AU752592B2 - 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.

,o 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, S osteoblasts, and hematopoietic cells.

Each member has functions overlapping with others and also has its unique spectrum 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:\OPER\MRO\27674-95. DIV 19/I/00 -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 *10 angiogenesis in vivo and may play important roles in early development (Burgess, W.H. and o 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 basi, 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 19/1/00 -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 acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a polynucleotide encoding a polypeptide of the present invention.

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:\OPER\MRO\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.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides 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.

Thus, 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\27674-9.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.

o 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 prosequence and a presequence (leader sequence).

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 19/1/00 -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).

44*** 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\2767495. 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 "o 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 S"polynucleotide or polypeptide could be part of a composition, and still be isolated in that such 15 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 19/1/00 -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 :i 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 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 -19/1/00 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 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 S9; 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:\OPER\MRO\27674-95.DIV 19/1/00 -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 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 the art.

In a further embodiment, the present invention relates to host cells containing the above-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 "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 15 sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous S* 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 GEM1 (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 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 19/1/00 -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 19/1/00 -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 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 S"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//O 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 S" 20 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 is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ug/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 cases, the dosage is from about 10 pg/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 Ag 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:\OPER\MRO\27674-95. DIV 19/1/00 -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 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 S. 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- 19/1/00 -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, if-AM, PA12, T19-14X, VT-19-17-H2, iCRE, iCRIP, 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 CaP04 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 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 may 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:\OPER\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 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 S1 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 19/1/00 -26assays, 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 0..

S- 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.

*o••000 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/Y 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.

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 i 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 primers, sublocalization can be achieved with panels of fragments from specific chromosomes 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\MRO\27674-95.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 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:\OPER\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 products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

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 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.

9 "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 /g of plasmid or DNA fragment is used with about 2 units of enzyme in about pl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, P:\OPER\MRO\27674-9S. DIV 19/1/00 typically 5 to 50 /2g 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 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 -31 contains 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.

o 10 pQE-60 was then digested with NcoI and BglII. The amplified sequences are ligated into 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 15 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 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 20 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 o 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 XbaI. The polyadenylation site of the simian virus (SV)40 is used P:\OPER\MRO\27674-95.DIV 19/1/00 -33for 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 pRGI, pAc373, pVL941 and pAcIMI (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).

The plasmid is digested with the restriction enzymes and dephosphorylated using calf 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 9 are confirmed by DNA sequencing.

5 ug of the plasmid pBacFGF-15 is co-transfected with 1.0 12g of a commercially 20 available linearized baculovirus ("BaculoGold T baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 12g of BaculoGold T virus DNA and 5 4sg of the plasmid is mixed in a sterile well of microtiter plates containing 50 4l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 kil Lipofectin plus 90 ul 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 27C 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).

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 /d 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 (MOI) of 2. Six hours later the medium is removed and replaced with SF900 I medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 UCi of 3 5 S-methionine and 5 Ci 35S 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 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 35 S-cysteine two days post transfection. Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCI, 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\27674-95.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.

ooo*o *i EDITORIAL NOTE FOR 12478/00 THE FOLLOWING SEQUENCE LISTING NUMBERED PAGE 48 68 IS PART OF THE DESCRIPTION THE CLAIMS FOLLOW ON PAGE NO. 37 P:\OPER\MRO\27674-95.DIV 19/1/00 -48- 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 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 i(ii) MOLECULE TYPE: DNA (genomic) c (ix) FEATURE: NAME/KEY:

CDS

P:\OPER\MRO\27674-95. DIV 19/1/00 49 LOCATION: 759 (xi) S'EQUENCE DESCRIPTION: SEQ ID NO:l1:

ATG

Met 1 GTA AAA CCG Val. Lys Pro

GTG

Val1 CCC CTC TTC AGG AGA ACT GAT TTC 1A.A TTA TTA Pro Leu ?he Ar; Arg Thr Asp The Lys Leu Leu TTA TGC AAC Leu Cys Asn AAG GAT CTC Lys Asp Leu TTC TTT Phe Phe .25 CTC AGG GTG Leu Arg Val TCT AAG CTG CTG Ser Lys Leu Leu GAT TGC TTT TCO CCC PAZ TCA ATO TGG TTT CTT TGG Asp Cys The Ser Pro Lys Ser Met Tro Phe Leu Tr-p ATT TTC AGC Ile Phe Ser AAA GGA Lys Gly s0 ACG CAT ATO CTG CAG TGT CTT TGT GGC Thr His Met Leu Gin Cys Leu Cys Gly 55

A.AG

Lys AGT CTT AAG AAA Ser Leu Lys Lys

AAC

Asn AAG, AAC CCA ACT Lys Asn Pro Thr

GAT

Asp CCC CAG CTC AAG Pro Gln Leu LYS ATA GTG ACC AGO Ile Val Thr Ar;

TTA

Leu TAT TGC AGG CAA Tyr Cys Arg Gin

GGC

Oly TAC TAC TTG, CAA Tyr Tyr Leu Gin

ATG

Met 90 CAC CCC GAT GOA His Pro Asp Oly GCT CTC Ala Leu GAT GGA ACC Aso Gly Thr

AAG

Lys 100 GOT GAC AOC ACT Gly Asp Ser Thr

AALT

Asn 105 TCT ACA CTC TTC Ser Thr Leu Phe AAC CTC ATA Asn Leu Ile ACA 000 TTG Thr Gly Leu *.e CCA GTG OGA CTA COT OTT OTT Pro Val Gly Leu Ar; Val Val 0CC Al a 120 ATC CAO GGA OTO le Gin Gly Val

AAA

Lys 125 336 384 432 480 TAT ATA Tlyr le 130 ACC ATO AAT OGA GAA GOT TAC CTC TAC CCA.n TCA-, OAAk CTT TTT Thr Met Asn Oiy 0Th Oly T-Yr Leu Tyr Pro Ser Olu Leu Phe ACC CCT GAA TOC PAOG Thr Pro Oiu Cys Lys 145 AAA GAA TCT OTT Lys 0Th Ser Vai GA.A AAT TAT TAT 0Th PAsn Tyr Tyr OT A Val 160 4* ATC TAC TCA TCC ATO TTO, TAC AGA CAA Ile Tyr Ser Ser Met Leu Tyr Ar; Gin

CAG

Gin 170 GAA TCT GOT AGA Glu Ser Gly Ar; GCC TOO Ala Tr-o 17 AGA OTA'b Ar; Val TTT TTO GA. TTA PAT AAO OPA 000 CAPA OCT ATO APA GO PAC Phe Leu Oiy Leu Asn Lys Olu Oly Gin Ala Met Lys Gly ASfl n r190 AG AA% ACC AAA CCA GC.A OCT CAT TTT CTA CCC P AG CCA TTG OPA OTT LysLysThrLyspro Ala Ala isTe Theu Pro Lys Pro Leu 0Th Val 624 P:\QPER\MROM674-.DIV 19/1100 195 200 205 GCC ATG TAC CGA GAA CCA TCT TTG CAT GAT GTT GGG GAA ACG GTC CCG Ala Met Tyr Arg Glu Pro Ser Leu His Asp Val Gly Glu Thr Val. Pro 210 215 220 AA 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 PAG 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 CHA.RACTEFRISTICS: LENGTH: 252 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: orotein Met Leu As-o Lys As n 5 Tyr AspD Pro Tyr Thr 145 (xi) SEQUENCE Val Lys Pro Val Cys Asn His Lys Cys ?he Ser Pro 35 Gly Thr His Met Lys Asn Pro Thr Cys Arg Gin Gly Gly Thr Lys Gly 100 Val Gly Leu Ar; DESCRIPTION: SEQ ID Pro Leu ?he Ar; Ar; 10 Asp Leu Phe Phe Leu 25 Lys Ser Met TroP ?he 40 Leu Gin Cys Leu Cys 55 Aso Pro Gin Leu Lys 70 Tyr Tyr Leu Gin Met 90 A s Ser Thr Asn Ser 105 Val. Val Ala Ile Gin N:2: rhr Asp Ax; Val Leu T rp Gly Lys Gly Ile 75 H is Pro Thr Leu Gly Val Ser Asn S er Val.

Aso Lys Lys Ile Thr Gly Asn.

Thr Lieu Phe Lys Arg a Lex2 G1-j Leu Leu Ser Lys Leu Leu Ile rLeu 125 le 130 Pro Thr Met Cys Asn Lys Gly Phe IS0 Gly G1u Tyr Ser Leu Val1 Tyr Phe 155 Pro 140 Glu Ser A~s n Leu Tyr Phe Val1 160 P:\0PER\MROM2674-95.DIV 19/1/00 -51 Ile Tyr Ser Ser Met Leu Tyr Arg Gin Gin Glu 5cr Gly Arg Ala Trp 165 170 .175 Phe Leu Gly Leu Asn Lys Glu Gly Gin Ala Met Lys Gly Asn Arg Val 130 185 190 Lys Lys Thr Lys Pro Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val 195 200 205 Ala Met Tyr Arg Glu 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 lie Met 225 230 235 240 Asn Gly Gly Lys Pro Val Asn Lys Ser Lys Thr Thr 245 250 INFOR.MATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 29 base oairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUEN11CE DESCRIPTION: SEQ ID NO:3: *.GCCAGACCAT GGTAAAACCG GTGCCCCTC 29 INFORMATION FOR SEQ ID NO: 4: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 33 base oairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: lin~ear (ii) MOLECULE TYPE: DNA (genornic) SEUEC DECITO:SQT O4 GGAGAA CTGTGC *ATTGTGC (x2) SEQUENC DESCRITION SEID: SE5D:O4 (i4) SEQUENCE C:-AACTERISTICS: P:\OPFR\MR0\27674-95. DIV' 19/1 /00 52 LENGTH: 35 base pairs TYPE: nucleic acid STPANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:* DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CTAGTGGATC CGCCATCATG GTAAAkCCGG TGCCC INFORMATION FOR SEQ ID NO:6: SEQUENCE

CHAPACTERISTICS:

LENGTIH: 32 base pairs TYPE: nucleic acid STRJANDED71TESS.: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGACTGGTAC CAGCCACGGA GCAGGAATGT CT 32 INFORPMATION FO0R SEQ ID NO:7: SEQUENCE CHARniCTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (i)MOLECULE TY1PE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTAGTGGATC CGCCATCATG GTAAACCGG TGCCC 3 INFORMATION FOR SEQ ID NO: 8: SEQUENCE

CHARACTERISTICS:

LENGTH: 30 base pairs TYPE: nucleic acid S TRANDEDNE S S: s ingl1e TOPOLOGY: linear P:\OPERWI10\27674-95. DIV 1911/00 53 MOLECULE TYPE: DN.- (xi) SEQUENCE DESCRIPTION: SEQ TD NO:B: GTCGACCTCG AGTGTGTGCT TACTCTTGTT INFORMATION FOR SEQ ID NO:9: SEQUENCE CHAR.ACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid STRANDEDN'ESS:. single TOPOLOGY: linear (ii) MOLECULE TYPE: Drotein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Ala Glu Gly Glu le Thr Thr ?he Thr Ala Le'a Thr Glu Lys ?he

I

Asn Asn Thr Ser Al a Glu Ile .as n Leu Gly AXrg 50 Val Met Cys 5cr Giy 130 Pro Gly Asp Gly Asp Leu Lys 115 5cr Pro His Arg ?he 100 Lys Cys Gly Phe Ser Val1 Aso 85 Leu His Lvs Asn Leu Asp Tyr 70 Gly Glu Ala A rg 'i'yr Ar; Gin 55 Ile Leu Arg Gl1u Gly Lys Ile His Lys Leu Leu Lys 120 Pro Lys 25 Leu Ile Ser T-yr Giu 105 Asn Arg Pro Pro Thr Gly 90 Glu T -p T17,r Lys Asp.: Leu Glu 7S Ser Asn Phe His Leu Gly Gin Thr Gin His Val Leu Tyr Thr Val Leu Ser Gly Gin Th r Pro T-rAsn Gly Leu 125 *Gly Gin Cys Asp Ala Tyr Asn T hr Lys Lys Ser Gly Glu Leu Giu Tyr Lvs Ala

S.

135 1.40 lie Leu Phe Leu Pro Leu Pro Val Ser Ser 145 2.50 INFO-RMATION FOR SEQ ID Asp 2.55 P:\OPER\NMRO\27674-95. DIV 19/1/00 54 SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: orotein (xi) SEQUENCE DESCRIPTION: sEQ ID NO:lO: Met Ala Ala Gly Ser Ile Thr Thr Leu Pro 1 Gly Tyr Val Gln Val1 Asn Ar; Ser Cys ASrP Ala Tyr Thr Thr Thr 130 Gly Lys Gly Glu Leu Asn TPyr 115 Gly Al a Asn Val Glu Al a Glu 100 Arg Gln Phe Gly .A r Arg met Cys Ser Tyr Leu Pro Gly Glu Gly Lys ?he Ar;- Lys Pro ?he Lys Val G1u Lys Leu 135 Met Gly P he Ser Val Aso Phe Tyr 120 Gly Hi s 25 Leu Asp Ser Gly Glui 105 Thr Ser- Phe Arg Pro Ile Arg 90 Arg Ser Lys Al a Lys TIle His Lys 75 Leu Leu Tr-) Thr Le Ile Gly Leu Glu Ty-r Gly 140 Pro P ro Lys Val1 Al a Ser Val 125 Pro Glu Lys Asp Leu Cys Ser Asn 110 Al a Gly Aso Ar; Gly Gln Al a Lys Asn Leu Gln Gly Leu Arg Leu Asn Cys ryr Lys Lys 0 00.

00* 6:*09 Ala Ile Leu Ser Ala LYS Ser 155 9 9 9* 9*9* 999 9999 INFORMATION FOR SEQ ID NO: !I: SEQUENCE

CHARACTERISTICS:

LENGTH: 239 amino acids TY'PE: amino acid STP-ANDEDNESS: sinrgle TOPOLOGY: linear (ii) MOLECULE TYPE: proteinl P:NOPER\MRO\27674-95. DIV -19/1/00 (xi) SEQUENCE DESCRIPTION: SEQ ID 140O1 Met Clv Leu Ile Trv Leu Leu Leu Leu Ser Leu Leu Giu Pro

I.

Pro Ala Gly Gly Tyr Cys Asn Gly Val Glu Leu Ala Ala Glu Tyr Ala Al a Val1 Ala Ser Val Met~ Cys 115 Ser Gly TIyr Thr Leu Gly Asfl 2100 Glu Akrg Pro Glu Lys Gi Ile Lys ?he Leki Gly His Tyr Asn 70 Va 1 Ar; Val Tyr Al a Leu Hi s 55 Ser Al a Gly Giu Arg Ar; Gly 40 Leu Al a Ile Ar; Ar; 2.20 Thr Leu 25 Gly Gin Tyr Ar; Leu 2.05 ile Val2 Ar; Al a Leu Gly 90 T-yr FHi s Ser Ar; pro His Ile 75 Leu Al a Glu 6cr Aksr Ala Ar; Ar; Pro Ser Leu Gin ?he 6cr Ser Giu Leu Gly 1252 Thr,. Pro 2.40 Ser Val Gly Ar; Gly Ilie Gly His 1120 Tyr Gi y Asr Gly Gly Lys Ar; Thr Ar; Tyr As n Ala Gly T ro Leu Val1 Al a Tyr Ser T h Ar; Lys 130 Ar; 1.45 Gin Pro Ser Ala Gin Ar; 2.50 Len T~r- Tyr Val 1r;

S

S S 5@ 0O *@jS

S

*SS* S 0S S

S

*5

S

0 0.0505 Gly Ar; Pro Akr; Ar; Giy Phe Lys T1- Len ?he Leu Pro Ar; Val Len Asp H 2.80 Gin Len Gin Ser Gly Leu Pro Ar; P 2.95 200 Ar; Akr; Ar; Ar; Gin Lys Gin Ser P 22.0 22.5 Val Gin Ala Ser Ar; Leu Giy Ser G 225 230 INFORMATION FOP. SE'Q ID NO:2.2: SEQUENCE

CHAR.ACTERISTICS:

LENGH: 206 amino acids TYPE: amino acid ST--A NDEDNTESS single TOPOLOGY: linear 85 ro ro 1 In Ar; 170 Ar; Pro AspD Leu As D Gly Asn Giu 23) Thr Gin His Gin Lys Gly 205 Leu Gln 220 Ala Scr Lys Ser Ser 2.75 met Val Ar; 2.90 V-al Gin Pro pro 5cr His A~la His S. S S S

SO

0 *00S 0@ S S

S.

0*SS

SSSSSS

S

*SSS

S

5555 P:\OPEP\,MRO\27674-95. DIV 19/11/DO 56 (ii) MOLECULE TYPE: Drotei- (xi) SEQUENCE DESCRIPTION: SEQ ID N~2 Met Ser Gly Pro Gly Thr Ala Ala Val. Ala Leu Leu Pro Ala Val Leu Leu Thr Ser Lys Lys Gin Asp Phe Leu 145 Leu Met Val Al a Al a Leu S0 Glu AXrg Al a Ser Gly 130 Tyr Leu Phe Set Leu Pro Val1 Al a Leu Leu Leu 115 Val1 Gly Pro le Pro Leu Asn Al a Ala Arg Pro 100 Leu Al a Ser As n Al a 180 Thr Al a Gly Leu Val Arg Aspo Glu 5cr Pro Asn 165 Leu Met Pro Thr Ser Gin 70 Leu Gly Leu Ar; Phe 150 Tyr 5cr Lys Trv Leu Leu 55 Ser Arg 5cr Phe 135 Phe Asn Lys Val1 Ala Glu 40 Al a Gly Cys Ile Pro 120 ?he Thr Ala Asn Gly 25 Al a Arg Ala Asn Gly 105 Val1 Val1 Asp ZTvr Gly 185 Arg Glu Leu Gly Val1 90 Gly Giu Ala G lu Glu 170 TLys Gly Gly Leu Glu Pro Val Asp Tyr 75 Gly Ile Ala H is Ar; Gly Met 5cr 140 Cys Thr 155 S3-cr Ty r Th r Lys Al a Arg .Al a Leu Gly Al a Val 125 3cr Phe Lys Lys Al a Ar; Ala Leu Phe AspD 110 Val Lys Lys T-yr Gly 190 Ala Tr-o Gin Gly His Thr Ser Gly Giu Pro 175 Asn Pro Glu Pro Ile Leu Ar; Ile Lys le 160 Gly Ar;- Thr His ?he Leu Pro Ar; Leu 200 205 INFORMYATION FOR SEQ ID NO:13: SE-QUENICvE CHJARACTERISTICS: LENGTH: 267 amino acids TYPE: amnino acid.

STRkNDE-DNESS: single TOPOLOGY: linear (ii) MOLECULE7 TYPE: protein P:\OPER\MRO\27674-95. DIV 19/1100 57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Met Ser Leu 5cr ?he Leu Leu Leu Leu The phe Ser His Leu Ile Leu

I

Ser Gly Ser Al a Tr-p Gly Glu Ile Lys 145 Phe le Al a Pro Ser S er Ser Phe A a Vai 130 Lys Arg His Trno Ala 3cr Leu Leu His Asn Gly Gly Glu Arg Al a Al a Ser Gly Gly Leu 100 Met le Lys Ar-g Thr His Thr Ala Ser Al a Gin Leu Arg Leu ?he 165 Giu Gly

ASP

MIet Gin 70 Arg Ile Ser Gly His 150 Gin Lys Giu Lys Ar 25 Arg Asn Pr 40 Ser Ser Se 55 Gly Ser G1 Thr Gly Se Tryr Pro As 10 Vai Leu G~ 120 Vai Phe Se 135 Ala Ser A.

Glu Asn S Th-r GlyA *0 Lu I a Arg Ser Leu Leu 90 Giy Ile ASn1 Lys Ala Pro Gly Ser Ala Ser Glui Gin 75 TLyr Cys Lys Vai Phe. Ala Lys ?he 140 Phe Thr 155 Asfl Thr Lys 5cr Ser 5cr Arg A-sn Vai 125 Leu Aso -Tyr Gly Ser Ser Ser Vali Gly 110 Ser Al a AspD Al a Gin Arg Pro Phe Gly Ser Gin Met Cys Ser Pro Gin Al a Gin Ile His Gly Ser Lys 160 Ala er Tyr Ala Leu Asn 190 .rg Glu T--O Tyr Val B5 1 180

S.

Lys His Giu 225 Pro Ser Arg Ile 210 Leu le Val Gly 195 Ser 5cr Lys Ly s Lys Thr Phe Ser Tyr Al a Hiis Lys 245 Arg Lys Phe V.al 230 le Arg Leu 215 Pro Lys Gly 200 Pro V a .1 Leu cy 5 Arg Pro Ser 2635 3cr Phe Glu Al.!a 250 ?he Pro Lys Lys 235 Pro

G

1 y Arg Gin 220 Lys Arg 205 S er Asn Lvs Glu Pro PAsn Gln Pro 255 Pro Ser 240 Asn P:\OPER\MR0\2767 4 -95. DIV 19/1/00 58 INFORMATION FOP. SEQ ID NO: 14: SEQUENCE

CILAPACTERISTICS:

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 Lei- Phre lie Thr Met Ser Arg Giy Ala Gly I is 1 A-rg Gly Leu Leu Gly Hi S Asn Ser Leu Met Asn Al a Ile Leu Pro L e' Gin Val 5cr Gly Lys Gin Tyr 115 Phe Gly Val A'r-g Glu ALrg Val1 100 5cr Gly Thr Pro Gly Ile Gin Leu Leu Val Leu 5cr TrD Al a 70 Arg Pro Leu Arg Tr-o pro Gly Gly Arg AspD Giu Ser Al a Al a 40 Thr Val1 Leu Gly ile 120 Aila Let' 25 Gly Leu Asn Tyr Arg 105 Ser Let' Val1 Thr Let' Trpo Cys 90 Ile Thr Phe Phe Arg Ser Glu 7S Asn Ser Val Val Glu Leu Al a Arg 5cr Val1 Gly Git' Al a 140 Git' Gly ASn 5cr Gly Gly Thr Arg 125 Met Cys le Asn Arg Tyr le i S 110 Gly Asn Lys Leu Thr Al a Let' Gly Giu Val Ser Phe Val1 Let' Gly Val Phe Glu Val Lys Arg 130 Pro 5cr Phe Gin Gly 145 Arg Leu Tyr Ala Thr 155 L 160Ty Git' Thr Let' Leu Gin Gly Thr T-vr 180 Ser Lys Val 3cr Pro Asfl 165 Ile A-la Pro le Asfl Let' -Met T'yr Asfl Ser Lys iB5 Thr Val 200 Ala Tyr 170 Tyr Giy Thr His G1u Ser ASp:: Arg Val Lys 190 Phne Let' Pro 205 175 A-rg Gly Ile P:\0PER\MM\27674-95.DV 1911100 -59- INFORMATION FOR SEQ ID SEQUENCE CHA?-ACTERISTICS: LENGTH: 194 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQ1JENCE DESCRIPTION: SEQ ID Met His Lys T=D Ile Leu Thr Tro lie Leu Pro Thr Leu Leu Tyr Arg 1 Ser Asn Pro Arg Lys Ile Val Ala Glu Cys Asn Glu Va1 Arg Met Glu Lys 130 Asn ?he Met A~r; Ar; Gly Glu Ser 115 Lys His His Thr His Ar; Lys Ile 100 Glu Glu Tyr 5 Ile Pro Thr Leu Val Arg Phe Cys Asn Ile Glu Arg Phe 70 Lys Thr Vy r Asn Thr Cys Gin Ser Cys Gly Val Leu Glu 135 Tyr Leu Met 40 T1yr Arg Thr Ala Ala 120 Asp) Ala Val 25 Ala Asp Thr Gin Val 105 Met Cys Ser Gly Thr Pyr Gin Glu 90 Gly Asn Asn Ala Thr Asn Met 75 Met Ile Lys Phe Lys Ile Val Glu Tyr Lys Va1 Glu Lys 140 Tmo Ser Asn Gly Leu Asn Ala Gly 125 Glu Thr Leu Cys Gly Ar; Asn Ile 110 Lys Leu His Ala Ser Asp Ile Tyr Lys Leu lie Asn Cys Ser Ile Asp Asn Gly Tyr Leu Gly Gly Glu Met Val Ala Leu As Gin Lys Gly Ile Pro Val Ar; Gly I 7 175 Lys Lys Thr Lys Lys Glu Gin Lys 180 Ile Thr INFORMATION FOR SEQ D NO:16 Thr 185 Ala His ?he Leu Pro Met Ala 190 P:\OPER\MRO\2767 4 -9 5 DIV 19/1 /00 60 SEQUENCE CHARACTERISTICS: LENGTH: 215 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECTULE TYPE: orotein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Gly Ser Pro Ar; Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 Val1 Thr Ar; Val AspD Val Lys P he Lys 145 Lys AIr; Phe Leu Gin Leu Gin Pro A-r; Gly Thr 130 Tyr Gly Leu Leu Cys His Ile Val Phe Val Lvs 115 Glu Glu Ser Pro Asn 195 Leu Val1 A-r; Leu Ala Ar; 100 Leu Ile Gly Lys, Ar; 150 Tyr Gin Ar; Thr Al a Lys Gly Ile Val T -p Thr 165 Gly Pro Al a Glu Tyr Asn 70 Leu Ala Al a Leu Tyr 150 Ar; His Pro Gln Gin Gin 55 Lys Ile Giu Lys Glu 135 Met Gin His ?he Val1 Ser 40 Leu Ar; Val Thr Ser 120 Asn Al a His Thr Thr Val 25 Leu Val Tyr Ser le Asn Glu Thr 90 Gly Leu 105 Asn Gly Asn Tyfr Phe Thr Gin Ar; 170 Thr Giu 185 Gin Thr Ar; Al a 75 Asn Tyr Lys Thr Ar; Glu Gin Ser Asp0 Thr Me t Thr Ile Gly Al a 140 Lys Val1 S er Ser Gin Ser Al a Phe C-Is Lv s 125 L eu Gly His Leu Pro Leu Gly Glu Gly Met 110 AsID Gln Ar; Ph e Ar; 190 As n Ser Lys Aso Ser Asn Cy s Asn Pro Me t 175 P'ne ?he Arg His Gly Ar; Lys Val Al a Arg 160 Lys Glu Thr Ar; Ser Leu Ar; Gly Ser Gin Ar; 200 205 Thr Trp 210 Ala Pro Glu Pro Ar; 215 P:\OP ER\MRO2i6-,4-95. DIV 19/1 /00 61 TNFZORMATION FOR. SEQ I 7D Nqo:17! SEQUE-NCE Ci-DaCTETSTICS: (A)1 LENGTH: 203 amino* acids TYPE: amino acid STP NDEDNESS: single TOPOLOGY: linar (ii) MOLECULE TYPE: orotein (x4) S-EQUENCE DESCRIPT7ION: SEQ ID INO: 17: Met- Ala Pro Lcau Cl-s lu Cly Asn Tyr Val Lau Pro Gin Phe Giv Tyr i43 A "-3 Pro 5cr Al a Li-, L eu Lau 130 Asn Th r ?he Aso Val1 T-yr Gi1n Ser Trr Tyr Lys Gly -z S Cy s Ciy Ile 100 Lau GC 1n Tyr Val Ar; Asn Lau Aso Arg Al a r Ala Val2 Cly Thr 70 Ar; Val1 Cys Scr 150 Le'i G 1n Pro Gin

AS-)

Cly Lys Gly Asn Ja 1 135 Asn As n Lvs Va 1 Ser 40 ?he Asp 120 Lau Lys Lau 25 Glu Lau :;is Val1 105 Lys Tyr Asno 7 hr Pro Lys Le~u Ser S r Cly Cl u Lys5 Gly 170

S

Val1 Cly 73 Ar; Clii 155 Thr Aso GCly lie Tie Le 140 Val1 ero S a 1 Lau Leu Cly Cly Tyr 123 Asn A-r; pr--o GC1n Pro Pro A-r; Pro Ile Val 110 Gly A-r; 130 As:: I1 Arg A-r; Asn Lau

AS-)

Ser Cly Al a Ar; Cly Clii 3cr C 1 u r V-a 1 Aso Pro Asp Lys 19; Val Pr--o GCiu Tyr Lys Asp Ilc 3cr Gin 3cr P:\0PER\MRO1,2767495.DIV 1911/00 62 INFORPMATION 7OP. SEQ TD NO:l3: SEQUENCE

CHARACTERISTICS:

LENGTH: 181 amino acids TYPE: amino acia STRUANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: orotC2in S S (xi) SE-:QUE-NCE DESCRIPT7ON: S-:Q 7D NO:13: Met Glu Ser Lys Glu pro Gln Lei- Lys Gly T e V Ser Gin Gin Gly Tyr ?he Leu Gln Met His Pro A 25 Gly Th Lys Asp Glu Asn 3cr As y Ir Thr Le--u

P

Val Gly Leu AIr; Val Val Ala j-e Gin Gly Val L Val Ala Met Asn. Gly Glju Gly -1-fr Leu Tlr 3cr S 70 73 pro 01': Cys Lys ?he Lys Gild 3cr Val G0T- Tyr 3cr 3cr T Leu Tyr Arg Gln Gin Glu 3cr 100 103 Leu Gl1y Leu A sn Lys 0l'a Gy Gin1 1 T1- Met Lys 115 120 Lys Thr Lys Pro Scr 3cr H i s p-he Val Pro Lys 130 133 Ve t T A Glu Pro Secr LeU Hi s GW 1_l T Gly 3cr Ar; Lys 3cr 3cr Gly Thr Pro ThrMeAs 163 170 A sfl Gin -As-p Ser 130) 7T--ORMATION FOR CZE:Q 7D, NO:3 SEQUE'NCE CEH.A-

CTERISTICS:

LENGTH: 233 amino acids a! Thn r A so GIN' T 3 ys A -a a :0 ;cr ASO ksn.- T Ifr G1ly P r i25 Pro 140 G u Lys 017 GlY rg 0 'Cu er Ta! Al a Arg 01: Ly ile A ile p Leu ?he Val Tr'0 Val n- Glv S Val 173 ro Lys Cys Arg 1a P:\OPBR\MR927674-95 DIV 19/1/00 63 TYPE: amrno acd STRANDEDNESS: sinlgle TOPOLOGY: li-a- (i)MOLECULE.- TYPE: :)roteinf (xi) SEQU7NCE DESCRIPTION: SEQ !D NO:19: Met Ser Gly Lys Val Thr Lys ?ro Lys Giu

S

0@ *5* 1 Val Gin Ar; Gly Is n hr Glu Asn Leu 225 Leu so Lys Lys Thr Leu Lys 130 Leu Ty r Gly Hiis Lys 210 Aso: Ser Lys Glu Leu Ile 1 15 Leu Phe V a I T r7 Va 1 135 Va 1 A S Ilie Cy s As n AspD 100 Tryr Thr T-Ir 13 0 Lys Al a 5 Gly Val2 Leu Lyvs Pro Glu Ahr Al a Gly As n 'I-Yr Pro Al a T1 Pro G 1 Lis Le- 135 Cys S er 215 Gly c- 1u Gly 120 A Sn Lys Po 200 G u Thr 2S G 1 Glu 105 Va 1 Ile 133 Al Pro G in Lys Cys Pro His Aso Gi u Lys 170 G 1 3cr Glu Lys 75 L eu AS a GiY GiU 15 Gl 3.

Lys T-yr Lys Leu le Tyr 140 C-1 n 220 Giu Lys Lys G 1 n 125 Leu 01-1 L eu 2-05 Ala Met Gly Gin Thr 110 190 P ro eu S -am Pro v1 Aa rLeu V-a 1 175 Ly s srh ?he Val Gin .As n 160 Gly Pro G 1 u *5

S

**SS

3cr Am; 3cr Gly 3cre_ Gly 7-r Pro Tnr- LYS 3cr Sr;3c Val 3cr 235 240 P:\0PER\VRO\276 74 -95 DIV 19/1100 64 Gly Val Leu Asn Gly Gly rYs 3cr Me- Ser Hu sf Glu 3cr Thr 2 45 250 255 INFORMA2-TION FOR SE-Q ID 2,7:20: (iSEQYE NCE CKARAPCTERISTICS: LENGTH-: 208 amino acids TYPE: amino acid STRANDEDITESS: single TOPOLOGY: linear (i4i) MOLECULE TYPE: oroteinf (xi) SE-7QUENCE D-ESCRIPTION: SEQ ID NO0: Met Trp Lys TrD ile Leu Thr- His C-is Ala 3cr Ala ?he Pro His Leu 1 510 Pro Gly Cys Cys Cys Cys Cys ?he Leu Leu Leu Phe Leu Val Ser Ser 25 Val Pro Val Thr Cys Gla Ala Len Gl 1y Gin Asp met Val Ser ?ro Glu 40 415 Ala Thr A sn 3cr 3cr 3cr 3cr 3cr ?he 3cr 3cr Pro 3cr Ser Ala Gly ArH-is Val At'; 3cr Tyr Asn H is Leu Gln G ly A5so Val Ar-; Tro- Arg S570 75 S.Lys Leu ?he 3cr Phe- T h r Lys Tyr The Len Lys 11-1e G lu Lys As rn G 1 y 5 90 3 *Lys Val 3cr G1,ly, T)hir Lys Lys Glu Asri Cys Pro 7yr 3cer le Leu G 1 0:600:Th Ser Val11 T-1 -l Gly Val Val Al a Val LsAl a le Asn 3cr 115 120 125 Asn Tyr r Leu Ala M-e t Asn Lys Lys Gly Lys Len Gy 1 y Ser Lys 130 135 4 6400.*Gn P he Asn Asn A'sp Cys Lys Len Lys Glu Ar; 71, U~ G 1 U Asf Gly @0 15150 153 Ty sn Thr Tyr Al-a 3cr The Asn Tr Gin His -1y Ar; Gin Me- 000015 170 173 Tyr Val Ala Leu Asn Gly Lys Gl l roA;A; 1 T-y Thr 180 135 Ar; Ar; Lys Asn Thr 3cr Ala His The Leu Pro Met VTal1 His c P:\OPER\),RO\.276'74-95. DIV 19/1100 65 TNFOP~AT±ON ORSE7Q ID N70:21: ()SVQUENCE C:zLRACTERIS

TICS:

LENGTH: 212 amrnlflO acids TYPE amnino acid STRNDEDNESS: s ingl= TOPOLOGY: linear (ii) MOLECiJE TYPE: orotainr (xi) SEQ-L7NCE DESCRTTON: S-EQ 1D 140:2 1 ~gLULeu Pro Asn Leu Thr- Let' Cys Leu Glfl Leu Leu Ile Leu

CYS

1~ g e 10 Cys Gin Thr Gin Gly Glu Asn 4s pro 5cr pro Asfl ?he Asn Gin T r 25 Val As-o Gin Gly Ala Met Thr A Gin Leu Ser Arg Arg Gin lie 35- 40 -45 P~r Glu Tyr Gin Leu Tyr ser Ar; Thr Ser Gly Lys -H is Val Gla Val 550s 7ro G.1 rgA;leSr la Thr Ala GJ2-1 Aso Gly ASrI Lys he Ala 6570750 Lys Leu Ile Val Glu Th r As Thr Pn e Gly Ser Arg Val Ar;T Tle Lys l l Gh5rGh y y Ie Cys Met Asn Lys Ar; Gly Lys Leu 100 10510 ile Gly Lys Pro Sr Gl1,y Lys Ser Lys Asp0 Cy -f V'al The Th r G 1,,1 T le- Val LUGhAnAnrr Th r Ala Phe Gin sn- Ala A- Hi s G1u Gly 130 135 14 0 To he Met V al Ph 7 Th -rP; Gin Gly Ar; Pro Arg Gi Ala Ser Ar 145 150 510 Ser A rg Gin A.sn Gin Arg Al a -i Pf l y r e. y i 165 170 G1ly G zln Leu Pro Pro As-. la Giu L ys Gln -,ys G1in The

GD,

Phe 'Jai Gly Ser '!la Pro T--r A r;g Ar; g Ly;s r P:\Oi'ER\MNRO\27674-95. DIV 19/1/100 -'66 195 200 205 Gin Pro Len Thr 210 IN-'O :MZrn.TON 7OP. SE:Q !D NO: 2 2 S-:QUENCE CzLARACTERTSTTCS: LENGT-H: 225 amino acids TYvPE: amino acid STRAINDEDNESS: single TOPOLOGY: linear (ii) MOLE'CTTE TYPE: protCefl (xi) SEQ 7NCE DESCRITbON, SE:Q 7D N0:22: Met Ala Ala Len Ala Ser Ser Len T1 Gin Lys Ar; Gln Val 1 510 Gin Pro Gly Gly Ser Pro Val Ser Ala Gin Ar; Arg Val Cys Pro 25 Ar; Gly Thr Lys Ser Len Cys Gin Lys Gin Len Leu lie Leu Leu Ser 40 Lys Val Ar LenU Cy.s Gly Gly Ar; Pro A! a A r Pro As-) Ag Gly Pro *Gin Pro Glh Ly ly le Val Thr Lys Len Cys A r; Gin Gly 657 3 Ph:"e Tyr Len G1n Al a Asn Pro Asp Gly. Se lie Gin Gl1y T 'ar Pro r-2I 90 0. ~AS-o Tr S-r Ser ?he Tnr~1 Phe Asn L en_ Lie Pro- '.al Gly Len A; 00100 10C)5 Val Val r lie Gin Scr Al a TLvs L en- Gl1y HJ s -r et Ala Met Asn 115 120 12 Ala Glu Gly Lena Len Tyr Scr Ser Pro His Pne Th r Al1a Gin Cys Ar; -130 135 140 Ph L-vs Gln Cys Val ?he Gln Asn Tyr Tyjr Vai Len_ Tyr Al a 5cr Al 1a 145 150 155 Le T-r g Gin, Ar; Ar; Ser Gly A-r; Ala T rp Ty r Len G-ly Len Asp i 65 170 173 Lys Gln Gly Gin Val Met Lys Gly A sn A-r; Val LsLys Thr Lys Al1a 130 195 150 P:\OPER\.MRO\276-74-95. DIV 19/1 /00 67 Ala Ala His Phe Leu Pro Lys Leu Leu Glu Val Ala Met Tyr Gin Gin 195 2 00 205 Pro Ser Leu His Ser Val Pro GIn Al-,a Ser Pro 5cr 5cr ?ro Pro Akla 210 215 220 Pro 225 INFORMATIONT FOR SEQ 7D NO:23: SEQUENCE C:-AACTERISTICS: LENGTH: 252 amino acids ~IP:amino acid STRANDEDNESS: single TOPOLOGY: line1-ar (i)MOLECULE,-= TY-PE o ro -2i (x)SEQUE=NCE-; DESCRITITON: SE-Q ID 1,O:23: Met Val Lys Pro Val Pro Leu ?he Arg Arg Thr As ?he Lys Len Leu 1 310 is Len Cys ASn Hi S LyS Asp Leu Phe Phe Len Ar; Val Ser Lys Len Leu 25 Asp Cys Phe S-:r P ro Lys 5cr Met Tro Phe Leu Tr- Asr i le Phe 40 :::Lys Gly Th-r His Met Leu Gin C- s Lenu Cys Gly Lys Ser Len Lys L-ys 55 0 Asn Lys Asn Pro ThInr A s Pro Gin Len Ly-,s fdl -r Ie Val Thr Akr Len 70 75 T* r Cys Ar; 0 Gi -ly Tyr Tyr n Te Gin M e s Pro Aspj Gly Ala Len .90 Aspn Gly Thr Lys Gly s p Ser Thr- Asni Ser Thr Leu Phe sn Len TIe 100 105 110 Pro Val Gly Leu Ar; Val1 Val Aa Ile0 l a y h l e 115 120 125.

Tyr I e Thr Me t Asn Gly G1, u lv r :1 Len Tyr Pro 5cr Glu Len ?he 130 135 140 **Thr Pro Glu Cys Lys Th e Lys Cln Ser V7al The Gn A P.S n Tyr Tyr Val &see* 145 0155 150 TIe !yr 5cr Ser M et Len T, /r Aro aC--n Gln Gln Ser Gly Ar; Ala Tr P:\OPER\MRO\27674-95. DIV 19/1 /00 68 165 170 173 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 Giu Val 195 200 205 Ala Met Tyr Arg Glu 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 *se

Claims (59)

1. -37- Claims Defining the Invention are as Follows An isolated antibody or fragment thereof that specifically binds to a protein selected from the group consisting of: a mature portion of the protein encoded by the amino acid sequence set forth as SEQ ID NO:2; a protein consisting of a portion of SEQ ID NO:2, wherein said portion comprises at least 30 contiguous amino acid residues of SEQ ID NO:2; and a protein consisting of a portion of SEQ ID NO:2, wherein said portion comprises at least 50 contiguous amino acid residues of SEQ ID NO:2. The antibody or fragment thereof of claim 1 that specifically binds protein

2.

3. The antibody or fragment thereof of claim 1 that specifically binds protein

4. The antibody or fragment thereof of claim 1 that specifically binds protein

5. The antibody or fragment thereof of claim 2 wherein said protein bound by said antibody or fragment thereof is glycosylated.

6. The antibody or fragment thereof of claim 2 which is a human antibody.

7. The antibody or fragment thereof of claim 2 which is a polyclonal antibody.

8. The antibody or fragment thereof of claim 2 which is selected from the group consisting of: -38 a chimeric antibody; a humanized antibody; a single chain antibody; and a Fab fragment.

9. The antibody or fragment thereof of claim 2 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot. The antibody or fragment thereof of claim 2 wherein said antibody or fragment thereof specifically binds to said protein in an ELISA.

11. An isolated cell that produces the antibody or fragment thereof of claim 2.

12. A hybridoma that produces the antibody or fragment thereof of claim 2.

13. A method of detecting fibroblast growth factor 15protein in a biological sample comprising: contacting the biological sample with the antibody or fragment thereof of claim 2; and 15 detecting the fibroblast growth factor 15protein in the biological sample. *oo

14. The method of claim 13 wherein the antibody or fragment thereof is a polyclonal antibody. An isolated antibody or fragment thereof obtained from an animal that has been immunized with a protein selected from the group consisting of: a mature portion of the protein encoded by the amino acid sequence Sset forth as SEQ ID NO:2; -39- a protein comprising the amino acid sequence of at least contiguous amino acid residues of SEQ ID NO:2; and a protein comprising the amino acid sequence of at least contiguous amino acid residues of SEQ ID NO:2; wherein said antibody or fragment thereof specifically binds to said amino acid sequence.

16. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein

17. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein

18. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein

19. The antibody or fragment thereof of claim 15 which is a monoclonal antibody.

20. The antibody or fragment thereof of claim 15 which is selected from the group consisting of: a chimeric antibody; a polyclonal antibody; a humanized antibody; a single chain antibody; and a Fab fragment.

21. An isolated monoclonal antibody or fragment thereof that specifically binds to a protein selected from the group consisting of: a mature portion of the protein encoded by the amino acid sequence set forth as SEQ ID NO:2; a protein consisting of a portion of SEQ ID NO:2, wherein said portion comprises at least 30 contiguous amino acid residues of SEQ ID NO:2; and a protein consisting of a portion of SEQ ID NO:2, wherein said portion comprises at least 50 contiguous amino acid residues of SEQ ID NO:2.

22. The antibody or fragment thereof of claim 21 that specifically binds protein

23. The antibody or fragment thereof of claim 21 that specifically binds protein

24. The antibody or fragment thereof of claim 21 that specifically binds protein

25. The antibody or fragment thereof of claim 22 wherein said protein bound by said antibody or fragment thereof is glycosylated.

26. The antibody or fragment thereof of claim 22 which is a human antibody. 20 27. The antibody or fragment thereof of claim 22 which is selected from the group consisting of: a chimeric antibody; a humanized antibody; -41- a single chain antibody; and a Fab fragment.

28. The antibody or fragment thereof of claim 22 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.

29. The antibody or fragment thereof of claim 22 wherein said antibody or fragment thereof specifically binds to said protein in an ELISA. An isolated cell that produces the antibody or fragment thereof of claim 22.

31. A hybridoma that produces the antibody or fragment thereof of claim 22.

32. A method of detecting fibroblast growth factor 15 protein in a biological sample comprising: contacting the biological sample with the antibody or fragment thereof of claim 22; and detecting the fibroblast growth factor 15 protein in the biological sample.

33. An isolated antibody or fragment thereof that specifically binds to a protein selected from the group consisting of: 0o.o0° a protein consisting of the mature form of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146; 0° a a protein consisting of a portion of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146, wherein said portion comprises at least 30 contiguous amino acid residues of the polypeptide encoded by the cDNA contained in ATCC Deposit 7 Number 97146; and -42- a protein consisting of a portion of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146, wherein said portion comprises at least 50 contiguous amino acid residues of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146.

34. The antibody or fragment thereof of claim 33 that specifically binds protein The antibody or fragment thereof of claim 33 that specifically binds protein

36. The antibody or fragment thereof of claim 33 that specifically binds protein

37. The antibody or fragment thereof of claim 34 wherein said protein bound by said antibody or fragment thereof is glycosylated. I

38. The antibody or fragment thereof of claim 34 which is a human antibody. 15 39. The antibody or fragment thereof of claim 34 which is a polyclonal antibody. The antibody or fragment thereof of claim 34 which is selected from the group consisting of: a chimeric antibody; a humanized antibody; a single chain antibody; and a Fab fragment.

41. The antibody or fragment thereof of claim 34 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot. -43-

42. The antibody or fragment thereof of claim 34 wherein said antibody or fragment thereof specifically binds to said protein in an ELISA.

43. An isolated cell that produces the antibody or fragment thereof of claim 34.

44. A hybridoma that produces the antibody or fragment thereof of claim 34.

45. A method of detecting fibroblast growth factor 15 protein in a biological sample comprising: contacting the biological sample with the antibody or fragment thereof of claim 34; and detecting the fibroblast growth factor 15 protein in the biological sample.

46. The method of claim 45 wherein the antibody or fragment thereof is a polyclonal antibody. 0

47. An isolated antibody or fragment thereof obtained from an animal that has been immunized with a protein selected from the group consisting of: a protein comprising the amino acid sequence of the mature form of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146; a protein comprising the amino acid sequence of at least contiguous amino acid residues of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146; and a protein comprising the amino acid sequence of at least contiguous amino acid residues the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146; -44- wherein said antibody or fragment thereof specifically binds to said amino acid sequence.

48. The antibody or fragment thereof of claim 47 obtained from an animal immunized with protein

49. The antibody or fragment thereof of claim 47 obtained from an animal immunized with protein The antibody or fragment thereof of claim 47 obtained from an animal immunized with protein

51. The antibody or fragment thereof of claim 48 which is a monoclonal antibody.

52. The antibody or fragment thereof of claim 48 which is selected from the group consisting of: a chimeric antibody; a polyclonal antibody; a humanized antibody; a single chain antibody; and e a Fab fragment.

53. An isolated monoclonal antibody or fragment thereof that specifically binds to a protein selected from the group consisting of: a protein consisting of the mature form of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146; a protein consisting of a portion of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146, wherein said portion comprises at least 30 contiguous amino acid residues of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146; and a protein consisting of a portion of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146, wherein said portion comprises at least 50 contiguous amino acid residues of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 97146.

54. The antibody or fragment thereof of claim 53 that specifically binds protein

55. The antibody or fragment thereof of claim 53 that specifically binds protein 15 56. The antibody or fragment thereof of claim 53 that specifically binds protein

57. The antibody or fragment thereof of claim 54 wherein said protein bound by *g said antibody or fragment thereof is glycosylated.

58. The antibody or fragment thereof of claim 54which is a human antibody.

59. The antibody or fragment thereof of claim 54 which is selected from the group consisting of: a chimeric antibody; a humanized antibody; a single chain antibody; and -46- a Fab fragment. The antibody or fragment thereof of claim 54 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.

61. The antibody or fragment thereof of claim 54 wherein said antibody or fragment thereof specifically binds to said protein in an ELISA.

62. An isolated cell that produces the antibody or fragment thereof of claim 54.

63. A hybridoma that produces the antibody or fragment thereof of claim 54.

64. A method of detecting fibroblast growth factor 15 protein in a biological sample comprising: 10 contacting the biological sample with the antibody or fragment thereof of claim 54; and detecting the fibroblast growth factor 15 protein in the biological sample. An isolated antibody or fragment thereof that specifically binds a fibroblast o" 15 growth factor 15 protein purified from a cell culture wherein the cells in said ~cell culture comprise a polynucleotide encoding amino acids 1 to 255 of SEQ ID NO:2 operably associated with a regulatory sequence that controls gene expression.

66. The antibody or fragment thereof of claim 65 which is a monoclonal antibody.

67. The antibody or fragment thereof of claim 65 which is a human antibody. -47-

68. The antibody or fragment thereof of claim 65 which is selected from the group consisting of: a chimeric antibody; a polyclonal antibody; a humanized antibody; a single chain antibody; and a Fab fragment.

69. The antibody or fragment thereof of claim 65 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.

70. The antibody or fragment thereof of claim 65 wherein said antibody or fragment thereof specifically binds to said protein in an ELISA. Dated this NINETEENTH day of JULY 2002. Human Genome Sciences, Inc. Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant