WO1996039497A1 - Transforming growth factor alpha hi - Google Patents

Transforming growth factor alpha hi Download PDF

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Publication number
WO1996039497A1
WO1996039497A1 PCT/US1996/009448 US9609448W WO9639497A1 WO 1996039497 A1 WO1996039497 A1 WO 1996039497A1 US 9609448 W US9609448 W US 9609448W WO 9639497 A1 WO9639497 A1 WO 9639497A1
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Prior art keywords
polypeptide
polynucleotide
tgfα
dna
compound
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PCT/US1996/009448
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French (fr)
Inventor
Paul S. Meissner
Rebecca A. Fuldner
Ying Fei Wei
Mark D. Adams
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Human Genome Sciences, Inc.
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Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to AU62604/96A priority Critical patent/AU717005B2/en
Priority to JP50176497A priority patent/JP2001517064A/en
Priority to EP96921363A priority patent/EP0856050A4/en
Publication of WO1996039497A1 publication Critical patent/WO1996039497A1/en
Priority to MXPA/A/1997/009237A priority patent/MXPA97009237A/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • 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.
  • the polypeptide of the present invention has been putatively identified as a human transforming growth factor alpha homolog. More particularly, the polypeptide of the present invention has been putatively identified as transforming growth factor alpha HII, sometimes hereafter referred to as "TGF ⁇ -HI" .
  • the invention also relates to inhibiting the action of such polypeptides.
  • Cellular growth and differentiation appear to be initiated, promoted, maintained and regulated by a multiplicity of stimulatory, inhibitory and synergistic factors and hormones.
  • the alteration and/or breakdown of the cellular homeostasis mechanism seems to be a fundamental cause of growth related diseases, including neoplasia.
  • Growth modular 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 (EGF) , transforming growth factor alpha (TGF ⁇ ) , betacellulin, amphiregulin, and vaccinia growth factor among other factors are growth and differentiation modulatory 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.
  • peptide growth factors influence wound cells through autocrine and paracrine mechanisms. They also play important roles in normal wound healing 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 activity in the receptor's cytoplasmic domain (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 GI tract. These peptide growth factors are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. These growth factors have also been implicated in both the stimulation of growth and differentiation of certain cells, for example, neoplasia, and the inhibition of other types of cells.
  • Betacellulin i ⁇ a 32-kilodalton glycoprotein that appears to be processed from a larger transmembrane precursor by proteolytic cleavage.
  • the carboxyl-terminal domain of betacellulin has 50% sequence similarity with that of rat transforming growth factor a .
  • Betacellulin 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 inhibitory 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.
  • 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 members of TGF ⁇ is synthesized by a number of oncogenically transformed fibroblasts (Ciardiello et al., .Cell.Bioche .. 42:45-57 (1990)) , as well as by a variety of tumors, including renal, breast and squamous carcinomas, melanomas and glioblastoma ⁇ (Derynck, R. et al. , Cancer Res.. 47:707-712 (1987)) .
  • 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 animals 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 macrophage ⁇ . Accordingly, TGF ⁇ is an important factor in controlling growth of epithelial cells and has a role in wound healing.
  • 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 as transforming growth factor TGF ⁇ -HI. Thi ⁇ identification has been made as a re ⁇ ult of amino acid sequence homology to human TGF ⁇ .
  • novel mature polypeptide as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • the polypeptide ⁇ of the present invention are of human origin.
  • nucleic acid molecules encoding the polypeptides of the pre ⁇ ent invention, including mRNAs, DNAs, cDNAs, genomic DNAs a ⁇ well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
  • proces ⁇ es for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention.
  • processes for utilizing such polypeptides, or polynucleotides encoding ⁇ uch polypeptides for therapeutic purposes for example, to stimulate wound healing to restore normal neurological functioning after trauma or AIDS dementia, to treat ocular disorder ⁇ , to target certain cell ⁇ , to treat kidney and liver disorder ⁇ and to promote hair follicular development, to ⁇ timulate angiogenesis for the treatment of burns, ulcers and corneal incisions and to ⁇ timulate embryogenesis.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid ⁇ equences of the present invention.
  • antibodies against such polypeptides there are provided antibodies against such polypeptides.
  • agonist ⁇ to the polypeptide of the present invention there are provided.
  • antagonist ⁇ to such polypeptide ⁇ which may be used to inhibit the action of such polypeptides, for example, in the treatment of corneal inflammation, neoplasia, for example, tumors and cancers and for psoriasis.
  • diagnostic assays for detecting diseases related to overexpres ⁇ ion of the polypeptide of the present invention and mutations in the nucleic acid sequences encoding such polypeptide are provided.
  • Figure 1 depicts the cDNA sequence in corresponding deduced amino acid sequence of TGF ⁇ -HI.
  • the standard one letter abbreviations for amino acids are used.
  • the putative signal sequence has been underlined and the putative soluble portion ha ⁇ been double underlined.
  • Figure 2 is an illustration of comparative amino acid sequence homology between human amphiregulin, human betacellulin, human epidermal growth factor, human heregulin and TGF ⁇ -HI (fifth row) . Shaded areas denotes the conserved EGF motif which i ⁇ shown to be conserved in the polypeptide of the present invention.
  • nucleic acid which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID N0:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75698 on March 4, 1994.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained from human brain and early stage brain tissue.
  • the polynucleotide of thi ⁇ invention wa ⁇ di ⁇ covered in a cDNA library derived from eight-week old embryo. It is structurally related to the TGF ⁇ gene family. It contains an open reading frame encoding a polypeptide of 380 amino acids, which exhibits significant homology to a number of members of the TGF ⁇ gene family; the ⁇ e members include TGF ⁇ itself a ⁇ well a ⁇ other members such as amphiregulin and cripto. Furthermore, the six cysteine residue ⁇ occurring in all member ⁇ in a characteristic motif are conserved in TGF ⁇ -HI.
  • the full-length polypeptide of the present invention as set forth in Figure 1 has a putative signal sequence which comprise ⁇ amino acid 1 through amino acid 39 of Figure 1 (SEQ ID NO:2) which aids in secretion of the polypeptide from the cell.
  • the polypeptide i ⁇ further processed wherein amino acid 40 through amino acid 266 of Figure 1 (SEQ ID NO:2) are cleaved from the polypeptide since this stretch of amino acids i ⁇ a putative precursor sequence.
  • amino acid 317 through amino acid 380 represents a putative transmembrane portion which is thought to be necessary to direct the polypeptide to particular target location ⁇ for the carrying out of biological functions as hereinafter described.
  • the transmembrane portion may also be cleaved from the polypeptide ⁇ uch that the putative ⁇ oluble portion of the polypeptide of the pre ⁇ ent invention comprises amino acid 267 through amino acid 316 of Figure 1 (SEQ ID NO:2) .
  • the polynucleotide of the pre ⁇ ent 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- ⁇ tranded or ⁇ ingle- ⁇ tranded, and if ⁇ ingle ⁇ tranded may be the coding strand or non-coding (anti- ⁇ en ⁇ e) strand.
  • the coding sequence which encode ⁇ the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID N0:1) or that of the deposited clone 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 mature polypeptide as the DNA of Figure 1 (SEQ ID N0:1) or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include, but is not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or ⁇ ecretory sequence or a proprotein ⁇ equence; 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.
  • 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 relate ⁇ to variant ⁇ of the hereinabove described polynucleotide ⁇ which encode for fragment ⁇ , analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non- naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) 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 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.
  • nucleotide variant ⁇ include deletion variant ⁇ , ⁇ ubstitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID N0:1) or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide ⁇ equence 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 polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode for a proprotein which i ⁇ the mature protein plu ⁇ additional 5' amino acid residues.
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the pro ⁇ equence i ⁇ cleaved an active mature protein remains.
  • 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 pre ⁇ ent invention may al ⁇ o 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- hi ⁇ tidine tag ⁇ upplied by a pQE-9 vector to provide for purification of the mature polypeptide fu ⁇ ed to the marker in the case of a bacterial ho ⁇ t, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. , Cell, 37:767 (1984)) .
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (intron ⁇ ) between individual coding ⁇ egment ⁇ (exons) .
  • Fragments of the full length TGF ⁇ -HI gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity.
  • Probe ⁇ of thi ⁇ type preferably have at least 30 ba ⁇ es and may contain, for example, 50 or more bases.
  • the probe may al ⁇ o be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete TGF ⁇ -HI gene including regulatory and promotor regions, exons, and introns.
  • An example of a screen comprises isolating the coding region of the gene by u ⁇ ing the known DNA ⁇ equence to synthesize an oligonucleotide probe.
  • Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the present invention further relates to polynucleotide ⁇ 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 hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequence ⁇ .
  • polypeptides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain sub ⁇ tantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:l) or the depo ⁇ ited cDNA( ⁇ ) .
  • the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, a ⁇ hereinabove described, and which may or may not retain activity.
  • such polynucleotide ⁇ may be employed as probes for the polynucleotide of SEQ ID NO:l, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • the present invention is directed to polynucleotide ⁇ having at lea ⁇ t a 70% identity, preferably at lea ⁇ t 90% and more preferably at lea ⁇ t a 95% identity to a polynucleotide which encode ⁇ the polypeptide of SEQ ID NO:2 a ⁇ well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptide ⁇ encoded by ⁇ uch polynucleotides.
  • the deposit ( ⁇ ) referred to herein will be maintained under the term ⁇ of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposit ⁇ are provided merely as convenience to those of skill in the art and are not an admi ⁇ sion that a deposit i ⁇ required under 35 U.S.C. ⁇ 112.
  • the ⁇ equence of the polynucleotide ⁇ contained in the deposited materials, a ⁇ well a ⁇ the amino acid ⁇ equence of the polypeptide ⁇ encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any de ⁇ cription of sequence ⁇ herein.
  • a licen ⁇ e may be required to make, u ⁇ e or ⁇ ell the depo ⁇ ited materials, and no such license is hereby granted.
  • the pre ⁇ ent invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or which ha ⁇ the amino acid ⁇ equence encoded by the deposited cDNA, as well as fragments, analogs and derivative ⁇ of ⁇ uch polypeptide.
  • fragment when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the depo ⁇ ited cDNA, mean ⁇ a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • 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 (SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid re ⁇ idues are ⁇ ubstituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid re ⁇ idue) and ⁇ uch ⁇ ub ⁇ tituted amino acid re ⁇ idue 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 fu ⁇ ed with another compound, ⁇ uch 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 i ⁇ employed for purification of the mature polypeptide or
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting material ⁇ in the natural ⁇ y ⁇ te , i ⁇ i ⁇ olated.
  • Such polynucleotide ⁇ could be part of a vector and/or ⁇ uch polynucleotide ⁇ or polypeptide ⁇ could be part of a compo ⁇ ition, and still be i ⁇ olated in that such vector or composition is not part of its natural environment.
  • polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well a ⁇ polypeptide ⁇ which have at lea ⁇ t 70% ⁇ imilarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at lea ⁇ t 95% ⁇ imilarity ( ⁇ till more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthe ⁇ i ⁇ ; therefore, the fragment ⁇ may be employed as intermediates for producing the full-length polypeptides.
  • Fragments or portions of the polynucleotides of the pre ⁇ ent invention may be u ⁇ ed to ⁇ ynthe ⁇ ize full-length polynucleotide ⁇ of the pre ⁇ ent invention.
  • the pre ⁇ ent 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 polypeptide ⁇ of the invention by recombinant technique ⁇ .
  • Ho ⁇ t 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 expre ⁇ ion vector.
  • the vector may be, for example, in the form of a pla ⁇ mid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified a ⁇ appropriate for activating promoter ⁇ , ⁇ electing tran ⁇ formant ⁇ or amplifying the gene ⁇ of the present invention.
  • the culture conditions, ⁇ uch 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 may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expre ⁇ ion vector ⁇ for expre ⁇ ing a polypeptide.
  • Such vector ⁇ include chromosomal, nonchromosomal and ⁇ ynthetic DNA ⁇ equence ⁇ , e.g., derivative ⁇ of SV40; bacterial pla ⁇ mids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such a ⁇ vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • 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.
  • the DNA sequence is inserted into an appropriate restriction endonuclea ⁇ e ⁇ ite( ⁇ ) 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 ⁇ equence in the expression vector is operatively linked to an appropriate expres ⁇ ion control sequence( ⁇ ) (promoter) to direct mRNA ⁇ ynthesis.
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoters known to control expres ⁇ ion of gene ⁇ in prokaryotic or eukaryotic cell ⁇ or their viruses.
  • the expression vector also contains a ribosome binding ⁇ ite for tran ⁇ lation initiation and a tran ⁇ cription terminator.
  • the vector may also include appropriate sequence ⁇ for amplifying expression.
  • the expres ⁇ ion vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reducta ⁇ e or neomycin resistance for eukaryotic cell culture, or such a ⁇ tetracycline or ampicillin re ⁇ istance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well a ⁇ an appropriate promoter or control ⁇ equence, may be employed to tran ⁇ form an appropriate ho ⁇ t to permit the ho ⁇ t to expre ⁇ the protein.
  • ho ⁇ t ⁇ As repre ⁇ entative example ⁇ of appropriate ho ⁇ t ⁇ , there may be mentioned: bacterial cells, such a ⁇ E. coli. Streptomvce ⁇ . Salmonella tvphimurium; fungal cell ⁇ , ⁇ uch as yeast; insect cells ⁇ uch as Dro ⁇ ophila S2 and Spodoptera Sf9; animal cell ⁇ such a ⁇ CHO, COS or Bowes melanoma; adenoviruses; plant cell ⁇ , etc.
  • bacterial cells such as a ⁇ E. coli. Streptomvce ⁇ . Salmonella tvphimurium
  • fungal cell ⁇ ⁇ uch as yeast
  • insect cells ⁇ uch as Dro ⁇ ophila S2 and Spodoptera Sf9
  • animal cell ⁇ such a ⁇ CHO, COS or Bowes melanoma
  • adenoviruses adenoviruses
  • the present invention also includes recombinant constructs comprising one or more of the sequence ⁇ a ⁇ broadly de ⁇ cribed above.
  • the constructs compri ⁇ e a vector, ⁇ uch a ⁇ a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phage ⁇ cript, psiX174, pbluescript SK, pb ⁇ ks, pNH8A, pNH16a, pNHl ⁇ A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, p K233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other plasmid or vector may be used as long as they are replicable and viable in the ho ⁇ t.
  • Promoter regions can be selected from any desired gene u ⁇ ing CAT (chloramphenicol tran ⁇ ferase) vectors or other vectors with selectable markers.
  • Two appropriate vector ⁇ are pKK232-8 and pCM7.
  • Particular named bacterial promoter ⁇ include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoter ⁇ include CMV immediate early, HSV thymidine kina ⁇ e, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter i ⁇ well within the level of ordinary ⁇ kill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the ho ⁇ t cell can be a higher eukaryotic cell, ⁇ uch as a mammalian 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 Method ⁇ in Molecular Biology, (1986) ) .
  • the con ⁇ truct ⁇ in host cell ⁇ can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cell ⁇ under the control of appropriate promoter ⁇ .
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA con ⁇ truct ⁇ of the pre ⁇ ent invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic host ⁇ are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. , (1989), the disclosure of which i ⁇ hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its tran ⁇ cription.
  • Example ⁇ including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancer ⁇ .
  • recombinant expre ⁇ ion vectors will include origin ⁇ of replication and ⁇ electable 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-expre ⁇ ed gene to direct transcription of a downstream structural sequence.
  • Such promoters can be derived from operon ⁇ encoding glycolytic enzymes such as 3-pho ⁇ phoglycerate kina ⁇ e (PGK) , ⁇ -factor, acid pho ⁇ phata ⁇ e, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled 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.
  • the heterologous sequence can encode a fu ⁇ ion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expres ⁇ ed recombinant product.
  • U ⁇ eful expre ⁇ sion vector ⁇ for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signal ⁇ in operable reading pha ⁇ e with a functional promoter.
  • the vector will compri ⁇ e one or more phenotypic selectable markers and an origin of replication to en ⁇ ure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis, Salmonella tvphimurium and various specie ⁇ within the genera Pseudomona ⁇ , Streptomyces, and Staphylococcu ⁇ , although other ⁇ may al ⁇ o be employed a ⁇ a matter of choice.
  • 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) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemical ⁇ , Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expre ⁇ ed.
  • the selected promoter is induced by appropriate means (e.g., temperature ⁇ hift or chemical induction) and cells are cultured for an additional period.
  • appropriate means e.g., temperature ⁇ hift or chemical induction
  • Cell ⁇ are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expre ⁇ ion of protein ⁇ can be di ⁇ rupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell ly ⁇ ing agents, such methods are well known to those skilled in the art.
  • mammalian cell culture system ⁇ can al ⁇ o be employed to expre ⁇ recombinant protein.
  • Example ⁇ of mammalian expression sy ⁇ tem ⁇ include the COS-7 lines of monkey kidney fibroblast ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981) , and other cell line ⁇ capable of expre ⁇ ing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell line ⁇ .
  • Mammalian expression vectors will compri ⁇ e an origin of replication, a ⁇ uitable promoter and enhancer, and al ⁇ o any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor site ⁇ , tran ⁇ criptional termination sequences, and 5' flanking nontranscribed ⁇ equence ⁇ .
  • DNA ⁇ equence ⁇ derived from the SV40 splice, and polyadenylation ⁇ ites may be used to provide the required nontranscribed genetic elements.
  • the polypeptide ⁇ can be recovered and purified from recombinant cell culture ⁇ by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulo ⁇ e chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • 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 ho ⁇ t (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic ho ⁇ t for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptide ⁇ of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may al ⁇ o include an initial methionine amino acid re ⁇ idue.
  • polypeptides and polypeptides of the present invention may be employed a ⁇ re ⁇ earch reagent ⁇ and materials for discovery of treatment ⁇ and diagno ⁇ tic ⁇ for human di ⁇ ea ⁇ e.
  • the polypeptide of the present invention may be employed for characterization of receptors.
  • the EGF family receptor ⁇ currently include ⁇ four EGF receptor ⁇ , denoted as EGFR1, EGFR2, EGFR3 and EGFR4.
  • the EGFR2 receptor may also be referred to as ERB-2 and this molecule i ⁇ u ⁇ eful for a variety of diagno ⁇ tic and therapeutic indication ⁇ (Prigent, S.A., and Lemoine, N.R. , Prog Growth Factor Res.. 4:1-24 (1992)) .
  • the TGF ⁇ -HI polypeptide is likely a ligand for one or more of these receptors a ⁇ well as for yet an identified new EGF-type receptor.
  • the EGF receptor gene represent ⁇ the cellular homolog of the v-erb-B oncogene of avian erythroblastosis virus.
  • Over expression of the EGF-receptor or deletion of kinase regulatory segment ⁇ 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 enhancement of neurological functions diminished a ⁇ a re ⁇ ult of trauma or other damaging pathologie ⁇ ( ⁇ uch a ⁇ AIDS dementia, ⁇ enile dementia, etc) .
  • TGF ⁇ and its homologs have been found to be the most abundant ligand for the EGF/TGF ⁇ receptor in most parts of the brain (Kaser, et al., Brain Res Mol Brain Res: 16:316-322, (1992)) .
  • TGF ⁇ and its homologs
  • TGF ⁇ has an important utility in promoting normal brain cell differentiation and function. Accordingly, in in ⁇ tance ⁇ where neurological functioning is diminished, an administration of the polypeptide of the pre ⁇ ent invention may stimulate the brain and enhance proper physiological functions.
  • TGF ⁇ -HI or soluble form thereof may al ⁇ o be employed to treat ocular disorders, for example, corneal inflammation.
  • ocular disorders for example, corneal inflammation.
  • a variety of experiments have implicated members of the TGF ⁇ 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 cell ⁇ to the ⁇ ubstantialitis of the eyes.
  • TGF ⁇ -HI or ⁇ oluble form ⁇ thereof can be coupled (by a wide variety of methods) to toxic molecules: for example, a radiopharmaceutical which inactivate target cells.
  • toxic molecules for example, a radiopharmaceutical which inactivate target cells.
  • These growth factor-toxin fusions kill the target cell (and in certain case ⁇ neighboring cells by a variety of "bystander” effects) .
  • a recent example of such toxin-fusion genes is published by Me ⁇ ri, et al. , J. Biol. Chem. 268:4853-62 (1993) .
  • TGF ⁇ -HI and related molecule ⁇ may al ⁇ o be encap ⁇ ulated in lipo ⁇ ome ⁇ and may be conjugated to antibodie ⁇ which recognize and bind to tumor or cell ⁇ pecific antigens, thereby provided a means for "targeting" cells.
  • TGF ⁇ -HI can be employed a ⁇ an anti- neopla ⁇ tic compound, ⁇ ince member ⁇ of the EGF family show anti-proliferative effects on transformed cells.
  • the subject polypeptide may be administered in a variety of ways, including but not limited to, injection, infusion, topically, parenterally, etc. Administration may be in any physiologically acceptable carrier, including pho ⁇ phate buffered ⁇ aline, ⁇ aline, ⁇ terilized water, etc.
  • T he TGF ⁇ -HI polypeptide fragment may also be employed to treat certain kidney disorder ⁇ , since it has been found that there ha ⁇ been expre ⁇ sion of these growth factor ⁇ in the kidney. Thu ⁇ , the ⁇ e factor ⁇ may be neces ⁇ ary for the proper phy ⁇ iological maintenance of thi ⁇ organ.
  • Treatments may also be related to liver regeneration or liver dysfunction, ⁇ ince TGF ⁇ and it ⁇ homolog ⁇ and hepatocyte growth factor trigger hepatocyte regeneration after partial hepatectomy and after acute liver cell necro ⁇ i ⁇ (Masuhara, M. et al, Hepatology 16:1241-1249 (1992)) .
  • a significant treatment involving TGF ⁇ -HI relates to wound healing.
  • the compositions of the present invention may be employed for treating a wide variety of wound ⁇ including ⁇ ub ⁇ tantially all cutaneou ⁇ wound ⁇ , corneal wound ⁇ , and injurie ⁇ to the epithelial-lined hollow organs of the body.
  • Wounds ⁇ uitable for treatment include those resulting from trauma such as burns, abrasions and cuts, as well as from surgical procedure ⁇ ⁇ uch a ⁇ ⁇ urgical 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 ⁇ -HI 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.
  • a cream or ointment base is usually preferred; suitable ba ⁇ es include lanolin, Silvadene (Marion) (particularly for the treatment of burns) , Aquaphor (Duke Laboratories, South Norwalk, Conn.), and the like.
  • it will be pos ⁇ ible to incorporate TGF ⁇ -HI containing compo ⁇ ition ⁇ in bandage ⁇ and other wound dre ⁇ sings to provide for continuous exposure of the wound to the peptide. Aerosol applications may also find use.
  • the concentration of TGF ⁇ -HI 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 a ⁇ cream ⁇ , ointment ⁇ or lotions to the skin. In the ca ⁇ e of the eye ⁇ , frequent treatment i ⁇ desirable, usually being applied at intervals of 4 hours or le ⁇ . On the ⁇ kin, 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 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 a ⁇ saline, phosphate-buffered ⁇ aline, or the like.
  • the amount of compound employed will be determined empirically, based on the respon ⁇ e of cell ⁇ in vitro and response of experimental animals to the subject polypeptides or formulations containing the subject polypeptide ⁇ .
  • TGF ⁇ -HI or ⁇ oluble fragment thereof may be employed in the modulation of angiogenesis, bone resorption, immune re ⁇ pon ⁇ e, and ⁇ ynaptic and neuronal effector functions.
  • TGF ⁇ -HI may also be used in the modulation of the arachidonic acid cascade.
  • TGF ⁇ -HI or ⁇ oluble fragment thereof may al ⁇ o be employed for application ⁇ related to terminal differentiation.
  • Many TGF ⁇ factor ⁇ , and their homolog ⁇ induce terminal differentiation in their target cell ⁇ . This property can be exploited in vivo by administering the factor and inducing target cell death.
  • This regimen is under con ⁇ ideration for di ⁇ orders related to the hyper-proliferation of medically undesirable cell types such a ⁇ cancers and other proliferative di ⁇ order ⁇ (eg inflammation, p ⁇ oria ⁇ is, etc) .
  • ⁇ ituation ⁇ where in vitro admini ⁇ tration may be warranted.
  • bone marrow can be purged of unde ⁇ irable cell populations in vitro by treating the cell ⁇ with growth factors and/or derivatives thereof.
  • Certain di ⁇ ea ⁇ e pathologies may be partially or completely ameliorated by the systemic clinical administration of the TGF ⁇ -HI growth factor.
  • This administration can be in the form of gene therapy (see below) ; or through the admini ⁇ tration of peptides or proteins synthesized from recombinant constructs of TGF ⁇ -HI DNA or from peptide chemical synthe ⁇ i ⁇ (Woo, et al., Protein Engineering 3:29-37 (1989) .
  • Thi ⁇ invention provide ⁇ a method of screening compounds to identify agonist or antagoni ⁇ t compounds to the polypeptide of the present invention.
  • a mammalian cell or membrane preparation expressing a TGF ⁇ -HI receptor i ⁇ incubated with a potential compound and the ability of the compound to generate a second signal from the receptor is measured to determine if it is an effective agonist.
  • second me ⁇ enger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoino ⁇ itide hydrolysis.
  • Effective antagonists are determined by the method above wherein an antagonist compound is detected which binds to the receptor but does not elicit a ⁇ econd messenger respon ⁇ e to thereby block the receptor from TGF ⁇ -HI.
  • Another a ⁇ say for identifying potential antagoni ⁇ t ⁇ ⁇ pecific to the receptor ⁇ to the polypeptide of the present invention i ⁇ a competition assay which compri ⁇ es isolating plasma membranes which over expre ⁇ a receptor to the polypeptide of the present invention, for example, human A431 carcinoma cell ⁇ .
  • Serially diluted test sample in a medium (volume i ⁇ approximately 10 microliters) containing 10 nM 125 I-TGF ⁇ -HI i ⁇ added to five microgram ⁇ of the pla ⁇ ma membrane in the presence of the potential antagonist compound and incubated for 4 hour ⁇ at 4°C.
  • the reaction mixture ⁇ are diluted and immediately passed through a millipore filter.
  • the filters are then rapidly washed and the bound radioactivity is measured in a gamma counter.
  • the amount of bound TGF ⁇ -HI is then measured.
  • a control as ⁇ ay i ⁇ al ⁇ o performed in the ab ⁇ ence of the compound to determine if the antagoni ⁇ t ⁇ reduce the amount of bound TGF ⁇ -HI.
  • Potential antagoni ⁇ t compound ⁇ include an antibody, or in ⁇ ome cases, an oligopeptide, which binds to the polypeptide.
  • 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 i ⁇ an anti ⁇ en ⁇ e con ⁇ truct prepared using antisense technology.
  • Antisense technology can be used to control gene expres ⁇ ion through triple-helix formation or anti ⁇ en ⁇ e DNA or RNA, both of which method ⁇ are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptide ⁇ of the present invention is used to de ⁇ ign an anti ⁇ en ⁇ e 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 Lee et al.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and block ⁇ tran ⁇ lation of the mRNA molecule into the polypeptide of the pre ⁇ ent invention (Anti ⁇ en ⁇ e - Okano, J.
  • oligonucleotides a ⁇ Anti ⁇ ense Inhibitors of Gene Expres ⁇ ion, CRC Press, Boca Raton, FL (1988)) .
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expre ⁇ ed in vivo to inhibit production of the polypeptide of the pre ⁇ ent invention.
  • Antagoni ⁇ t 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.
  • the ⁇ mall molecule ⁇ may also bind the receptor to the polypeptide to prevent binding. Examples of small molecules include but are not limited to small peptides or peptide-like molecule ⁇ .
  • the antagonists may be employed to treat neoplasia, for example, cancers and tumor ⁇ . It is known that inhibition of secretion or production of members of the EGF family by tumor cell ⁇ in mice causes regre ⁇ sion of tumors.
  • the antagonists to the polypeptides of the present invention may also be u ⁇ ed therapeutically for the treatment of certain skin di ⁇ order ⁇ , for example, p ⁇ oriasis. Elevated levels of expression of member ⁇ of this family of growth factors in skin biopsie ⁇ taken from di ⁇ ea ⁇ e ⁇ ⁇ uch a ⁇ p ⁇ oriatic lesions have been found to be elevated (Cook, et al., Cancer Research. 52:3224-3227 (1992)) .
  • the antagonists may be employed in a compo ⁇ ition with a pharmaceutically acceptable carrier, e.g., a ⁇ hereinafter described.
  • polypeptides of the present invention or agoni ⁇ t or antagoni ⁇ t compound ⁇ may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier includes but i ⁇ not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • a carrier includes but i ⁇ not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention also provide ⁇ a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ itions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ itions of the invention.
  • Associated with such container(s) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, use or ⁇ ale of pharmaceutical ⁇ or biological products, which notice reflect ⁇ approval by the agency of manufacture, u ⁇ e or ⁇ ale for human admini ⁇ tration.
  • the polypeptide ⁇ or compound ⁇ of the present invention may be employed in conjunction with other therapeutic compound ⁇ .
  • the pharmaceutical compo ⁇ ition ⁇ may be administered in a convenient manner ⁇ uch a ⁇ by the oral, topical, intravenou ⁇ , intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
  • the pharmaceutical compo ⁇ ition ⁇ are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at lea ⁇ t about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in exces ⁇ of about 8 mg/Kg body weight per day. In mo ⁇ t cases, the dosage is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptom ⁇ , etc.
  • 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 i ⁇ often referred to a ⁇ "gene therapy.”
  • cell ⁇ from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cell ⁇ then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • the engineered cell ⁇ then being provided to a patient to be treated with the polypeptide.
  • Such method ⁇ are well-known in the art and are apparent from the teaching ⁇ herein.
  • cells may be engineered by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention.
  • cell ⁇ may be engineered in vivo for expre ⁇ sion of a polypeptide in vivo by, for example, procedures known in the art.
  • a packaging cell is tran ⁇ duced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particle ⁇ containing the gene of intere ⁇ t.
  • the ⁇ e producer cells may be admini ⁇ tered to a patient for engineering cell ⁇ in vivo and expression of the polypeptide in vivo.
  • Retroviruse ⁇ 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 viru ⁇ , gibbon ape leukemia viru ⁇ , human immunodeficiency viru ⁇ , adenoviru ⁇ , Myeloproliferative Sarcoma Viru ⁇ , and mammary tumor viru .
  • the retroviral pla ⁇ mid vector is derived from Moloney Murine Leukemia Virus.
  • the vector include ⁇ one or more promoter ⁇ .
  • Suitable promoter ⁇ which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegaloviru ⁇ (CMV) promoter de ⁇ cribed in Miller, et al., Biotechni ⁇ ue ⁇ . Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoter ⁇ ⁇ uch a ⁇ eukaryotic cellular promoters including, but not limited to, the histone, pol III, and /3-actin promoters) .
  • CMV cytomegaloviru ⁇
  • viral promoter ⁇ which may be employed include, but are not limited to, adenoviru ⁇ promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoter ⁇ .
  • TK thymidine kinase
  • B19 parvovirus promoter ⁇ The ⁇ election of a suitable promoter will be apparent to those skilled in the art from the teaching ⁇ contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such a ⁇ the adenoviral major late promoter; or heterologous promoter ⁇ , ⁇ uch as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such a ⁇ the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoter ⁇ ; viral thymidine kina ⁇ e promoter ⁇ , ⁇ uch a ⁇ the Herpe ⁇ Simplex thymidine kina ⁇ e promoter; retroviral LTR ⁇ (including the modified retroviral LTR ⁇ hereinabove de ⁇ cribed) ; the /3-actin promoter; and human growth
  • CMV cytomegalovirus
  • RSV respiratory syncytial virus
  • inducible promoters such a ⁇ the MMT promoter,
  • Example ⁇ of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , ⁇ -iW, PA12, T19-14X, VT-19-17-H2, ⁇ CKE , ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell line ⁇ as de ⁇ cribed in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990) , which i ⁇ incorporated herein by reference in its entirety.
  • the vector may tran ⁇ duce the packaging cell ⁇ through any mean ⁇ known in the art.
  • retroviral pla ⁇ mid vector may be encap ⁇ ulated 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 ⁇ equence ( ⁇ ) encoding the polypeptide ⁇ .
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo.
  • the tran ⁇ duced eukaryotic cell ⁇ will express the nucleic acid sequence( ⁇ ) encoding the polypeptide.
  • Eukaryotic ⁇ ell ⁇ which may be tran ⁇ duced include, but are not limited to, embryonic stem cell ⁇ , embryonic carcinoma cell ⁇ , a ⁇ well a ⁇ hematopoietic stem cell ⁇ , hepatocyte ⁇ , fibrobla ⁇ t ⁇ , myobla ⁇ t ⁇ , keratinocyte ⁇ , endothelial cells, and bronchial epithelial cells.
  • This invention i ⁇ also related to the use of the gene of the present invention a ⁇ a diagnostic. Detection of a mutated form of the gene of the present invention will allow a diagnosi ⁇ of a disease or a susceptibility to a disea ⁇ e which results from underexpression of the polypeptide of the pre ⁇ ent invention for example, improper wound healing, improper neurological functioning, ocular disorders, kidney and liver disorder ⁇ , hair follicular development, angiogenesis and embryogene ⁇ i .
  • Nucleic acids for diagnosi ⁇ may be obtained from a patient's cells, such a ⁇ from blood, urine, ⁇ aliva, ti ⁇ sue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by u ⁇ ing PCR (Saiki et al . , Nature, 324:163-166 (1986)) prior to analy ⁇ i ⁇ .
  • RNA or cDNA may al ⁇ o be used for the same purpo ⁇ e.
  • PCR primer ⁇ complementary to the nucleic acid encoding 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 mutation ⁇ can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled anti ⁇ ense DNA sequence ⁇ . Perfectly matched sequence ⁇ can be distinguished from mismatched duplexe ⁇ by RNa ⁇ e A dige ⁇ tion or by differences in melting temperatures.
  • Sequence differences between the reference gene and genes having mutation ⁇ may be revealed by the direct DNA sequencing method.
  • cloned DNA segments may be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic ⁇ equencing procedures with fluore ⁇ cent-tag ⁇ .
  • DNA ⁇ equence 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 ⁇ equence ⁇ may be di ⁇ tinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different po ⁇ ition ⁇ according to their ⁇ pecific melting or partial melting temperature ⁇ ( ⁇ ee, e.g., Myer ⁇ et al . , Science, 230:1242 (1985) ) .
  • Sequence change ⁇ at specific locations may al ⁇ o be revealed by nuclease protection assays, ⁇ uch as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)) .
  • the detection of a specific DNA sequence may be achieved by method ⁇ ⁇ uch a ⁇ hybridization, RNa ⁇ e protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphisms (RFLP)
  • RFLP Restriction Fragment Length Polymorphisms
  • mutation ⁇ can also be detected by in situ analysis.
  • the present invention also relates to diagnostic assays for detecting altered levels of the polypeptide of the present invention in variou ⁇ tissue ⁇ ⁇ ince an over-expression of the proteins compared to normal control tis ⁇ ue ⁇ amples can detect the presence of certain disease conditions ⁇ uch a ⁇ neoplasia, skin disorder ⁇ , ocular disorders and inflammation.
  • As ⁇ ay ⁇ u ⁇ ed to detect level ⁇ of the polypeptide of the pre ⁇ ent invention in a ⁇ ample derived from a ho ⁇ t are well- known to tho ⁇ e of ⁇ kill in the art and include radioimmunoa ⁇ ay ⁇ , competitive-binding assays, Western Blot analy ⁇ i ⁇ and preferably an ELISA a ⁇ ay.
  • An ELISA assay initially comprises preparing an antibody specific to an antigen of the polypeptide of the present invention, preferably a monoclonal antibody.
  • a reporter antibody i ⁇ prepared again ⁇ t the monoclonal antibody.
  • a detectable reagent such as radioactivity, fluore ⁇ cence or in thi ⁇ example a horseradish peroxidase enzyme.
  • any free protein binding ⁇ ite ⁇ on the dish are then covered by incubating with a non ⁇ specific protein such a ⁇ bovine serum albumin.
  • a non ⁇ specific protein such as a ⁇ bovine serum albumin.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodie ⁇ attach to any polypeptide ⁇ of the present invention attached to the polystyrene di ⁇ h. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to polypeptides of the pre ⁇ ent invention. Unattached reporter antibody i ⁇ then wa ⁇ hed out.
  • Peroxida ⁇ e ⁇ ub ⁇ trate ⁇ are then added to the di ⁇ h and the amount of color developed in a given time period i ⁇ a measurement of the amount of protein present in a given volume of patient sample when compared again ⁇ t a ⁇ tandard curve.
  • a competition a ⁇ ay may also be employed to determine level ⁇ of the polypeptide of the present invention in a sample derived from the ho ⁇ t ⁇ .
  • Such an assay comprise ⁇ isolating plasma membranes which over-expres ⁇ the receptor for the polypeptide of the pre ⁇ ent invention.
  • a te ⁇ t ⁇ ample containing the polypeptide ⁇ of the present invention which have been labeled, are then added to the plasma membranes and then incubated for a set period of time.
  • Al ⁇ o added to the reaction mixture i ⁇ a sample derived from a host which is su ⁇ pected of containing the polypeptide of the pre ⁇ ent invention.
  • the reaction mixtures are then pas ⁇ ed through a filter which is rapidly washed and the bound radioactivity 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.
  • Antibodie ⁇ ⁇ pecific to TGF ⁇ -HI may be used for cancer diagnosi ⁇ and therapy, ⁇ ince many type ⁇ of cancer cells up ⁇ regulate various members of the TGF ⁇ family during the proces ⁇ of neopla ⁇ ia or hyperpla ⁇ ia.
  • the ⁇ e antibodie ⁇ bind to and inactivate TGF ⁇ -HI.
  • Monoclonal antibodies against TGF ⁇ -HI (and/or its family member ⁇ ) are in clinical use for both the diagnosis and therapy of certain disorders including (but not limited to) hyperplastic and neopla ⁇ tic growth abnormalitie ⁇ .
  • Upregulation of growth f ctor expression by neopla ⁇ tic tissues forms the basis for a variety of serum assay ⁇ which detect increa ⁇ e ⁇ in growth factor in the blood of affected patient ⁇ .
  • serum assay ⁇ which detect increa ⁇ e ⁇ in growth factor in the blood of affected patient ⁇ .
  • malignant cells expressing the TGF ⁇ -HI receptor may be detected by using labeled TGF ⁇ -HI in a receptor binding as ⁇ ay, or by the use of antibodies to the TGF ⁇ -HI receptor it ⁇ elf.
  • Cell ⁇ may be di ⁇ tingui ⁇ hed in accordance with the presence and density of receptors for TGF ⁇ -HI, thereby providing a means for predicting the susceptibility of such cells to the biological activitie ⁇ of TGF ⁇ -HI.
  • the sequences of the present invention are al ⁇ o valuable for chromo ⁇ ome identification.
  • the ⁇ equence i ⁇ ⁇ pecifically targeted to and can hybridize with a particular location on an individual human chromo ⁇ ome.
  • there i ⁇ a current need for identifying particular ⁇ ite ⁇ on the chromo ⁇ ome.
  • Few chromo ⁇ ome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention i ⁇ an important fir ⁇ t step in correlating those ⁇ equence ⁇ with gene ⁇ associated with disease.
  • sequence ⁇ can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysi ⁇ of the 3' untran ⁇ lated region of the gene i ⁇ used to rapidly ⁇ elect primer ⁇ 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 hybrid ⁇ containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of somatic cell hybrids i ⁇ a rapid procedure for a ⁇ igning a particular DNA to a particular chromo ⁇ ome.
  • U ⁇ ing the present invention with the same oligonucleotide primer ⁇ ⁇ ublocalization can be achieved with panels of fragments from specific chromosome ⁇ or pool ⁇ of large genomic clone ⁇ in an analogou ⁇ manner.
  • Other mapping ⁇ trategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow- ⁇ orted chromo ⁇ ome ⁇ and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluore ⁇ cence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one ⁇ tep.
  • Thi ⁇ technique can be used with cDNA as short as 50 or 60 bases.
  • the phy ⁇ ical po ⁇ ition of the sequence on the chromosome can be correlated with genetic map data.
  • genetic map data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins Univer ⁇ ity Welch Medical Library) .
  • the relation ⁇ hip between gene ⁇ and di ⁇ ease ⁇ that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent gene ⁇ ) .
  • a cDNA precisely localized to a chromosomal region a ⁇ ociated with the di ⁇ ease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping re ⁇ olution and one gene per 20 kb) .
  • the polypeptide ⁇ , their fragment ⁇ or other derivatives, or analogs thereof, or cell ⁇ expressing them can be used a ⁇ an immunogen to produce antibodie ⁇ thereto.
  • the ⁇ e antibodie ⁇ can be, for example, polyclonal or monoclonal antibodie ⁇ .
  • the pre ⁇ ent invention al ⁇ o includes chimeric, single chain, and humanized antibodie ⁇ , a ⁇ well a ⁇ Fab fragment ⁇ , or the product of an Fab expre ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be u ⁇ ed for the production of ⁇ uch antibodies and fragments.
  • Antibodies generated against the polypeptide ⁇ corre ⁇ ponding to a sequence of the present invention can be obtained by direct injection of the polypeptide ⁇ into an animal or by administering the polypeptide ⁇ to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptide ⁇ can be u ⁇ ed to generate antibodie ⁇ binding the whole native polypeptide ⁇ . Such antibodies can then be used to i ⁇ olate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodie ⁇ , any technique which provide ⁇ antibodies produced by continuous cell line culture ⁇ 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. Lis ⁇ , Inc., pp. 77-96) .
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmid ⁇ herein are either commercially available, publicly available on an unre ⁇ tricted basis, or can be constructed from available plasmids in accord with published procedure ⁇ .
  • 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 act ⁇ only at certain ⁇ equence ⁇ in the DNA.
  • the variou ⁇ re ⁇ triction enzyme ⁇ u ⁇ ed herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • analytical purpo ⁇ e ⁇ typically 1 ⁇ g of pla ⁇ mid or DNA fragment i ⁇ used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of i ⁇ olating DNA fragment ⁇ for pla ⁇ mid con ⁇ truction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume.
  • ⁇ ub ⁇ trate amounts for particular re ⁇ triction enzyme ⁇ are ⁇ pecified by the manufacturer. Incubation time ⁇ of about 1 hour at 37 * C are ordinarily used, but may vary in accordance with the supplier's instructions. After dige ⁇ tion the reaction i ⁇ electrophore ⁇ ed directly on a polyacrylamide gel to i ⁇ olate the de ⁇ ired fragment.
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strand ⁇ which may be chemically ⁇ ynthe ⁇ ized. Such synthetic oligonucleotides have no 5' pho ⁇ phate and thu ⁇ will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kina ⁇ e. A ⁇ ynthetic oligonucleotide will ligate to a fragment that ha ⁇ not been depho ⁇ phorylated.
  • Ligase refers ⁇ to the process of forming pho ⁇ phodie ⁇ ter bond ⁇ between two double ⁇ tranded nucleic acid fragment ⁇ (Maniatis, T., et al., Id., p. 146) . Unles ⁇ otherwise provided, ligation may be accomplished using known buffers and condition ⁇ with 10 unit ⁇ of T4 DNA liga ⁇ e ("ligase") per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragment ⁇ to be ligated.
  • ligase T4 DNA liga ⁇ e
  • the DNA sequence encoding TGF ⁇ -HI was initially amplified using PCR oligonucleotide primers corresponding to the 5' sequence ⁇ of the processed TGF ⁇ -HI protein (minus the signal peptide sequence) and the vector sequences 3' to the TGF ⁇ -HI gene. Additional nucleotides corresponding to TGF ⁇ -HI were added to the 5' and 3' sequence ⁇ re ⁇ pectively.
  • the 5' oligonucleotide primer ha ⁇ the ⁇ equence 5' CCCGGATCCGCACGAGACATACCTTGTCCG 3' (SEQ ID NO:3) contains a BamHI restriction enzyme site (in bold) followed by 21 nucleotides of TGF ⁇ -HI coding sequence starting from the presumed terminal amino acid of the proces ⁇ ed protein codon.
  • the 3' ⁇ equence 5' GGGAAGCTTTTAATACTGAAATCGTACAGGAC 3' (SEQ ID NO:4) contain ⁇ complementary sequence ⁇ to a Hind III site and i ⁇ followed by 23 nucleotide ⁇ of TGF ⁇ -HI.
  • pQE-9 encode ⁇ antibiotic re ⁇ istance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribosome binding site (RBS) , a 6-Hi ⁇ tag and re ⁇ triction enzyme ⁇ ites.
  • the amplified sequence ⁇ were ligated into pQE-9 and were in ⁇ erted in frame with the sequence encoding for the histidine tag and the RBS.
  • the ligation mixture wa ⁇ 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 Manual, Cold Spring Laboratory Press, (1989) .
  • M15/rep4 contain ⁇ multiple copie ⁇ of the pla ⁇ mid pREP4, which expre ⁇ e ⁇ the lacl repre ⁇ sor and al ⁇ o confers kanamycin resi ⁇ tance (Kan r ) .
  • Transformants were identified by their ability to grow on LB plate ⁇ and ampicillin/kanamycin re ⁇ i ⁇ tant colonie ⁇ were ⁇ elected. Pla ⁇ mid DNA wa ⁇ i ⁇ olated and confirmed by re ⁇ triction analy ⁇ i ⁇ . Clone ⁇ containing the de ⁇ ired con ⁇ tructs were grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The 0/N culture wa ⁇ used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • O.D. 600 optical density 600
  • IPTG Isopropyl-B-D-thiogalacto pyranoside
  • IPTG induces by inactivating the lacl repressor, clearing the P/0 leading to increased gene expres ⁇ ion.
  • Cell ⁇ were grown an extra 3 to 4 hour ⁇ .
  • Cell ⁇ were then harve ⁇ ted by centrifugation.
  • the cell pellet wa ⁇ ⁇ olubilized in the chaotropic agent 6 Molar Guanidine HCl.
  • TGF ⁇ -HI 85 % pure wa ⁇ eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpo ⁇ e of renaturation adju ⁇ ted to 3 molar guanidine HCl, lOOmM ⁇ odium pho ⁇ phate, 10 molar glutathione (reduced) and 2 molar glutathione (oxidized) . After incubation in thi ⁇ ⁇ olution for 12 hour ⁇ the protein was dialyzed to 10 molar sodium phosphate.
  • the first set of primers are, 5' CGCGGATCCGCCATCATGGGCGCCGCAGCCGC 3' (SEQ ID NO:5) and 5' GCGTCTAGACTAGTATAGAACACTGTAGTCC 3' (SEQ ID NO:6) ;
  • the ⁇ econd ⁇ et of primer ⁇ are:
  • All 5' primers have a BamHI re ⁇ triction enzyme site (in bold) followed by nucleotides re ⁇ embling an efficient ⁇ ignal for the initiation of tran ⁇ lation in eukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) (the initiation codon for translation is "ATG”) .
  • the 3' primer sequence ⁇ contain the cleavage site for the restriction endonuclea ⁇ e Xbal and have nucleotide ⁇ complementary to the 3' TGF ⁇ ⁇ oluble domain of the TGF ⁇ -HI gene.
  • the amplified sequence ⁇ were i ⁇ olated from a 1% agaro ⁇ e gel u ⁇ ing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) .
  • the fragment wa ⁇ then digested with the endonucleases BamHI and Xbal and then purified again on a 1% agarose gel. This fragment was designated F2.
  • the vector pA2 wa ⁇ used (modification of pVL941 vector, discu ⁇ sed below) for the expres ⁇ ion of the TGF ⁇ -HI protein u ⁇ ing the baculoviru ⁇ expression sy ⁇ tem (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
  • This expression vector contain ⁇ the ⁇ trong polyhedrin promoter of the Autographa californica nuclear polyhedro ⁇ i ⁇ viru ⁇ (AcMNPV) followed by the recognition ⁇ ite ⁇ for the re ⁇ triction endonuclea ⁇ e .
  • the polyhedrin ⁇ equence ⁇ were flanked at both ⁇ ides by viral ⁇ equence ⁇ for the cell-mediated homologou ⁇ recombination of co-transfected wild-type viral DNA.
  • baculovirus vector ⁇ could be u ⁇ ed in place of pRGl such a ⁇ pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31- 39) .
  • the plasmid was dige ⁇ ted with the re ⁇ triction enzyme ⁇ BamHI and Xbal and then depho ⁇ phorylated u ⁇ ing calf intestinal phosphata ⁇ e by procedure ⁇ known in the art.
  • the DNA wa ⁇ then i ⁇ olated from a 1% agaro ⁇ e gel u ⁇ ing the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) .
  • Fragment F2 and the depho ⁇ phorylated pla ⁇ mid V2 were ligated with T4 DNA ligase.
  • E.coli HB101 cells were then tran ⁇ formed and bacteria identified that contained the pla ⁇ mid (pBacTGF ⁇ -HI) with the TGF ⁇ -HI gene u ⁇ ing the re ⁇ triction enzyme ⁇ BamHI and Xbal.
  • the ⁇ equence of the cloned fragment wa ⁇ confirmed by DNA ⁇ equencing.
  • the viru ⁇ wa ⁇ added to the cell ⁇ and blue stained plaques were picked with the tip of an Eppendorf pipette.
  • the agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
  • the agar was removed by a brief centrifugation and the supernatant containing the recombinant baculoviru ⁇ wa ⁇ used to infect Sf9 cells seeded in 35 mm dishes.
  • the supernatant ⁇ of the ⁇ e culture di ⁇ hes were harvested and then stored at 4°C.
  • Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculoviru ⁇ V-TGF ⁇ -HI at a multiplicity of infection (MOI) of 2. Six hour ⁇ later the medium wa ⁇ removed and replaced with SF900 II medium minu ⁇ methionine and cy ⁇ teine (Life Technologie ⁇ Inc., Gaither ⁇ burg) . 42 hour ⁇ later 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 3S S cy ⁇ teine (Amersham) were added. The cell ⁇ were further incubated for 16 hour ⁇ before they were harve ⁇ ted by centrifugation and the labelled protein ⁇ vi ⁇ ualized by SDS-PAGE and autoradiography.
  • MOI multiplicity of infection
  • TGF ⁇ -HI HA was derived from a vector pcDNA3/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site.
  • the HA tag corre ⁇ pond ⁇ to an epitope derived from the influenza hemagglutinin protein a ⁇ previou ⁇ ly de ⁇ cribed I. Wil ⁇ on, H. Niman, R. Heighten, A Cheren ⁇ on, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984).
  • the infu ⁇ ion of HA tag to the target protein allow ⁇ ea ⁇ y detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy wa ⁇ de ⁇ cribed a ⁇ follows:
  • the pcDNA3/Amp vector contain ⁇ BamHI/XhoI cloning ⁇ ite ⁇ which bring the PCR insert in frame with the 3' HA tag followed by a ⁇ top codon. Therefore, the PCR product contain ⁇ a BamHI ⁇ ite, 936 base pair coding sequence and an Xhol site.
  • the PCR amplified DNA fragment and the vector, pcDNA3/Amp, were digested with BamHI and Xhol re ⁇ triction enzyme and ligated. The ligation mixture wa ⁇ tran ⁇ formed into E.
  • Fibroblast ⁇ are obtained from a ⁇ ubject by skin biopsy.
  • the resulting ti ⁇ ue is placed in tissue-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 piece ⁇ are placed in each fla ⁇ k.
  • the fla ⁇ k i ⁇ turned up ⁇ ide down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tis ⁇ ue remain fixed to the bottom of the fla ⁇ k and fre ⁇ h media (e.g., Ham' ⁇ F12 media, with 10% FBS, penicillin and ⁇ treptomycin, is added. This is then incubated at 37°C for approximately one week.
  • fre ⁇ h media e.g., Ham' ⁇ F12 media, with 10% FBS, penicillin and ⁇ treptomycin
  • a monolayer of fibroblast ⁇ emerge.
  • the monolayer is trypsinized and scaled into larger flask ⁇ .
  • pMV-7 (Kir ⁇ chmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma viru ⁇ , i ⁇ dige ⁇ ted with EcoRI and Hindlll and subsequently treated with calf intestinal phosphata ⁇ e.
  • the cDNA encoding a polypeptide of the present invention i ⁇ amplified u ⁇ ing PCR primer ⁇ which corre ⁇ pond to the 5' and 3' end sequences respectively.
  • the 5' primer containing an EcoRI site and the 3' primer further includes a Hindlll site.
  • Equal quantities of the Moloney murine ⁇ arcoma virus linear backbone and the amplified EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase.
  • the resulting mixture is maintained under conditions appropriate for ligation of the two fragments.
  • the ligation mixture i ⁇ u ⁇ ed to transform bacteria HBlOl, which are then plated onto agar-containing 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 Eagle ⁇ Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagle ⁇ Medium
  • CS calf serum
  • the MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector.
  • the packaging cells now produce infectiou ⁇ viral particles containing the gene (the packaging cells are now referred to a ⁇ producer cell ⁇ ) .
  • the spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media i ⁇ then used to infect fibroblast cell ⁇ .
  • Thi ⁇ media is removed and replaced with fre ⁇ h media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the 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 fibroblast ⁇ are then injected into the ho ⁇ t, either alone or after having been grown to confluence on cytodex 3 microcarrier bead ⁇ .
  • the fibrobla ⁇ t ⁇ now produce the protein product.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • CGCGTCCAAC CAGCCCCCGG GTGGTGGCGG CGGCAGCGGC GGGGACTGTC CCGGCGGCAA 240
  • a ⁇ n Tyr lie Pro Val Cy ⁇ Gly Ser Asn Gly Asp Thr Tyr Gin Asn 70 75 80

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Abstract

The present invention discloses transforming growth factor alpha HII 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 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

TRANSFORMING GROWTH FACTOR ALPHA HI
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. The polypeptide of the present invention has been putatively identified as a human transforming growth factor alpha homolog. More particularly, the polypeptide of the present invention has been putatively identified as transforming growth factor alpha HII, sometimes hereafter referred to as "TGFα-HI" . The invention also relates to inhibiting the action of such polypeptides.
Cellular growth and differentiation appear to be initiated, promoted, maintained and regulated by a multiplicity of stimulatory, inhibitory and synergistic factors and hormones. The alteration and/or breakdown of the cellular homeostasis mechanism seems to be a fundamental cause of growth related diseases, including neoplasia. Growth modular 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 (EGF) , transforming growth factor alpha (TGFα) , betacellulin, amphiregulin, and vaccinia growth factor among other factors are growth and differentiation modulatory 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.
These peptide growth factors influence wound cells through autocrine and paracrine mechanisms. They also play important roles in normal wound healing 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 activity in the receptor's cytoplasmic domain (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 GI tract. These peptide growth factors are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. These growth factors have also been implicated in both the stimulation of growth and differentiation of certain cells, for example, neoplasia, and the inhibition of other types of cells.
Betacellulin iε a 32-kilodalton glycoprotein that appears to be processed from a larger transmembrane precursor by proteolytic cleavage. The carboxyl-terminal domain of betacellulin has 50% sequence similarity with that of rat transforming growth factor a . Betacellulin 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 inhibitory 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.
TGFα has pleiotropic biological effects. The production of certain members of TGFα is synthesized by a number of oncogenically transformed fibroblasts (Ciardiello et al., .Cell.Bioche .. 42:45-57 (1990)) , as well as by a variety of tumors, including renal, breast and squamous carcinomas, melanomas and glioblastomaε (Derynck, R. et al. , Cancer Res.. 47:707-712 (1987)) . There is direct evidence that TGFα 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 TGFα. TGFα transgenic animals display a variety of neoplastic lesions, depending on the strain of mouse and the choice of promotor regulating TGFα expression (Sandgren, et al., Cell. 61:1121-1135 (1990)) .
TGFα also plays a role in normal embryonic development and adult physiology (Derynck, R. Adv.Cancer Res. , 58:27-5 (1992) ) . TGFα has been expressed in many tissues including skin, brain, gastrointestinal mucosa and activating macrophageε. Accordingly, TGFα is an important factor in controlling growth of epithelial cells and has a role in wound healing. TGFα 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 as transforming growth factor TGFα-HI. Thiε identification has been made as a reεult of amino acid sequence homology to human TGFα. In accordance with one aspect of the present invention, there are provided novel mature polypeptide , as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptideε 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 preεent invention, including mRNAs, DNAs, cDNAs, genomic DNAs aε well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with yet a further aspect of the present invention, there are provided procesεes for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing 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 εuch polypeptides for therapeutic purposes, for example, to stimulate wound healing to restore normal neurological functioning after trauma or AIDS dementia, to treat ocular disorderε, to target certain cellε, to treat kidney and liver disorderε and to promote hair follicular development, to εtimulate angiogenesis for the treatment of burns, ulcers and corneal incisions and to εtimulate 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 εequences 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 aspect of the present invention, there are provided agonistε to the polypeptide of the present invention.
In accordance with yet another aspect of the present invention, there are provided antagonistε to such polypeptideε, which may be used to inhibit 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 overexpresεion of the polypeptide of the present invention and mutations in the nucleic acid sequences encoding such polypeptide.
In accordance with yet a further aspect of the present invention, there is provided a procesε for utilizing εuch polypeptides, or polynucleotides encoding εuch polypeptideε, for in vitro purposes related to scientific research, syntheεiε of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawingε are illuεtrative of embodimentε of the invention and are not meant to limit the εcope of the invention aε encompassed by the claims.
Figure 1 depicts the cDNA sequence in corresponding deduced amino acid sequence of TGFα-HI. The standard one letter abbreviations for amino acids are used. The putative signal sequence has been underlined and the putative soluble portion haε been double underlined.
Figure 2 is an illustration of comparative amino acid sequence homology between human amphiregulin, human betacellulin, human epidermal growth factor, human heregulin and TGFα-HI (fifth row) . Shaded areas denotes the conserved EGF motif which iε shown to be conserved in the polypeptide of the present invention.
In accordance with an aspect of the present invention, there iε provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID N0:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75698 on March 4, 1994.
A polynucleotide encoding a polypeptide of the present invention may be obtained from human brain and early stage brain tissue. The polynucleotide of thiε invention waε diεcovered in a cDNA library derived from eight-week old embryo. It is structurally related to the TGFα gene family. It contains an open reading frame encoding a polypeptide of 380 amino acids, which exhibits significant homology to a number of members of the TGFα gene family; theεe members include TGFα itself aε well aε other members such as amphiregulin and cripto. Furthermore, the six cysteine residueε occurring in all memberε in a characteristic motif are conserved in TGFα-HI.
The full-length polypeptide of the present invention as set forth in Figure 1 (SEQ ID NO:2) has a putative signal sequence which compriseε amino acid 1 through amino acid 39 of Figure 1 (SEQ ID NO:2) which aids in secretion of the polypeptide from the cell. The polypeptide iε further processed wherein amino acid 40 through amino acid 266 of Figure 1 (SEQ ID NO:2) are cleaved from the polypeptide since this stretch of amino acids iε a putative precursor sequence. Further, amino acid 317 through amino acid 380 represents a putative transmembrane portion which is thought to be necessary to direct the polypeptide to particular target locationε for the carrying out of biological functions as hereinafter described. The transmembrane portion may also be cleaved from the polypeptide εuch that the putative εoluble portion of the polypeptide of the preεent invention comprises amino acid 267 through amino acid 316 of Figure 1 (SEQ ID NO:2) .
The polynucleotide of the preεent 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- εtranded or εingle-εtranded, and if εingle εtranded may be the coding strand or non-coding (anti-εenεe) strand. The coding sequence which encodeε the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID N0:1) or that of the deposited clone 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 mature polypeptide as the DNA of Figure 1 (SEQ ID N0:1) or the deposited cDNA.
The polynucleotide which encodes for the mature polypeptide of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include, but is not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or εecretory sequence or a proprotein εequence; 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 relateε to variantε of the hereinabove described polynucleotideε which encode for fragmentε, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone. 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 as shown in Figure 1 (SEQ ID NO:2) 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 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variantε include deletion variantε, εubstitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID N0:1) or of the coding sequence of the deposited clone. Aε known in the art, an allelic variant is an alternate form of a polynucleotide εequence 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 polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which iε the mature protein pluε additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the proεequence iε cleaved an active mature protein 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 preεent invention may alεo 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- hiεtidine tag εupplied by a pQE-9 vector to provide for purification of the mature polypeptide fuεed to the marker in the case of a bacterial hoεt, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. , Cell, 37:767 (1984)) .
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (intronε) between individual coding εegmentε (exons) .
Fragments of the full length TGFα-HI gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probeε of thiε type preferably have at least 30 baεes and may contain, for example, 50 or more bases. The probe may alεo be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete TGFα-HI gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by uεing the known DNA εequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The present invention further relates to polynucleotideε 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 hereinabove-described polynucleotides. Aε 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 sequenceε. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain subεtantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:l) or the depoεited cDNA(ε) .
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, aε hereinabove described, and which may or may not retain activity. For example, such polynucleotideε may be employed as probes for the polynucleotide of SEQ ID NO:l, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
Thus, the present invention is directed to polynucleotideε having at leaεt a 70% identity, preferably at leaεt 90% and more preferably at leaεt a 95% identity to a polynucleotide which encodeε the polypeptide of SEQ ID NO:2 aε well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptideε encoded by εuch polynucleotides.
The deposit (ε) referred to herein will be maintained under the termε of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These depositε are provided merely as convenience to those of skill in the art and are not an admiεsion that a deposit iε required under 35 U.S.C. §112. The εequence of the polynucleotideε contained in the deposited materials, aε well aε the amino acid εequence of the polypeptideε encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any deεcription of sequenceε herein. A licenεe may be required to make, uεe or εell the depoεited materials, and no such license is hereby granted.
The preεent invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or which haε the amino acid εequence encoded by the deposited cDNA, as well as fragments, analogs and derivativeε of εuch polypeptide.
The termε "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the depoεited cDNA, meanε a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thuε, 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 (SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid reεidues are εubstituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid reεidue) and εuch εubεtituted amino acid reεidue 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 fuεed with another compound, εuch 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 iε employed for purification of the mature polypeptide or a proprotein εequence. Such fragments, derivatives and analogs are deemed to be within the scope of those εkilled 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 polypeptide, separated from some or all of the coexisting materialε in the natural εyεte , iε iεolated. Such polynucleotideε could be part of a vector and/or εuch polynucleotideε or polypeptideε could be part of a compoεition, and still be iεolated 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 aε polypeptideε which have at leaεt 70% εimilarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at leaεt 95% εimilarity (εtill more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. As known in the art "εimilarity" between two polypeptideε iε determined by comparing the amino acid sequence and itε conεerved amino acid εubstitutes of one polypeptide to the εequence 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 syntheεiε; therefore, the fragmentε may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the preεent invention may be uεed to εyntheεize full-length polynucleotideε of the preεent invention.
The preεent 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 polypeptideε of the invention by recombinant techniqueε.
Hoεt 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 expreεεion vector. The vector may be, for example, in the form of a plaεmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified aε appropriate for activating promoterε, εelecting tranεformantε or amplifying the geneε of the present invention. The culture conditions, εuch 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 may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expreεεion vectorε for expreεεing a polypeptide. Such vectorε include chromosomal, nonchromosomal and εynthetic DNA εequenceε, e.g., derivativeε of SV40; bacterial plaεmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such aε 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 sequence is inserted into an appropriate restriction endonucleaεe εite(ε) 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 εequence in the expression vector is operatively linked to an appropriate expresεion control sequence(ε) (promoter) to direct mRNA εynthesis. 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 expresεion of geneε in prokaryotic or eukaryotic cellε or their viruses. The expression vector also contains a ribosome binding εite for tranεlation initiation and a tranεcription terminator. The vector may also include appropriate sequenceε for amplifying expression.
In addition, the expresεion vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductaεe or neomycin resistance for eukaryotic cell culture, or such aε tetracycline or ampicillin reεistance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well aε an appropriate promoter or control εequence, may be employed to tranεform an appropriate hoεt to permit the hoεt to expreεε the protein.
As repreεentative exampleε of appropriate hoεtε, there may be mentioned: bacterial cells, such aε E. coli. Streptomvceε. Salmonella tvphimurium; fungal cellε, εuch as yeast; insect cells εuch as Droεophila S2 and Spodoptera Sf9; animal cellε such aε CHO, COS or Bowes melanoma; adenoviruses; plant cellε, etc. The selection of an appropriate hoεt 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 sequenceε aε broadly deεcribed above. The constructs compriεe a vector, εuch aε a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of εuitable vectorε and promoters are known to those of skill in the art, and are commercially available. The following vectorε are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phageεcript, psiX174, pbluescript SK, pbεks, pNH8A, pNH16a, pNHlβA, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, p K233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, 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 hoεt.
Promoter regions can be selected from any desired gene uεing CAT (chloramphenicol tranεferase) vectors or other vectors with selectable markers. Two appropriate vectorε are pKK232-8 and pCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoterε include CMV immediate early, HSV thymidine kinaεe, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter iε well within the level of ordinary εkill in the art. In a further embodiment, the present invention relates to host cells containing the above-described constructs. The hoεt cell can be a higher eukaryotic cell, εuch as a mammalian 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 Methodε in Molecular Biology, (1986) ) .
The conεtructε in host cellε can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cellε under the control of appropriate promoterε. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA conεtructε of the preεent invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hostε are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. , (1989), the disclosure of which iε hereby incorporated by reference.
Tranεcription of the DNA encoding the polypeptideε of the present invention by higher eukaryotes is increaεed by inεerting an enhancer εequence 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 tranεcription. Exampleε including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancerε. Generally, recombinant expreεεion vectors will include originε of replication and εelectable 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-expreεεed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operonε encoding glycolytic enzymes such as 3-phoεphoglycerate kinaεe (PGK) , α-factor, acid phoεphataεe, or heat shock proteins, among others. The heterologous structural sequence is assembled 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 fuεion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expresεed recombinant product.
Uεeful expreεsion vectorε for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signalε in operable reading phaεe with a functional promoter. The vector will compriεe one or more phenotypic selectable markers and an origin of replication to enεure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis, Salmonella tvphimurium and various specieε within the genera Pseudomonaε, Streptomyces, and Staphylococcuε, although otherε may alεo be employed aε a matter of choice.
Aε a repreεentative 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 Chemicalε, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expreεεed.
Following tranεformation of a εuitable hoεt strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature εhift or chemical induction) and cells are cultured for an additional period.
Cellε are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expreεεion of proteinε can be diεrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lyεing agents, such methods are well known to those skilled in the art.
Various mammalian cell culture systemε can alεo be employed to expreεε recombinant protein. Exampleε of mammalian expression syεtemε include the COS-7 lines of monkey kidney fibroblastε, deεcribed by Gluzman, Cell, 23:175 (1981) , and other cell lineε capable of expreεεing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε. Mammalian expression vectors will compriεe an origin of replication, a εuitable promoter and enhancer, and alεo any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor siteε, tranεcriptional termination sequences, and 5' flanking nontranscribed εequenceε. DNA εequenceε derived from the SV40 splice, and polyadenylation εites may be used to provide the required nontranscribed genetic elements.
The polypeptideε can be recovered and purified from recombinant cell cultureε by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptideε 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 hoεt (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the hoεt employed in a recombinant production procedure, the polypeptideε of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may alεo include an initial methionine amino acid reεidue.
The polynucleotides and polypeptides of the present invention may be employed aε reεearch reagentε and materials for discovery of treatmentε and diagnoεticε for human diεeaεe.
The polypeptide of the present invention may be employed for characterization of receptors. The EGF family receptorε currently includeε four EGF receptorε, denoted as EGFR1, EGFR2, EGFR3 and EGFR4. The EGFR2 receptor may also be referred to as ERB-2 and this molecule iε uεeful for a variety of diagnoεtic and therapeutic indicationε (Prigent, S.A., and Lemoine, N.R. , Prog Growth Factor Res.. 4:1-24 (1992)) . The TGFα-HI polypeptide is likely a ligand for one or more of these receptors aε well as for yet an identified new EGF-type receptor. Use of the TGFα-HI can assist with the identification, characterization and cloning of such receptors. For example, the EGF receptor gene representε the cellular homolog of the v-erb-B oncogene of avian erythroblastosis virus. Over expression of the EGF-receptor or deletion of kinase regulatory segmentε 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 enhancement of neurological functions diminished aε a reεult of trauma or other damaging pathologieε (εuch aε AIDS dementia, εenile dementia, etc) . TGFα and its homologs have been found to be the most abundant ligand for the EGF/TGFα 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 TGFα in various regions of the brain in contrast to EGF which is only present in εmaller, more diεcrete areas, εuggeεting that TGF- alpha might play a physiological role in brain tisεueε . Theεe numerous receptor sites for TGFα in the brain suggest that TGF has an important utility in promoting normal brain cell differentiation and function. Accordingly, in inεtanceε where neurological functioning is diminished, an administration of the polypeptide of the preεent invention may stimulate the brain and enhance proper physiological functions.
TGFα-HI or soluble form thereof may alεo be employed to treat ocular disorders, for example, corneal inflammation. A variety of experiments have implicated members of the TGFα 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 TGFα gene displayed corneal inflammation due to an infiltration of leukocytes and other cellε to the εubstantia propria of the eyes.
In addition, the specificity of the TGFα growth factors for their target cells can be exploited as a mechanism to destroy the target cell. For example, TGFα-HI or εoluble formε thereof can be coupled (by a wide variety of methods) to toxic molecules: for example, a radiopharmaceutical which inactivate target cells. These growth factor-toxin fusions kill the target cell (and in certain caseε neighboring cells by a variety of "bystander" effects) . A recent example of such toxin-fusion genes is published by Meεri, et al. , J. Biol. Chem. 268:4853-62 (1993) . TGFα-HI and related moleculeε may alεo be encapεulated in lipoεomeε and may be conjugated to antibodieε which recognize and bind to tumor or cell εpecific antigens, thereby provided a means for "targeting" cells.
In thiε εame manner, TGFα-HI can be employed aε an anti- neoplaεtic compound, εince memberε of the EGF family show anti-proliferative effects on transformed cells. 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. Administration may be in any physiologically acceptable carrier, including phoεphate buffered εaline, εaline, εterilized water, etc. T he TGFα-HI polypeptide fragment may also be employed to treat certain kidney disorderε, since it has been found that there haε been expreεsion of these growth factorε in the kidney. Thuε, theεe factorε may be necesεary for the proper phyεiological maintenance of thiε organ.
Treatments may also be related to liver regeneration or liver dysfunction, εince TGFα and itε homologε and hepatocyte growth factor trigger hepatocyte regeneration after partial hepatectomy and after acute liver cell necroεiε (Masuhara, M. et al, Hepatology 16:1241-1249 (1992)) .
A significant treatment involving TGFα-HI relates to wound healing. The compositions of the present invention may be employed for treating a wide variety of woundε including εubεtantially all cutaneouε woundε, corneal woundε, and injurieε to the epithelial-lined hollow organs of the body. Wounds εuitable for treatment include those resulting from trauma such as burns, abrasions and cuts, as well as from surgical procedureε εuch aε εurgical 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.
TGFα-HI 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 baεes include lanolin, Silvadene (Marion) (particularly for the treatment of burns) , Aquaphor (Duke Laboratories, South Norwalk, Conn.), and the like. If desired, it will be posεible to incorporate TGFα-HI containing compoεitionε in bandageε and other wound dreεsings to provide for continuous exposure of the wound to the peptide. Aerosol applications may also find use.
The concentration of TGFα-HI 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 aε creamε, ointmentε or lotions to the skin. In the caεe of the eyeε, frequent treatment iε desirable, usually being applied at intervals of 4 hours or leεε. On the εkin, 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 manner of administration, the employment of other active compounds, and the like, generally being in the range of about 1 μg to 100 μg. The subject polypeptide may be employed with a physiologically acceptable carrier, such aε saline, phosphate-buffered εaline, or the like. The amount of compound employed will be determined empirically, based on the responεe of cellε in vitro and response of experimental animals to the subject polypeptides or formulations containing the subject polypeptideε.
The TGFα-HI or εoluble fragment thereof may be employed in the modulation of angiogenesis, bone resorption, immune reεponεe, and εynaptic and neuronal effector functions. TGFα-HI may also be used in the modulation of the arachidonic acid cascade.
TGFα-HI or εoluble fragment thereof may alεo be employed for applicationε related to terminal differentiation. Many TGFα factorε, and their homologε, induce terminal differentiation in their target cellε. This property can be exploited in vivo by administering the factor and inducing target cell death. This regimen is under conεideration for diεorders related to the hyper-proliferation of medically undesirable cell types such aε cancers and other proliferative diεorderε (eg inflammation, pεoriaεis, etc) . In addition to in vivo administration, there are a variety of εituationε where in vitro adminiεtration may be warranted. For example, bone marrow can be purged of undeεirable cell populations in vitro by treating the cellε with growth factors and/or derivatives thereof.
Applicationε are also related to alopecia, hair losε and to other εkin conditionε which affect hair follicular developmen . Several lineε of evidence implicate the involvement TGFα growth factors in such conditions. Aε described above, "knockout" mice engineered to contain a null mutation in the TGFα gene display abnormalitieε related to quantitative and qualitative hair εyntheεiε. In addition, mapping εtudies in mice have shown that some mutationε affecting hair growth map to the TGFα gene locuε (Mann et al, Cell 73:249-261(1993)) . Topical or systemic applications of TGFα-HI or derivatives thereof may be employed to treat some forms of alopecia and hair loss and these claims fall within the εcope of thiε invention.
Certain diεeaεe pathologies may be partially or completely ameliorated by the systemic clinical administration of the TGFα-HI growth factor. This administration can be in the form of gene therapy (see below) ; or through the adminiεtration of peptides or proteins synthesized from recombinant constructs of TGFα-HI DNA or from peptide chemical syntheεiε (Woo, et al., Protein Engineering 3:29-37 (1989) .
Thiε invention provideε a method of screening compounds to identify agonist or antagoniεt compounds to the polypeptide of the present invention. As an example, a mammalian cell or membrane preparation expressing a TGFα-HI receptor iε incubated with a potential compound and the ability of the compound to generate a second signal from the receptor is measured to determine if it is an effective agonist. Such second meεεenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinoεitide hydrolysis. Effective antagonists are determined by the method above wherein an antagonist compound is detected which binds to the receptor but does not elicit a εecond messenger responεe to thereby block the receptor from TGFα-HI.
Another aεsay for identifying potential antagoniεtε εpecific to the receptorε to the polypeptide of the present invention iε a competition assay which compriεes isolating plasma membranes which over expreεε a receptor to the polypeptide of the present invention, for example, human A431 carcinoma cellε. Serially diluted test sample in a medium (volume iε approximately 10 microliters) containing 10 nM 125I-TGFα-HI iε added to five microgramε of the plaεma membrane in the presence of the potential antagonist compound and incubated for 4 hourε at 4°C. The reaction mixtureε are diluted and immediately passed through a millipore filter. The filters are then rapidly washed and the bound radioactivity is measured in a gamma counter. The amount of bound TGFα-HI is then measured. A control asεay iε alεo performed in the abεence of the compound to determine if the antagoniεtε reduce the amount of bound TGFα-HI.
Potential antagoniεt compoundε include an antibody, or in εome 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 iε an antiεenεe conεtruct prepared using antisense technology. Antisense technology can be used to control gene expresεion through triple-helix formation or antiεenεe DNA or RNA, both of which methodε 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 polypeptideε of the present invention, is used to deεign an antiεenεe 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 Lee et al. , Nucl. Acids Res., 6:3073 (1979) ; Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991) ) , thereby preventing transcription and the production of the polypeptide of the present invention. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockε tranεlation of the mRNA molecule into the polypeptide of the preεent invention (Antiεenεe - Okano, J. Neurochem., 56:560 (1991) ; Oligodeoxynucleotides aε Antiεense Inhibitors of Gene Expresεion, CRC Press, Boca Raton, FL (1988)) . The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expreεεed in vivo to inhibit production of the polypeptide of the preεent invention. Antagoniεt 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. The εmall moleculeε may also bind the receptor to the polypeptide to prevent binding. Examples of small molecules include but are not limited to small peptides or peptide-like moleculeε.
The antagonists may be employed to treat neoplasia, for example, cancers and tumorε. It is known that inhibition of secretion or production of members of the EGF family by tumor cellε in mice causes regreεsion of tumors.
The antagonists to the polypeptides of the present invention may also be uεed therapeutically for the treatment of certain skin diεorderε, for example, pεoriasis. Elevated levels of expression of memberε of this family of growth factors in skin biopsieε taken from diεeaεeε εuch aε pεoriatic lesions have been found to be elevated (Cook, et al., Cancer Research. 52:3224-3227 (1992)) . The antagonists may be employed in a compoεition with a pharmaceutically acceptable carrier, e.g., aε hereinafter described.
The polypeptides of the present invention or agoniεt or antagoniεt compoundε may be employed in combination with a suitable pharmaceutical carrier. Such compositionε compriεe a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but iε not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation εhould εuit the mode of administration.
The invention also provideε a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredientε of the pharmaceutical compoεitions of the invention. Associated with such container(s) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, use or εale of pharmaceuticalε or biological products, which notice reflectε approval by the agency of manufacture, uεe or εale for human adminiεtration. In addition, the polypeptideε or compoundε of the present invention may be employed in conjunction with other therapeutic compoundε.
The pharmaceutical compoεitionε may be administered in a convenient manner εuch aε by the oral, topical, intravenouε, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compoεitionε are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at leaεt about 10 μg/kg body weight and in most cases they will be administered in an amount not in excesε of about 8 mg/Kg body weight per day. In moεt cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptomε, 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 iε often referred to aε "gene therapy."
Thus, for example, cellε from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cellε then being provided to a patient to be treated with the polypeptide. Such methodε are well-known in the art and are apparent from the teachingε herein. For example, cells may be engineered by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention.
Similarly, cellε may be engineered in vivo for expreεsion of a polypeptide in vivo by, for example, procedures known in the art. For example, a packaging cell is tranεduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particleε containing the gene of intereεt. Theεe producer cells may be adminiεtered to a patient for engineering cellε in vivo and expression of the polypeptide in vivo. These and other methodε for adminiεtering a polypeptide of the preεent invention by εuch method should be apparent to those εkilled in the art from the teachings of the present invention.
Retroviruseε 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 viruε, gibbon ape leukemia viruε, human immunodeficiency viruε, adenoviruε, Myeloproliferative Sarcoma Viruε, and mammary tumor viru . In one embodiment, the retroviral plaεmid vector is derived from Moloney Murine Leukemia Virus.
The vector includeε one or more promoterε. Suitable promoterε which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegaloviruε (CMV) promoter deεcribed in Miller, et al., Biotechniαueε. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoterε εuch aε eukaryotic cellular promoters including, but not limited to, the histone, pol III, and /3-actin promoters) . Other viral promoterε which may be employed include, but are not limited to, adenoviruε promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoterε. The εelection of a suitable promoter will be apparent to those skilled in the art from the teachingε contained herein.
The nucleic acid εequence encoding the polypeptide of the preεent invention iε under the control of a εuitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such aε the adenoviral major late promoter; or heterologous promoterε, εuch as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such aε the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoterε; viral thymidine kinaεe promoterε, εuch aε the Herpeε Simplex thymidine kinaεe promoter; retroviral LTRε (including the modified retroviral LTRε hereinabove deεcribed) ; the /3-actin promoter; and human growth hormone promoter . The promoter alεo may be the native promoter which controlε the gene encoding the polypeptide.
The retroviral plaεmid vector iε employed to tranεduce packaging cell lineε to form producer cell lineε. Exampleε of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, φ-2 , ψ-iW, PA12, T19-14X, VT-19-17-H2, ψCKE , ^CRIP, GP+E-86, GP+envAml2, and DAN cell lineε as deεcribed in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990) , which iε incorporated herein by reference in its entirety. The vector may tranεduce the packaging cellε through any meanε known in the art. Such meanε include, but are not limited to, electroporation, the use of liposomeε, and CaP04 precipitation. In one alternative, the retroviral plaεmid vector may be encapεulated 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 εequence (ε) encoding the polypeptideε. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo. The tranεduced eukaryotic cellε will express the nucleic acid sequence(ε) encoding the polypeptide. Eukaryotic σellε which may be tranεduced include, but are not limited to, embryonic stem cellε, embryonic carcinoma cellε, aε well aε hematopoietic stem cellε, hepatocyteε, fibroblaεtε, myoblaεtε, keratinocyteε, endothelial cells, and bronchial epithelial cells.
This invention iε also related to the use of the gene of the present invention aε a diagnostic. Detection of a mutated form of the gene of the present invention will allow a diagnosiε of a disease or a susceptibility to a diseaεe which results from underexpression of the polypeptide of the preεent invention for example, improper wound healing, improper neurological functioning, ocular disorders, kidney and liver disorderε, hair follicular development, angiogenesis and embryogeneεi .
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 diagnosiε may be obtained from a patient's cells, such aε from blood, urine, εaliva, tiεsue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by uεing PCR (Saiki et al . , Nature, 324:163-166 (1986)) prior to analyεiε. RNA or cDNA may alεo be used for the same purpoεe. Aε an example, PCR primerε complementary to the nucleic acid encoding 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 mutationε can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antiεense DNA sequenceε. Perfectly matched sequenceε can be distinguished from mismatched duplexeε by RNaεe A digeεtion or by differences in melting temperatures.
Sequence differences between the reference gene and genes having mutationε may be revealed by the direct DNA sequencing method. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic εequencing procedures with fluoreεcent-tagε.
Genetic testing based on DNA εequence 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 εequenceε may be diεtinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different poεitionε according to their εpecific melting or partial melting temperatureε (εee, e.g., Myerε et al . , Science, 230:1242 (1985) ) .
Sequence changeε at specific locations may alεo be revealed by nuclease protection assays, εuch as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)) .
Thus, the detection of a specific DNA sequence may be achieved by methodε εuch aε hybridization, RNaεe protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresiε and DNA εequencing, mutationε can also be detected by in situ analysis.
The present invention also relates to diagnostic assays for detecting altered levels of the polypeptide of the present invention in variouε tissueε εince an over-expression of the proteins compared to normal control tisεue εamples can detect the presence of certain disease conditions εuch aε neoplasia, skin disorderε, ocular disorders and inflammation. Asεayε uεed to detect levelε of the polypeptide of the preεent invention in a εample derived from a hoεt are well- known to thoεe of εkill in the art and include radioimmunoaεεayε, competitive-binding assays, Western Blot analyεiε and preferably an ELISA aεεay. 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 iε prepared againεt the monoclonal antibody. To the reporter antibody iε attached a detectable reagent such as radioactivity, fluoreεcence or in thiε example a horseradish peroxidase enzyme. A sample iε now removed from a hoεt and incubated on a εolid εupport, e.g. a polyεtyrene diεh, that bindε the proteinε in the εample. Any free protein binding εiteε on the dish are then covered by incubating with a non¬ specific protein such aε bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodieε attach to any polypeptideε of the present invention attached to the polystyrene diεh. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to polypeptides of the preεent invention. Unattached reporter antibody iε then waεhed out. Peroxidaεe εubεtrateε are then added to the diεh and the amount of color developed in a given time period iε a measurement of the amount of protein present in a given volume of patient sample when compared againεt a εtandard curve.
A competition aεεay may also be employed to determine levelε of the polypeptide of the present invention in a sample derived from the hoεtε. Such an assay compriseε isolating plasma membranes which over-expresε the receptor for the polypeptide of the preεent invention. A teεt εample containing the polypeptideε of the present invention which have been labeled, are then added to the plasma membranes and then incubated for a set period of time. Alεo added to the reaction mixture iε a sample derived from a host which is suεpected of containing the polypeptide of the preεent invention. The reaction mixtures are then pasεed through a filter which is rapidly washed and the bound radioactivity 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.
Antibodieε εpecific to TGFα-HI may be used for cancer diagnosiε and therapy, εince many typeε of cancer cells up¬ regulate various members of the TGFα family during the procesε of neoplaεia or hyperplaεia. Theεe antibodieε bind to and inactivate TGFα-HI. Monoclonal antibodies against TGFα-HI (and/or its family memberε) are in clinical use for both the diagnosis and therapy of certain disorders including (but not limited to) hyperplastic and neoplaεtic growth abnormalitieε. Upregulation of growth f ctor expression by neoplaεtic tissues forms the basis for a variety of serum assayε which detect increaεeε in growth factor in the blood of affected patientε. These asεayε are typically applied not only in diagnoεtic εettingε, but are applied in prognoεtic settings as well (to detect the presence of occult tumor cells following εurgery, chemotherapy, etc) .
In addition, malignant cells expressing the TGFα-HI receptor may be detected by using labeled TGFα-HI in a receptor binding asεay, or by the use of antibodies to the TGFα-HI receptor itεelf. Cellε may be diεtinguiεhed in accordance with the presence and density of receptors for TGFα-HI, thereby providing a means for predicting the susceptibility of such cells to the biological activitieε of TGFα-HI.
The sequences of the present invention are alεo valuable for chromoεome identification. The εequence iε εpecifically targeted to and can hybridize with a particular location on an individual human chromoεome. Moreover, there iε a current need for identifying particular εiteε on the chromoεome. Few chromoεome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention iε an important firεt step in correlating those εequenceε with geneε associated with disease.
Briefly, sequenceε can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysiε of the 3' untranεlated region of the gene iε used to rapidly εelect primerε 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 hybridε containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids iε a rapid procedure for aεεigning a particular DNA to a particular chromoεome. Uεing the present invention with the same oligonucleotide primerε, εublocalization can be achieved with panels of fragments from specific chromosomeε or poolε of large genomic cloneε in an analogouε manner. Other mapping εtrategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-εorted chromoεomeε and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluoreεcence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one εtep. Thiε technique can be used with cDNA as short as 50 or 60 bases. For a review of thiε technique, see Verma et al. , Human Chromosomeε: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
Once a sequence has been mapped to a preciεe chromoεomal location, the phyεical poεition 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 Univerεity Welch Medical Library) . The relationεhip between geneε and diεeaseε that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent geneε) .
Next, it is neceεεary to determine the differences in the cDNA or genomic εequence between affected and unaffected individualε. If a mutation iε obεerved in εome or all of the affected individualε but not in any normal individualε, then the mutation is likely to be the causative agent of the disease.
With current reεolution of phyεical mapping and genetic mapping techniqueε, a cDNA precisely localized to a chromosomal region aεεociated with the diεease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping reεolution and one gene per 20 kb) .
The polypeptideε, their fragmentε or other derivatives, or analogs thereof, or cellε expressing them can be used aε an immunogen to produce antibodieε thereto. Theεe antibodieε can be, for example, polyclonal or monoclonal antibodieε. The preεent invention alεo includes chimeric, single chain, and humanized antibodieε, aε well aε Fab fragmentε, or the product of an Fab expreεεion library. Variouε procedureε known in the art may be uεed for the production of εuch antibodies and fragments.
Antibodies generated against the polypeptideε correεponding to a sequence of the present invention can be obtained by direct injection of the polypeptideε into an animal or by administering the polypeptideε to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptideε can be uεed to generate antibodieε binding the whole native polypeptideε. Such antibodies can then be used to iεolate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodieε, any technique which provideε antibodies produced by continuous cell line cultureε 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. Lisε, Inc., pp. 77-96) .
Techniqueε deεcribed for the production of single chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodieε to immunogenic polypeptide products of thiε invention. Also, transgenic mice may be used to expresε humanized antibodieε to immunogenic polypeptide products of this invention.
The present invention will be further described with reference to the following examples; however, it iε to be underεtood that the preεent invention is not limited to such examples. All partε or amounts, unlesε otherwiεe εpecified, are by weight.
In order to facilitate understanding of the following examples certain frequently occurring methodε and/or termε will be deεcribed.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmidε herein are either commercially available, publicly available on an unreεtricted basis, or can be constructed from available plasmids in accord with published procedureε. 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 actε only at certain εequenceε in the DNA. The variouε reεtriction enzymeε uεed 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 purpoεeε, typically 1 μg of plaεmid or DNA fragment iε used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of iεolating DNA fragmentε for plaεmid conεtruction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and εubεtrate amounts for particular reεtriction enzymeε are εpecified by the manufacturer. Incubation timeε of about 1 hour at 37*C are ordinarily used, but may vary in accordance with the supplier's instructions. After digeεtion the reaction iε electrophoreεed directly on a polyacrylamide gel to iεolate the deεired fragment.
Size εeparation of the cleaved fragmentε iε performed uεing 8 percent polyacrylamide gel deεcribed by Goeddel, D. et al . , Nucleic Acidε Reε. , 8:4057 (1980) .
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strandε which may be chemically εyntheεized. Such synthetic oligonucleotides have no 5' phoεphate and thuε will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that haε not been dephoεphorylated.
"Ligation" referε to the process of forming phoεphodieεter bondε between two double εtranded nucleic acid fragmentε (Maniatis, T., et al., Id., p. 146) . Unlesε otherwise provided, ligation may be accomplished using known buffers and conditionε with 10 unitε of T4 DNA ligaεe ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragmentε to be ligated.
Unless otherwise εtated, tranεformation waε performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973) . Example 1 Bacterial Expresεion and Purification of the snlnhle form of TGFα-HI
The DNA sequence encoding TGFα-HI, ATCC # 75698, was initially amplified using PCR oligonucleotide primers corresponding to the 5' sequenceε of the processed TGFα-HI protein (minus the signal peptide sequence) and the vector sequences 3' to the TGFα-HI gene. Additional nucleotides corresponding to TGFα-HI were added to the 5' and 3' sequenceε reεpectively. The 5' oligonucleotide primer haε the εequence 5' CCCGGATCCGCACGAGACATACCTTGTCCG 3' (SEQ ID NO:3) contains a BamHI restriction enzyme site (in bold) followed by 21 nucleotides of TGFα-HI coding sequence starting from the presumed terminal amino acid of the procesεed protein codon. The 3' εequence 5' GGGAAGCTTTTAATACTGAAATCGTACAGGAC 3' (SEQ ID NO:4) containε complementary sequenceε to a Hind III site and iε followed by 23 nucleotideε of TGFα-HI. The reεtriction enzyme εiteε correεpond to the reεtriction enzyme εites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA, 91311) . pQE-9 encodeε antibiotic reεistance (Ampr) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribosome binding site (RBS) , a 6-Hiε tag and reεtriction enzyme εites. pQE-9 waε then digested with BamHI and Hindlll. The amplified sequenceε were ligated into pQE-9 and were inεerted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture waε 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 Manual, Cold Spring Laboratory Press, (1989) . M15/rep4 containε multiple copieε of the plaεmid pREP4, which expreεεeε the lacl repreεsor and alεo confers kanamycin resiεtance (Kanr) . Transformants were identified by their ability to grow on LB plateε and ampicillin/kanamycin reεiεtant colonieε were εelected. Plaεmid DNA waε iεolated and confirmed by reεtriction analyεiε. Cloneε containing the deεired conεtructs were grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The 0/N culture waε used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside" ) was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacl repressor, clearing the P/0 leading to increased gene expresεion. Cellε were grown an extra 3 to 4 hourε. Cellε were then harveεted by centrifugation. The cell pellet waε εolubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized TGFα-HI waε purified from thiε εolution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al. , J. Chromatography 411:177-184 (1984)) . TGFα-HI (85 % pure) waε eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpoεe of renaturation adjuεted to 3 molar guanidine HCl, lOOmM εodium phoεphate, 10 molar glutathione (reduced) and 2 molar glutathione (oxidized) . After incubation in thiε εolution for 12 hourε the protein was dialyzed to 10 molar sodium phosphate.
Example 2 Cloning and expression of εoluble TGFα-HI using the baculovirus expression εvstem
The DNA sequence encoding the TGFα-HI protein, ATCC # 75698, was amplified using PCR oligonucleotide primers correεponding to the 5' and 3' εequenceε of the gene for expreεεing from the firεt amino acid of Figure 1 to the end of the active domain are:
Three sets of primers were used:
The first set of primers are, 5' CGCGGATCCGCCATCATGGGCGCCGCAGCCGC 3' (SEQ ID NO:5) and 5' GCGTCTAGACTAGTATAGAACACTGTAGTCC 3' (SEQ ID NO:6) ;
The εecond εet of primerε are:
5' CGCGGATCCAGTTTATATTGGAAACCACATGCC 3' (SEQ ID NO:7)
5' GCGTCTAGACTAATAGAGAATACTAAAGTC 3' (SEQ ID NO: 8) , theεe primerε are uεed to expreεs the putative active (soluble) domain;
All 5' primers have a BamHI reεtriction enzyme site (in bold) followed by nucleotides reεembling an efficient εignal for the initiation of tranεlation in eukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) (the initiation codon for translation is "ATG") .
The 3' primer sequenceε contain the cleavage site for the restriction endonucleaεe Xbal and have nucleotideε complementary to the 3' TGFα εoluble domain of the TGFα-HI gene. The amplified sequenceε were iεolated from a 1% agaroεe gel uεing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) . The fragment waε then digested with the endonucleases BamHI and Xbal and then purified again on a 1% agarose gel. This fragment was designated F2.
The vector pA2 waε used (modification of pVL941 vector, discuεsed below) for the expresεion of the TGFα-HI protein uεing the baculoviruε expression syεtem (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) . This expression vector containε the εtrong polyhedrin promoter of the Autographa californica nuclear polyhedroεiε viruε (AcMNPV) followed by the recognition εiteε for the reεtriction endonucleaεe . The polyadenylation εite of the εimian viruε (SV)40 waε uεed for efficient polyadenylation. For an easy selection of recombinant virus the beta- galactosidase gene from E.coli waε inεerted in the same orientation as the polyhedrin promoter followed by the polyadenylation εignal of the polyhedrin gene. The polyhedrin εequenceε were flanked at both εides by viral εequenceε for the cell-mediated homologouε recombination of co-transfected wild-type viral DNA. Many other baculovirus vectorε could be uεed in place of pRGl such aε pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31- 39) .
The plasmid was digeεted with the reεtriction enzymeε BamHI and Xbal and then dephoεphorylated uεing calf intestinal phosphataεe by procedureε known in the art. The DNA waε then iεolated from a 1% agaroεe gel uεing the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . Thiε vector DNA waε deεignated V2.
Fragment F2 and the dephoεphorylated plaεmid V2 were ligated with T4 DNA ligase. E.coli HB101 cells were then tranεformed and bacteria identified that contained the plaεmid (pBacTGFα-HI) with the TGFα-HI gene uεing the reεtriction enzymeε BamHI and Xbal. The εequence of the cloned fragment waε confirmed by DNA εequencing.
5 μg of the plaεmid pBacTGFα-HI waε co-tranεfected with 1.0 μg of a commercially available linearized baculoviruε ("BaculoGold™ baculoviruε DNA", Pharmingen, San Diego, CA. ) uεing the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)) . lμg of BaculoGold™ viruε DNA and 5 μg of the plaεmid pBacTGFα-HI were mixed in a εterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) . Afterwards 10 μl Lipofectin plus 90 μl Grace'ε medium were added, mixed and incubated for 15 minuteε at room temperature. Then the tranεfection mixture waε 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 waε rocked back and forth to mix the newly added εolution. The plate waε then incubated for 5 hourε at 27°C. After 5 hourε the tranεfection εolution waε removed from the plate and 1 ml of Grace's insect medium εupplemented with 10% fetal calf serum waε added. The plate waε put back into an incubator and cultivation continued at 27°C for four dayε.
After four dayε the εupernatant waε collected and a plaque assay performed similar aε described by Summers and Smith (εupra) . Aε a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue εtained plaques. (A detailed deεcription of a "plaque aεεay" can alεo be found in the user'ε guide for inεect cell culture and baculovirology diεtributed by Life Technologieε Inc., Gaitherεburg, page 9- 10) .
Four dayε after the εerial dilution, the viruε waε added to the cellε and blue stained plaques were picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculoviruε waε used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatantε of theεe culture diεhes were harvested and then stored at 4°C.
Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculoviruε V-TGFα-HI at a multiplicity of infection (MOI) of 2. Six hourε later the medium waε removed and replaced with SF900 II medium minuε methionine and cyεteine (Life Technologieε Inc., Gaitherεburg) . 42 hourε later 5 μCi of 35S-methionine and 5 μCi 3SS cyεteine (Amersham) were added. The cellε were further incubated for 16 hourε before they were harveεted by centrifugation and the labelled proteinε viεualized by SDS-PAGE and autoradiography.
Example 3 Expression of Recombinant TGFα-HI in COS cells The expression of plasmid, TGFα-HI HA was derived from a vector pcDNA3/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site. A DNA fragment encoding the entire TGFα-HI precursor and a HA tag fused in frame to itε 3' end waε cloned into the polylinker region of the vector, therefore, the recombinant protein expreεεion waε directed under the CMV promoter. The HA tag correεpondε to an epitope derived from the influenza hemagglutinin protein aε previouεly deεcribed (I. Wilεon, H. Niman, R. Heighten, A Cherenεon, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)) . The infuεion of HA tag to the target protein allowε eaεy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy waε deεcribed aε follows:
The DNA εequence encoding TGFα-HI, ATCC # 75698, waε conεtructed by PCR on the original EST cloned uεing two primerε: the 5' primer 5' CGCGGATCCGCCATCATGGTGCTGTGGGAGTCC 3' (SEQ ID NO:12) containε a BamHI site (in bold) followed by 18 nucleotides of TGFα-HI coding sequence starting from the initiation codon; the 3' sequence 5' GCGCTCGAGGTATAGAAC ACTGTAGTCC 3' (SEQ ID NO:13) contains complementary εequenceε to an Xhol εite, the laεt 19 nucleotideε of the TGFα domain and an Xhol εite. The pcDNA3/Amp vector containε BamHI/XhoI cloning εiteε which bring the PCR insert in frame with the 3' HA tag followed by a εtop codon. Therefore, the PCR product containε a BamHI εite, 936 base pair coding sequence and an Xhol site. The PCR amplified DNA fragment and the vector, pcDNA3/Amp, were digested with BamHI and Xhol reεtriction enzyme and ligated. The ligation mixture waε tranεformed into E. coli εtrain SURE (available from Stratagene Cloning Systems, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plateε and reεiεtant colonieε were selected. Plasmid DNA was isolated from transformants and examined by restriction analysiε for the preεence of the correct fragment. For expression of the recombinant TGFα-HI, COS cellε were tranεfected with the expreεεion vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Presε, (1989)) . The expreεεion of the TGFα-HI HA protein waε detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Presε, (1988)) . Cellε were labelled for 8 hourε with 35S-cyεteine two dayε post transfection. Culture media was then collected and cells were 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 were precipitated with an HA εpecific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.
Example 4 Expression via Gene Therapy
Fibroblastε are obtained from a εubject by skin biopsy. The resulting tiεεue is placed in tissue-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 pieceε are placed in each flaεk. The flaεk iε turned upεide down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tisεue remain fixed to the bottom of the flaεk and freεh media (e.g., Ham'ε F12 media, with 10% FBS, penicillin and εtreptomycin, is added. This is then incubated at 37°C for approximately one week. At this time, freεh media iε added and εubεequently changed every εeveral dayε. After an additional two weekε in culture, a monolayer of fibroblastε emerge. The monolayer is trypsinized and scaled into larger flaskε. pMV-7 (Kirεchmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma viruε, iε digeεted with EcoRI and Hindlll and subsequently treated with calf intestinal phosphataεe. The linear vector iε fractionated on agarose gel and purified, using glass beadε.
The cDNA encoding a polypeptide of the present invention iε amplified uεing PCR primerε which correεpond to the 5' and 3' end sequences respectively. The 5' primer containing an EcoRI site and the 3' primer further includes a Hindlll site. Equal quantities of the Moloney murine εarcoma virus linear backbone and the amplified EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture iε uεed to transform bacteria HBlOl, which are then plated onto agar-containing 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 Eagleε Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectiouε viral particles containing the gene (the packaging cells are now referred to aε producer cellε) .
Freεh media iε added to the transduced producer cellε, and subsequently, the media iε harvested from a 10 cm plate of confluent producer cellε. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media iε then used to infect fibroblast cellε. Media iε removed from a εub-confluent plate of fibroblaεtε and quickly replaced with the media from the producer cells. Thiε media is removed and replaced with freεh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the 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 fibroblastε are then injected into the hoεt, either alone or after having been grown to confluence on cytodex 3 microcarrier beadε. The fibroblaεtε now produce the protein product.
Numerouε modificationε and variationε of the 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 deεcribed.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: MEISSNER, ET AL.
(ii) TITLE OF INVENTION: Tranεforming Growth Factor αHI
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07068
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: MS -DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/468,846
(B) FILING DATE: June 6, 1995
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER:
(B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134
(C) REFERENCE/DOCKET NUMBER: 325800-465
(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: 1565 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TGGGCGCGCG GCTGGATGCC CCCGGCCTGC GGCTCCCTGC GCTTCCCGCC GTGGAGGGGC 60
ACCAGTCATG GGCGCCGCAG CCGCTGAGGC GCCGCTCCGG CTGCCTGCCG CGCCTCCGCT 120
CGCCTTCTGC TGCTACACGT CGGTGCTTCT GCTCTTCGCC TTCTCTCTGC CCGGGAGCCG 180
CGCGTCCAAC CAGCCCCCGG GTGGTGGCGG CGGCAGCGGC GGGGACTGTC CCGGCGGCAA 240
AGGCAAGAGC ATCAACTGCT CAGAATTAAA TGTGAGGGAG TCTGACGTAA GAGTTTGTGA 300
TGAGTCATCA TGTAAATATG GAGGAGTCTG TAAAGAAGAT GGAGATGGTT TGAAATGTGC 360
ATGCCAATTT CAGTGCCATA CAAATTATAT TCCTGTCTGT GGATCAAATG GGGACACTTA 420
TCAAAATGAA TGCTTTCTCA GAAGGGCTGC TTGTAAGCAC CAGAAAGAGA TAACAGTAAT 480
AGCAAGAGGA CCATGCTACT CTGATAATGG ATCTGGATCT GGAGAAGGAG AAGAGGAAGG 540
GTCAGGGGCA GAAGTTCACA GAAAACACTC CAAGTGTGGA CCCTGCAAAT ATAAAGCTGA 600
GTGTGATGAA GATGCAGAAA ATGTTGGGTG TGTATGTAAT ATAGATTGCA GTGGATACAG 660
TTTTAATCCT GTGTGTGCTT CTGATGGGAG TTCCTATAAC AATCCCTGTT TTGTTCGAGA 720
AGCATCTTGT ATAAAGCAAG AACAAATTGA TATAAGGCAT CTTGGTCATT GCACAGATAC 780
AGATGACACT AGTTTGTTGG GAAAGAAAGA TGATGGACTA CAATATCGAC CAGATGTGAA 8 0
AGATGCTAGT GATCAAAGAG AAGATGTTTA TATTGGAAAC CACATGCCTT GCCCTGAAAA 900
CCTCAATGGT TACTGCATCC ATGGAAAATG TGAATTCATA TATTCTACTC AGAAGGCTTC 960
TTGTAGATGT GAATCTGGCT ACACTGGACA GCACTGTGAA AAGACAGACT TTAGTATTCT 1020 CTATGTAGTG CCAAGTAGGC AAAAGCTCAC TCATGTTCTT ATTGCAGCAA TTATTGGAGC 1080 TGTACAGATT GCCATCATAG TAGCAATTGT AATGTGCATA ACAAGAAAAT GCCCCAAAAA 1140 CAATAGAGGA CGTCGACAGA AGCAAAACCT AGGTCATTTT ACTTCAGATA CGTCATCCAG 1200 AATGGTTTAA ACTGATGACT TTTATATGTA CACTGACCAT GTGATGTACA TTTATTATGT 1260 CTTTTTTTAA AGAATGGAAA TATTTATTTC AGAGGCCTTA TTTTTGGACA TTTTTAGTGT 1320 AGTACTGTTG GCTCGTATTT AGAATATTCA GCTACGACAG TTTTGGACTG TTTAGTAGTC 1380 TTTGTTTTAT GTTTTTAAAT ACAGAAATTG CTTTCACAAA TTTGTACCAC ATGGTAATTC 1440 TAAGACTTGT TCTTTACCCA TGGAATGTAA TATTTTTGCA AAGATGGACT ACTTCACAAA 1500 TGGTTATAAA GTCATATCCA CTTCTTCCAC AATGACCACA GCAAATGACC AAGCATGAAC 1560 TAAAG 1565
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 380 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Gly Ala Ala Ala Ala Glu Ala Pro Leu Arg Leu Pro Ala Ala
-35 -30 -25
Pro Pro Leu Ala Phe Cys Cys Tyr Thr Ser Val Leu Leu Leu Phe
-20 -15 -10
Ala Phe Ser Leu Pro Gly Ser Arg Ala Ser Asn Gin Pro Pro Gly
-5 1 5
Gly Gly Gly Gly Ser Gly Gly Asp Cyε Pro Gly Gly Lys Gly Lys
10 15 20
Ser lie Asn Cyε Ser Glu Leu Asn Val Arg Glu Ser Asp Val Arg
25 30 35
Val Cys Asp Glu Ser Ser Cyε Lyε Tyr Gly Gly Val Cyε Lyε Glu
40 45 50
Aεp Gly Aεp Gly Leu Lyε Cyε Ala Cyε Gin Phe Gin Cyε Hiε Thr
55 60 65
Aεn Tyr lie Pro Val Cyε Gly Ser Asn Gly Asp Thr Tyr Gin Asn 70 75 80
Glu Cyε Phe Leu Arg Arg Ala Ala Cyε Lyε Hiε Gin Lyε Glu lie
85 90 95
Thr Val lie Ala Arg Gly Pro Cyε Tyr Ser Asp Asn Gly Ser Gly
100 105 110
Ser Gly Glu Gly Glu Glu Glu Gly Ser Gly Ala Glu Val His Arg
115 120 125
Lyε His Ser Lyε Cyε Gly Pro Cyε Lyε Tyr Lyε Ala Glu Cyε Aεp
130 135 140
Glu Aεp Ala Glu Asn Val Gly Cyε Val Cyε Asn lie Asp Cys Ser
145 150 155
Gly Tyr Ser Phe Asn Pro Val Cys Ala Ser Asp Gly Ser Ser Tyr
160 165 170
Asn Asn Pro Cys Phe Val Arg Glu Ala Ser Cys lie Lys Gin Glu
175 180 185
Gin lie Asp lie Arg His Leu Gly Hiε Cyε Thr Aεp Thr Aεp Aεp
190 195 200
Thr Ser Leu Leu Gly Lyε Lyε Aεp Aεp Gly Leu Gin Tyr Arg Pro
205 210 215
Aεp Val Lyε Asp Ala Ser Asp Gin Arg Glu Asp Val Tyr lie Gly
220 225 230
Asn His Met Pro Cyε Pro Glu Asn Leu Aεn Gly Tyr Cyε lie His
235 .240 245
Gly Lyε Cyε Glu Phe lie Tyr Ser Thr Gin Lys Ala Ser Cys Arg
250 255 260
Cyε Glu Ser Gly Tyr Thr Gly Gin Hiε Cyε Glu Lyε Thr Aεp Phe
265 270 275
Ser lie Leu Tyr Val Val Pro Ser Arg Gin Lyε Leu Thr Hiε Val
280 285 290
Leu lie Ala Ala lie lie Gly Ala Val Gin lie Ala lie lie Val
295 300 305
Ala lie Val Met Cyε lie Thr Arg Lyε Cyε Pro Lyε Asn Asn Arg
310 315 320
Gly Arg Arg Gin Lys Gin Asn Leu Gly Hiε Phe Thr Ser Asp Thr
325 330 335 Ser Ser Arg Met Val
340 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CCCGGATCCG CACGAGACAT ACCTTGTCCG 30
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 32 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGGAAGCTTT TAATACTGAA ATCGTACAGG AC 32
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 32 BASE PAIRS
(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CGCGGATCCG CCATCATGGG CGCCGCAGCC GC 32
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 31 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCGTCTAGAC TAGTATAGAA CACTGTAGTC C 31
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 33 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CGCGGATCCA GTTTATATTG GAAACCACAT GCC 33 (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GCGTCTAGAC TAATAGAGAA TACTAAAGTC 30

Claims

WHAT IS CLAIMED IS:
1. An iεolated polynucleotide compriεing a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide as set forth in Figure 1;
(b) a polynucleotide encoding the polypeptide comprising amino acids 1 to 380 of Figure 1;
(c) a polynucleotide encoding the polypeptide comprising amino acids 1 to 316 of Figure 1;
(d) a polynucleotide encoding the polypeptide comprising amino acids 267 to 316 of Figure 1;
(e) a polynucleotide encoding the polypeptide comprising amino acids 40 to 316 of Figure 1; and
(f) a polynucleotide capable of hybridizing to and which is at leaεt 70% identical to the polynucleotide of (a) , (b) , (c) , (d) or (e) ; and
(h) a polynucleotide fragment of the polynucleotide of (a) , (b) , (c) , (d) , (e) or (f) .
2. The polynucleotide of Claim 1 wherein the polynucleotide iε DNA.
3. The polynucleotide of Claim 2 encoding the polypeptide comprising amino acids 267 to 316 aε set forth in Figure 1.
4. An iεolated polynucleotide compriεing a member selected from the group consisting of:
(a) a polynucleotide which encodes a mature polypeptide having the amino acid sequence expressed by the DNA contained in ATCC Deposit No. 75698;
(b) a polynucleotide capable of hybridizing to and which iε at leaεt 70% identical to the polynucleotide of (a) ; and
(c) a polynucleotide fragment of the polynucleotide of (a) or (b) .
5. A vector containing the DNA of Claim 2.
6. A host cell transformed or transfected with the vector of Claim 5.
7. A procesε for producing a polypeptide compriεing: expreεsing from the host cell of Claim 6 the polypeptide encoded by said DNA.
8. A procesε for producing cellε capable of expreεεing a polypeptide compriεing genetically engineering cellε with the vector of Claim 5.
9. A polypeptide comprising a member selected from the group consisting of:
(a) a polypeptide having the deduced amino acid εequence of Figure 1;
(b) a polypeptide comprising amino acids 1 to 380 aε set forth in Figure 1;
(c) a polypeptide comprising amino acids 267 to 316 as set forth in Figure 1;
(d) a polypeptide comprising amino acids 40 to 316 aε set forth in Figure 1;
(e) a polypeptide comprising amino acids 1 to 316 aε εet forth in Figure 1;
(f) fragmentε, analogε and derivatives of the polypeptide of (a) , (b) , (c) , (d) or (e) ; and
(g) a polypeptide encoded by the cDNA of ATCC Deposit No. 75698 and fragmentε, analogε and derivativeε of εaid polypeptide.
10. The polypeptide of claim 9 comprising amino acid 267 to amino acid 316 of Figure 1.
11. An antibody against the polypeptide of claim 9.
12. A compound which inhibitε activation of the polypeptide of claim 9.
13. A compound which activates the polypeptide of claim 9.
14. A method for the treatment of a patient having need of TGFα-HI comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 9.
15. A method for the treatment of a patient having need to inhibit TGFα-HI compriεing: administering to the patient a therapeutically effective amount of the compound of Claim 12.
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 expresεing εaid polypeptide in vivo.
17. A process for identifying compoundε active aε agonists to the polypeptide of Claim 9 comprising: contacting a reaction mixture containing a cell type which expreεses a TGFα-HI receptor and a compound to be screened; and determining if the compound generateε a εignal from εaid receptor to identify if the compound iε an effective agoniεt.
18. A procesε for identifying compoundε active aε antagoniεtε to the polypeptide of Claim 9 compriεing: contacting a reaction mixture containing a cell type which expreεεeε the TGFα-HI receptor and a compound to be εcreened; 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.
19. A process for diagnoεing a diεeaεe or a εuceptibility to a disease comprising: determining a mutation in the polynucleotide of claim 1.
20. A diagnostic procesε comprising: analyzing for the presence of the polypeptide of Claim 9 in a sample derived from host.
PCT/US1996/009448 1995-06-06 1996-06-06 Transforming growth factor alpha hi WO1996039497A1 (en)

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EP96921363A EP0856050A4 (en) 1995-06-06 1996-06-06 Transforming growth factor alpha hi
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EP0873360A4 (en) * 1996-01-04 2000-06-21 Human Genome Sciences Inc Transforming growth factor alpha hiii

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US6074839A (en) 2000-06-13
KR19990022316A (en) 1999-03-25
EP0856050A4 (en) 2000-06-21
JP2001517064A (en) 2001-10-02
CA2223957A1 (en) 1996-12-12
MX9709237A (en) 1998-03-31
AU717005B2 (en) 2000-03-16
AU6260496A (en) 1996-12-24
CN1240836C (en) 2006-02-08
CN1186515A (en) 1998-07-01
EP0856050A1 (en) 1998-08-05
US6265543B1 (en) 2001-07-24

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