective amount of the polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of P:\OPER\MRO\27674-95.DIV 19/1/00 -22the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides, agonists and antagonists of the present invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which 10 is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 zg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most 9 cases, the dosage is from about 10 /g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc. In the specific case of topical administration, dosages are preferably administered from about 0.1 /g to 9 mg per cm 2 The polypeptide of the invention and agonist and antagonist compounds which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptide in vivo, which is often referred to as "gene therapy." Thus, for example, cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding for the polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of the polypeptide in vivo, for example, by procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present P:OPERMRO\27674-95.DIV 1911/00 -23invention by such methods should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retroviral particle, for example, an adenovirus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative 10 Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector :is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and p-actin promoters).

Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the p-actin promoter; and human growth hormone promoters. The P:\OPER\MRO\27674-95.DIV 19/100 -24promoter also may be the native promoter which controls the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ij-2, i-AM, PA12, T19-14X, VT-19-17-H2, iCRE, *iiCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.

The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 10 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may 15 be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.

Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the genes of the present invention as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences encoding the polypeptide of the present invention.

Individuals carrying mutations in a gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or nay be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding a polypeptide of the present invention can be used to identify and analyze P:AOPER\MRO\27674-95.DIV 19/1/00 mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel 10 electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, Myers et al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detecting altered levels of proteins in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of abnormal cellular proliferation, for example, a tumor. Assays used to detect levels of protein in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding P:\OPER\MRO\27674-95.DIV- 1911/00 26 assays, Western Blot analysis, ELISA assays and "sandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Immunology, Chapter 6, (1991)) initially comprises preparing an antibody specific to an antigen to the polypeptides of the present invention, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme. A sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein like bovine serum albumen. Next, the monoclonal 10 antibody is incubated in the dish during which time the monoclonal antibodies attach to any polypeptides of the present invention attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the protein of interest.

Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of a polypeptide of the present invention present in a given volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to a polypeptide of the present invention are attached to a solid support and labelled FGF-13 and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of a polypeptide of the present invention in the sample.

A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay a polypeptide of the present invention is passed over a solid support and binds to antibody attached to a solid support. A second antibody is then bound to the polypeptide of interest. A third antibody which is labelled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.

P:\OPER\MRO\27674-95.DIV 19/1/00 -27- The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphism's) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

S. 10 Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is :"used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide S. primers, sublocalization can be achieved with panels of fragments from specific chromosomes 9• or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labelled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical P:\OPER\MROX27674-95.DIV 19/1/00 -28position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the 10 causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution 15 and one gene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

P:\PER\MRO\27674-95.DIV 19/1/00 -29- For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBVhybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide 10 products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

*o The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such oo examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples, certain frequently occurring methods and/or terms will be described.

20 "Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 fg of plasmid or DNA fragment is used with about 2 units of enzyme in about l 1 of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, P:\OPER\MRO\27674-95.DIV 19/1100 typically 5 to 50 pg of DNA are digested with 20 to 250 units of enzyme in a larger volume.

Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 0 C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).

10 "Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A -synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 /g of approximately equimolar amounts of the DNA fragments to be 20 ligated.

Unless otherwise stated, transformation was performed as described by the method of Graham, F. and Van der Eb, Virology, 52:456-457 (1973).

Example 1 Bacterial Expression and Purification of FGF-15 protein The DNA sequence encoding FGF-15 ATCC 97146, is initially amplified using PCR oligonucleotide primers corresponding to the 5' sequences of the processed protein (minus the signal peptide sequence) and the vector sequences 3' to the gene. Additional nucleotides corresponding to the gene are added to the 5' and 3' sequences. The 5' oligonucleotide primer has the sequence 5'GCCAGACCATGGTAAAACCGGTGCCCCTC 3' (SEQ ID NO:3) and P:OPER\MRO\27674-95.DIV 19/1/00 -31contains an NcoI restriction enzyme site (in bold). The 3' sequence GGCAGGAGATCTTGTTGTCTTACTCTTGTTGAC 3' (SEQ ID NO: 4) contains complementary sequences to a BglII site (in bold) and is followed by 21 nucleotides of coding sequence.

The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE60 (Qiagen, Inc. Chatsworth, CA 91311). pQE-60 encodes antibiotic resistance a bacterial origin of replication (ori), an IPTG-regulatable promoter operator a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites.

10 pQE-60 was then digested with NcoI and BgllI. The amplified sequences are ligated into pQE-60 and are inserted in frame with the sequence encoding for the histidine tag and the ribosome binding site (RBS). The ligation mixture is then used to transform E. coli strain 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan). Transformants are identified by their ability to grow on LB S plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight in liquid culturein LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.

6 of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration of 1 mM.

IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation.

The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, solubilized FGF-15 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). The proteins are eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HC1, 100mM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours P:\OPER\MRO\27674-95.DIV 19/1/00 -32the proteins are dialyzed to 10 mmolar sodium phosphate.

Example 2 Cloning and expression of FGF-15 using the baculovirus expression system The DNA sequence encoding the full length FGF-15 protein, ATCC 97146, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene: The FGF-15 5' primer has the following sequence and contains a BamHI restriction 10 enzyme site (in bold) followed by 4 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, J. Mol. Biol., 196:947-950 (1987) which is just behind the first 18 nucleotides of the gene (the initiation codon for translation "ATG" is CTAGTGGATCCGCCATCATGGTAAAACCGGTGCCC 3' (SEQ ID The 3' primer has the following sequence and contains the cleavage site for the restriction endonuclease Asp718 (in bold) and 21 nucleotides complementary to the 3' non-translated sequence of the gene: CGACTGGTACCAGCCACGGAGCAGGAATGTCT 3' (SEQ ID NO:6).

20 The amplified sequences are isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, The fragment is then digested with the respective endonucleases and purified again on a 1% agarose gel. This fragment is designated F2.

The vector pA2 (modifications of pVL941 vector, discussed below) is used for the expression of the proteins using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHI and Xbal. The polyadenylation site of the simian virus (SV)40 is used P:\OPER\MRO\27674-95.DIV 19/1100 -33 for efficient polyadenylation. For an easy selection of recombinant virus the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pA2 such as pRG1, pAc373, pVL941 and pAcIM (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).

The plasmid is digested with the restriction enzymes and dephosphorylated using calf 10 intestinal phosphatase by procedures known in the art. The DNA is then isolated from a 1% :agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).

This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase.

15 E.coli DH5a cells are then transformed and bacteria identified that contained the plasmid using the respective restriction enzymes. The sequence of the cloned fragment S are confirmed by DNA sequencing.

5 /g of the plasmid pBacFGF-15 is co-transfected with 1.0 /tg of a commercially 20 available linearized baculovirus ("BaculoGold baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 kIg of BaculoGold T virus DNA and 5 /2g of the plasmid is mixed in a sterile well of microtiter plates containing 50 pzl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 plI Lipofectin plus 90 pl Grace's medium are added mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711) seeded in 35 mm tissue culture plates with 1 ml Grace's medium without serum. The plates are rocked back and forth to mix the newly added solution. The plates are then incubated for 5 hours at 27 0 C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium P:\OPER\MRO\27674.95.DIV 19/1/00 -34supplemented with 10% fetal calf serum is added. The plates are put back into an incubator and cultivation continued at 27 0 C for four days.

After four days the supernatant is collected and plaque assays performed similar as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A detailed description of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).

p Four days after the serial dilution the virus is added to the cells and blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 p~ of Grace's medium. The agar is removed by a brief centrifugation and the supernatant 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 stored at 40 0

C.

Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-FGF-15 at a multiplicity of infection 20 (MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 /Ci of 3 "S-methionine and 5 iUCi 35 S cysteine (Amersham) are added. The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Example 3 Expression of Recombinant FGF-15 in COS cells The expression of plasmids, FGF-15-HA derived from a vector pcDNA3/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site. DNA fragments encoding the entire FGF-15 precursor and an HA tag P:\OPER\MRO\27674-95. DIV 19/1/00 fused in frame to the 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I.

Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows: The DNA sequence encoding FGF-15, ATCC 97146, is constructed by PCR using 10 two primers: the 5' primer 5' CTAGTGGATCCGCCATCATGGTAAAACCGGTGCCC 3' (SEQ ID NO:7) contains a BamHI site followed by 18 nucleotides of coding sequence starting from the initiation codon; the 3' sequence 5' GTCGACCTCGAGTGTGTGCTTACTCTTGTT 3' (SEQ ID NO:8) contains complementary sequences to an XhoI site, translation stop codon, HA tag and the last 18 nucleotides of the FGF-15 coding sequence (not including the stop 15 codon). Therefore, the PCR product contains a BamHI site, coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site.

The PCR amplified DNA fragments and the vector, pcDNA3/Amp, are digested with the respective restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates and resistant colonies are selected.

Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant FGF-15 COS cells are transfected with the expression vector by DEAE-DEXTRAN method Sambrook, E.

Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the FGF-15-HA protein is detected by radiolabelling and immunoprecipitation method Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with "S-cysteine two days post transfection. Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaC1, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are P:\OPER\MRO\27674-9S.DIV 19/1/00 -36precipitated with an HA specific monoclonal antibody. Proteins precipitated are analyzed on SDS-PAGE gels.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will 10 be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Pages 37 to 40 are claims pages They appear after sequence listing P:\OPER\MRO\27674-95.DIV 19/1/00 -41- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Human Genome Sciences, Inc.

(ii) TITLE OF INVENTION: Fibroblast Growth Factor (iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: AU Divisional I FILING DATE: 19-JAN-2000 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: AU 27674/95 FILINE DATE: 05-JUN-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: SLATTERY MR, JOHN M REFERENCE/DOCKET NUMBER: JMS/MRO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +61 3 254 2777 TELEFAX: +61 3 254 2770 TELEX: AA31787 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 759 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS P:\OPER\MROX27674-95. DIV 19/1/00 42 LOCATION: 1. .756 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATG GTA AAA CCG GTG CCC CTC TTC AGG AGA ACT GAT TTC AAA TTA TTA Met Val Lys Pro Val Pro Leu Phe Arg Arg Thr Asp Phe Lys Leu Leu TTA TGC AAC CAC AAG GAT CTC TTC Lys Asp Leu Phe Leu Cys Asn GAT TGC TTT Asp Cys Phe

TTT

Phe CTC AGG GTG TCT Leu Arg Val Ser AAG CTG CTG Lys Leu Leu ATT TTC AGC Ile Phe Ser TCG CCC AAA TCA Ser Pro Lys Ser TGG TTT CTT TGG Trp, Phe Leu Trp S S *5*S S. S S S

S.

5 AAA GGA Lys Gly 50 AAC AAG Asn Lys ACG CAT ATG CTG Thr His Met Leu AAC CCA ACT GAT Asn Pro Thr Asp TGT CTT TGT GGC Cys Leu Cys Gly

AAG

Lys AGT CTT AAG AAA Ser Leu Lys Lys 144 192 240 CCC CAG CTC AAG Pro Gin Leu Lys ATA GTG ACC AGG Ile Val Thr Arg TAT TGC AGG CAA GGC TAC TAC TTG CAA Tyr Cys Arg Gin Gly Tyr Tyr Leu Gin

ATG

Met 90 CAC CCC GAT GGA His Pro Asp Gly GCT CTC Ala Leu GAT GGA ACC Asp Giy Thr CCA GTG GGA Pro Val Gly 115

AAG

Lys 100 GGT GAC AGC ACT Gly Asp Ser Thr TCT ACA CTC TTC AAC CTC ATA Ser Thr Leu Phe Asn Leu Ile 110 S *5 5

S

CTA CGT GTT GTT Leu Arg Val Val GOC ATC CAG GGA GTG Ala Ile Gin Gly Val 120 GGT TAC CTC TAC CCA Gly Tyr Leu Tyr Pro 140 ACA GGG TTG Thr Gly Leu 384 TAT ATA Tyr Ile 130 ACC ATG AAT GGA Thr Met Asn Gly

GAA

Giu 135 TCA GAA CIT TTT Ser Giu Leu Phe 432 480

ACC

Thr 145 CCT GAA TGC AAG Pro Giu Cys Lys

TTT

Phe 150 AAA GAA TCT GTT Lys Giu Ser Val GAA AAT TAT TAT Giu Asn Tyr Tyr

GTA

Val 160

TGG

Trp ATC TAC TCA TCC Ile Tyr Ser Ser ATG TTG TAC Met Leu Tyr 165 AGA CAA CAG GAA TCT GOT AGA GCC 528 Arg Gin Gin Giu 170 Ser Gly Arg Ala 175 TTT TTG GGA TTA AAT AAG GAA GGG CAA GCT ATG AAA GGG AAC AGA GTA Phe Leu Gly Leu Asn Lys Glu Giy Gin Ala Met Lys Gly Asn Arg Val 180 185 190 AAG AAA ACC AAA CCA GCA GCT CAT TTT CTA CCC AAG CCA TTG GAA GTT Lys Lys Thr Lys Pro Ala Ala His Phe Leu Pro Lys Pro Leu Giu Vai 624 P:\OPERW RO\27674-95. DIV 191/00G -43 195 200 205 GCC ATG TAC CGA GAA CCA TCT TTG CAT GAT GTT GGG GAA ACG GTC CCG Ala Met Tyr Arg Giu Pro Ser Leu His Asp Val Gly Giu Thr Val Pro 210 215 220 AAG CCT GGG GTG ACG CCA AGT AAA AGC ACA AGT GCG TCT GCA ATA ATG Lys Pro Gly Val Thr Pro Ser Lys Ser Thr Ser Ala Ser Ala Ile Met 225 230 '235 240 AAT GGA GGC AAA CCA GTC AAC AAG AGT AAG ACA ACA TAG Asn Gly Gly Lys Pro Val Asn Lys Ser Lys Thr Thr 245 250 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 252 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Vai Lys Pro Val Pro Leu Phe Arg Arg Thr Asp Phe Lys Leu Leu is 1 Leu Asp Lys Asn Tyr Asp Pro Tyr Cys Cys Gly Lys Cys Gly Val Ile Asn Phe 35 Thr Asn Arg Thr Gly 115 Thr His 20 S er His Pro Gin Lys 100 Leu 5 Lys Asp Pro Lys Met Leu Thr Asp 70 Gly Tyr Gly Asp Arg Val 10 Leu Ser Gin 55 Pro Tyr Ser Val Phe Met 40 Cys Gin Leu Thr Ala Phe 25 Trp Leu Leu Gin Asn 105 Ile Leu Phe Cys Lys Met 90 Ser Gin Arg Leu Gly Gly 75 His Thr Gly Val Trp Lys Ile Pro Leu Val Ser Asn Ser Val1 Asp Phe Lys Lys Ile Leu Thr Gly Asn 110 Thr Leu Phe Lys Axg Al a Leu Gly Leu Ser Lys Leu Leu Ile Leu 120 125 Met Asn Giy Giu Gly Tyr Leu Tyr Pro Ser Giu Leu Phe 135 140 130 Thr 145 Pro Giu Cys Lys Phe 150 Lys Giu Ser Val Phe 155 Glu Asn Tyr Tyr Val1 160 P:\OPERkNlRO\27674-95. DIV 1911/00 44 Ile Tyr Ser Ser Met Leu Tyr Arg Gin Gin Giu Ser Gly Arg Ala Trp 165 170 175 Phe Leu Gly Leu Asn Lys Glu Gly Gin Ala Met Lys Gly Asn Arg Val 180 185 190 Lys Lys Thr Lys Pro Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val 195 200 205 Ala Met Tyr Arg Giu Pro Ser Leu His Asp Val Gly Glu Thr Val Pro 210 215 220 Lys Pro Gly Val Thr Pro Ser Lys Ser Thr Ser Ala Ser Ala Ile Met 225 230 235 240 Asn Gly Gly Lys Pro Val Asn Lys Ser Lys Thr Thr 245 250 INFORMATION FOR SEQ ID NO:3: SEQ~UENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid 9 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCCAGACCAT GGTAAAACCG GTGCCCCTC 29 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDbTESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GOCAGGAGAT CTTGTTGTCT TACTCTTGTT GAC 3 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: P:\OPER\MR0\27674-95.DIV 19/1 00 LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CTAGTGGATC CGCCATCATG GTAAAACCGG TGCCC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGACTGGTAC CAGCCACGGA GCAGGAATGT CT 32 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARXCTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTAGTGGATC CGCCATCATG GTAAAACCGG TGCCC INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PA\OPERWROU\7674-95 DIV 19/1100 -46- (ii) MOLECULE TYPE: DNA kgenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GTCGACCTCG AGTGTGTGCT TACTCTTGTT INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid STRANDEDNESS- single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe cc r o r r c or r Asn Asn Thr Ser Ala Glu Ile Asn Ile 145 Leu Gly Arg so Val Met Cys Ser Gly 130 Leu Pro Pro 20 Gly His Asp Arg Gly Glu Asp Thr Leu Phe 100 Lys Lys 115 Ser Cys Phe Leu Gly Phe Ser Val Asp Leu His Lys Pro Asn Tyr Leu Arg Asp Gln 55 Tyr Ile 70 Gly Leu Glu Arg Ala Glu Arg Gly 135 Leu Pro 150 Lys Ile 40 His Lys Leu Leu Lys 120 Pro Lys 25 Leu Ile Ser Glu 105 Asn Arg 10 Pro Lys Pro Asp Gln Leu Thr Glu 75 Gly Ser 90 Glu Asn Trp Phe Thr His Leu Gly Gln Thr Gtln His Val 140 Leu Tyr Thr Val Leu Ser Gly Gln Thr Pro Tyr Asn 110 Gly Leu 125 Gly Gln is Cys Ser Asp Gly Aa Glu Tyr Leu Asn Glu Thr Tyr Lys Lys Lys Ala Val Ser Ser Asp 155 INFORMATION FOR SEQ ID NO: P:\OPERXMRO\27674-95. DIV 19/1/00 -47 SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 04 a.

1 Gly Tyr Val Gin Val Asn Arg Ser Cys Asp 50 Ala Tyr Thr Thr Thr 130 Gly Lys 35 Gly Glu Leu Asp Tyr 115 Gly Al a 20 Asn Val Glu Ala Giu 100 Arg Gin Phe Phe Gi y Arg Arg Met Cys Ser Tyr Leu Pro Gly Giu Gly 70 Lys Phe Arg Lys Pro Pro Phe Lys 55 Val Glu Phe Lys Leu 135 Met Gly Phe 40 Ser Val Asp Phe Tyr 120 Gly Ser His 25 Leu Asp Ser Gly Glu 105 Thr Ser Al a 10 Phe Arg Pro Ile Arg 90 Arg Ser Lys Lys Lys Ile His Lys 75 Leu Leu Trp Thr Ser Asp His Ile Gly Leu Glu Tyr Gly 140 Pro Pro Lys Val Ala Ser Val 125 Pro Lys Asp Leu Cys Ser Asn 110 Ala Gly Arg Leu Gly Arg Gin Leu Ala Asn Lys Cys Asn Tyr Leu Lys Gin Lys Ala Ile Leu 145 INFORMATION FOR SEQ ID NO:l1: SEQUENCE CHARACTERISTICS: LENGTH: 239 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein P:\0PER\MRO\27674-95. DIV 19/1/00 -48- (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:l1: Met Gly Leu Ile Trp Leu Leu Leu Leu Ser Leu Leu Giu Pro Gly Trp 5 0 0 0 00* Pro Ala Gly Giy Tyr Cys Asn Gly Vai Giu Leu Ala Ala Giu Tyr Ala 130 Arg Gin 145 Gly Arg Leu Phe Ala Gly Val Tyr Ala Thr Ser Leu Val Gly Met Asn 100 Cys Giu 115 Ser Arg Pro Ser Pro Arg Leu Pro 180 Pro Glu Lys Glu Ile 85 Lys Phe Leu Ala Arg Arg Gly His Tyr Asn 70 Val1 Arg Val Tyr Glu 150 Gly Val Ala Leu His 55 Ser Ala Gly Giu Arg 135 Arg Phe Leu Arg Gly 40 Leu Ala Ile Arg Arg 120 Thr Leu Lys Asp Leu 25 Gly Gin Tyr Arg Leu 105 Ile Val T rp Thr His Arg Al a Leu Ser Giy 90 Tyr His Ser Tyr Arg 170 Arg Arg Pro His Ile 75 Leu Ala Giu Ser Val 155 Arg Asp Asp Arg Pro Leu Phe Ser Leu Thr 140 Ser Thr His Ala Arg Ser Giu Ser Giu Giy 125 Pro Val Gin Glu Gly Arg Gi y Ile Gly His 110 Tyr Gly Asf Lys Met Gly Lys Arg Thr Arg Tyr Asn Ala Gly Ser 175 Val Arg Leu Val Ala Tyr Ser Thr Arg Lys 160 Ser Arg 190 Gln Leu Gin Ser Giy Leu Pro Arg Pro Pro Gly Lys Gly Val Gin Pro 205 195 200 Arg Arg Arg Arg Gin Lys Gin Ser Pro 210 215 Val Gin Aia Ser Arg Leu Gly Ser Gin 225 230 INFORMATION FOR SEQ ID NO:12: SEQUENCE

CHARACTERISTICS:

LENGTH: 206 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Asp Leu Asn Giu 235 Leu 220 Ala Glu Ser Pro Ala Ser His His P:\OPER\M RO\27674-95. DIV 19/1/00 49 (ii) MOLECULE TYPE: prote~fl (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Thr Ala Ser Leu Lys Glu Lys Arg Gin Ala Asp Ser Phe Gly 130 Leu Tyr 145 Leu Leu Met Phe Val Ser Leu Leu Pro Asn Val Ala Ala Ala Leu Arg Leu Pro 100 Leu Leu 115 Val Ala Gly Ser Pro Asn Ile Ala 180 Pro Thr Ala Gly Leu Val1 Arg 85 Asp Glu Ser Pro Asn 165 Leu Met Pro Thr Ser Gin 70 Leu Gly Leu Arg Phe 150 Tyr Ser Lys Trp Leu Leu 55 Ser Tyr Arg Ser Phe 135 Phe Asn Lys Val Al a Glu 40 Ala Gly Cys Ile Pro 120 Phe Thr Al a Asn Thr 200 Gly 25 Ala Arg Ala Asn Gly 105 Val Val Asp Tyr Gly 185 His Arg Glu Leu Gly Val 90 Gly Giu Ala Giu Giu 170 Lys Phe Gly Leu Pro Asp 75 Gly Ala Arg Met (Cys 155 Ser Thr Leu Gly Ala Glu Arg Val Ala Tyr Leu Ile Gly His Ala Gly Val 125 Ser Ser 140 Thr Phe Tyr Lys Lys Lys Pro Arg 205 Ala Ala Arg Trp Ala Gin Leu Gly Phe His Asp Thr 110 Val Ser Lys Gly Lys G1lu Tyr Pro 175 Gly Asn 190 Leu Pro Glu Pro Ile Leu Arg Ie Lys £l e.

160 Gly Arg 195 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 267 amino acids TYPE: amnino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein P:\OPER\MRO\27674-95. DIV 19/1/00 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i3: Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu 1:1 1 Ser Gly Ser Ala Trp Gly Glu Ile Lys 145 Phe Ile Lys His Ala Pro Ser Ser Ser Phe Ala Val 130 Lys Arq His Arg Ile Trp Ala Ser Leu Leu His Asfl 115 Gly Giy Giu Arg Gly 195 Ser Ala Ala Ser Gly Gly Leu 100 Met Ilie Lys Arg Thr 180 Lys Thr 5 His Thr Ala Ser Aia 85 Gin Leu Arg Leu Phe 165 Giu Ala His Giy Asp Met Gin 70 Arg Ile Ser Gly His i50 Gin Lys Lys Giu Lys Arg 25 Arg Asn Pro 40 Ser Ser Ser 55 Gly Ser Gly Thr Gly Ser Tyr Pro Asp 105 Val Leu Glu 120 Val Phe Ser 135 Ala Ser Ala Giu Asn Ser Thr Gly Arg 185 Arg Gly Cys Leu Arg Ser Leu Leu 90 Giy Ile Asn Lys Tyr i7 0 Giu Ala Gly Ala Giu 75 Tyr Lys Phe.

Lys Phe 155 Asf Trp Pro Ser Ser Gin Cys Val Ala Phe 140 Thr Thr Tyr Lys Ser Ser Ser Arg Asn Val 125 Leu Asp Tyr Val Val1 Gly Ser Ser Ser Val Gly 110 Ser Ala Asp Ala Ala 190 Lys Gin Arg Pro Phe Gly Ser Gin Met Cys Set Leu Pro Pro Gin Ala Gin Ile His Gly Ser Lys 160 Ala Asn Gin Ser Pro Arg Gin Ser Gilu Gin Pro 220 Phe Leu Pro Arg Phe Lys 215 210 Giu Leu 225 Pro Ile Ser Vai Ser Lys Lys Phe Ser Tyr 260 Thr Val 230 Lys Ile 245 Arg Leu Thr Pro Lys Val Leu Phe Pro Giu Lys 235 Ser Ala Pro 250 Arg Phe Gly 265 Lys Arg Asn Lys Pro Pro Ser 240 Asfl Thr Asn 255 P:\OPER\MRO\27674-95. DIV 19/1/00 51 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 208 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Ala Leu Gly Gin Lys Leu Phe Ile Thr Met 5cr Arg Gly Ala Gly a. a.

a a.

0 1 Arg Gly Leu Leu Giy His Asn Ser Gly Leu Met Asp Ala Ile Leu Pro Leu 130 Arg Gin Val Ser Gly Lys Gin Tyr 115 Phe Leu Gly Val Arg Glu Arg Val1 100 Scr Gly Tyr Thr Pro Gly Ilie Gin 85 Leu Leu Val1 Al a Leu Ser Trp Ala 70 Arg Pro Leu Arg Thr Trp, Pro Gly 55 Giy Arg Asp Giu Ser 135 Pro Al a Ala 40 Thr Val Leu Gly ile 120 Al a Ser Leu 25 Gly Leu Asn Tyr Arg 105 Ser Leu Phe Val Phe Thr Arg Leu Ser Trp Giu 75 Cys Asfl 90 ile 5cr Thr Val Phe Val Gin Glu Leu Ala Arg Ser Val Giu Ala 140 Giu Gly Asn Ser Gly Gly Arg 125 Met Cys Ile Asn Arg Tyr Ile Him 110 Gly Asn Lys Leu Val Thr Leu Ala Gly Leu Val Gly Phe r-1~ ir.

Val Val Ser Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr 175 170 165 Gin Ser Gly Lys Thr Val 195 Tyr Ile 180 Ser Pro Al a Ile 5cr Thr 200 Tyr Thr Gly His Arg Phe Val Lys 190 Leu Pro 205 Arg Arg Gly Ile P:\OPERWMR\2767495. DIV 19/1/0 52 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 194 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg a.

a.

S

1 Ser Asn Pro Arg Lys Ile Val Ala Giu 145 Gly Cys Phe Asp Met 35 Glu Arg so Val Arg Arg Gly Met Glu Glu Ser 115 Lys Lys 130 Asn His Glu Met 5 His Ile Thr Pro His Thr Arg Leu Lys Val le Arg 100 Giu Phe Giu Cys Tyr Asri Phe Val Ile Glu Arg Phe 70 Lys Thr Tyr Asn Thr 150 Ala Cys Gin Ser 55 Cys Gly Val Leu Giu 135 Tyr Leu Leu Met 40 Tyr Arg Thr Ala Ala 120 Asp Ala Asn Val1 25 Ala Asp Thr Gin Vadl 105 Met Cys Ser Gin Giy Thr Thr Asn Tyr Met Gin Trp 75 Giu Met 90 11 Asn Lys Asfl Phe Ala Lys 155 Lys Gly Ile Val Glu Tyr Lys Giu Lys 140 Trp Ser Leu Asn Cys Gly Gly Leu Arg Asn Asri T1 110 Gly Lys 125 Giu Leu Thr His Al a Ser Asp Ile Tyr Leu Ile Asn Cys Ser Ile Asp Asn r-1 Tyr Leu Gly 160 Ile Pro 165 170 Ala His Phe Leu Val Arg Gly 175 Pro Met Ala 190 Lys Lys Thr Lys Lys Giu Gin Lys Thr 180 185 Ile Thr INFORMATION FOR SEQ ID NO:16: P:\OPER\MRO\27674-95. DIV 19/II/00 -53- SEQUENCE CHARACTERISTICS: LENGTH: 215 amino acids TYPE: amino acid STPANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu sees.

1 Val Leu Thr Gin Arg Leu Val Gin 65 Asp Pro Val Arg Lys Gly Phe Thr 130 Lys Tyr 145 Lys Gly Arg Leu Phe Leu Cys Leu His Val Ile Arg Val Leu Phe Ala Val Arg 100 Lys Leu 115 Giu Ile Glu Gly Ser Lys Pro Arg 180 Asn Tyr 195 5 Gin Arg Thr Ala Lys Gly Ile Val Ti-p Thr 165 Gly Pro Al a Glu Tyr Asn 70 Leu Ala Ala Leu Tyr 150 Arg His Pro Gin Gin Gin 55 Lys Ile Glu Lys Glu 135 Met Gin His Phe Vai Ser 40 Leu Arg Val Thr Ser 120 Asn Ala His Thr Thr 25 Leu Tyr Ile Giu Gly 105 Asn Asn Phe Gin Thr 185 10 Val1 Val1 Ser Asn Thr Leu Gly Tyr Thr Arg 170 Glu Gin Thr Arg Ala 75 Asp Tyr Lys Thr Arg 155 Glu Gin Ser Ser Asp Gin Thr Ser Met Ala Thr Phe Ile Cys Gly Lys 125 Ala Leu 140 Lys Gly Val His Ser Leu Pro Asn Leu Ser Gly Lys Giu Asp Gly Ser Met Asn 110 Asp Cys Gin Asn Arg Pro Phe Met 175 Arg Phe 190 Phe Arg His Gly Arg Lys Val Ala A-rg 160 Lys Glu Thr Arg Ser Leu Arg Gly Ser Gin Arg 205 Thr Ti-p 210 Ala Pro Glu Pro Arg 215 P: %OPER\MRO\27674-95.DIV 19/1/00 54 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 208 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i7: Met Ala Pro Leu Gly Giu Val Gly Asn Tyr Phe Gly Val Gin Asp Ala S. S *5

S

S

55.

555 1 Val Pro Leu Ser Pro Al1a Gin Leu Thr Ile Phe Ile Giy Leu Lys Leu 130 Tyr Asn 145 Arg Tyr Arg Thr Phe Asp Val Tyr Gin Ser Tyr 115 Thr Thr Tyr Lys Gly His Thr Cys Gly Ile 100 Leu Gin Tyr Val Arg 180 5 Asn Leu Asp Arg Thr 85 Ala Gly Giu Ser Al a 165 His Val1 Gly Leu Thr 70 Arg Val1 Met Cys Ser 150 Leu Gin Pro Gin Asp 55 Gly Lys Gly Asn Val 135 Asn Asn Lys Val Ser 40 His Phe Asp Leu Glu 120 Phe Leu Lys Phe Leu 25 Giu Leu His His Val 105 Lys Arg Tyr Asp Thr 185 Pro Val Ala Gly Lys Gly Leu Glu 75 Ser Arg 90 Ser Ile Gly Giu Glu Gin Lys His 155 Gly Thr 170 His Phe Asp Ser Gly Leu Ile Leu Ile Phe Phe Gly Arg Gly Leu Tyr 125 Phe Giu 140 Val Asp Pro Arg Leu Pro Pro Val Pro Arg Arg Arg Pro Asn le Leu Val Asp 110 Gly Ser Giu Asn Thr Gly Giu Gly 175 Arg Pro 190 Leu Gly Arg Gly s0 Giu Ser Giu Trp, Arg 160 Thr Val1 Asp Pro Asp Lys Val Pro Giu Leu Tyr Lys Asp Ile Leu Ser Gin Ser 195 200 205 P:\0PER\MR0\27674-95. DIV 19/1/00 55 INFORMATION FOR SEQ ID NO:18: SEQUENCE

CHARACTERISTICS:

LENGTH: 18i amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i8: Met Glu Ser Lys Glu Pro Gin Leu Lys Gly Ile Val Thr Arg Leu Phe so so* 0 1.

Ser Gly Val Val Pro Tyr Leu Gin Thr Gly so Ala Giu Ser Gly Gin Lys Leu Met Cys Ser Leu Gly 20 Asp Arg Asn Lys Thr 100 Asn Tyr Glu Val Gly Phe Leu Lys Phe Asn Val Giu 70 Lys Tyr Giu Leu Ser Ala 55 Gly Glu Arg Gly Gln Asp 40 Ile Tyr Ser Gin Gln 120 Phe M4et 25 Tyr Gin Leu Val Gin 105 Ile Val His Thr Gly Tyr Phe 90 Giu Met Pro Pro Leu Val Ser 75 Giu Ser Lys Lys A~sp Phe Lys Ser Asn Gly Gly Pro Gly Asn A-1a Asp Tyr Arg Asfl 125 Ile Thr Leu Ser Val Tyr Ala 110 Arg Glu Ile Ile Leu Phe Val Trp Val Val.

Asp Pro Tyr Thr Ile Phe Lys Cys 115 Lys Thr Lys 130 Pro Ser Ser His 140 135 Met Tyr Arg Giu Pro Ser Leu His Giu 145 150 Ser Arg Lys Ser Ser Gly Thr Pro Thr 165 Asn Gin Asp Ser Thr 180 INFORMATION FOR SEQ ID NO:19: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 255 amino acids Ile Met 170 Gly Giu 155 Asfl Giy Lys Gln Gly Lys Gly Val 175 Arg 160 Val P:\OPERWMRO\27674-95. DIV 1911/W0 -56- TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Met Val1 Gin Arg His Thr Gly Asn Thr Giu 145 Tyr Arg Asn Leu SEQUENCE DESCRIPTION: SEQ ID NO:19: Ser Gly Lys Val Thr Lys Pro Lys Giu Le.

Asp Al a 50 Lys Lys Thr Leu Lys 130 Leu Tyr Sly H{is Lys 210 Asp *Ser 35 Lys Giu Leu Ile Ile 115 Leu Phe Val Trp, Val 195 Val Asp Ile Cys Asn Tyr Asp 1-00 Pro Tyr rhr rhr Tyr 180 Lys Aa Ala Pro Pro Gly Thr Gin Giu Tyr Ile Met Leu Arg Giu Lys Asp Ala Ser Lys Gin His Thr Ser 85 Gly Val Leu Pro Tyr 165 Leu Lys Met Ser Giu Giu 70 Arg Thr Gly Ala Giu 150 Ser Gly Asn Tyr Ala Giu 40 Ile Phe 55 Pro Giu Gin Giy Lys Asp Leu Arg 120 Met Asn 135 Cys Lys Ser Met Leu Asn Lys Pro 200 Lys Glu 215 Leu Lys Cys Cys Glu Pro Tyr His 90 Glu Asp 105 Val Val Ser Glu Phe Lys Ile Tyr 170 Lys Glu 185 Ala Ala Pro Ser Lys Lys Pro Leu Gin Leu 75 Leu Gin Ser Thr Ala Ile Gly Tyr 140 Giu Ser 155 Arg Gin Gly Glu His Phe Leu His 220 Glu Ser Lys Gin Lys Gly Leu Gin Tyr Thr 110 Gin Gly 125 Leu Tyr Val Phe Gin Gin Ile Met 190 Leu Pro 205 Asp Leu Pro Val Ile Ala Leu Val Thr Giu Ser 175 Lys Lys Thr Phe His Val Asp Phe Gin Ser Asn 160 Gly Gly Pro Glu Phe Ser Arg Ser Giy Ser Gly Thr Pro Thr Lys Ser Arg Ser Val Ser 225 230 235 240 PAC)PERWI10\27674-95. DIV 19/1/00 -57- Gly Val Leu Asn Gly Gly Lys Ser Met Ser HisAsGlSrTh 245 250 255 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 208 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 555555

S

*5*5 0e 5* (xi) SEQUENCE DESCRIPTION: SEQ ID Met Trp, Lys Trp Ile Leu Thr His Cys Ala 1 Pro Val Ala Arg 65 Lys Lys Ile Asn Giu 145 Tyr Tyr Gly Pro Thr His Leu Val Thr Tyr 130 Phe Asn Val1 Cys Val 35 Asn Val Phe Ser Ser 115 Tyr Asn Thr Al a Cys Thr Ser Arg Ser Gly 100 Val Leu Asn Tyr Leu Cys Cys Ser Ser Phe 85 Thr Glu Al a Asp Ala 165 Asn Cys Gin Ser 70 Thr Lys Ile Met Cys 150 Ser Gly Cl's Ala Ser Aisn Lys Lys Gly Asn 135 Lys Phe Lys Phe Leu Ser His Giu Val 120 Lys Leu Asn Gly Leu 25 Gly Phe Leu Phe Asn 105 Val Lys Lys Trp Ala 10 Leu Gin Ser Gin Leu 90 Cys A1a Gly Giu Gin 170 Pro Ser Leu Asp Ser Gly 75 Lys Pro Val1 Lys Arg His Arg kla Phe 4et Pro Ile Tyr Lys Leu 140 Ile Asn Xrg Phe Leu Val Ser V'ai Ser Ala 125 Tyr Glu Pro Val Ser Ser Arg Lys Ile 110 Ile Gly Glu His Ser Pro Ala Trp Asn Leu Asn Ser Asn Gin 175 Lys Leu Ser Glu Gly Arg Gly Glu Ser Lys Gly 160 Met Thr Gly Arg Gly Gin 185 190 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser P:\OPER\MRO\27674-95. DIV 19/1/00I -58- INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 212 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2i: Arg Leu Leu Pro Asn Leu Thr Leu Cys Leu Gin Leu Leu Ile

I

I

*1*I *1I**I 1 Cys Val Arg Pro Lys Gly I le Gln Thr Arg Asp 35 Giu Tyr 50 Gly Arg Leu Ile Ala Glu Gly Lys Gin Gly Gin Gly Gin Leu Arg Ile Val Glu 85 Ser Glu 100 Pro Ser Giu Ala Tyr Ser 70 Thr Lys Gly Asn Met Ser 55 Ala Asp Tyr Lys Thr His Thr 40 Arg Thr Thr Ile Ser 120 Ala Pro 25 Asp Thr Ala Phe Cys 105 Lys Phe Ser Gin Ser Giu Gly 90 Met Asp Gin Pro Leu G1y Asp 75 Ser Asn Cys Asn As n Ser Lys Gly Arg Lys Val Ala Phe Arg His Asn Val Arg Phe 125 Arg Asn Arg Val Lys Arg Gly 110 Thr His Leu Gin Gin Gin Phe Ile Lys Giu Glu Cys Tyr Ile Val Ala Lys Leu Ile Gly 115 Val Leu Giu Asn Asn Tyr 130 140 135 Trp 145 Ser Gly Phe Phe Arg Gin Val1 Met Val Phe Gin Asn Gin 165 Leu Pro Phe 180 Gly Ser Ala Thr Arg Gin 150 Arg Giu Ala Pro Asn His Pro Thr Arg Giy His Ala 185 Arg Arg Phe 170 Giu Thr Pro 155 Ile Lys Lys Arg Lys Gin Arg Gin Arg Lys Thr Ala Leu Gin 190 Arg Ser Arg 160 Tyr Gin 175 Phe Giu Arg Pro PACOPER\MROX27674-95. DIV 1911/00 59 Gin Pro Leu Thr 210 INFORMATION FOR SEQ ID, NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 225 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Ala Ala Leu Ala Ser Ser Leu Ile Arg 9 1 Glu Arg Lys Glu Phe Asp Val Ala Phe 145 Leu Pro Gly Val 50 Pro Tyr Thr Val Glu 130 Lys Tyr Gly Gly Ser Thr Lys Ser 35 Arg Leu Cys Gin Leu Lys Leu Gin Ala 85 Ser Ser Phe 100 Thr Ile Gin 115 Gly Leu Leu Giu Cys Val Arg Gin Arg 165 Arg Leu Gly Gly 70 Asn Thr Ser Tyr Phe 150 Arg Pro Cys Giy 55 Ile Pro His Ala Ser 135 Glu Val Gin 40 Arg Val1 Asp Phe Lys 120 Ser Asn Ser 25 Lys Pro Thr Gly Asn 105 Leu Pro Tyr Al a Gin Al a Lys Ser 90 Leu Gly His Tyr Gin Gin Leu Arg Leu 75 Ile Ile His Phe Val Lys Arg Leu Pro Phe Gin Pro.

Tyr Thr 140 Leu Arg Arg Ile Asp.

Cys Gly Val Met 125 Ala Tyr Glu Val1 Leu A.rg Arg Thr Gly 110 Al1 a Glu Ala Val1 Cys Leu Gly Gin Pro Leu Met Cys Ser Arg Pro Ser Pro Gly so Giu Arg Asn Arg Ala Ser Gly Arg Ala Trp, Tyr Leu Gly 170 Lys Gly Asn Arg Val Lys Lys Thr Leu Asp 173 Lys Ala Lys Giu Gly Gin Val Met 180 185 190 P:\OPERN110\27674-95. DIV 19/1/00 60 Ala Ala His Phe Leu Pro Lys Leu Leu Glu Val Ala Met Tyr Gln Glu 195 200 205 Pro Ser Leu His Ser Val Pro Giu Ala Ser Pro Ser Ser Pro Pro Ala 210 215 220 Pro 225 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 252 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

S.

Met Val Lys Pro Val Pro Leu Phe Arg

S

S

S

*SSS

S S S. S

S

Leu Asp Lys Asn Tyr Asp Pro Tyr Thr Cys Cys Gly 50 Lys Cys Gly Val Ile 130 Pro Asn Phe 35 Thr Asn Arg Thr Gly 115 Thr Glu His 20 Ser His Pro Gin Lys 100 Leu Met Cys Lys Pro Met Thr Gly Gly Arg Asn Lys Asp Lys Leu Asp 70 Tyr Asp Val Gly Phe Leu Ser Gin 55 Pro Tyr Ser Val Giu 135 Lys Phe Met 40 Cys Gin Leu Thr Ala 120 Gly Glu Phe 25 Trp Leu Leu Gin Asn 105 Ile Tyr Ser Arg Thr 10 Leu Arg Phe Leu Cys Gly Lys Gly 75 Met His 90 Ser Thr Gin Giy Leu Tyr Val Phe Asp Val Trp Lys Ile Pro Leu Val1 Pro 140 Glu Phe Ser Asn Ser Val Asp Phe Lys 125 Ser Asn Lys Lys Ile Leu Thr Gly Asn 110 Thr Giu Tyr Leu is Leu Phe Lys Arg Ala Leu Gly Leu Tyr Leu Leu Ser Lys Leu Leu Ile Leu Phe Val1 160 145 150 Ile Tyr Ser Ser Met Leu Tryr Arg Gin Gin Giu Ser Gly Arg Ala Trp P:\OPER\MR\27674-95. DIV 19/1100 -61 Phe Lys Ala Lys 225 Asn 165 Leu Gly Leu Asn Lys 180 Lys Thr Lys Pro Ala 195 Met Tyr Arg Glu Pro 210 Pro Gly Val Thr Pro 230 Gly Gly Lys Pro Val 245 Glu Ala Ser 215 Ser Asn 170 175 Gly Gln Ala Met Lys Gly Asn Arg 185 190 His Phe Leu Pro Lys Pro Leu Glu 200 205 Leu His Asp Val Gly Glu Thr Val 220 Lys Ser Thr Ser Ala Ser Ala Ile 235 Lys Ser Lys Thr Thr 250 Val Val Pro Met 240 cc r r r r

Claims (24)

1. An isolated polynucleotide comprising a member selected from the group consisting of: a polynucleotide encoding the polypeptide as set forth in Figure 1; a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of and a polynucleotide fragment of the polynucleotide of or

2. The polynucleotide of Claim 1 encoding the polypeptide comprising amino acid 1 to amino acid 252 as set forth in SEQ ID NO:2.

3. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

4. The polynucleotide of Claim 1 wherein said polynucleotide is RNA.

5. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

6. The polynucleotide of Claim 1 comprising from nucleotide 1 to nucleotide 759 as set forth in SEQ ID NO:1.

7. An isolated polynucleotide comprising a member selected from the group consisting of: a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Deposit No. 97146; a polynucleotide encoding the polypeptide expressed by the DNA contained in ATCC Deposit No. 97146; a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of or and a polynucleotide fragment of the polynucleotide of or P:\OPER\MRO\27674-95.DIV 19/1/00 -38-

8. The polynucleotide of Claim 7 wherein said polynucleotide encodes a polypeptide expressed by the DNA contained in ATCC Deposit No. 97146.

9. The polynucleotide of Claim 7 wherein said polynucleotide encodes a polypeptide expressed by the DNA contained in ATCC Deposit No. 97146. The polynucleotide of Claim 7 wherein said polynucleotide encodes a polypeptide expressed by the DNA contained in ATCC Deposit No. 97146.

11. A vector containing the DNA of Claim 2. .i

12. A host cell genetically engineered with the vector of Claim 11.

13. A process for producing a polypeptide comprising expressing from the host cell of Claim 12 the polypeptide encoded by said DNA.

14. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 11.

15. A polypeptide selected from the group consisting of: a polypeptide having the deduced amino acid sequence- of Figure 1 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC Deposit No. 97146 and fragments, analogs and derivatives of said polypeptide.

16. The polypeptide of Claim 15 wherein the polypeptide has the deduced amino acid sequence of Figure 1.

17. An antibody against the polypeptide of claim

18. A compound which inhibits the polypeptide of claim P:\OPER\MRO\27674-95.DIV 19/I/0 -39-

19. A compound which activates a receptor to the polypeptide of claim A method for the treatment of a patient having need of an FGF-15 polypeptide comprising administering to the patient a therapeutically effective amount of the polypeptide of claim

21. A method for the treatment of a patient having need to inhibit an FGF-15 polypeptide comprising administering to the patient a therapeutically effective amount of the compound of claim 18.

22. The method of claim 20 wherein said therapeutically effective amount of said polypeptide is administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.

23. The method of claim 21 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA encoding said antagonist and expressing said antagonist in vivo.

24. A process for identifying compounds active as agonists to the polypeptide of claim comprising: combining a compound to be screened and a reaction mixture containing cells under conditions where the cells are normally stimulated by said polypeptide, said reaction mixture containing a label incorporated into the cells as they proliferate; and determining the extent of proliferation of the cells to identify if the compound is an effective agonist. A process for identifying compounds active as antagonists to the polypeptide of claim comprising: combining a compound to be screened, the polypeptide and a reaction mixture containing cells under conditions where the cells are normally stimulated by a a a P:\OPERMRO\27674-95.ODV 19/1/00 said polypeptide, said reaction mixture containing a label incorporated into the cells as they proliferate; and determining the extent of proliferation of the cells to identify if the compound is an effective antagonist.

26. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 15 comprising determining a mutation in the nucleic acid sequence encoding said polypeptide.

27. A diagnostic process comprising analyzing for the presence of the polypeptide of claim in a sample derived from a host. S DATED this NINETEENTH day of JANUARY, 2000 Human Genome Sciences, Inc. by DAVIES COLLISON CAVE Patent Attorneys for the applicant(s) Patent Attorneys for the applicant(s) o