This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well aε 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-ll". The invention also relates to inhibiting the action of such polypeptides.

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

Each member has functions overlapping with others and also has its unique spectrum 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-l 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 cellε.

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

The FGF family presently consiεts 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 memberε, FGF-l and FGF-2, have been characterized under many nameε, but most often aε 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 angiogenesis in vivo and may play important roles in early development (Burgesε, W.H. and Maciag, T. , 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 tranεfer into porcine arterieε. Thiε model defines gene function in the arterial wall in vivo. FGF-1 expresεion induced intimal thickening in porcine arteries 21 days after gene transfer (Nabel, E.G., et al., Nature, 362:844-6 (1993)). It has further been demonstrated that baεic fibroblaεt growth factor may regulate glioma growth and progression independent of its role in tumor angiogenesiε and that baεic fibroblaεt growth factor releaεe or secretion may be required for theεe actionε (Morrison, R.S., 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'ε sarcoma cells in vitro (Huang, Y.Q., et al., J. Clin. Invest., 91:1191-7 (1993)). Also, the cDNA sequence encoding human baεic fibroblaεt growth factor has been cloned downstream of a tranεcription promoter recognized by the bacteriophage T7 RNA polymerase. Basic fibroblaεt growth factorε εo obtained have been εhown to have biological activity indistinguiεhable from human placental fibroblaεt growth factor in mitogenicity, εyntheεis of plasminogen activator and angiogeneεiε aεεays (Squireε, C.H., et al., J. Biol. Chem., 263:16297-302 (1988)).

U.S. Patent No. 5,155,214 diεcloses subεtantially pure mammalian baεic fibroblaεt growth factors and their production. The amino acid sequences of bovine and human basic fibroblast growth factor are discloεed, as well as the DNA sequence encoding the polypeptide of the bovine εpecieε.

Newly discovered FGF-9 haε around 30% sequence εimilarity to other memberε of the FGF family. __,Two cyεteine reεidues and other conεenεuε sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found

to have no typical signal εequence in its N terminuε like those in acidic and basic FGF. However, FGF-9 was found to be secreted from cells after syntheεiε deεpite itε lack of a typical εignal εequence FGF (Miyamoto, M. et al., Mol. and Cell. Biol., 13(7) :4251-4259 (1993). Further, FGF-9 waε found to εtimulate 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, K., et al., J. Biol. Chem., 268:2857-2864 (1993).

Baεic FGF and acidic FGF are potent modulatorε of cell proliferation, cell motility, differentiation, and εurvival 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 aε oncogeneε. , expreεsion of which waε reεtricted to embryogeneεiε and certian typeε of cancerε. FGF-9 waε demonεtrated to be a mitogen againεt glial cellε. Memberε of the FGF family are reported to have oncogenic potency. FGF-9 haε εhown tranεforming potency when tranεformed into BALB/c3T3 cells (Miyamoto, M. , 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 mouεe mammary carcinoma cellε (SC-3) εimulated with teεtoεterone. AIGF iε a diεtinctive FGF-like growth factor, having a putative εignal peptide and εharing 30-40% homology with known memberε of the FGF family. Mammalian cellε tranεformed with AIGF shows a remarkable stimulatory effect on the growth of SC-3 cells in the absence of androgen. Therefore, AIGF mediates androgen-induced growth of SC-3 cells, and perhaps other cells, since it iε 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

reεult 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 polypeptideε aε well aε biologically active and diagnostically or therapeutically useful fragmentε, analogε and derivativeε thereof. The polypeptideε of the preεent invention are of human origin.

In accordance with another aεpect of the preεent invention, there are provided iεolated nucleic acid moleculeε encoding the polypeptideε of the present invention, including mRNAs, DNAε, cDNAε, genomic DNA, aε well aε antiεenεe analogε thereof and biologically active and diagnoεtically or therapeutically uεeful fragmentε thereof.

In accordance with εtill another aεpect of the preεent invention, there are provided proceεεeε for producing εuch polypeptideε by recombinant techniqueε through the uεe of recombinant vectorε, εuch aε cloning and expreεεion plaεmids 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 iε provided a procesε for utilizing such polypeptideε, or polynucleotideε encoding εuch polypeptides, for εcreening for agoniεtε and antagoniεtε thereto and for therapeutic purpoεeε, for example, promoting wound healing for example aε a reεult of burnε and ulcerε, to prevent neuronal damage aεεociated with εtroke and due to neuronal diεorderε and promote neuronal growth, and to prevent εkin aging and hair loεε, to εtimulate angiogeneεiε, mesodermal induction in early embryos and limb regeneration.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptideε.

In accordance with yet another aεpect of the preεent invention, there are provided antagonists against such polypeptides and procesεeε for their use to inhibit the action of εuch polypeptideε, for example, in the treatment of cellular tranεformation, for example, tumorε, to reduce εcarring and treat hyper-vaεcular diεeaεes.

In accordance with another aspect of the present invention, there are provided nucleic acid probes compriεing nucleic acid moleculeε of εufficient length to εpecifically hybridize to a polynucleotide encoding a polypeptide of the preεent invention

In accordance with yet another aεpect of the preεent invention, there are provided diagnostic asεayε for detecting diεeaεeε or εusceptibility to diseases related to mutations in a nucleic acid sequence of the present invention and for detecting over-expresεion of the polypeptideε encoded by εuch εequenceε.

In accordance with another aεpect of the preεent invention, there iε provided a proceεε for utilizing εuch polypeptideε, or polynucleotideε encoding εuch polypeptides, for in vitro purpoεeε related to εcientific reεearch, synthesiε of DNA and manufacture of DNA vectorε.

Theεe and other aεpectε of the preεent invention εhould be apparent to thoεe εkilled in the art from the teachingε herein.

The following drawingε are meant only aε illuεtrationε of εpecific embodimentε of the preεent invention and are not meant aε limitations in any manner.

Figure 1 depicts the cDNA sequence and correεponding deduced amino acid εequence of FGF-11. The amino acid εequence εhown repreεentε the mature form of the protein. The standard one letter abbreviation for amino acids is uεed. Sequencing was performed uεing a 373 Automated DNA εequencer (Applied Biosystems, Inc.) .

Figure 2 illuεtrateε the amino acid εequence homology between FGF-11 and the other FGF family memberε. Conεerved amino acids are readily ascertainable.

In accordance with one aεpect of the preεent invention, there are provided iεolated nucleic acidε moleculeε (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. 97150 on May 12, 1995.

The polynucleotide encoding FGF-11 of this invention waε diεcovered initially in a cDNA library derived from 9 week old early εtage human tiεεue. The FGF-11 polypeptide iε structurally related to all memberε of the fibroblaεt growth factor family and containε an open reading frame encoding a polypeptide of 255 amino acidε. Among the top matcheε are: 1) 42 % identity and 65 % εequence εimilarity to FGF-9 over a εtretch of 127 amino acidε; 2) 37 % identity and 64 % similarity with FGF-7 (keratinocyte growth factor) in a region of 87 amino acidε; 3) 38 % identity and 64 % similarity with FGF-1 (acidic FGF) over a εtretch of 120 amino acidε.

The FGF/HBGF family εignature, GXLX(S,T,A,G)X6 (D,E)CXFXE iε conεerved in the polypeptide of the preεent invention, (X means any amino acid residue; (D,E) meanε either D or E reεidue; X6 meanε any 6 amino acid reεidueε) .

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 double- stranded or single-stranded. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NOS:l) or that of the deposited clone or may be a different coding εequence, aε a

result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1, (SEQ ID NOS:l) or the deposited cDNA.

The polynucleotides which encodes for the mature polypeptide of Figure 1 (SEQ ID NOS:2) or for the mature polypeptides encoded by the deposited cDNA(ε) may include: only the coding εequence for the mature polypeptide; the coding εequence for the mature polypeptide and additional coding εequence εuch aε a leader or εecretory εequence 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 5' and/or 3' of the coding sequence for the mature polypeptide.

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

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 εequence of Figure 1 (SEQ ID NOS:2) or the polypeptideε encoded by the cDNA(ε) of the depoεited clone(ε). The variantε of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the preεent invention includeε polynucleotides encoding the same mature polypeptide aε εhown in Figure 1 (SEQ ID NOS:2) or the εame mature polypeptides encoded by the cDNA(s) of the depoεited clone(ε) aε well aε variantε of εuch polynucleotideε which variantε encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NOS:2) or the polypeptideε encoded by the cDNA(ε) of the depoεited clone(ε). Such nucleotide variantε include

deletion variants, subεtitution variantε and addition or inεertion variantε.

As hereinabove indicated, the polynucleotide may have a coding εequence which is a naturally occurring allelic variant of the coding εequence shown in Figure 1 (SEQ ID NOS:l) 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 εequence which may have a εubstitution, deletion or addition of one or more nucleotides, which does not εubεtantially alter the function of the encoded polypeptideε.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptides may be fuεed in the same reading frame to a polynucleotide sequence which aidε in expreεεion and εecretion of a polypeptide from a host cell, for example, a leader sequence which functionε 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 reεidueε. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the proεequence iε cleaved an active mature protein remainε.

Thuε, for example, the polynucleotideε of the preεent invention may encode for a mature protein, or for a protein having a proεequence or for a protein having both a proεequence and a preεequence (leader εequence) .

The polynucleotideε of the preεent invention may alεo have the coding εequence fuεed in frame to a marker εequence which allowε for purification of the polypeptide of the preεent invention. The marker εequence may be a hexa- hiεtidine 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 εequence may be a hemagglutinin (HA) tag when a mammalian hoεt, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., 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 aε intervening εequenceε (intronε) between individual coding εegmentε (exonε) .

Fragmentε of the full length FGF-11 gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other geneε which have a high εequence εimilarity to the gene or εimilar biological activity. Probeε of thiε type preferably have at least 30 baseε and may contain, for example, 50 or more baεeε. The probe may alεo be uεed to identify a cDNA clone correεponding to a full length tranεcript and a genomic clone or cloneε that contain the complete FGF-11 gene including regulatory and promotor regionε, exonε, and intronε. An example of a εcreen comprises isolating the coding region of the FGF-ll gene by uεing the known DNA εequence to εynthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are uεed to εcreen a library of human cDNA, genomic DNA or mRNA to determine which memberε of the library the probe hybridizes to.

The present invention further relates to polynucleotideε which hybridize to the hereinabove-deεcribed εequenceε if there iε at leaεt 70%, preferably at leaεt 90%, and more preferably at least 95% identity between the εequenceε. The preεent invention particularly relateε to polynucleotideε which hybridize under stringent conditions to the hereinabove-deεcribed polynucleotideε. Aε herein uεed,

the term "stringent conditions" means hybridization will occur only if there is at leaεt 95% and preferably at least 97% identity between the sequenceε. The polynucleotideε which hybridize to the hereinabove deεcribed polynucleotideε in a preferred embodiment encode polypeptideε which either retain εubεtantially the same biological function or activity aε the mature polypeptide encoded by the cDNAε of Figure l (SEQ ID N0:1) or the depoεited cDNA(ε) .

Alternatively, the polynucleotide may have at leaεt 20 bases, preferably at leaεt 30 baεeε, and more preferably at leaεt 50 bases which hybridize to a polynucleotide of the present invention and which haε an identity thereto, aε hereinabove deεcribed, and which may or may not retain activity. For example, εuch polynucleotideε may be employed aε probeε for the polynucleotide of SEQ ID NO:l, for example, for recovery of the polynucleotide or as a diagnoεtic probe or aε a PCR primer.

Thus, the preεent invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at leaεt a 95% identity to a polynucleotide which encodeε the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at leaεt 30 bases and preferably at least 50 bases and to polypeptides encoded by εuch polynucleotideε.

The depoεit(ε) referred to herein will be maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganiεmε for the purposes of Patent Procedure. These depositε are provided merely aε a convenience and are not an admiεεion that a depoεit iε required under 35 U.S.C. § 112. The εequence of the polynucleotideε contained in the depoεited materialε, aε well aε the amino acid εequence of the polypeptideε encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the deεcription of εequenceε herein. A licenεe may be required to make, uεe

or εell the deposited materialε, and no εuch licenεe is hereby granted.

The present invention further relates to an FGF polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NOS:2) or which has the amino acid sequence encoded by the deposited cDNA(s) , aε well aε fragmentε, analogε and derivativeε of εuch polypeptideε.

The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 (SEQ ID NOS:2) or those encoded by the deposited cDNA(s) , meanε polypeptideε which retainε essentially the same biological function or activity aε εuch polypeptideε. 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 εynthetic polypeptideε, preferably recombinant polypeptideε.

The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NOS:2) or that encoded by the depoεited cDNA(ε) may be (i) one in which one or more of the amino acid reεidueε are εubεtituted with a conεerved or non-conεerved amino acid reεidue (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 residueε includeε a substituent group, or (iii) one in which the mature polypeptide iε fuεed with another compound, εuch aε a compound to increaεe the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acidε are fuεed to the mature polypeptide, such as a leader or εecretory εequence or a sequence which iε employed for purification of the mature polypeptide or a proprotein εequence. Such fragmentε, derivativeε and analogε are deemed to be within the εcope of thoεe εkilled in the art from the teachings herein.

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

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

The polypeptideε of the preεent invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) aε well aε polypeptideε which have at least 70% similarity (preferably at leaεt 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at leaεt 90% identity) to the polypeptide of SEQ ID NO:2 and εtill more preferably at leaεt 95% εimilarity (εtill more preferably at leaεt 95% identity) to the polypeptide of SEQ ID NO:2 and alεo include portionε of εuch polypeptideε with εuch portion of the polypeptide generally containing at leaεt 30 amino acidε and more preferably at leaεt 50 amino acidε.

Aε known in the art "εimilarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substituteε of one polypeptide to the εequence of a εecond polypeptide.

Fragmentε or portions of the polypeptides of the present invention may be employed for producing the correεponding full-length polypeptide by peptide εyntheεiε,- therefore, the fragmentε may be employed aε intermediateε for producing the full-length polypeptideε. Fragmentε or portionε of the

polynucleotideε of the present invention may be uεed to εyntheεize full-length polynucleotideε of the preεent invention.

The preεent invention alεo relateε to vectorε 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 tranεformed or tranεfected) with the vectorε of thiε invention which may be, for example, a cloning vector or an expreεεion vector. The vector may be, for example, in the form of a plaεmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformantε or amplifying the FGF geneε. The culture conditionε, εuch aε temperature, pH and the like, are thoεe previouεly uεed with the hoεt cell εelected for expreεεion, and will be apparent to the ordinarily εkilled 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 expresεion vehicles, in particular vectors or plaεmidε for expreεεing a polypeptide. Such vectors include chromosomal, nonchromoεomal and εynthetic DNA εequenceε, e.g., derivativeε of SV40; bacterial plaεmidε; phage DNA; yeaεt plaεmidε; vectors derived from combinationε of plaεmids and phage DNA, viral DNA such aε vaccinia, adenoviruε, fowl pox viruε, and pεeudorabieε. However, any other vector or plaεmid may be uεed 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 procedureε. In general, the DNA

sequence is inεerted into an appropriate reεtriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the εcope of thoεe εkilled in the art.

The DNA εequence in the expreεεion vector iε operatively linked to an appropriate expreεsion control sequence(s) (promoter) to direct mRNA synthesis. Aε repreεentative exampleε of εuch promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P _L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expresεion vector alεo containε a riboεome binding εite for tranεlation initiation and a tranεcription terminator. The vector may alεo include appropriate εequenceε for amplifying expreεεion.

In addition, the expreεεion vectorε preferably contain a gene to provide a phenotypic trait for εelection of tranεformed hoεt cellε εuch aε dihydrofolate reductaεe or neomycin reεiεtance for eukaryotic cell culture, or εuch aε tetracycline or ampicillin reεiεtance in E. coli.

The vector containing the appropriate DNA εequence aε herein above deεcribed, aε well aε an appropriate promoter or control εequence, may be employed to tranεform an appropriate hoεt to permit the host to expresε the protein. Aε repreεentative exampleε of appropriate hoεtε, there may be mentioned: bacterial cellε, εuch aε E. coli. Salmonella typhimurium, Streptomyceε; fungal cellε, εuch aε yeaεt; inεect cellε, εuch as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruεeε,- plant cellε, etc. The εelection of an appropriate hoεt iε deemed to be within the εcope of those skilled in the art from the teachingε herein.

More particularly, the preεent invention alεo includeε recombinant conεtructε compriεing one or more of the εequenceε as broadly deεcribed above. The conεtructε

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 εuitable vectorε and promoterε 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, pεiX174, pBlueεcript SK, pBsKS, pNHδa, pNH16a, pNHlβa, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) . Eukaryotic: pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plaεmid or vector may be uεed aε long aε they are replicable and viable in the hoεt.

Promoter regionε can be εelected from any deεired gene using CAT (chloramphenicol transferaεe) vectorε or other vectorε with εelectable markerε. Two appropriate vectorε are pKK232-8 and pCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda P _R , P _L and trp. Eukaryotic promoterε include CMV immediate early, HSV thymidine kinaεe, early and late SV40, LTRε from retrovirus, and mouse metallothionein-I. 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 conεtruct. The hoεt cell can be a higher eukaryotic cell, εuch aε a mammalian cell, or a lower eukaryotic cell, εuch aε a yeaεt cell, or the hoεt cell can be a prokaryotic cell, εuch aε a bacterial cell. Introduction of the conεtruct into the hoεt cell can be effected by calcium phoεphate tranεfection, DEAE- Dextran mediated tranεfection, or electroporation (Daviε, L.,

Dibner, M. , Battey, I., Basic Methods in Molecular Biology, 1986)) .

The conεtructε in hoεt cellε can be uεed in a conventional manner to produce the gene product encoded by the recombinant εequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide syntheεizerε.

Mature proteinε can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoterε. Cell-free translation syεtems can alεo be employed to produce εuch proteinε uεing RNAε derived from the DNA conεtructε of the preεent invention. Appropriate cloning and expreεεion vectorε for uεe with prokaryotic and eukaryotic hoεtε are deεcribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y. , 1989), the diεcloεure of which iε hereby incorporated by reference.

Tranεcription of a DNA encoding the polypeptideε of the preεent invention by higher eukaryotes is increased by inεerting an enhancer εequence into the vector. Enhancerε are ciε-acting elementε of DNA, uεually about from 10 to 300 bp, that act on a promoter to increaεe itε tranεcription. Exampleε include the SV40 enhancer on the late εide of the replication origin (bp 100 to 270) , a cytomegaloviruε early promoter enhancer, a polyoma enhancer on the late εide of the replication origin, and adenoviruε enhancerε.

Generally, recombinant expreεsion vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resiεtance gene of E. coli and S. cereviεiae TRPl gene, and a promoter derived from a highly-expreεεed gene to direct tranεcription of a downεtream εtructural εequence. Such promoterε can be derived from operonε encoding glycolytic enzymeε εuch aε 3-phoεphoglycerate kinaεe (PGK) , a factor, acid phoεphatase, or heat shock proteinε, among otherε. The

heterologous structural sequence is asεerabled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic εpace or extracellular medium. Optionally, the heterologouε sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristicε, e.g., εtabilization or εimplified purification of expreεεed recombinant product.

Uεeful expression vectors for bacterial uεe are conεtructed by inεerting a εtructural DNA εequence encoding a deεired protein together with εuitable tranεlation, initiation and termination εignals in operable reading phaεe with a functional promoter. The vector will compriεe one or more phenotypic εelectable markerε and an origin of replication to enεure maintenance of the vector and to, if deεirable, provide amplification within the hoεt. Suitable prokaryotic hoεtε for tranεformation include E. coli. Bacillus subtilis. Salmonella typhimurium and variouε εpecieε within the genera Pεeudomonaε, Streptomyceε, and Staphylococcuε, although otherε may alεo be employed aε a matter of choice.

Aε a repreεentative but nonlimiting example, uεeful expression vectors for bacterial uεe can compriεe a εelectable marker and bacterial origin of replication derived from commercially available plaεmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppεala, Sweden) and GEM1 (Promega Biotec, Madiεon, WI, USA) . Theεe pBR322 "backbone" εectionε are combined with an appropriate promoter and the εtructural sequence to be expresεed.

Following tranεformation of a suitable host strain and growth of the hoεt εtrain to an appropriate cell denεity, the εelected promoter iε derepreεεed by appropriate means (e.g.,

temperature shift or chemical induction) and cellε are cultured for an additional period.

Cellε are typically harveεted by centrifugation, diεrupted by phyεical or chemical meanε, and the resulting crude extract retained for further purification.

Microbial cells employed in expresεion of proteinε can be diεrupted by any convenient method, including freeze-thaw cycling, εonication, mechanical diεruption, or use of cell lysing agents.

Various mammalian cell culture systemε can also be employed to expresε recombinant protein. Exampleε of mammalian expreεεion εyεtemε include the COS-7 lineε of monkey kidney fibroblaεtε, deεcribed by Gluzman, Cell, 23:175 (1981) , and other cell lineε capable of expreεεing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε. Mammalian expreεεion vectorε will compriεe an origin of replication, a εuitable promoter and enhancer, and alεo any necessary ribosome binding εiteε, polyadenylation εite, εplice donor and acceptor εiteε, tranεcriptional termination εequenceε, and 5' flanking nontranεcribed εequenceε. DNA εequenceε derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, εplice, and polyadenylation εites may be used to provide the required nontranεcribed genetic elementε.

The polypeptide of the preεent invention may be recovered and purified from recombinant cell cultureε by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding εtepε can be uεed, aε neceεεary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification εtepε.

The polypeptide of the preεent invention may be a naturally purified product, or a product of chemical εynthetic procedureε, or produced by recombinant techniqueε from a prokaryotic or eukaryotic hoεt (for example, by bacterial, yeast, higher plant, inεect and mammalian cellε in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycoεylated with mammalian or other eukaryotic carbohydrateε or may be non-glycoεylated. Polypeptideε of the invention may alεo include an initial methionine amino acid residue.

The polypeptide of the present invention, as a result of the ability to stimulate vaεcular endothelial cell growth, may be employed in treatment for εtimulating re- vaεcularization of iεchemic tiεεueε due to variouε diεeaεe conditionε εuch as thromboεiε, arteriosclerosiε, and other cardiovascular conditions. Theεe polypeptide may also be employed to stimulate angiogenesiε and limb regeneration.

The polypeptide may alεo be employed for treating woundε due to injuries, burns, poεt-operative tissue repair, and ulcers εince they are mitogenic to various cells of different originε, εuch aε fibroblaεt cellε and εkeletal muεcle cellε, and therefore, facilitate the repair or replacement of damaged or diseased tiεεue.

The polypeptide of the preεent invention may alεo be employed εtimulate neuronal growth and to treat and prevent neuronal damage aεεociated with εtroke and which occurε in certain neuronal diεorderε or neuro-degenerative conditions such as Alzheimer's diseaεe, Parkinεon'ε diεease, and AIDS- related complex. FGF-11 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.

The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

The FGF-11 polypeptide may also be employed for preventing hair losε, εince FGF family memberε activate hair- forming cells and promoteε melanocyte growth. Along the εame lineε, the polypeptideε of the present invention may be employed to εtimulate growth and differentiation of hematopoietic cellε and bone marrow cellε when uεed in combination with other cytokineε.

The FGF-11 polypeptide may also be employed to maintain organs before transplantation or for εupporting cell culture of primary tiεεueε.

The polypeptide of the preεent invention may alεo be employed for inducing tissue of mesodermal origin to differentiate in early embryoε.

In accordance with yet a further aεpect of the preεent invention, there iε provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptideε, for in vitro purposes related to scientific research, synthesis of DNA, manufacture of DNA vectors and for the purpoεe of providing diagnoεticε and therapeuticε for the treatment of human diεeaεe.

Thiε invention provideε a method for identification of the receptorε for the polypeptideε of the preεent invention. The geneε 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. , 1(2), Chapter 5, (1991)). Preferably, expresεion cloning iε employed wherein polyadenylated RNA iε prepared from a cell reεponεive to the polypeptides, for example, NIH3T3 cellε which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from thiε RNA iε divided into poolε and uεed to transfect COS cells or other cellε that are not

responεive to the polypeptideε. Tranεfected cellε which are grown on glaεε εlideε are expoεed to the the polypeptide of the preεent invention, after they have been labelled. The polypeptideε can be labeled by a variety of meanε including iodination or incluεion of a recognition εite for a site- specific protein kinase.

Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and εub-poolε are prepared and re-tranεfected uεing an iterative εub-pooling and re-εcreening proceεs, eventually yielding a single cloneε that encodeε the putative receptor.

Aε an alternative approach for receptor identification, the labeled polypeptideε can be photoaffinity linked with cell membrane or extract preparationε that express the receptor molecule. Crosε-linked material iε reεolved by PAGE analyεiε and expoεed to X-ray film. The labeled complex containing the receptorε of the polypeptideε can be exciεed, reεolved into peptide fragmentε, and εubjected to protein microεequencing. The amino acid sequence obtained from microsequencing would be used to deεign a εet of degenerate oligonucleotide probeε to εcreen a cDNA library to identify the geneε encoding the putative receptorε.

Thiε invention provideε a method of εcreening compoundε to identify thoεe which modulate the action of the polypeptide of the preεent invention. An example of such an asεay compriεeε combining a mammalian fibroblaεt cell, a the polypeptide of the present invention, the compound to be screened and ³ [H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control aεεay may be performed in the abεence of the compound to be εcreened and compared to the amount of fibroblaεt proliferation in the preεence of the compound to determine if the compound εtimulateε proliferation by determining the uptake of [H] thymidine in each case. The amount of

fibroblast cell proliferation is measured by liquid scintillation chromatography whichmeasures 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 or block this interaction could then be measured. Alternatively, the reεponεe of a known second mesεenger εyεtem following interaction of a compound to be εcreened and the FGF-11 receptor iε meaεured and the ability of the compound to bind to the receptor and elicit a εecond meεεenger reεponεe iε measured to determine if the compound is a potential agoniεt or antagonist. Such second meεεenger εyεtemε include but are not limited to, cAMP guanylate cyclaεe, tyroεine phoεphorylation, ion channelε or phoεphoinoεitide hydrolysis.

Examples of antagonist compounds include antibodies, or in some cases, oligonucleotides, which bind to the receptor for the polypeptide of the preεent invention but elicit no εecond meεεenger reεponεe or bind to the FGF-ll polypeptide itεelf. Alternatively, a potential antagoniεt may be a mutant form of the polypeptide which bindε to the receptorε, however, no εecond messenger responεe iε elicited and, therefore, the action of the polypeptide is effectively blocked.

Another antagonist compound to the FGF-11 gene and gene product is an antisenεe conεtruct prepared uεing antiεenεe technology. Antiεenεe technology can be uεed to control gene expreεεion 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 5' coding portion of the polynucleotide sequence, which encodeε for the mature polypeptides of the present invention, is used to

design an antisense RNA oligonucleotide of from about 10 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 -εee Lee et al., Nucl. Acidε Reε., 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 blockε tranεlation of the mRNA molecule into the polypeptide (Antiεenεe - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotideε aε Antiεenεe Inhibitorε of Gene Expreεεion, CRC Preεε, Boca Raton, FL (1988)). The oligonucleotideε deεcribed above can alεo be delivered to cellε εuch that the antiεenεe RNA or DNA may be expreεεed in vivo to inhibit production of the polypeptide.

Potential antagoniεt compoundε alεo include εmall moleculeε which bind to and occupy the binding εite of the receptorε thereby making the receptor inacceεεible to itε polypeptide εuch that normal biological activity iε prevented. Examples of small molecules include, but are not limited to, small peptides or peptide-like moleculeε.

Antagonist compounds may be employed to inhibit the cell growth and proliferation effectε of the polypeptideε of the present invention on neoplastic cellε and tiεεueε, i.e. εtimulation of angiogeneεis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

The antagoniεtε may alεo be employed to prevent hyper- vaεcular diεeaεeε, and prevent the proliferation of epithelial lenε cellε after extracapεular cataract εurgery. Prevention of the mitogenic activity of the polypeptideε of the preεent invention may alεo be deεirouε in caεeε εuch aε restenosiε after balloon angioplaεty.

The antagonistε may alεo be employed to prevent the growth of εcar tissue during wound healing.

The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., aε hereinafter deεcribed.

The polypeptideε, agonists and antagonistε of the preεent invention may be employed in combination with a suitable pharmaceutical carrier to compriεe a pharmaceutical compoεition for parenteral adminiεtration. Such compoεitionε compriεe a therapeutically effective amount of the polypeptide, agoniεt or antagonist and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to εaline, buffered εaline, dextroεe, water, glycerol, ethanol, and combinationε thereof. The formulation εhould εuit the mode of administration.

The invention also provides a pharmaceutical pack or kit compriεing one or more containerε filled with one or more of the ingredientε of the pharmaceutical compoεitionε of the invention. Aεεociated with such container(s) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, uεe or εale of pharmaceuticalε or biological productε, which notice reflectε approval by the agency of manufacture, uεe or εale for human adminiεtration. In addition, the polypeptides, agonistε and antagoniεtε of the preεent invention may be employed in conjunction with other therapeutic compoundε.

The pharmaceutical compoεitionε may be adminiεtered in a convenient manner such as by the oral, topical, intravenouε, intraperitoneal, intramuεcular, εubcutaneouε, intranaεal or intradermal routeε. The pharmaceutical compoεitionε are adminiεtered in an amount which iε effective for treating and/or prophylaxiε of the εpecific indication. In general, they are adminiεtered in an amount of at leaεt about 10 μg/kg body weight and in moεt caεes they will be administered in an amount not in exceεε of about 8 mg/Kg body

weight per day. In most cases, the dosage iε from about 10 μg/kg to about l mg/kg body weight daily, taking into account the routeε of adminiεtration, εymptoms, etc. In the specific case of topical administration, dosageε are preferably adminiεtered from about 0.1 μg to 9 mg per cm ² .

The polypeptide of the invention and agoniεt and antagoniεt compoundε which are polypeptideε, may alεo be employed in accordance with the preεent invention by expreεεion of εuch polypeptide in vivo, which iε 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, cellε may be engineered by procedureε known in the art by uεe of a retroviral particle containing RNA encoding for the polypeptide of the preεent invention.

Similarly, cells may be engineered in vivo for expresεion 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 expresεion of the polypeptide in vivo. Theεe and other methodε for adminiεtering a polypeptide of the preεent invention by εuch methodε εhould be apparent to thoεe εkilled 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 εuitable 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

viruε, retroviruses such as Rouε Sarcoma Viruε, Harvey Sarcoma Viruε, avian leukoεiε viruε, gibbon ape leukemia viruε, human immunodeficiency viruε, adenoviruε, Myeloproliferative Sarcoma Viruε, and mammary tumor viruε. In one embodiment, the retroviral plaεmid vector iε derived from Moloney Murine Leukemia Viruε.

The vector includeε one or more promoterε. Suitable promoterε 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., Biotechniσues. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and 0-actin promoters) . Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoterε. 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 iε under the control of a suitable promoter. Suitable promoterε which may be employed include, but are not limited to, adenoviral promoterε, such as the adenoviral major late promoter,- or hetorologous promoters, εuch aε the cytomegaloviruε (CMV) promoter; the reεpiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter,- heat εhock promoterε; the albumin promoter; the ApoAI promoter,- human globin promoterε; viral thymidine kinaεe promoterε, such aε the Herpeε Simplex thymidine kinaεe promoter; retroviral LTRε (including the modified retroviral LTRε hereinabove deεcribed) ; the jβ-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controlε the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cellε which may be tranεfected include, but are not limited to, the PE501, PA317, ψ-2 , ψ-2Wl, PA12, T19-14X, VT-19-17-H2, ^CRE, ^CRIP, GP+E-86, GP+envAml2, and DAN cell lineε aε deεcribed in Miller, Human Gene Therapy. Vol. 1, pgε. 5-14 (1990), which iε incorporated herein by reference in itε entirety. The vector may transduce the packaging cells through any meanε known in the art. Such means include, but are not limited to, electroporation, the use of lipoεomes, and CaP0 ₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a lipoεome, or coupled to a lipid, and then adminiεtered to a hoεt.

The producer cell line generateε infectiouε retroviral vector particleε which include the nucleic acid εequence(ε) encoding the polypeptideε. Such retroviral vector particleε then may be employed, to tranεduce eukaryotic cellε, either in vi tro or in vivo. The tranεduced eukaryotic cellε will expreεs the nucleic acid sequence(ε) encoding the polypeptide. Eukaryotic cellε which may be tranεduced include, but are not limited to, embryonic εtem cellε, embryonic carcinoma cells, as well as hematopoietic εtem cells, hepatocytes, fibroblastε, myoblaεts, keratinocytes, endothelial cells, and bronchial epithelial cells.

Thiε invention iε alεo related to the uεe of the geneε of the preεent invention as part of a diagnostic asεay for detecting diseaseε or εuεceptibility to diεeaεeε related to the preεence of mutationε in the nucleic acid εequenceε 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 diagnoεiε may be obtained from a patient'ε cellε, εuch aε from blood, urine, εaliva, tiεεue biopεy and autopsy material. The genomic DNA may be used

directly for detection or may be amplified enzymatically by uεing PCR (Saiki et al . , Nature, 324:163-166 (1986)) prior to analyεiε. RNA or cDNA may also be used for the εame 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 mutations. For example, deletions and inεertionε can be detected by a change in εize of the amplified product in compariεon to the normal genotype. Point mutationε can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antiεense DNA sequences. Perfectly matched sequences can be distinguiεhed from miεmatched duplexeε by RNaεe A digeεtion or by differenceε in melting temperatureε.

Genetic teεting baεed on DNA εequence differenceε may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agentε. Small εequence deletionε and inεertionε can be visualized by high resolution gel electrophoreεiε. DNA fragmentε of different εequenceε may be diεtinguiεhed on denaturing formamide gradient gelε in which the mobilitieε of different DNA fragmentε are retarded in the gel at different poεitionε according to their εpecific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985)).

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

Thuε, the detection of a εpecific DNA εequence may be achieved by methodε such as hybridization, RNaεe protection, chemical cleavage, direct DNA εequencing or the uεe of reεtriction enzymeε, (e.g., Reεtriction Fragment Length Polymorphiεmε (RFLP) ) and Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoreεiε and DNA sequencing, mutationε can alεo be detected by in si tu analysis.

The present invention also relates to a diagnostic asεay for detecting altered levelε of FGF-11 proteinε in variouε tiεεueε εince an over-expreεεion of the proteinε compared to normal control tiεεue samples may detect the presence of abnormal cellular proliferation, for example, a tumor. Asεayε uεed to detect levelε of protein in a εample derived from a hoεt are well-known to thoεe of εkill in the art and include radioimmunoaεsays, competitive-binding assayε, Weεtern Blot analyεiε, ELISA aεεayε and "εandwich" aεεay. An ELISA aεεay (Coligan, et al., Current Protocolε in Immunology, 1(2), Chapter 6, (1991)) initially compriεeε preparing an antibody εpecific to an antigen to the polypeptideε of the preεent invention, preferably a monoclonal antibody. In addition a reporter antibody iε prepared againεt the monoclonal antibody. To the reporter antibody iε attached a detectable reagent εuch aε radioactivity, fluorescence or, in this example, a horseradiεh peroxidaεe enzyme. A εample iε removed from a hoεt and incubated on a εolid εupport, e.g. a polyεtyrene diεh, that bindε the proteinε in the εample. 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 antibody is incubated in the diεh during which time the monoclonal antibodieε attach to any polypeptideε of the preεent invention attached to the polyεtyrene diεh. All unbound monoclonal antibody iε waεhed out with buffer. The reporter antibody linked to horεeradiεh peroxidaεe is now placed in the dish reεulting in binding of the reporter antibody to any monoclonal antibody bound to the protein of intereεt.

Unattached reporter antibody iε then waεhed out. Peroxidaεe εubεtrateε are then added to the diεh and the

amount of color developed in a given time period is a measurement of the amount of a polypeptide of the present invention present in a given volume of patient sample when compared against a standard curve.

A competition asεay may be employed wherein antibodieε εpecific to a polypeptide of the preεent invention are attached to a εolid εupport and labeled FGF-11 and a εample derived from the hoεt are paεεed over the εolid εupport and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of a polypeptide of the preεent invention in the εample.

A "εandwich" assay is similar to an ELISA asεay. In a "sandwich" asεay a polypeptide of the preεent invention iε paεεed over a εolid 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 iε labeled and εpecific to the εecond antibody iε then paεεed over the εolid εupport and binds to the second antibody and an amount can then be quantified.

The sequenceε of the present invention are also valuable for chromosome identification. The εequence iε εpecifically targeted to and can hybridize with a particular location on an individual human chromoεome. Moreover, there iε a current need for identifying particular εiteε on the chromosome. Few chromosome marking reagentε baεed on actual εequence data (repeat polymorphiεm'ε) are preεently available for marking chromoεomal location. The mapping of DNAε to chromoεomes according to the present invention is an important firεt εtep in correlating thoεe εequenceε with geneε asεociated with disease.

Briefly, sequenceε can be mapped to chromoεomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analyεiε of the 3' untranεlated region is used to rapidly εelect primerε that do not εpan more than one exon in the genomic DNA, thuε complicating the amplification proceεε.

These primers are then used for PCR screening of somatic cell hybrids containing individual human chromoso eε. 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 chromoεome. Uεing the preεent invention with the εame oligonucleotide primers, sublocalization can be achieved with panelε of fragmentε from εpecific chromoεomeε or poolε of large genomic cloneε in an analogouε manner. Other mapping εtrategieε that can εimilarly be uεed to map to itε chromosome include in situ hybridization, prescreening with labeled flow-εorted chromoεomeε and preεelection by hybridization to conεtruct chromoεome εpecific-cDNA librarieε.

Fluoreεcence in situ hybridization (FISH) of a cDNA clone to a metaphaεe chromoεomal εpread can be uεed to provide a preciεe chromoεomal location in one εtep. Thiε technique can be uεed with cDNA aε εhort aε 50 or 60 baεeε. For a review of thiε technique, εee Verma et al. , Human Chromoεomeε: a Manual of Baεic Techniqueε, Pergamon Preεε, New York (1988) .

Once a εequence haε been mapped to a preciεe chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKuεick, Mendelian Inheritance in Man (available on line through Johnε Hopkinε Univerεity Welch Medical Library) . The relationεhip between genes and diseaεeε that have been mapped to the εame chromosomal region are then identified through linkage analysiε (coinheritance of physically adjacent genes) .

Next, it iε neceεεary to determine the differenceε in the cDNA or genomic εequence between affected and unaffected individualε. If a mutation iε obεerved in εome or all of the affected individualε but not in any normal individualε, then

the mutation iε likely to be the cauεative agent of the diεeaεe.

With current reεolution of phyεical mapping and genetic mapping techniqueε, a cDNA precisely localized to a chromosomal region asεociated with the diεeaεe could be one of between 50 and 500 potential causative genes. (This assumeε 1 megabaεe mapping resolution and one gene per 20 kb) .

The polypeptides, their fragments or other derivatives, or analogs thereof, or cellε expressing them can be used as an immunogen to produce antibodies thereto. Theεe antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, εingle chain, and humanized antibodieε, aε well aε Fab fragmentε, or the product of an Fab expreεsion library. Various procedureε known in the art may be uεed for the production of such antibodieε and fragmentε.

Antibodieε generated against the polypeptides corresponding to a sequence of the preεent invention can be obtained by direct injection of the polypeptideε into an animal or by adminiεtering the polypeptideε to an animal, preferably a nonhuman. The antibody εo obtained will then bind the polypeptideε itεelf. In thiε manner, even a εequence encoding only a fragment of the polypeptideε can be uεed to generate antibodieε binding the whole native polypeptideε. Such antibodieε can then be uεed to iεolate the polypeptide from tiεεue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provideε antibodieε produced by continuouε cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milεtein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodieε

(Cole, et al., 1985, in Monoclonal Antibodieε and Cancer Therapy, Alan R. Liεs, Inc., pp. 77-96).

Techniques described for the production of εingle chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodieε to immunogenic polypeptide productε of thiε invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

The preεent 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 amountε, unleεε otherwiεe specified, are by weight.

In order to facilitate understanding of the following examples, certain frequently occurring methodε and/or termε will be deεcribed.

"Plaεmidε" are deεignated by a lower caεe p preceded and/or followed by capital letterε and/or numberε. The εtarting plaεmids herein are either commercially available, publicly available on an unrestricted basiε, or can be constructed from available plasmidε in accord with publiεhed procedureε. In addition, equivalent plaεmidε to thoεe deεcribed are known in the art and will be apparent to the ordinarily εkilled artiεan.

"Digeεtion" of DNA referε to catalytic cleavage of the DNA with a reεtriction enzyme that actε only at certain εequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactorε and other requirementε were uεed aε would be known to the ordinarily skilled artisan. For analytical purposeε, typically 1 μg of plasmid or DNA fragment is uεed with about 2 unitε of enzyme in about 20 μl of buffer εolution. For the purpoεe of iεolating DNA fragmentε for plaεmid conεtruction, typically 5 to 50 μg of DNA are digeεted with 20 to 250 unitε of enzyme in a larger

volume. Appropriate buffers and subεtrate amountε for particular reεtriction enzymeε are εpecified by the manufacturer. Incubation timeε of about 1 hour at 37°C are ordinarily uεed, but may vary in accordance with the supplier's instructionε. After digeεtion the reaction iε electrophoreεed directly on a polyacrylamide gel to iεolate the deεired fragment.

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

"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide εtrandε which may be chemically εyntheεized. Such εynthetic oligonucleotideε have no 5' phoεphate and thuε will not ligate to another oligonucleotide without adding a phoεphate with an ATP in the preεence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that haε not been dephoεphorylated.

"Ligation" referε to the proceεε of forming phosphodiester bonds between two double εtranded nucleic acid fragments (Maniatis, T. , et al., Id., p. 146). Unleεε otherwiεe provided, ligation may be accompliεhed uεing known bufferε and conditionε with 10 unitε of T4 DNA ligaεe ("ligaεe") per 0.5 μg of approximately equimolar amountε of the DNA fragmentε to be ligated.

Unless otherwise εtated, tranεformation waε performed aε deεcribed by the method of Graham, F. and Van der Eb, A. , Virology, 52:456-457 (1973).

Example 1 Bacterial Expression and Purification of FGF-11 proteinε

The DNA εequence encoding FGF-11, ATCC # 97150, is initially amplified using PCR oligonucleotide primers corresponding to the 5' εequenceε of the proceεεed protein (minuε the εignal peptide εequence) and the vector εequenceε

3' to the gene. Additional nucleotideε correεponding to the gene are added to the 5' and 3' sequences respectively. The 5 ' o l i g o n u c l e o t i d e p r i m e r 5 ' CGCGGATCCΑTCΑTGAGTGGAAAGGTGACCAAG 3' (SEQ ID NO:3) contains a BamHI reεtriction enzyme site. The 3' sequence 5' CGCGGATCCCGTTGATTCATTGTGGCTCAT 3' (SEQ ID NO:4) contains complementary sequenceε to a BamHI site and is followed by 21 nucleotides of FGF-11 coding sequence.

The restriction enzyme sites correspond to the restriction enzyme siteε on the bacterial expreεεion vector pQE-60 (Qiagen, Inc. Chatεworth, CA 91311) . pQE-60 encodeε antibiotic reεiεtance (Amp ^r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a riboεome binding εite (RBS) , a 6-Hiε tag and restriction enzyme εiteε. pQE-60 waε then digeεted with Ncol and BamHI. The amplified sequences are ligated into pQE-60 and are inserted in frame with the sequence encoding for the histidine tag and the riboεome binding εite (RBS) . The ligation mixture iε then uεed to tranεform E. coli εtrain M15/rep 4 (Qiagen, Inc.) by the procedure deεcribed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989) . M15/rep4 containε multiple copieε of the plaεmid pREP4, which expreεεeε the lad repreεεor and alεo conferε kanamycin reεiεtance (Kan ^r ) . Tranεformantε are identified by their ability to grow on LB plateε and ampicillin/kanamycin reεiεtant colonieε were εelected. Plaεmid DNA iε iεolated and confirmed by reεtriction analyεiε. Cloneε containing the deεired conεtructε are grown overnight (0/N) in liquid culturein LB media εupplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/N culture iε 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. ^βOD ) of between 0.4 and 0.6. IPTG ("isopropyl-B-D-thiogalacto pyranoεide") iε then added to a final concentration of 1 mM. IPTG induceε by inactivating

the lad repreεεor, clearing the P/O leading to increaεed gene expreεεion. Cellε are grown an extra 3 to 4 hourε. Cellε are then harveεted by centrifugation. The cell pellet iε εolubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized FGF-11 iε purified from this solution by chromatography on a Nickel-NAT resin under conditions that allow for tight binding by proteinε containing the 5-Hiε tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)) . The proteinε are eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpoεe of renaturation adjuεted to 3 molar guanidine HCl, lOOmM εodium phoεphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in thiε solution for 12 hours the proteins are dialyzed to 10 mmolar sodium phosphate.

Example 2 Expreεεion of FGF-11 by in vi tro tranεcription and tranεlation.

The FGF-11 cDNA, ATCC # 97150, waε transcribed and translated in vitro to determine the size of the translatable polypeptide encoded by the full length FGF-11 cDNA. The full length cDNA inεertε of FGF-11 in the pBlueεcript SK vector.

The in vi tro tranεcription/tranεlation reaction waε performed in a 25 ul volume, uεing the T _N T™ Coupled Reticulocyte Lyεate Systerns (Promega, CAT# L4950) . Specifically, the reaction contains 12.5 ul of T _N T rabbit reticulocyte lysate, 2 μl of T _N T reaction buffer, 1 μl of T3 polymeraεe, 1 μl of 1 mM amino acid mixture (minuε methionine) , 4 μl of ³⁵ S-methionine (>1000 Ci/mmol, 10 mCi/ml) , 1 μl of 40 U/μl; RNaεin ribonucleaεe inhibitor, 0.5 or 1 μg of pBlueεcript FGF-11 plaεmid. Nucleaεe-free H ₂ 0 waε added to bring the volume to 25 ul. The reaction waε incubated at 30°C for 2 hourε. Five microliterε of the reaction product waε analyzed on a 4-20% gradient SDS-PAGE

gel. After fixing in 25% isopropanol and 10% acetic acid, the gel was dried and exposed to an X-ray film overnight at 70°C.

Example 3 Cloning and expreεεion of FGF-11 uεing the baculoviruε expreεεion εyεtem

The DNA εequence encoding the full length FGF-11 protein, ATCC # 97150, iε amplified uεing PCR oligonucleotide primerε correεponding to the 5' and 3' εequenceε of the gene:

The FGF-115' primer haε the εequence 5' CGCGGATCCATCATG AGTGGAAAGGTGACCAAG 3' (SEQ ID NO:5) and containε a BamHI reεtriction enzyme εite (in bold) εuch that cloning at thiε site will put the baculovirus signal sequence in frame with 21 nucleotides of the FGF-ll gene downstream of the putative FGF-11 signal peptide cleavage site.

The 3' primer has the sequence 5' CGCGGTACCCTACGTTGA TTCATTGTGGCT 3' (SEQ ID NO:6) and contains the cleavage εite for the reεtriction endonucleaεe Aεp718 and 21 nucleotideε complementary to the 3' non-tranεlated εequence of the gene.

The amplified εequenceε are iεolated from a 1% agaroεe gel uεing a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.) . The fragment is then digested with the respective endonucleaseε and purified again on a 1% agaroεe gel. Thiε fragment iε deεignated F2.

The vector pA2 (modification of pVL94l vector, diεcussed below) is used for the expresεion of the proteinε uεing the baculoviruε expreεεion εyεtem (for review εee: Summerε, M.D. and Smith, G.E. 1987, A manual of methodε for baculoviruε vectorε and inεect cell culture procedureε, Texas Agricultural Experimental Station Bulletin No. 1555) . This expresεion vector containε the εtrong polyhedrin promoter of the Autographa califomica nuclear polyhedroεiε viruε (AcMNPV) followed by the recognition εiteε for the reεtriction endonucleaεeε BamHI and Aεp7l8. The

polyadenylation site of the εimian viruε (SV)40 iε uεed for efficient polyadenylation. For an eaεy εelection of recombinant virus the beta-galactosidaεe gene from E.coli iε inεerted in the εame orientation aε the polyhedrin promoter followed by the polyadenylation εignal of the polyhedrin gene. The polyhedrin sequences are flanked at both sideε by viral εequenceε for the cell-mediated homologouε recombination of co-tranεfected wild-type viral DNA. Many other baculoviruε vectorε could be uεed in place of pA2 εuch aε pRGl, pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summerε, M.D., Virology, 170:31-39).

The plaεmid iε digested with the restriction enzymes and dephosphorylated using calf inteεtinal phoεphataεe by procedureε known in the art. The DNA iε then isolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA iε deεignated V2.

Fragment F2 and the dephoεphorylated plaεmid V2 are ligated with T4 DNA ligase. E.coli DH5α cells are then transformed and bacteria identified that contained the plasmid (pBacFGF-11) using the respective reεtriction enzymeε. The sequence of the cloned fragment are confirmed by DNA sequencing.

5 μg of the plasmid pBacFGF-li are co-transfected with 1.0 μg of a commercially available linearized baculoviruε ("BaculoGold™ baculoviruε DNA", Pharmingen, San Diego, CA.) uεing the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)). lμg of BaculoGold™ viruε DNA and 5 μg of the plaεmidε, in each caεe, are mixed in a εterile well of microtiter plateε containing 50 μl of εerum free Grace'ε medium (Life Technologies Inc., Gaithersburg, MD) . Afterwardε 10 μl Lipofectin pluε 90 μl Grace'ε medium are added, mixed and incubated for 15 minuteε at room temperature. Then the tranεfection mixture iε added drop-wiεe to the Sf9 inεect

cellε (ATCC CRL 1711) seeded in 35 mm tiεεue culture plateε 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°C. After 5 hourε the tranεfection solution is removed from the plate and 1 ml of Grace's insect medium εupplemented with 10% fetal calf εerum iε added. The plateε are put back into an incubator and cultivation continued at 27°C for four dayε.

After four dayε the εupernatant iε collected and plaque aεεayε performed similar as described by Summers and Smith (εupra) . Aε a modification an agarose gel with "Blue Gal" (Life Technologies Inc. , Gaithersburg) is used which allows an eaεy iεolation of blue εtained plaqueε. (A detailed deεcription of a "plaque aεεay" can alεo be found in the uεer'ε guide for inεect cell culture and baculovirology diεtributed by Life Technologieε Inc., Gaitherεburg, page 9- 10) .

Four dayε after the serial dilution the virus iε added to the cellε and blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruseε iε then reεuεpended in an Eppendorf tube containing 200 μl of Grace'ε medium. The agar iε removed by a brief centrifugation and the εupernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm disheε. Four dayε later the εupernatantε of theεe culture dishes are harvested and then stored at 4°C.

Sf9 cells are grown in Grace'ε medium εupplemented with 10% heat-inactivated FBS. The cellε are infected with the recombinant baculoviruε V-FGF-ll at a multiplicity of infection (MOD of 2. Six hourε later the medium iε removed and replaced with SF900 II medium minuε methionine and cyεteine (Life Technologieε Inc. , Gaitherεburg) . 42 hourε later 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cyεteine (Amerεham) are added. The cellε are further incubated for 16 hourε

before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Example 4 Expresεion of Recombinant FGF-11 in COS cellε

The expreεεion of plaεmidε, FGF-ll-HA derived from a vector pcDNA3/Ainp (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 εite. DNA fragmentε encoding the entire FGF-11 precursor and an HA tag 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 correspondε to an epitope derived from the influenza hemagglutinin protein aε previouεly described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infuεion of HA tag to the target protein allowε eaεy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy iε deεcribed aε followε:

The DNA εequence encoding FGF-11, ATCC # 97150, iε conεtructed by PCR uεing two primerε: the 5' primer 5' CGCGGATCCATCATGAGTGGAAAGGTGACCAAG 3' (SEQ ID NO:7) containε a BamHI εite followed by 21 nucleotideε of coding εequence εtarting from the initiation codon; the 3' sequence 5' CTCGAG CGTTGATTCATTGTGGCTCAT 3' (SEQ ID NO:8) contains complementary εequenceε to an Xbal εite and the last 21 nucleotideε of the FGF-11 coding εequence (not including the εtop codon) . Therefore, the PCR product containε a Xhol εite, coding εequence followed by HA tag fuεed in frame, a tranεlation termination εtop codon next to the HA tag, and an Xhol εite.

The PCR amplified DNA fragmentε and the vector, pcDNA3/Amp, are digeεted with the reεpective 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 iε iεolated from tranεformants and examined by restriction analysis for the presence of the correct fragment. For expresεion of the recombinant FGF-ll COS cellε are tranεfected with the expreεεion vector by DEAE- DEXTRAN method (J. Sambrook, E. Fritεch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expresεion of the FGF-ll-HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Presε, (1988)). Cells are labelled for 8 hours with ³⁵ S-cysteine two days post transfection. Culture media is then collected and cells are lyεed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5) (Wilεon, I. et al., Id. 37:767 (1984)). Both cell lyεate and culture media are precipitated with an HA εpecific monoclonal antibody. Proteinε precipitated are analyzed on 15% SDS-PAGE gelε.

Example 5 Expreεεion via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resulting tisεue iε placed in tiεεue-culture medium and εeparated into small pieceε. Small chunks of the tiεεue are placed on a wet εurface of a tiεεue culture flaεk, approximately ten pieceε are placed in each flaεk. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added.

This is then incubated at 37°C for approximately one week. At this time, fresh media iε added and εubεequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblastε emerge. The monolayer iε trypεinized and scaled into larger flaskε. pMV-7 (Kirεchmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeatε of the Moloney murine sarcoma virus, is digeεted with EcoRI and Hindlll and subεequently treated with calf inteεtinal phosphatase. The linear vector iε fractionated on agarose gel and purified, uεing glaεε beads.

The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer containing an EcoRI site and the 3' primer further includeε a Hindlll εite. Equal quantitieε of the Moloney murine εarcoma viruε linear backbone and the amplified EcoRI and Hindlll fragment are added together, in the preεence of T4 DNA ligaεe. The reεulting mixture iε maintained under conditionε appropriate for ligation of the two fragmentε. The ligation mixture iε uεed to tranεform bacteria HBlOl, which are then plated onto agar-containing kanamycin for the purpoεe of confirming that the vector had the gene of intereεt properly inεerted.

The amphotropic pA317 or GP+aml2 packaging cellε are grown in tiεεue culture to confluent denεity in Dulbecco'ε Modified Eagleε Medium (DMEM) with 10% calf εerum (CS) , penicillin and εtreptomycin. The MSV vector containing the gene iε then added to the media and the packaging cellε are tranεduced with the vector. The packaging cellε now produce infectiouε viral particleε containing the gene (the packaging cellε are now referred to aε producer cellε) .

Freεh media is added to the transduced producer cellε, and εubsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore

filter to remove detached producer cells and thiε media iε then used to infect fibroblaεt cells. Media iε removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cellε. Thiε media iε removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection iε required. If the titer iε very low, then it iε necessary to use a retroviral vector that has a selectable marker, εuch aε neo or hiε.

The engineered fibroblaεtε are then injected into the hoεt, either alone or after having been grown to confluence on cytodex 3 microcarrier beadε. The fibroblaεtε now produce the protein product.

Numerouε modificationε and variationε of the preεent invention are poεεible in light of the above teachingε and, therefore, within the εcope of the appended claimε, the invention may be practiced otherwise than as particularly described.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: HU, ET AL.

(ii) TITLE OF INVENTION: Fibroblast Growth Factor-11

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Concurrently

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER: 08/207,412

(B) FILING DATE: 8 MAR 1994

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 768 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGAGTGGAA AGGTGACCAA GCCCAAAGAG GAGAAAGATG CTTCTAAGGT TCTGGATGAC 60

GCCCCCCCTG GCACACAGGA ATACATTATG TTACGACAAG ATTCCATCCA ATCTGCGGAA 120

TTAAAGAAAA AAGAGTCCCC CTTTCGTGCT AAGTGTCACG AAATCTTCTG CTGCCCGCTG 180

AAGCAAGTAC ACCACAAAGA GAACACAGAG CCGGAAGAGC CTCAGCTTAA GGGTATAGTT 240

ACCAAGCTAT ACAGCCGACA AGGCTACCAC TTGCAGCTGC AGGCGGATGG AACCATTGAT 300

GGCACCAAAG ATGAGGACAG CACTTACACT CTGTTTAACC TCATCCCTGT GGGTCTGCGA 360

GTGGTGGCTA TCCAAGGAGT TCAAACCAAG CTGTACTTGG CAATGAACAG TGAGGGATAC 420

TTGTACACCT CGGAACTTTT CACACCTGAG TGCAAATTCA AAGAATCAGT GTTTGAAAAT 480

TATTATGTGA CATATTCATC AATGATATAC CGTCAGCAGC AGTCAGGCCG AGGGTGGTAT 540

CTGGGTCTGA ACAAAGAAGG AGAGATCATG AAAGGCAACC ATGTGAAGAA GAACAAGCCT 600

GCAGCTCATT TTCTGCCTAA ACCACTGAAA GTGGCCATGT ACAAGGAGCC ATCACTGCAC 660

GATCTCACGG AGTTCTCCCG ATCTGGAAGC GGGACCCCAA CCAAGAGCAG AAGTGTCTCT 720

GGCGTGCTGA ACGGAGGCAA ATCCATGAGC CACAATGAAT CAACGTAG 768

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 255 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ser Gly Lyε Val Thr Lyε Pro Lyε Giu Giu Lyε Aεp Ala Ser

5 10 15 Lyε Val Leu Aεp Aεp Ala Pro Pro Gly Thr Gin Giu Tyr lie Met

20 25 30

Leu Arg Gin Aεp Ser lie Gin Ser Ala Giu Lue Lyε Lyε Lyε Giu

35 40 45

Ser Pro Phe Arg Ala Lyε Cyε Hiε Giu lie Phe Cyε Cyε Pro Leu

50 55 60

Lyε Gin Val Hiε Hiε Lyε Giu Aεn Thr Giu Pro Giu Giu Pro Gin

65 70 75

Leu Lyε Gly lie Val Thr Lyε Leu Tyr Ser Arg Gin Gly Tyr Hiε

80 85 90

Leu Gin Leu Gin Ala Aεp Gly Thr lie Aεp Gly Thr Lyε Aεp Giu

95 100 105

Aεp Ser Thr Tyr Thr Leu Phe Aεn Leu lie Pro Val Gly Leu Arg

110 115 120

Val Val Ala He Gin Gly Val Gin Thr Lyε Leu Tyr Leu Ala Met

125 130 135

Aεn Ser Giu Gly Tyr Leu Tyr Thr Ser Giu Leu Phe Thr Pro Giu

140 145 150

Cyε Lyε Phe Lys Giu Ser Val Phe Giu Asn Tyr Tyr Val Thr Tyr

155 160 165

Ser Ser Met He Tyr Arg Gin Gin Gin Ser Gly Arg Gly Trp Tyr

170 175 180

Leu Gly Leu Asn Lyε Giu Gly Giu He Met Lyε Gly Aεn Hiε Val

185 190 195

Lyε Lyε Aεn Lyε Pro Ala Ala Hiε Phe Leu Pro Lyε Pro Leu Lyε

200 205 210

Val Ala Met Tyr Lyε Giu Pro Ser Leu Hiε Asp Leu Thr Giu Phe

215 220 225

Ser Arg Ser Gly Ser Gly Thr Pro Thr Lys Ser Arg Ser Val Ser

230 235 240

Gly Val Leu Asn Gly Gly Lyε Ser Met Ser Hiε Aεn Giu Ser Thr

245 250 255

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 33 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CGCGGATCCA TCATGAGTGG AAAGGTGACC AAG 33

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 30 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CGCGGATCCC GTTGATTCAT TGTGGCTCAT 30

(2) INFORMATION FOR SEQ ID NO:5

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8