CA2249213A1

CA2249213A1 - Extracellular/epidermal growth factor-like protein

Info

Publication number: CA2249213A1
Application number: CA002249213A
Authority: CA
Inventors: Henrik S. Olsen; Haodong Li
Original assignee: Individual
Current assignee: Human Genome Sciences Inc
Priority date: 1996-04-10
Filing date: 1996-04-10
Publication date: 1997-10-16
Also published as: WO1997038002A1; AU5549296A; EP0910569A1; JP2000508531A; EP0910569A4

Abstract

The present invention discloses extracellular/epidermal growth factor polypeptides and polynucleotides encoding such polypeptides. Also provided is a procedure for producing such polypeptides by recombinant techniques and therapeutic uses of the polypeptides which include induction of DNA synthesis, stimulating wound healing, treating neurological disorders, treating ocular disorders, treating kidney and liver disorders and stimulating embryogenesis and angiogenesis. Also disclosed are antagonists against such polypeptide and their use as a therapeutic to treat neoplasia. Also disclosed are diagnostic assays for detecting altered levels of the polypeptide of the present invention and mutations in the nucleic acid sequences which encode the polypeptides of the present invention.

Description

W097/38002 PCT~S96/05247 ~TP~rFT-T~ /EPIDER~AL GROWT~ F~CTOR ~I~E PROTEIN

This invention relates to newly identified polynucleotides, polypeptides PnCoAe~ by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been putatively identified as a human extracellular protein-like/Epidermal Growth Factor-like protein, hereafter referred to as "EEGF". The invention also relates to i nhihi ting the action of such polypeptides.
Cellular growth and differentiation appear to be initiated, promoted, ~-int~ine~ and regulated by a multlplicity of stimulatory, i nhihi tory and synergistic factors and hor~L~nes. The alteration and/or breakdown of the cellular ho~leostasls mechanism seems to be a fl~nA~m~ntal cause of growth rel~ed diseases, including neoplasia. Growth moAl~l~tory factors are implicated in a wide variety of pathological and physiological processes including signal transduction, cell communication, growth and development, embryogenesis, immune response, hematopoiesis cell survival and differentiation, inflammation, tissue repair and remodeling, atherosclerosis and cancer. Epidermal growth factor (BGF), transforming growth factor alpha (TGF~), betacellulin, hiregulin, and vaccinia growth factor among other factors are growth and differentiation moA~ tory proteins produced by a variety of cell types either under normal physiological conditions or in response to exogenous stimuli and are members of the EGF
family.

W O 97/38002 PCT/U~/05247 These peptide growth factors influence influence epithelial and epidermal cells through autocrine and paracrine mechanisms.
They also play important roles in normal wound heAl ing in tissues such as skin, cornea and gastrointestinal tract and all share substantial amino acid sequence homology including the conserved placement of three intra-chain disulfide bonds. In addition, all the factors of this family bind to a 170,000 molecular weight transmembrane glycoprotein receptor and activate the tyrosine kinase acti~ity in the receptor's cytoplasmic ~ i n (Buhrow, S.A.
et al., J. Bio.Chem., 258:7824-7826 (1983)).
The receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the gastro-intestinal tract (GI) tract. These peptide growth factors are synthesized by several cells in~olved in wound healing including platelets, keratinocytes, and activated macrophages. These growth factors have also been implicated in both the st~ tion of growth and differentiation of certain cells, for example, neoplasia, and the i nhihi tion of other types of cells.
Betacellulin is a 32-kilodalton glycoprotein that appears to be processed from a larger transmembrane precursor by proteolytic cleavage. The cArhq~cyl-terminal ~ i n of betacellulin has 50%
sequence similarity with that of rat transforming growth factor a.
~etacellulin is a potent mitogen for retinal pigment epithelial cells and vascular smooth muscle cells.
Amphiregulin is a bifunctional cell growth regulatory factor which exhibits potent i nhihi tory activity on DNA synthesis in neoplastic cells, yet promotes the growth of certain normal cells.
A wide variety of uses for amphiregulin have been assigned including the treatment of wounds and cancers. For example, amphiregulin has potent anti-proliferative effects in vitro on several human cancer cell lines of epithelial origin. Amphiregulin also induces the proliferation of human foreskin fibroblasts as shown in United States Patent Application No. 5,115,096.
TGF~ has pleiotropic biological effects. The production of certain m~mh~rs of TGF~ is synthesized by a number of oncogenically transformed fibroblasts (Ciardiello et al., J. Cell. Biochem., 42:45-57 (1990)), as well as by a variety of tumors, including renal, breast and squamous carcino~a.s, melAnomA.s and glioblastomas PCT~S96/OS247 ~Derynck, R. et al., Cancer Res., 47:707-712 (1987)). There is direct evidence that TGF~ expression can be a contributing factor in the conversion of a normal cell to its tumorigenic counterpart by analyzing transgenic mice in which tumor cells express high ~ levels of TGF~. TGF~ transgenic ~nim~l S display a variety of neoplastic lesions, depending on the strain of mouse and the choice of promotor regulating TGF~ expression ~Sandgren, et al., Cell, 61:1121-1135 (1990)).
TGF~ also plays a role in normal embryonic development and adult physiology (Derynck, R. Adv. Cancer Res., 58:27-5 (1992)).
TGF~ has been expressed in many tissues including skin, brain, gastrointestinal mucosa and activating macrophages. Accordingly, TGF~ is an important factor in controlling growth of epithelial cells and has a role in wound ~eAli~g. TGF~ has also been found to be angiogenic (Schreiber, et al., Science, 232:1250-1253 (1986)).
The polypeptide of the present invention has been putatively identified a~ an Extracellular/Bpidermal Growth Factor. This identification has been made a~ a result of amino acid sequence homology to human extracellular protein which is a secreted protein with EGF-like ~nm~inS that is abundant in heart tissue.
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, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with another aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC
Deposit No. 97285.
In accordance with yet a further aspect of the present invention, there are provided processes for producing such polypeptide by recombinant technigues comprising culturing rec~inAnt prokaryotic and~or eukaryotic host cells, cont~i ni ng PCT~S96/05247 a nucleic acid sequence encoding a polypeptide of the present invention.
In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, to regulate vascular smooth muscle cell proliferation, to treat Marfan syndrome, to stimulate wound healing, to restore normal neurological functioning after trauma or AIDS dementia, to treat ocular disorders, to treat kidney and liver disorders, to promote hair follicular development, to sti~nl~te growth and differentiation of various epidermal and epithP~ ~Al cells in vivo and in vitro and for the treatment of burns, ulcers and cor~e~l incisions, to stimulate embryogenesis.
In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid sequences of the present invention.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with yet a further a~pect of the present invention, there are provided agonists to the polypeptide of the present invention.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to i nhl hl t the action of such polypeptides, for example, in the treatment of corneal inflammation, neoplasia, for example, tumors and cancers and for psoriasis.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to overexpression of the polypeptide of the present invention and mutations in the nucleic acid sequences PncoAl ng such polypeptide.
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
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.

PCT~S96/05247 The following drawings are illustrative of PmhoAi~ents of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1 depicts the cDNA sequence and correspon~i ng deduced amino acid sequence of EEGF. Both the st~n~Ard one letter and three letter ab~reviations for amino acids are used.
Figure 2 is an illustration of comparative amino acid sequence homology between the polypeptide of the present invention (lower line) and human extracellular protein (upper line) (SEQ ID NO:9).
In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2).
A polynucleotide encoding a polypeptide of the present invention may be obtained from human brain and early stage brain tissue. The polynucleotide of this invention was discovered in a human fetal heart cDNA li~rary. It has homology to the characteristic EGF domains. It contains an open reading frame encoding a polypeptide of 392 amino acids. EEGF eyhih~ts the highest degree of homology at the amino acid level to human extracellular protein with 45% identity and 34~ similarity over a 392 amino acid stretch. Northern blot analysis of this protein shows high levels of expression in heart tissue with the transcript being approximately 2 kb. In accordance with another aspect of the present invention there are provided isolated polynucleotides encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97285, deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, USA, on September 26, 1995. The deposited material is an a plasmid that contAin~ the full-length EEGF cDNA which has been transformed into a viable host.
The deposit(s) have been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-org~n~s~c for purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. These deposits are provided merely as convenience to those of skill in the art 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 W O 97/38002 rCTrUS96/05247 materials, as well as the amino acid sequence of the polypeptides encoded thereby, are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted. References to "polynucleotides" throughout this specification includes the DNA of the deposit referred to above.
The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDN~, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 ~SEQ ID NO:l) or may be a different coding sequence which coding sequence, as a result of the reA-~n~ncy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA
of Figure 1 ~SEQ ID NO:1).
The polynucleotide which ~ncq~es for the mature polypeptide of Figure 1 (SBQ ID NO:2) may include, but is not limited to: only the coAin~ sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional ro~ ng 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 5' 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 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 polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2). The variant 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 PncoAing the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) as well as variants of such polynucleotides which variants encode for W097/38002 PCT~S96/05247 a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As her~in~hove 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). 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 subst~nti Al ly alter the function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same r~A~i ng frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a eader sequence which functions as a secretory sequence for contro].ling 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 enco~e 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 r~m~i n~ Thus, for example, the polynucleotide 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 hexa-histidine 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 ~ ian host, 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 W O 97/38002 PCTrUS96/05247 the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Fragments of the full length EEGF gene may be used as a hybridization probe for a cDNA library to isolate the full length ~ene and to isolate other genes which have a high sequence similarity to the gene or S jmi 1 ~r 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 be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete EEGF gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a ~equence 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 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 her~inAhove-described polynucleotides.
As herein used, the term "stringent conditionsll 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).
Alternatively, the polynucleotide may have at least 15 bases, preferably at least 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 hereinAhove described, and which may or may not retain activity. ~or 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 dia~nostic probe or as a PCR primer.

W 097/38002 PCT/U~ 247 Thus, the present invention is directed to polynucleotides having at least a 70% 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 and polynucleotides complementary thereto as well as portions thereof, which portions have at least 15 consecutive bases preferably at least 30 consecutive bases and preferably at least 50 consecutive bases and to polypeptides encoded by such polynucleotides.
The present invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ I~ NO:2), as well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 (SEQ ID NO:2), means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a reComhin~nt polypeptide.
The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) may be (i) 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 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 hu...oyel,eity.

The term "isolated" means that the material is removed from its original environment ~e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~ni~-l is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at lea~t 70% identity) to the polypeptide of SEQ ID
NO:2 and mor-_ preferably at least 90~ similarity (more preferably at least 90% identity) to the polypeptide of SBQ ID NO:2 and still more preferably at least 95~ simil~rity (still more preferably at least 95~ identity) to the polypeptide of SBQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally cont~ning at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "s~milArity" 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 fragments 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 techni~ues.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention W097/38~2 PCT~S96/05247 which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as a~pLGpriate for activating promoters, selecting transformants or am.plifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention m~y be employed for producing polypeptides by recomhin~nt techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirusi yeast plasmids; vectors derived from com~binations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is 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 se~uence is inserted into an ~ o~Liate restriction Pn~ lease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector s 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 tr~, the phage l~mh~ P~ promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also cont~tn~ a ribosome htn~tng site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors prefera~ly cont~tn one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resifitance for eukaryotic cell culture, or such as tetracycline or am~icillin resistance in E. coli.

W O 97138002 PCT~US96/05247 The vector contAining the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an a~-u~iate 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, Streptomyces, Salmonella tY~himllrium; fungal cells, such as yeast; insect cells such as Drosophila S2 and S~odo~tera Sf9; ~nim~l cells such as CHO, COS or Bowes melanoma; adenoviruses; plant 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 recomh~n~nt 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 em~o~im~nt, 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, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene)i ptrc99a, pKK223-3, pKK233-3, pDR540, pRlT5 (Pharmacia); Eukaryotic: pWLN~O, pSV2CAT, pOG44, pXT1, 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 (chlor~m~h~nicol transferase~ 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, l;~mh~l~ PRI PL and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs 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 emhoAiment, the present invention relates to host cells contAining the above-described constructs. The host cell can W O 97/380U2 PCTrUS96/05247 be a higher eukaryotic cell, such as a m~ l ian 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, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional m~nner to produce the gene product encoded by the recomhinAnt sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in m;~ ian 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. A~Lo~iate cloning and expression vectors for u~e with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory ~ml~l, Second Bdition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an Pnh~ncer sequence into the vector. Rnh~ncers are cis-acting elements of DNA, u~ually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter Pnh~ncer, the polyoma Pnh~ncer on the late side of the replication origin, and adeno~irus Pnh~ncers.
Generally, reCo~hln~nt expression vectors will include origins of replication and selecta~le markers permitting transformation of the host cell, e.g., the ampicillin resistance 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), ~-factor, acid phosphatase, or heat shock proteins, among others.
The heterologous structural sequence is assembled in appropriate pha~e with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of W O 97/38002 PCT/U~ v3~47 translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can ~nco~ a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., 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 ~rk~rs 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, S~?~nella tY~himl~rium and various species within the genera Pse~l~nmon~.s, 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 cu..~lise a selectable marker and bacterial origin of replication derived from commercially av~ hle plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKR223-3 tPharmacia Fine Chemicals, Ur~s~l~, Sweden) and GEMl (Plo...e~d Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are combined with an a~lo~iate promoter and the structural seguence 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 induced by appropriate means (e.g., 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 ret~in~ for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
Various ~ ian cell culture systems can also be employed to express reco~hin~nt protein. Examples of mammalian expression W O 97/38002 PCTrUS96/05247 systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines c~p~hle 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 ~ i n~ sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination se~uences, and 5~ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The polypeptides can be recovered and purified from reCo~hinAnt cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation ~rh~nge chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, a~ necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography tHPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by reco~ nt 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 procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics for human disease.
The polypeptide of the present invention may be employed to to regulate vascular smooth muscle cell proliferation.
The polypeptide of the present invention may also be employed for characterization of receptors. The EGF family of receptors ~UL ' e~.tly includes four EGF receptors, denoted as EGFR1, EGFR2, EGFR3 and EGFR4. The EGFR2 receptor may also be referred to as W O 97138002 PCTrUS96/05247 ERB-2 and this molecule is useful for a variety of diagnostic and therapeutic indications (Prigent, S.A., and Lemoine, N.R., Proq Growth Factor Res., 4:1-24 (1992)). The EEGF polypeptide is likely a ligand for one or more of these receptors as well as for an unidentified new EGF-type receptor. Use of the EEGF can assist with the identification, characterization and cloning of such receptors. For example, the EG~ receptor gene represents the cellular homolog of the v-erb-B oncogene of avian erythroblastosis virus. Over expression of the E~F-receptor or deletion of kinase regulatory segments of the protein can bring about tumorigenic transformation of cells (Manjusri, D. et al., Human CYtokines, 364 and 381 ~1991)).
The polypeptides of the present invention may also be employed for restoration or ~nh~ncement of neurological functions ~i~inished as a result of trauma or other damaging pathologies (such as AIDS
~ , senile dementia, etc). TGF~ and its homologs have been found to be the most Ahl~n~nt ligand for the EGF/TGF~ receptor in most parts of the brain (Raser, et al., Mol Brain Res: 16:316-322, (1992)). EBGF or soluble form thereof may also be employed to treat ocular disorders, for example, corneal inflammation. A
variety of experiments have implicated m~mh~rs of the TGFa gene family in such pathologies. A recent paper summarizes some of the data related to the role these growth factors play in eye disease (Mann, et al, Cell, 73:249-261 (1993)). Recent experiments have shown that a number of mice lacking the TGF~ gene displayed corneal inflammation due to an infiltration of leukocytes and other cells to the substantia propria of the eyes.
In addition, the specificity of certain growth factors for their target cells can be exploited as a me~h~ni~~ to destroy the target cell. For example, EEGF or soluble forms thereof can be coupled, by a wide variety of methods known in the art, to toxic molecules: for example, a radiopharmaceutical which inactivate target cells. These growth factor-toxin fusions kill the target cell (and in certain cases neighboring cells by a variety of "byst~nA~n effects). A recent example of such toxin-fusion genes is pllhli~hed by Mesri, et al., J. Biol. Chem. 268:48~3-62 (1993).
~EGF and related molecules may also be encapsulated in liposomes and may be conjugated to antibodies which recognize and bind to W O 97/38002 PCT/Ug31/0',247 tumor or cell specific antigens, thereby provided a means for "targeting~ cells.
The EBGF polypeptide may also be employed to treat certain kidney disorders, since it has been found that there has been expression of these growth factors in the kidney. Thus, these factors may be necessary for the proper physiological maintenance of this organ. Treatments may also be related to liver regeneration or liver dysfunction.
A significant treatment involving EEGF relates to wound healing. The compositions of the present invention may be employed for treating a wide variety of wounds including substantially all cutaneous wounds, corneal wounds, and injuries to the epithelial-lined hollow organs of the body. Wounds suitable for treatment include those resulting from trauma such as burns, abrasions and cuts, as well as from surgical procedures such as surgical incisions and skin grafting. Other conditions suitable for treatment with the polypeptide of the present invention include chronic conditions, such as chronic ulcers, diabetic ulcers, other non-h~Al ;ng ~trophic) conditions, to treat Marfan syndrome, to promote hair follicular development, to stimulate growth and differentiation of various epidermal and epit~eli~l cells in vivo and in vitro and to st~ te embryogenesis.
E~GF or soluble fragment thereof may be incorporated in physiologically-acceptable carriers for application to the affected area. The nature of the carriers may vary widely a.d will depend on the intended location of application. For application to the skin, a cream or o~ntm~nt base is usually preferred; suitable bases include lanolin, Silvadene (Marion) (particularly for the treatment of burns), Aquaphor (Duke Laboratories, South Norwalk, Conn.), and the like. If desired, it will be possible to incorporate EEGF cont~ ni ng compositions in bandages and other wo~nd dressings to provide for continuous exposure of the wound to the peptide. Aerosol applications may also find use.
The concentration of EEGF in the treatment composition is not critical but should be enough to induce epithelial cell proliferation. ~he compositions may be applied topically to the affected area, typically as eye drops to the eye or as creams, ointments or lotions to the skin. In the case of the eyes, frequent treatment is desirable, usually being applied at intervals W097/38002 PCT~S96/05247 of 4 hours or less. On the skin, it is desirable to continually maintain the treatment composition on the affected area during the healing, with applications of the treatment composition from two to four times a day or more frequently.
The amount employed of the subject polypeptide will vary with the m~nner of ~mi ni stration, the employment of other active compounds, and the like, generally being in the range of about l ~g to 100 ~g. The subject polypeptide may be employed with a physiologically acceptable carrier, such as saline, phosphate-buffered saline, or the like. The amount of compound employed will be determined empirically, based on the response of cells in vitro and response of ex~perimental ~ni~-l S to the subject polypeptides or formulations ront~i~tn~ the subject polypeptides.
The EEGF or soluble fragment thereof m~y be employed in the moA~ tion cr angiogenesis, bone resorption, im~ln~ response, and synaptic and neuronal effector functions. EEGF may also be used in the moiulation of the arachidonic acid cascade.
Applic-.ions are also related to alopecia, hair loss and to other skin conditions which affect hair follicular development.
Several lines of evidence implicate the involvement of growth factors in such conditions. As described above, "knockoutll mice engineered to CQntA i n a null mutation in the TGF~ gene display abnormalities related to quantitative and qualitative hair synthesis. In addition, mapping studies in mice have shown that some mutations affecting hair growth map to the TGF~ gene locus (Mann et al, Cell, 73:249-261~1993)). Topical or systemic applications of EEGF or derivatives thereof may be employed to treat some forms of alopecia and hair loss and these claims fall within the scope of this invention.
Certain disease pathologies may be partially or completely ameliorated by the systemic clinical ~mi ni stration of the EEGF
growth factor. This ~mtnistration can be in the form of gene therapy (see below); or through the ~mi~istration of peptides or proteins synthesized from recomhin~nt constructs of EEGF DNA or from peptide chemical synthesis (Woo, et al., Protein Engineering 3:29-37 (1989).
This invention provides a method for identification of EEGF
receptors. The gene encoding a receptor can be identified by numerous methods known to those of skill in the art, for example, W 097138002 PCTrUS96/05247 ligand pAnning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1~2), Chapter 5, tl991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to EEGF, 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 EEGF. Transfected cells which are grown on glass slides are exposed to labeled EEGF, which can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific 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 clone that encodes the putative receptor. As an alternative approach for receptor identification, labeled ligand can be photoaffinity linked with cell l,-e-.~ne or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex cont~ining the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microse~Pnri n~. The amino acid sequence obtained from microse~lencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene ~nCo~in~ the putative receptor.
This invention also provides a method of screening compounds to identify antagonist compounds to the polypeptide of the present invention. As an example, a mammalian cell or membrane preparation expressing a EEGF receptor is incubated with EEGF and a potential antagonist compound and the ability of the compound to inhihi t a second signal from the receptor is measured to determine if it is an effective antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion ch~nnels or phosphoinositide hydrolysis.
Another assay for identifying potential antagonists specific to the receptors to the polypeptide of the present invention is a competition assay which comprises isolating plasma l.le..~anes which over-express a receptor to the polypeptide of the present invention, for example, human A431 carcinoma cells. Serially diluted test sample in a medium (volume is approximately 10 microliters) containing 10 nM '25I-EEGF is added to five micrograms W O 97/38002 PCTAUs96/0~247 of the plasma membrane in the presence o~ the potential antagonist compound and incubated for 4 hours at 4~C. The reaction ~ixtures are diluted and immediately passed throu~h a millipore filter. The filters are then rapidly washed and the bound radioactivity is measured in a gamma counter. The amount of bound EEGF is then measured. A control assay is also performed in the absence of the compound to determine if the antagonists reduce the amount of bound EEGF.
Potential antagonist compounds include an antibody, or in some cases, an oligopeptide, which binds to the polypeptide.
Alternatively, a potential antagonist may be a closely related protein which binds to the receptor which is an inactive forms of the polypeptide and thereby prevent the action of the polypeptide of the present invention.
Another antagonist compound 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 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to desi~n 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 -see ~ee et al., Nucl. Acids Res., ~: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 polypeptide of the present invention. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide of the present invention (Antisense - Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynuc~eotides 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 DN~ may be expressed in vivo to inhihi t production of the polypeptide of the present invention.
Antagonist compounds include a small molecule which binds to the polypeptide of the present invention and blocks its action at the receptor such that normal biological activity is prevented.

W097/38002 PCT~S96/05247 The small molecules may also bind the receptor to the polypeptide to prevent hi n~i n~. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.
The antagonists may be employed to treat neoplasia, for example, cancers and tumors. It is known that inhibition of secretion or production of me~hers of the EGF family by tumor cells in mice causes regression of tumors, since these proteins stimulate induction of DNA synthesis in all cells including neoplastic cells.
The antagonists to the polypeptides of the present invention may also be used therapeutically for the treatment of certain skin disorders, for example, psoriasis. ~levated levels of expression of mPmhers of this family of growth factors in skin biopsies taken from diseases such as psoriatic lesions have been found to be elevated (Cook, et al., Cancer Research, 52:3224-3227 (1992)). The antagonists may be employed in a composi.~sn with a pharmaceutiCally acceptable carrier, e.g., as hereinafter described.
The polypeptides of the present invention or agonist or antagonist compounds may be employed in comhin~tion with a suitable pharmaceutical carrier. Such compositions cG...~lise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to sAlinp~ buffered saline, dextrose, water, glycerol, ethanol, and com~in~tions thereof. The ~ormulation should suit the mode of ~dmi ni stration.
The invention also provides a pharmaceutical pac~ or kit comprising one or more cont~iners filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
Associated with such cont~in~r(s) can be a notice in the form prescribed by a governm~ntAl 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 a~mi ni stration. In addition, the polypeptides or compounds of the present invention may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient m~nn~r such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intr~n~R~l 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 ~mi ni stered in an amount of at least about 10 ~g/kg body weight and in most cases they will be ~mi n i stered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ~g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
The polypeptides, and agonists and antagonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.ll Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art and are apparent from the teachings herein. For example, cells may be engineered by the use of a retroviral plasmid vector cont~ining RNA
Pnco~i n~ a polypeptide of the present invention.
si~i 1 ~rly, cells may be engineered in VlVO for expression of a polypeptide in vivo by, for example, procedures known in the art.
~or example, a packaging cell is transduced with a retroviral rl~mid vector con~nin~ RNA Pnco~in~ a polypeptide of the present invention such that the packaging cell now produces infectious viral particles cQntA~ning the gene of interest. These producer cells may be ~mj nistered 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 invention by such method should be apparent to those skilled in the art from the teachings of the present invention.
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 em~odiment, 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 W097/38002 PCT~S96105247 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 (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and Bl9 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 heterologous promoters, such as the cytomegalovirus tCMV) promoter; the respiratory syncytial virus (RSV) promoter;
inAllcihle 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 her~ln~hove described); the ~-actin promoter; and human growth hormone promoters. The promoter 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, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Thera~Y, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 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 W O 97/38002 PCTrUS96/05247 polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vlvo. 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, f ibroblasts, myoblasts, keratinocytes, endothelial cells, and broncht~l epithPli~l cells.
This invention is also related to the use of the gene of the present invention as a diagnostic. Detection of a mutated fonm of the gene of the present invention will allow a diagnosis of a disease or a susceptibility to a disease which results f rom underexpression of the polypeptide of the present invention, for example, improper wound heAl ing, improper neurological functioning, ocular disorders, kidney and li~er disorders, hair f ollicular development, angiogenesis and en~Lyoy~lesis.
Individuals carrying mutations in the human 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 ~n~o~ing a polypeptide of the present invention can be used to identify and analyze mutations thereof. 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.
Seguence differences between the reference gene and genes having mutations may be revealed by the direct DNA se~pncing method. In addition, cloned DNA segments may be employed as probes to detect specific DNA se~Pnts~ The sensitivity of this method is greatly ~nh~nced when cQ~ined with PCR. For example, a seqllencing primer is used with double-stranded PCR product or a W O 97138002 PCT/U',"3'247 single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
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 se~l~nce~ may be distinguished on denaturing formamide gradient gels in which the mn~i ~ i ties of different DNA fragments are retarded in the gel at different positions according to their specific 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 RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (19~5)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA se~)~ncin~ or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorph~ F~ (RFLP)) and SollthPrn 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 ~ nostic assays for detecting altered levels of the polypeptide of the present invention in various tissues since an over-expression of the proteins comr~red to normal control tissue samples can detect the presence of certain disease conditions such as neoplasia, skin disorders, ocular disorders and inflammation. Assays used to detect levels of the polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and include radioimm~lno~csays, competitive-binding assays, Western Blot analysis and preferably an ELISA assay. An ELISA
assay initially comprises preparing an antibody specific to an antigen of the polypeptide of the present invention, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is W O 97/38002 PCT~US96/05247 attached a detectable reagen~ such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid suy~or L, e.g. a polystyrene dish, that binds the proteins in the sam~le. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum Alhnmin Next, the monoclonal 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 un~ound monoc~Qn~
antibody is washed out with buffer. The reporter Antihody l;nke~
to horseradish peroxidase is now placed in the dish resulting in hintli ng of the reporter antibody to any monoclonal Antihody bound to polypeptides of the present invention. UnattAcheA Le~o~Ler antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of protein present in a given volume of patient sample when compared against a st~n~Ard cur~e.

A competition assay may also be employed to determine levels of the polypeptide of the present invention in a sample derived from the hosts. Such an assay comprises isolating plasma membranes which over-express the receptor for the polypeptide of the present invention. A test sample cnntAin~ng the polypeptides of the present invention which have been labeled, are then added to the plasma membranes and then incubated for a set period of time. Also added to the reaction mixture is a sample derived from a host which is suspected of containing the polypeptide of the present invention. The reaction mixtures are then passed through a filter which is rapidly washed and the bound radioacti~ity is then measured to determine the amount of competition for the receptors and therefore the amount of the polypeptides of the present invention in the sample.
Antibodies specific to EEGF may be used for cancer diagnosis and therapy, since many types of cancer cells up-regulate various members of the TGF~ family during the process of neoplasia or hyperplasia. These antibodies bind to and inactivate EEGF.
Monnrlonal Antiho~ies against EEGF (and/or its family members) are in clinical use for both the diagnosis and therapy of certain disorders including (~ut not limited to) hyperplastic and neoplastic growth abnormalities. Upregulation of growth factor expression by neoplastic tissues forms the basis for a variety of serum assays which detect increases in growth factor in the blood of affected patients. These assays are typically applied not only in diagnostic settings, but are applied in prognostic settings as well (to detect the presence of occult tumor cells following surgery, chemotherapy, etc).
In addition, malignant cells expressing the EEGF receptor may be detected by using labeled ~EGF in a receptor binding assay, or by the use of antibodies to the EEGF receptor it~elf. Cells may be distinguished in accordance with the presence and density of receptors for EEGF, thereby providing a means for predicting the susceptibility of such cells to the biological activities of EEGF.
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 seguence data (repeat polymorrhi ,eTnC) are presently av~ hl e for marking chromosomal location. The mapping of DNAs to chromosomes according to the present inYention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to c},l~...osomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids cont~inin~
individual human chromosomes. Only those hybrids cont~ining 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 in~ention 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 ~-nner. Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, W O 97/38002 PCTrUS96105247 prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a meeAph~se 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 ~Aml~l of Basic Techniques, PeL~dl..oll Press, New York (1988).
Once a sequence has been mapped to a precise 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. McKusick, Men~Pl i ~n Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).
~he 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 se~uence between affected and unaffected individuals.
If a m.utation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the 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 ~nti~odies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes rhim~ric, single chain, and hnm~nized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures ~nown in the art m.ay be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides correspon~i n~
to a se~uence of the present invention can be obtained by direct injection of the polypeptides into an ~ni ~1 or by administering the polypeptides to an ~ni~l, preferably a nnnhllm~n. The antibody so obt~ine~ will then bind the polypeptides itself. In this W O 97/38002 PCTrUS96/05247 m~nner~ even a seguence encoding only a fragment of the polypeptides can be used to generate antibodies bi~i n~ the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Bxamples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:49~-497), the trioma technigue, the human B-cell hybridoma technique (Kozbor et al., 1983, Jm~lnology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies ~Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Technigues described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to t mmllnogeniC 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; howe~er, 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 underst~n~ng of the following examples certain frequently occurring methods and/or terms will be described.
"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 estricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, eguivalent 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 seguences 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 20 ~l of buffer solution. For the purpose of isolating DNA

W O 97/38002 PCT~US96/05247 fragments for plasmid construction, typically 5 to 50 ~g 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 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).
IlOligonucleotides'' 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, T., et al., Id., p. 146). Unless otherwise pro~ided, ligation may be accompl;~;hP~l 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 in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

~xample 1 Bacterial ExDression and Purification of EEGF
The DNA sequence encoding EBGF, ATCC # 97285, is initially amplified using PCR oligonucleotide primers corresponding to the 5' sequences of the EEGF protein and the sequences 3' of the EEGF gene. The 5' oligonucleotide primer has the sequence 5' GACTTCATGA~ lAACCAAAATGG 3' ~SEQ ID NO:3) contains a Bsp HI
restriction enzyme site followed by 19 nucleotides of E~GF coding sequence starting from the presumed terminal amino acid. The 3' sequence 5' GACTGGATCCGAATGGGTACTGCGACACATATATC 3' (SEQ ID NO:4) contains complementary sequences to a BamHI site and is followed W O 97/38002 PCT/Ub,~)47 by 25 nucleotides of EEGF. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, CA). pQE-60 ~n~oA~ antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-60 is then digested with BamHI. The amplified sequences are ligated into pQE-60 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory ~n~
Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on ~3 plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA
is isolated and confirmed by restriction analysis. Clones cont~i ni n~ the desired constructs are grown o~ernight (OJN) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.~) 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 le~i n~ to increased gene ~-xpression.
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 HCl. After clarification, solubilized EEGF is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight bin~i~g by proteins cont~ining the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-134 (1984)). EEGF is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adju~ted to 3 molar guanidine HCl, 100mM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

Exam~le 2 W O 97/38002 PCT~US96/OS247 Cloninq and exDression of EBGF usin~ the baculovirus ex~ression sYstem The DNA sequence encoding the EEGF protein, ATCC X 97285, is amplified using PCR oligonucleotide primers corresp~n~i n~ to the 5' and 3' sequences of the gene.
The primer sequences are as follows: 5' CAGTGGATCCGCCATC
ATGAl~lb~ AACCAAAATGGCG 3' (SEQ ID NO:5), has a BamHI restriction enzyme site (in bold) followed by 6 nucleotides res~m~ling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) (the initiation codon for translation is "ATGn).
The 3' primer 5' GCATGGT~CrC~l-C~A&GCTCCAGCCCGAGG 3~ (SEQ ID
NO:6) contains the cleavage site for the re~triction Pn~nnllrlea5e Asn718 (bold) and 22 nucleotides complementary to the 3~ end of the ~G~ gene. The amplified sequences are isolated from a 1% agarose gel using a ~om~rcially av~ hle kit ("Geneclean,'l BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with the Pn~on~lCleaseS BamHI and Asp718 and then purified again on a 1%
agarose gel. This fragment is designated F2.
The vector pA2 is used (modification of pVL941 vector, discussed below) for the expression of the EEGF protein using the baculovirus expression system (for review see: Summer~ M.D. and Smith, G.E. 1987, A manual of methods for baculo~ir_~ vectors and insect cell culture procedures, Texas Agricultural ~xperimental Station Bulletin No. 1555). This expression ve~tor ~ -nt~i ns the strong polyhedrin promoter of the Autographa cali__rnica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction ~ leases. The polyadenylation site of the ~ n virus (SV)40 is used for efficient polyadenylation. For an ea~y 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 sequPnces are flanked at both sides by viral sequences for the cell-mediated homologous recom~in~tion of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used such as pAc373, pRG1, pVL941 and pAcIM1 (Luckow, V.A.
and Summers, M.D., Virology, 170:31-39).
The plasmid is digested with the restriction enzymes BamHI and Asp718 and then 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~
BI0 ~01 Inc., ~a Jolla, Ca.). This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid (p~acEEGF) with the ~EGF gene using the restriction enzymes BamHI and Asp718. The sequence of the cloned fragment is confirmed by DNA seqllPncing.
5 ~g of the plasmid pBacEEGF is co-transfected with 1.0 ~g of a commercially available linearized baculovirus ("BaculoGold~
baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid pBacEEGF
are mixed in a sterile well of a microtiter plate cont~ining 50 ~l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l Lipofectin plu~ 90 ~l 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 a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27~C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10~ fetal calf serum is added. The plate is put back into an incubator and cultivation continued at 27~C for four days.
After four days the supernatant is collected and a plaque assay 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 st~inP~ 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 st~i nP~ plaques are picked with the tip of an Bppendorf pipette. The agar cont~i ni n~ the recombinant viruses is then resuspended in an Eppendorf tube cont~ining 200 ~l of Grace~s medium. The agar is removed by a brief centrifugation and the supernatant cont~ining 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 4~C.
Sf9 cells are grown in Grace's medium supplemented with 10~
heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-EEGF at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technoloyies Inc., Gaithersburg). 42 hours later 5 ~Ci of 35S-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.

Exam~le 3 Bx~ression of Recombinant REGF in COS cells The expression of plasmid, EEGF HA is deri~ed from a vector pcDNA3/Amp (In~itrogen) cont~inin~ 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV
promoter followed by a polylinkPr region, an SV40 intron and polyadenylation site. A DNA fragment ~nco~;ng the entire EBGF
precursor and a HA tag fused in frame to its 3' end is cloned into the polylinkpr region of the vector, therefore, the reco~inAnt 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. T~ern~r~ 1984, Cell 37:767, (1984)). The infusion of HA tag to the target protein allows easy detection of the recor~in~nt protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding EEGF, ATCC # 97285, is constructed by PCR using two primers: the 5~ primer 5' GACTGGATCCGCCACCATGA~ lAACCAAAATG 3' ~SEQ ID NO:7) contains a BamHI site (in bold) followed by 6 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells and 22 nucleotides of EEGF coding sequence starting from the i n i t i a t i o n c o d o n ; t h e 3 ' s e q u e n c e 5 ' GA~-~ r.~A~ ACTGCGACACATAT 3~ (SEQ ID NO:B) contains complementary sequences to an XbaI site, 22 nucleotides of the EEGF
gene followed by sequences encoding the HA tag. The pcDNA3JAmp vector contains BamHI/XbaI cloning sites which bring the PCR insert in frame with the 3' HA tag followed by a stop codon. The PCR
amplified DNA fragment and the vector, pcDNA3/Amp, are digested with BamHI and XbaI restriction enzyme and ligated. The ligation mixture is transformed into E. coli strain SURE (avAilAhle 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 P~AminP~ by restriction analysis for the presence of the correct fragment. For expression of the reComhinAnt EFGF, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J.
Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the EBGF HA protein is detected by radiolabelling and i~mllnoprecipitation method (E. Harlow, D. Lane, Antibodies: A
Laboratory ~Anll~l, Cold Spring Harbor Laboratory Press, ~1988)).
Cells are lAhPlled for 8 hours with 35S-cysteine two days post transfection. Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 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 precipitated with an HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.

Exam~le 4 ExDression via Gene Thera~
Fibroblasts are obtAinP~ from a subject by skin biopsy. The resulting tissue is placed in tis~ue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. 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~ F~S, penicillin and streptomycin, is added. This is then incubated at 37~C for approximately one week.

W O 97/38002 PCT~US96/05247 At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and ~indIII and subsequently treated with calf intestinal phosphatase. The ~n~r vector is fractionated on agarose gel and purified, using glass beads.
The cDNA ~nco~i ng 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 includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus ~lne~r backhonP and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maint~ine~
under conditions appropriate for ligation of the two fragments.
The ligation mixture is used to transform bacteria H~101, which are then plated onto agar-contAjning kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector c~nt~ining the gene is then added to the media and the packaging cells are transduced with the vector.
The packaging cells now produce infectious viral particles cont~ning the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, cont~ini ng the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the W097t38~2 PCT~S96/05247 titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
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.

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a member selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:2;
(b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 15 bases of the polynucleotide of (a) or (b).

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

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

4. The polynucleotide of claim 1 comprising a nucleotide sequence set forth in SEQ ID NO:1.

5. The polynucleotide of claim 1 comprising a nucleotide 38 to nucleotide 1228 of SEQ ID NO:1.

6. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a member selected from the group consisting of:
(a) a polynucleotide encoding the mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97285;
(b) a polynucleotide complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 15 bases of the polynucleotide of (a) or (b).

7. A vector comprising the DNA of Claim 2.

8. A host cell comprising the vector of Claim 7.

9. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 8 the polypeptide encoded by said DNA.

10. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 7.

11. A polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO:2.

12. An antibody against the polypeptide of claim 11.

13. An antagonist for the polypeptide of claim 11.

14. A method for the treatment of a patient having need of EEGF comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 11.

15. A method for the treatment of a patient having need to inhibit EEGF comprising: administering to the patient a therapeutically effective amount of the antagonist of Claim 13.

16. The method of Claim 14 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.

17. A process for identifying compounds active as antagonists to the polypeptide of Claim 11 comprising:
contacting a reaction mixture containing a cell type which expresses the EEGF receptor and a compound to be screened; and detecting the absence of a signal generated from said receptor after binding of said compound to identify if the compound is an effective antagonist.

18. A process for diagnosing a disease or a susceptibility to a disease comprising:
determining a mutation in the polynucleotide of claim 1.

19. A diagnostic process comprising:
analyzing for the presence of the polypeptide of Claim 11 in a sample derived from host.