CA2184583A1 - Transforming growth factor alpha h1 - Google Patents

Abstract

Disclosed is a human TGF.alpha.-H1 polypeptide and DNA (RNA) encoding such TGF.alpha.-H1 polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and for producing antibodies and antagonists against such polypeptide. Such polypeptides may be combined with a suitable pharmaceutical carrier or diluent to provide diagnostic, therapeutic and/or prophylactic effects against various diseases. Also provided are methods of using the antibodies and antagonists to inhibit TGF.alpha.-H1 for therapeutic purposes.

Description

W O 95/24466 2 1 8 4 5 ~ 3 PCTrUS94/05476 -T~UUNS F OFU~ING G ~O~rTH F A CTO R FiLPHL~ Hl This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is human transforming growth factor alpha-H1 (TGFa-H1). The invention also relates to inhibiting the action of such polypeptides.
TGFa-H1 i8 a novel member of the epidermal growth factor (EGF) family. The EGF growth factor supergene family enro~pAsses a large number of medically important growth factors including the subfamily of Alpha Transforming Growth Factors (TGFa).
The EGF family of growth factors includes amphiregulin, cripto, heregulin, and heparin-binding EGF in addition to TGF-alpha. The most recently discovered member of this family is betacellulin, which was purified from conditioned media of mouse pancreatic beta tumor cell6 (sasada, et al.,B.B.R.C. 190:1173-9 (1993)). This gene was found to be expressed in kidney and liver tissues as well as in various tumor cell lines.
Purified and structurally and functionally characterized amphiregulin is disclo6ed in U.S. Patent No.
5,115,096, issued May 19, 1992. Amphiregulin is a bifunctional cell growth regulatory factor which exhibits potent inhibitory 21 ~45~3 wossl24466 PCT~S94/05476 activity on DNA synthesis in neoplastic cells, yet promotes the growth of certain normal cells. The amphiregulin gene has been cloned and used to construct plasmids which direct the expression of bioactive amphiregulin in transformed E. coli cells.
TGFa has pleiotropic biological effects. The production of certain members of the TGFa family is often elevated in certain disease conditions such as cancers, skin disorders, ocular disorders, and at the site of inflammation or wound healing.
Members of the TGFa family and their cognate receptors have been intensively studied for several years and comprehensive reviews have recently been published (Prigent and Lemoine, Progress in Growth Factor Research 4:1-24 (1992)); Schultz et al., J. Cell.
Biochem 45:346-352 (1991); Derynck, R., Mol. Reprod. and Dev.
27:3-9 (1990)).
TGFa in normal adult is expressed in a wide variety of tissues including skin, brain, gastrointestinal mucosa, breast tissues (including virgin, pregnant and lactating breast)~
activated macrophages, keratinocytes, and TGF possesses angiogenic activity as well, (Kudlow, J.E., and Bjorge, J.D., Seminars in Cancer Biology, 1:293-302 (1990).
In addition to their involvement in the control of cellular proliferation in various disorders, TGF~ growth factors are important for embryogenesis and the maintenance of normal adult physiologies. These growth factors influence a diversity of processes; there is evidence which suggests TGFa is involved in several aspects of embryogenesis as it is expres~ed in unfertilized oocytes, in preimplantation embryos, and in the maternal decidua where it may play a role in implantation or placental development. Transgenic mice ("knockout" mice) lacking a functional TGFa gene have abnormal skin architecture, wavy hair, curly whiskers, and they often develop corneal inflammation, these observations suggest that TGFa plays a pivotal role in determining skin architecture and regulating hair development (Mann et al., Cell 73:249-261 (1993)). Many members of the alpha transforming growth factor family are autocrine w095/24466 ~ 3 ~ `5 ~ 3 PCT~S94/05476 and/or paracrine growth factors for cancer cells from many tissues such as breast (TGFa), colon (cripto), and pancreas (betacellulin). Betacellulin is a potent mitogen for retinal pigment epithelial cells and vascular smooth muscle cells (Shing et al, Science, 259:1604-1607 (1993)); and amphiregulin (AR) possesses either growth stimulatory or growth inhibitory properties depending on the target cell which is tested and the concentration of AR applied to the cells (Shoyab et al Science 243:1074-1076 (1989)). For example, AR stimulates the proliferation of human foreskin fibroblasts yet AR inhibits the growth of the A431 cell line.
Further, TGFa growth factors are related to the following disease conditions: a) tumors; recent studies have shown that administration of agents which antagonize TGFa ( and/or its family members) activity in mice causes regression of the tumor, (Cook et al., Cancer Research, 52:3224-3227 (1992)); b) skin disorders, for example, psoriasis, (Cook et al., Cancer Research, 52:3224-3227 (1992)); and c) wound healing (Schultz et al., J. Cell. Biochem 45:346-352 (1991)) .
Human type-alpha transforming growth factor (TGFa) is a small, 6 Kda mitogenic protein containing 50 amino acids and 3 disulfide bonds. TGFa interacts with the EGF receptor and activates its intrinsic protein kinase. The role of TGFa in normal physiology has been described in Kudlow, J.E. and Bjorge, J.D., Cancer Biology, 1:293-302 (1990). Expression of TGFa i6 most prevalent and abundant in transformed cells and tumors, but it is also detectable at relatively low or moderate levels in certain normal adult tissues (brain, keratinocytes, epithelial cells, activated macrophages, pituitary). Expression of TGFa is also detectable in developing embryos at specific times and in specific tissues, most notably in the developing brain, kidney and liver.
Nearly all of the members of this family which have been purified and cloned to date have been found to contain six conserved cysteine residues which form disulfide bonds to create 2i 34583 W O 95/24466 PC~rrUS94/OS476 three peptide loops, thereby possessing a similar secondary structure. In addition, all of these growth factors are synthesized as a much larger membrane-bound, glycosylated precursor. TGFa-Hl contains all six of these conserved cysteine residues.
This family of growth factors interacts with the EGF
receptor family which also includes c-erb-2 and c-erb-3, the ligands for which have not been identified (Prigent and Lemoine, Prog. in Growth Factor Res., 4:1-24 (1992)). The involvement of these receptors in human neopla~ia has been widely studied and overexpression of these receptors has been found to be associated with poor prognosis for some forms of cancer (Holmes et al., Science, 256:1205-1210 (1992)). Some tumor cells have also been found tc synthesize significantly elevated levels of TGFa and/or other members of this family.
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is a human transforming growth factor alpha H1 (TGFa-H1), as well as fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.
In accordance with another aspect of the present invention, there is provided a polypeptide which is a soluble fragment of TGFa-H1, i.e. TGFa-Hl without the trAns~mhrane portion.
In accordance with still another aspect of the present invention, there is provided a procedure for producing such polypeptides by recombinant techniques.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotide encoding such polypeptides, for diagnostic and therapeutic purposes, for example, to stimulate wound healing, to restore normal neurological functioning after trauma or AIDS dementia, to treat ocular disorders, to target and W095/24466 2 1 ~ 4 5 8 3 PCT~S94105476 kill certain cells, to treat kidney and liver disorders, and to promote hair follicular development.
In accordance with another aspect of the present invention, there is provided an antibody against the TGFc-Hl or a soluble fragment thereof.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of tumors and psoriasis and diagno6tically to detect cancer.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawings are meant only as illustrations of specific embodiments of the present invention and are not meant as limitations in any manner.
Fig. l shows the predicted amino acid translation of the open reading frame of the TGF~-Hl cDNA. Numbering starts at position 7 because of the synthetic BamXI linker at positions 1-6 (not shown) which was used to clone the gene. The final stop codon at position 406 is also shown. The sequence shown encodes l32 amino acids. By comparison with the other members of the TGFa gene family, it can be concluded that only the 50 amino acids which are underlined are necessary for production of a soluble biologically active growth factor (see Figure 3).
Fig. 2 show~ the complete nucleotide sequence of the 3,286 nucleotide TGF~-Hl cDNA. The synthetic BamHI linker at position l and the synthetic XhoI linker at position 3,286 are shown in bold. The open reading frame which encodes the TGFa-Hl protein is underlined followed by the final stop codon ~shown in bold). In the long 3' untranslated region uncertainties in the sequence are shown using standard IUPAC codes.
Fig. 3 presents the alignment of TGF~-Hl with other members of the TGF~ gene family. A6terisks show the po6itions of the six critical conserved cysteine residues necessary for

2 1 ~45~3 -- PCr/llS 94~05 ~7 IPEA/US O ~ OCT 19~
biological activity for this family of growth factor molecules.
The 50 amino acid residues of TGF~ which are underlined are those residues which have been shown to be necessary for activity of the soluble growth factor. (Amphi= Amphiregulin) Fig. 4 illustrates the results of a northern blot analysis showing the RNA expression pattern of the polypeptide of the present invention.
In accordance with one 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 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75698 on March 4, 1994.
The polynucleotide of this invention was discovered in cDNA
libraries derived from human brain and fetal tissue. It is structurally related to the TGF~ gene family. It contains an open reading frame encoding a mature polypeptide of 132 amino acids, which exhibits significant homology to a number of members of the TGF~ gene family; these members include TGF~ itself as well as other members such as amphiregulin and cripto. Furthermore, the six cysteine residues occurring in all members in a characteristic motif are conserved in TGF~-H1.
In Figure 1 the 50 amino acids which are underlined are a soluble fragment of TGFa-H1, i.e., without the transmembrane portion. Like TGFa, the soluble form of TGF~-H1 is released from a larger amino acid integral membrane glycoprotein precursor via proteolytic cleavage. Derynck, R., Mol. Repro. and Dev., 27:3-9 (1990). ~Derynck, Mol. Reprod. Devel., 27:3-9 (1990)).
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-strar.ded 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 or that of the deposited clone O S~tEE~

W O 95/24466 2 1 ~ 4 ~ ~ 3 PCTrUS94/05476 or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, msture polypeptide as the DNA of Figure 1 or the deposited cDNA.
- The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by - the deposited cDNA or a soluble fragment thereof may include:
only the coding sequence for the mature polypeptide or a soluble form thereof; the coding sequence for the mature polypeptide or a soluble form thereof and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide or soluble form thereof (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 or the polypeptide encoded by the cDNA of the deposited clone or soluble form thereof. 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 encoding the same mature polypeptide or soluble form thereof as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide 21 ~45~3 variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a po~lypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional S' 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 remains.
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 21 ~C`~3 w095/2~66 PCT~Ss4/os476 _g_ (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95~ and preferably at least 97~ identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.
The deposit(s) referred to herein will be maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure.
These deposits are provided merely as a convenience and are not an admission that a deposit is required under 35 U.S.C. 112.
The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
The present invention further relates to a TGF~-H1 polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure l or that encoded by the W095/24466 PCT~S94/05476 deposited cDNA, 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 recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA 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 i6 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 se~uence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term isolated" means that the material is removed from its original environment (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 W095/24466 ~1 8 4 5 ~ 3 PCT~S94/05476 of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the TGF~-H1 gene. 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 polynucleotide6 of the pre6ent invention may be employed for producing a polypeptide by recombinant techniques.
Thus, for example, the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmlds; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

W095/24466 2 ~ 8 ~ 5 ~ ~ PCT~S94/05476 The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expres 5 ion vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
The vector containing the appropriate DNA sequence as herein above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Salmonella typhimurium; Streptomyces;
fungal cells, such as yeast; infect cells, such as Drosophila and Sf9; ~nim~l cells such as CH0, COS or Bowes melanoma; 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 recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a Woss/2~66 2 1 38~ S ~ 3 PCT~S94/05476 promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, PQE60, PQE-9 (Qiagen), Pbs, phagescript, PsiX174, Pbluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKR223-3, pKR233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVg3, pBPV, pMSG, pSVL
(Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pRR232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV ;m~eA;ate 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 embodiment, the present invention relates to host cells cont~i n i~g the above-described construct. The host cell can be a higher eukaryotic cell, such as a mA~Alian 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 manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the present invention can be synthetically produced by conventional peptide synthesizers.

W095/24466 2 1 8 4 5 ~ 3 PCT~Sg4/05476 Mature proteins can be expressed in mA~lian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems csn also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expres6ion vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook. et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 19~9), the disclosure of which is hereby incorporated by reference.
Transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will inc~ude origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl 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 assem~led in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

W095/24466 2 1 8 4 5 ~ 3 PCT~S94/0~76 Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden~ and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 ~backbone~ sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means (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 retained 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.
Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian woss/24466 PCT~S94/05476 expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. ~mm~lian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
TGF~-Hl or soluble form thereof is recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography ~e.g., using DNA or nucleotides on a solid support), hydroxyapatite chromatography and lectin chromatography. It i6 preferred to have low concentrations (approximately 0.1-5 mM) of calcium ion present during purification (Price, et al., J. Biol. Chem., 244:917 (1969)). Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-W095/24466 2 1 ~ 4 5 ~ 3 PCT~S94/0~476 glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
The polypeptides of the present invention may be used for characterization of receptors in the EGFR family of EGF
receptors. This family currently includes the EGFR1, EGFR2, EGFR3 and EGFR4 receptors. The EGFR2 receptor is also referred to as erb-2 and thi~ molecule is useful for a variety of diagnostic and therapeutic indications (Prigent, S.A., and Lemoine, N.R., Prog. in Growth Factor Res., 4:1-24 (1992)). The TGFa-H1 polypeptide is likely a ligand for one or more of these receptors as well as for yet unidentified new EGF-type receptors.
Use of the TGFa-H1 polypeptide can assist with the identification, characterization and cloning of such receptors.

The polypeptides of the present invention may also be used for restoration or enhancement of neurological functions ~;mini shed as a result of trauma or other damaging pathologies (such as AIDS dementia, senile dementia, etc). TGF and its homologs have been found to be the most abundant ligand for the EGF/TGFa receptor in most parts of the brain (Kaser, et al., Brain Res Mol Brain Res: 16:316-322, (1992)). There appears to be a widespread distribution of TGF in various regions of the brain in contrast to EGF which is only present in smaller, more discrete aress, suggesting that TGF-alpha might play a physiological role in brain tissues. These numerous receptor sites for TGFa in the brain suggest that TGF has an important utility in promoting normal brain cell differentiation and function. Accordingly, in instances where neurological functioning is ~;m; n; shed, an administration of the polypeptide of the present invention may stimulate the brain and enhance proper physiological functions.
TGFa-Hl or soluble form thereof may also be employed to treat ocular disorders, for example, corneal inflammation. A
variety of experiments have implicated members of the TGFa gene family in such pathologies. A recent paper summarizes some of W095/24466 2 1 8 4 5 8 3 PCT~S94/05476 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 TGFa gene dispiayed corneal inflammation due to an infiltration of leukocytes and other cells to the substantia propria of the eyes.
In addition, the specificity of the TGFa growth factors for their target cells can be exploited as a mechanism to destroy the target cell. For example, TGFa-H1 or soluble forms thereof can be coupled (by a wide variety of methods) to toxic molecules:
for example, radiopharmaceuticals which inactivate target cells.
These growth factor-toxin fusions kill the target cell (and in certain cases neighboring cells by a variety of "bystander"
effects). A recent example of such toxin-fusion genes is published by Mesri, et al., J. Biol. Chem. 268:4853-62 (1993).
In this same manner, TGFa-Hl can be used as an anti-neoplastic compound. For in vivo use, the subject polypeptide may be administered in a variety of ways, including but not limited to, injection, infusion, topically, parenterally, etc.
AAm;n;stration may be in any physiologically acceptable carrier, including phosphate buffered saline, saline, sterilized water, etc. TGFa-Hl and related molecules may also be encapsulated in liposomes and may be conjugated to antibodies which recognize and bind to tumor or cell specific antigens, thereby provided a means for "targeting" cells.
The TGFa-Hl polypeptide fragment may also be used to treat certain kidney disorders, since it has been found that there has been expression of these growth factors in kidney.
Thus, these factors may be necessary for the proper physiological maintenance of this organ.
Treatments may also be related to liver regeneration/liver dysfunction, since TGFa and its homologs and hepatocyte growth factor trigger hepatocyte regeneration after partial hepatectomy and after acute liver cell necrosis (Masuhara, M. et al, Hepatology 16:1241-1249 (1992)).

woss/2~66 2 1 ~ 4 5 ~ 3 PCT~ss4/o5476 -A significant use for TGF~-Hl relates to wound healing.
The compositions of the present invention msy be used 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, and other non-healing (trophic) conditions.
TGF~-H1 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 and will depend on the intended location of application. For application to the skin, a cream or ointment 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 TGFa-Hl containing compositions in bandages and other wound dressings to provide for continuous exposure of the wound to the peptide. Aerosol applications may also find use.
The concentration of TGFc-Hl in the treatment composition is not critical but should be enough to induce epithelial cell proliferation. The compositions may be applied topically to the affected area, typically as eye drops to the eye or as creams, ointments or lotions to the s~in. In the case of the eyes, frequent treatment is desirable, usually being applied at intervals 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.

woss/24466 2 1 8 4 5 ~ ~ PCT~S94/05476 The amount employed of the subject polypeptide will vary with the manner of administration, the employment of other active compounds, and the like, generally being in the range of about 1 ~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 experimental animals to the subject polypeptides or formulations contA;n;ng the subject polypeptides.
The TGFa-Hl or soluble fragment thereof may be used in the modulation of angiogenesis, bone resorption, immune response, and synaptic and neuronal effector functions. TGF~-Hl may also be used in the modulation of the arachidonic acid cascade.
TGFa-~l or soluble fragment thereof may also be used for applications related to terminal differentiation. Many TGFa factors, and their homologs, induce terminal differentiation in their target cells. This property can be exploited in vivo by ~m;n; stering the factor and inducing target cell death. This regimen is under consideration for disorders related to the hyperproliferation of medically undesirable cell types such as cancers and other proliferative disorders (eg inflammation, psoriasis, etc). In addition to in vivo administration, there are a variety of situations where in vitro administration may be warranted. For example, bone marrow can be purged of undesirable cell populations in vitro by treating the cells with growth factors and/or derivatives thereof.
Applications are also related to alopecia, hair loss and to other skin conditions which affect hair follicular development. Several lines of evidence implicate the involvement TGFa growth factors in such conditions. As described above, "knockout' mice engineered to contain a null mutation in the TGFa 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 - -21 ~45~3 W095/24466 PCT~S94/0~76 TGF~ gene locus ~Mann et al, Cell 73:249-261(1993)). Topical or systemic applications of TGFa-H1 or derivatives thereof may be used 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 sy6temic clinical administration of the TGF~-H1 growth factor. This administration can be in the form of gene therapy (see below); or through the administration of peptides or proteins synthesized from recombinant constructs of TGF~-Hl DNA or from peptide chemical synthesis (Woo, et al., Protein Engineering 3:29-37 (1989).
Gene therapy is the expression of the polypeptide of the present invention in vivo.
Thus, for example, cells such as bone marrow cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle cont~;ning RNA encoding for the polypeptide of the present nventlon .
Similarly, cells may be engineered in vivo for expression of the polypeptide in vivo, for example, by procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
These and other methods for administering a polypeptide of the present invention by such methods should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retroviral particle, for example, an adenovirus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

~1 ~45~3 wossl24466 PCT~S94/05476 In an alternative method of gene therapy, administration of the polypeptide may be accomplished through direct injection of naked or encapsulated (e.g. liposomes, etc TGFa-Hl DNA.
The polypeptide of the present invention msy be employed in combination with a suitable pharmaceutical carrier.
Such compositions comprise a therapeutically effective amount of the protein, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptide of the present invention may be employed on conjunction with other therapeutic compounds.
The most effective concentration for inducing or inhibiting the proliferation of target cell populations sensitive to TGFa-Hl can be determined by adding various amounts of TGFa-Hl to the cells and monitoring their responses. In addition, pharmacological substances which enhance or depress the production of TGFa-Hl can be assessed by monitoring the synthesis of TGFa-Hl message or protein by cells treated with the agents of interest.
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.

21 8$5~3 Few chromosome marking reagents based on actual sequence data (repeat polymorphism~s) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the cDNA i5 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~;ning 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 invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in sltU hybridization (FISH) of a cDNA
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clone from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human 21 845&3 W095/24466 PCT~S94/0~76 Chromosomes: a Manual of Basic Techniques. Pergamon 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, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the 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 re601ution and one gene per 20 kb).
Co~rArison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
The present invention is further directed to inhibiting TGFa-H1 in vivo by the use of 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 polypeptide of the present W O 95124466 2 1 8 4 ~ ~ 3 PCTAUS94/05476 invention, is u6ed to de6ign an anti6en6e ~NA oligonucleotide of from 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 Lee et al, Nucl. Acids Res., 6 :3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al, Science, 251: 1360 (1991), thereby preventing tran6cription and the production of TGFa-Hl. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks translation of an mRNA molecule into the TGFa-H1 (antisense -Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, 30ca Raton, FL ( 1988)).
Alternatively, the oligonucleotides described above can be delivered to cells by procedures in the art such that the anti-sense RNA or DNA may be expressed in vivo to inhibit production of TGFa-H1 in the manner described above.
Antisense constructs to TGFa-H1, therefore, may be used in anti-tumor therapy, since a recent study has shown that inhibition of secretion or production of TGFa (or its homologs) by tumor cells in mice causes regression of the tumor. Such inhibitors can be antisense oligonucleotides, monoclonal antibodies, etc. Antisense oligonucleotides prevent production of the growth factor by the cell, whereas antibodies bind to and neutralize 6urface bound or secreted growth factor.
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptide corresponding to a sequence of the present invention or its in vivo receptor can be obtained by direct injection of the 21 ~45~3 polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then be u6ed 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. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the 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).
Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
Antibodies specific to TGFa may be used for cancer diagnosis and therapy, since many types of cancer cells upregulate various members of the TGFa family during the process of neoplasia or hyperplasia. These antibodies bind to and inactivate TGF-Hl. Monoclonal antibodies against TGFa (and/or its family members) are in clinical use for both the diagnosis and therapy of certain disorders including (but 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).

wossl24466 2 1 8 4 5 8 3 PCT~S94/05476 In addition, malignant cells expressing the TGFa-Hl receptor may be detected by using labeled TGFa-Hl or TGFa-Hl-related molecules in a receptor binding assay, or by the use of antibodies to the TGF-Hl receptor itself. Cells may be distinguished in accordance with the presence and density of receptors for TGF~-Hl, thereby providing a means for predicting the susceptibility of such cells to the biological activities of TGFa-Hl.
The present invention is also directed to antagonist/inhibitors of the polypeptides of the present invention. The antagonist/inhibitors are those which inhibit or eliminate the function of the polypeptide.
Thus, for example, antagonist6 bind to a polypeptide of the present invention and inhibits or eliminates its function.
The antagonist, for example, could be an antibody against the polypeptide which binds to the polypeptide or, in some cases, an oligonucleotide. An example of an inhibitor is a small molecule which binds to and occupies the catalytic site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.
Alternatively, antagonists to the polypeptides of the present invention may be employed which bind to the receptors to which a polypeptide of the present invention normally binds. The antagonists may be closely related proteins such that they recognize and bind to the receptor sites of the natural protein, however, they are inactive forms of the polypeptide and thereby prevent the action of TGFa-Hl since receptor sites are occupied.
In these ways, the antagonist/inhibitors may be used therapeutically for the treatment of certain skin disorders, for example, psoriasis. Recent studies have found elevated levels of expression of these growth factors in skin biopsies taken from diseases such as psoriatic lesions (Cook et al Cancer Research -52:3224-3227 ~1992)).

W095/24466 PCT~S94/0~76 The antagonist/inhibitors may also be used diagnostically to detect cancer, since TGFa-Hl may be upregulated by some types of cancer cells and the antagonist can be used in an assay to determine elevated levels of TGFa-Hl. These antagonist/inhibitors can also be used to treat cancer, since they block TGFa-Hl receptor sites on tumors and inhibition of the activity of TGFa or its homologs in mice causes regression of tumors.
The antagonist/inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinabove described.
The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to ~uch examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate understanding of the following examples, certain frequently occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~g of pla~mid or DNA fragment is used with about 2 units of enzyme in about 20 ~1 of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to wossl24466 2 1 8 ~ 5 ~ 3 PCT~S94/05476 _ -29-50 ~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 37C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a poly-acrylamide 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).
"Oligonucleotides~ refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5~ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ~"ligase") per 0.5 ~g of approximately equimolar amounts of the DNA fragments to be ligated.
Unle6s otherwise stated, tran6formation was performed as described in the method of Graham F. and Van der Eb, A., Virol., 52:456-457 (1973).

Example 1 Bacterial Expression and Purification of TGFa-Hl The open reading frame from TGF~-Hl can be removed from the Bluescript-based vector in which it is inserted and placed into a new type of cloning vector called pQE9 ~see below). Thi~
vector can accept BamHI-HindIII fragments. A BamHI-HindIII
compatible restriction fragment can be generated from TGF~-Hl by 21 845~3 Wossl24466 PCTNS~4/05476 using PCR oligonucleotide primers corresponding to the 5l and 3 end of the DNA sequence to synthesize insertion fragments. The 5' oligonucleotide primer has the sequence 5'-ATTCTAGTTGGATCCGATGGACTACAATATCGACCA-3', contains a BamHI
restriction site (underlined) followed by 21 nucleotides of TGFa-H1 coding sequence; the 3' sequence 5'-CTCCCTCAAAGGAAGCTTTTAAGAGC-3~ contains complementary sequences to a naturally occurring HindIII site (underlined) within the 3' untranslated portion of the TGFa-H1 gene. The BamHI and HindIII
sites are compatible with the BamHI and HindIII sites on the bacterial expression vector pQE9 (Qiagen, Inc. 9259 Eton Ave, Chatsworth CA 91311). The plasmid vector encodes antibiotic resistance (Ampt), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His) and restriction enzyme cloning sites. The ligation mixture was then used to transform the E.
coli strain M15/rep4 (available from Qiagen under the trademark ml5/rep4). 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 LB plates containing both Amp and Kan.
Clones cont~;n;ng the desired constructs were grown overnight (O/N) in liquid culture in either LB media supplemented with both Amp (100 ~g/ml) and Kan (25 ~g/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density of 600 (O.D.~) between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of lmM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3-4 hours.
Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCL. After clsrificstion, solubilized TGF~-H1 was purified from this solution by chromatography on a Nickel-Chelate column under wossl24466 2 3 18 4 5 8 3 PCT~Sg4/05476 conditions that allow for tight binding by proteins cont~ining the 6-His tag. (Hochuli, E. et al., Genetic Engineering, Principle & Methods, 12:87-98 Plenum Press, New York (1990)).
TGF~-Hl (95~ pure) was eluted from the column in 6 molar guanidine HCL pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCL, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar gluthatione (oxidized). After incubation in this solution for 12 hours the protein was dialyzed to 50 mmolar sodium phosphate.
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.

21 845~3 - 9 4 ~ J" 76 IP~A/US l~ r f SEQUENCE LISTING

(1) GENE2AL INFORMATION:
(i) APPLICANT: MEISSNER, ET AL.

(ii) TITLE OF INVENTION: Transfonming Growth Factor Alpha - H1 (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCB 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 READA}3LE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COM~u-l~: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US94/05476 (B) FILING DATE: 12 MAY 94 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/208,008 (B) FILING DATE: 08 MAR 94 ~ 1 8~3 (viii) ATTORNEY/AGENT INFORMATION~
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134 (C) REFERENCE/DOCKET NUMBER: 32'QJ0-154 (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: 400 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

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

ATTGGAAACC ACATGCCTTG CCCTGAAAAC CTCAATGGTT ACTGCATCCA TGG~AAATGT 120 GAATTCATCT ATTCTACTCA GAA~GGCTTCT TGTAGATGTG AATCTGGCTA CACTGGACAG 180 CAl~l~ lA TTGCAGCAAT TATTGGAGCT GTACAGATTG CCATCATAGT AGCAATTGTA 300 AM~NDED SHEEr 21 ~4~3 Pcr~s 94/05 4 7~

(2) INFORMATION FOR SEQ ID NO:2: IP ~ /Us 05 ocr ~gg5 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 132 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUEN~E DESCRIPTION: SEQ ID NO:2:
sp Gly Leu Gln Tyr Arg Pro Asp Val Lys Asp Ala Ser Asp Gln 15rg Glu Asp Val Tyr Ile Gly Asn His Met Pro Cys Pro Glu Asn 30eu Asn Gly Tyr Cys Ile His Gly Lys Cys Glu Phe Ile Tyr Ser 45hr Gln Lys Ala Ser Cys Arg Cys Glu Ser Gly Tyr Thr Gly Gln 60is Cys Glu Lys Thr Asp Phe Ser Ile Leu Tyr Val Val Pro Ser 75rg Gln Lys Leu Thr His Val Leu Ile Ala Ala Ile Ile Gly Ala 90al Gln Ile Ala Ile Ile Val Ala Ile Val Met Cys Ile Thr Arg 100 105ys Cys Pro Lys Asn Asn Arg Gly Arg Arg Gln Lys Gln Asn Leu 110 115 120ly His Phe Thr Ser Asp Thr Ser Ser Arg Met Val

(3) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 3288 BASE PAIRS
(B) TYPE: NUCLEIC ACID

,", ;... . .

21 3$~3 .

(C) STRANDEDNESS: SINGLE IP ~ /U~ ~ 5 ~CT J~
(D) TOPOLOGY: LINEAR
.

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

A~ATGTGAAT TCATCTATTC TACTCAGAAG G~ 11~1A GATGTGAATC TGGCTACACT 180 GGACAGCACT GTGAAAAGAC AGACTTTAGT Al-L~l~-lATG TAGTGCCAAG TAGGCAAAAG 240 AACCTAGGTC ATTTTACTTC AGATACGTCA TCCAGAATGG m AAACTGA TGACTTTTAT 420 ATGTACACTG ACCATGTGAT GTACATTTAT TAl~l~l-l-l-l TTTAAAGAAT GGAAATATTT 480 ATTCAGCTAC GACAGTTTTG GA~-l~l-l-lAG TA~`l~-l-l-l~L TTTAl~l-l-l-l TAAATACAGA 600 AATTGCTTTC ACAAATTTGT ACCACATGGT AATTCTAAGA ~-l-l~l-l~-ll-l ACCCATGGAA 660 CTTCCACAAA TGACCACAGG AAATTGACCA AGCATGAACT TAARGAATCG C~-l~lCGKGR 780 AATGGAAAAT GCCATAAAAT CC~lCCAGAT TGAC~l~l-lG RAGCTTTCAC AGTCGCATAG 960 GCAAGAGGAA ATAATGAAGA AAAACAAATT CCGCG`l~GlA ATATTCCAGG AGAAGTTTCG 1140 ~l~lCCGACA 'lCC~ -lG 'l-l ~h l-lAAAG ACAGAAACCT AACTGAGAAC CAAGAAGAGG 1200 ATGATGATG ATAL~-l-l-LGGA TCCCCCAGTA GGAL--l-LGlC TTCGGATGAA GATTATTATG 1260 TTGAAGAAA GCAGGTCTTGC CAGG~-l-lAGG AAGTCCGGCC AGGAGCCCCT TGATAATATC 1320 A m CTAAT GCAG~-lCC~-lC AAAGGAAGCT m AAGATGC GCAGCCTCAG GAAAGGTAAG 1560 SCAGGAGCG AGTC-llCTKGG SCCCATCAGK GAGCTCTWCT CTGATGAGTC ARTGACCAAA 1680 RCCTTTAAA AGTTACTTTTA AATCTCAGGT GAAAGTAGAG GATGATGAAT ~ l-l-lGGT 1800 TAGA m AA AGCACTCATCG TAAAGAGGGA ATTAAGTATA TCCTAAATAT GAATCTCCTA 1860 ATCATGCAG TTTTAG m GA ATAGTGTAGT CGTCYACATT TCTGTGCCAT GTAGGAAAAC 1920 ATAAATGTA Al-l-l-l-l-l-lCTT ATA m AAAA TCTTGAAGAT AATATAAATA TTATTATCAC 1980 21 ~45~3 PCT,'U~ 94~05~

T~ -lCA TGGCAGCTGTG GAl-l-l-l-l-lAG TTCCll-lCTC TTGTCCACCA GAAAAATAGT 2040 TTCCTAGGT TGGGCCAGTTA C~l~l-l-lGGT AAGGGCAACT TTGCGSCCGT CATTTGCAGG 2100 ACCCCTTTT TTGATG~-l-l-lC ACATTAAAAA ATTGAAGTTT TGGACTTGAG CATTTGGCTC 2280 TAGTATCAT AGCTTTACTTA AAAGAAAACC CTGGSCAAGT CATCATCTGC TTATTCTCA~ 2340 A~l-lC~-lCC CAAACAGAATG GAAAGAGTTA CACCTTCTGA WAAGCCCTCA GCACCAATCA 2520 GTAAGGTCC TAGGTTGGAGA GAAACTAAAG ~-lG~l~-l-lCA GAARCCTTTT CACAGAATCA 2580 AGAGTGAAA AATAAGTAAAT GTTTGGGTGW CCA~-l-l-l-l-lC ATCAGACTAA CTATATCTTG 2640 GGTTTTAGT TGGGTCCAAAT GTTCCCCAGC CAGACC~-l-l-l CTAATTTCCT TTTGATTAAG 2700 Al~-l-l-lG~l GGACTATAGCR CNTAAATTTG m AAGCRGT ATGRGGCATA AAATTGTGAC 2760 TAl~l-l-l~-l AAAGTCGGCCC TGATGCATTG GGTTTGGAAA TGACCACAAA TATTCCTGTT 2820 ACCTCCTTT GGGAGGAAGCC AAGATTCTCC TTAl~-l-l-l-lR GCTTTAAGRT CCGTGGARTC 2940 TACTGGGCP TGGTGGCATGC GC~1~1CC~A GCTACTCRGG AGGCTGAGAC AGGRGAATTG 3180 CCAAAGAGC CRGA -l~-l~l-l TCCAAAAAAA AAiUUUUUAAA AACTCGAG 3288 4J~IENDED SHEE~

Claims (23)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide encoding for TGF.alpha.-H1, said polynucleotide selected from the groups consisting of (a) a polynucleotide encoding for the TGF.alpha.-H1 polypeptide having the deduced amino acid sequence of Figure 1 or an active fragment, analog or derivative of said polypeptide;
(b) a polynucleotide encoding for the TGF.alpha.-H1 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75698 or an active fragment analog or derivative of said polypeptide.

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

3. The polynucleotide of Claim 1 wherein the polynucleotide encodes for a soluble fragment of TGF.alpha.-H1.

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

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

6. The polynucleotide of Claim 2 wherein said polynucleotide encodes for TGF.alpha.-H1 having the deduced amino acid sequence of Figure 1.

7. The polynucleotide of Claim 2 wherein said polynucleotide encodes for the TGF.alpha.-H1 polypeptide encoded by the cDNA of ATCC Deposit No. 75698.

8. The polynucleotide of Claim 1 having the coding sequence for TGF.alpha.-H1 as shown in Figure 1.

9. The polynucleotide of Claim 2 having the coding sequence for TGF.alpha.-H1 deposited as ATCC Deposit No. 75698.

10. A vector containing the DNA of Claim 2.

11. A host cell genetically engineered with the vector of Claim 10.

12. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 11 the polypeptide encoded by said DNA.

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

14. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having TGF.alpha.-H1 activity.

15. A polypeptide selected from the group consisting of (i) a TGF.alpha.-H1 polypeptide having the deduced amino acid sequence of Figure 1 and active fragments, analogs and derivatives thereof and (ii) a TGF.alpha.-H1 polypeptide encoded by the cDNA of ATCC Deposit No. 75698 and active fragments, analogs and derivatives of said polypeptide.

16. The polypeptide of Claim 15 wherein the polypeptide is TGF.alpha.-H1 having the deduced amino acid sequence of Figure 1.

17. An antibody against the polypeptide of claim 15.

18. An antagonist/inhibitor against the polypeptide of claim 15.

19. A method for the treatment of a patient having need of TGF.alpha.-H1 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 15.

20. A method for the treatment of a patient having need to inhibit TGF.alpha.-H1 comprising: administering to the patient a therapeutically effective amount of the antagonist/inhibitor of Claim 18.

21. A pharmaceutical composition comprising the polypeptide of Claim 15 and a pharmaceutically acceptable carrier.

22. The polypeptide of claim 15 wherein the polypeptide is a soluble fragment of TGF.alpha.-H1.

23. The method of Claim 19 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.