WO1997038002A1 - Extracellular/epidermal growth factor-like protein - Google Patents
Extracellular/epidermal growth factor-like protein Download PDFInfo
- Publication number
- WO1997038002A1 WO1997038002A1 PCT/US1996/005247 US9605247W WO9738002A1 WO 1997038002 A1 WO1997038002 A1 WO 1997038002A1 US 9605247 W US9605247 W US 9605247W WO 9738002 A1 WO9738002 A1 WO 9738002A1
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- WIPO (PCT)
- Prior art keywords
- polypeptide
- polynucleotide
- dna
- cells
- polypeptides
- Prior art date
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Classifications
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- C07—ORGANIC CHEMISTRY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- 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
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- 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
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- 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
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- 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
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- 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
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- 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
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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
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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 extracellular protein-like/Epidermal Growth Factor-like protein, hereafter referred to as "EEGF" .
- 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 modulatory 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.
- TGF ⁇ transforming growth factor alpha
- betacellulin betacellulin
- amphiregulin vaccinia growth factor among other factors
- vaccinia growth factor 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 influence epithelial and epidermal 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-c ain disulfide bonds.
- the receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the gastro-intestinal tract (GI) tract.
- GI gastro-intestinal 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 is 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 or.
- 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., J. Cell. Biochem.. 42:45-57 (1990)), as well as by a variety of tumors, including renal, breast and squamous carcinomas, melanomas and glioblastomas (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 macrophages. 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 an Extracellular/Epidermal Growth Factor. This identification has been made as a result of amino acid sequence homology to human extracellular protein which is a secreted protein with EGF-like domains that is abundant in heart tissue.
- novel mature polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
- the polypeptides of the present invention are of human origin.
- nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
- an isolated nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97285.
- processes 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 such polypeptides for therapeutic purposes for example, to regulate vascular smooth muscle cell proliferation, to treat Marfan syndrome, to stimulate wound healing, to restore normal neurological functioning after trauma or AIDS dementia, to treat ocular disorders, to treat kidney and liver disorders, to promote hair follicular development, to stimulate growth and differentiation of various epidermal and epithelial cells in vivo and in vi tro and for the treatment of burns, ulcers and corneal incisions, to stimulate embryogenesis.
- nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid sequences of the present invention.
- antagonists to such polypeptides 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 overexpression 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 and corresponding deduced amino acid sequence of EEGF. Both the standard one letter and three letter abbreviations for amino acids are used.
- Figure 2 is an illustration of comparative amino acid sequence homology between the polypeptide of the present invention (lower line) and human extracellular protein (upper line) (SEQ ID NO:9) .
- nucleic acid which encodes for the mature polypeptide having the deduced amino acid sequence of Figure l (SEQ ID NO.-2) .
- a polynucleotide encoding a polypeptide of the present invention may be obtained from human brain and early stage brain tissue.
- the polynucleotide of this invention was discovered in a human fetal heart cDNA library. It has homology to the characteristic EGF domains. It contains an open reading frame encoding a polypeptide of 392 amino acids. EEGF exhibits the highest degree of homology at the amino acid level to human extracellular protein with 45% identity and 34% similarity over a 392 amino acid stretch. Northern blot analysis of this protein shows high levels of expression in heart tissue with the transcript being approximately 2 kb.
- isolated polynucleotides encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97285, deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, USA, on September 26, 1995.
- the deposited material is an a plasmid that contains the full-length EEGF cDNA which has been transformed into a viable host.
- the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
- the DNA may be double-stranded or single- stranded, and if single stranded may be the coding strand or non ⁇ coding (anti-sense) strand.
- the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID N0:1) 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 NO:l) .
- the polynucleotide which encodes for the mature polypeptide of Figure 1 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 secretory sequence or a proprotein sequence,- the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non ⁇ coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
- polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
- the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) .
- the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
- the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) as well as variants of such polynucleotides which variants encode for ⁇ »wire , ⁇ -,
- nucleotide variants include deletion variants, substitution 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 NO:l) .
- an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
- the present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
- the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
- the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues.
- a mature protein having a prosequence is a proprotein and is an inactive form of the protein.
- the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
- the marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a 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 (introns) between individual coding segments (exons) .
- Fragments of the full length EEGF gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity.
- Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases.
- the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete EEGF gene including regulatory and promotor regions, exons, and introns.
- An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
- the present invention further relates to polynucleotides which hybridize to the 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 sequences.
- polypeptides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure l (SEQ ID NO:l) .
- the polynucleotide may have at least 15 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity.
- such polynucleotides 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 polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 and polynucleotides complementary thereto as well as portions thereof, which portions have at least 15 consecutive bases preferably at least 30 consecutive bases and preferably at least 50 consecutive bases and to polypeptides encoded by such polynucleotides.
- the present invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) , as well as fragments, analogs and derivatives of such polypeptide.
- fragment when referring to the polypeptide of Figure 1 (SEQ ID NO:2) , means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
- 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.
- 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 materials in the natural system, is isolated.
- Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and mor: preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still 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.
- similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
- the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
- 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 expression vectors for expressing a polypeptide.
- Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as 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 endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
- the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
- the vector containing Che appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
- bacterial cells such as E. coli. Streptomyces. Salmonella typhimurium
- fungal cells such as yeast
- insect cells such as Drosophila S2 and Spodootera Sf9
- animal cells such as CHO, COS or Bowes melanoma
- adenoviruses plant cells, etc.
- the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
- the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
- 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, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNHl ⁇ A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
- Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
- Two appropriate vectors are pKK232-8 and pCM7.
- Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
- Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
- constructs in host cells 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 cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. , (1989), the disclosure of which is hereby incorporated by reference.
- Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
- the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
- Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
- 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 Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) .
- pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
- the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
- Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
- Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysmg agents, such methods are well known to those skilled in the art.
- mammalian cell culture systems can also be employed to express recombinant protein.
- mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
- Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
- polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose 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
- polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
- a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
- the polypeptides of the present invention may be glycosylated or may be non- glycosylated.
- Polypeptides of the invention may also include an initial methionine amino acid residue.
- the polypeptide of the present invention may be employed to to regulate vascular smooth muscle cell proliferation.
- the polypeptide of the present invention may also be employed for characterization of receptors.
- the EGF family of receptors currently includes four EGF receptors, denoted as EGFRl, EGFR2, EGFR3 and EGFR4.
- the EGFR2 receptor may also be referred to as ERB-2 and this molecule is useful for a variety of diagnostic and therapeutic indications (Prigent, S.A., and Lemoine, N.R., Prog Growth Factor Res.. 4:1-24 (1992)) .
- the EEGF polypeptide is likely a ligand for one or more of these receptors as well as for an unidentified new EGF- ype receptor. Use of the EEGF can assist with the identification, characterization and cloning of such receptors.
- the EGF receptor gene represents the cellular homolog of the v-erb-B oncogene of avian erythroblastosis virus.
- Over expression of the EGF-receptor or deletion of kinase regulatory segments of the protein can bring about tumorigenic transformation of cells (Manjusri, D. et al., Human Cytokines. 364 and 381 (1991)) .
- the polypeptides of the present invention may also be employed for restoration or enhancement of neurological functions diminished as a result of trauma or other damaging pathologies (such as AIDS dementia, senile dementia, etc) .
- TGF ⁇ and its homologs have been found to be the most abundant ligand for the EGF/TGF ⁇ receptor in most parts of the brain (Kaser, et al., Mol Brain Res: 16:316-322, (1992) ) .
- EEGF or soluble form thereof may also be employed to treat ocular disorders, for example, corneal inflammation.
- a variety of experiments have implicated members of the TGF ⁇ gene family in such pathologies.
- EEGF or soluble forms thereof can be coupled, by a wide variety of methods known in the art, to toxic molecules: for example, a radiopharmaceutical which inactivate target cells. These growth factor-toxin fusions kill the target cell (and in certain cases neighboring cells by a variety of "bystander” effects) .
- toxic molecules for example, a radiopharmaceutical which inactivate target cells.
- EEGF and related molecules may also be encapsulated in liposomes and may be conjugated to antibodies which recognize and bind to tumor or cell specific antigens, thereby provided a means for "targeting" cells.
- the EEGF polypeptide may also be employed to treat certain kidney disorders, since it has been found that there has been expression of these growth factors in the kidney. Thus, these factors may be necessary for the proper physiological maintenance of this organ. Treatments may also be related to liver regeneration or liver dysfunction.
- a significant treatment involving EEGF relates to wound healing.
- the compositions of the present invention may be employed for treating a wide variety of wounds including substantially all cutaneous wounds, corneal wounds, and injuries to the epithelial- lined hollow organs of the body.
- Wounds suitable for treatment include those resulting from trauma such as burns, abrasions and cuts, as well as from surgical procedures such as surgical incisions and skin grafting.
- conditions suitable for treatment with the polypeptide of the present invention include chronic conditions, such as chronic ulcers, diabetic ulcers, other non-healing (trophic) conditions, to treat Marfan syndrome, to promote hair follicular development, to stimulate growth and differentiation of various epidermal and epithelial cells m vivo and in vitro and to stimulate embryogenesis.
- chronic conditions such as chronic ulcers, diabetic ulcers, other non-healing (trophic) conditions
- EEGF 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.
- suitable bases include lanolin, Silvadene (Marion) (particularly for the treatment of burns) , Aquaphor (Duke Laboratories, South Norwalk, Conn.), and the like.
- EEGF containing compositions in bandages and other wound dressings to provide for continuous exposure of the wound to the peptide. Aerosol applications may also find use.
- the concentration of EEGF in the treatment composition is not critical but should be enough to induce epithelial cell proliferation.
- the compositions may be applied topically to the affected area, typically as eye drops to the eye or as creams, ointments or lotions to the skin. In the case of the eyes, frequent treatment is desirable, usually being applied at intervals of 4 hours or less. On the skin, it is desirable to continually maintain the treatment composition on the affected area during the healing, with applications of the treatment composition from two to four times a day or more frequently.
- the amount employed of the subject polypeptide will vary with the manner of administration, the employment of other active compounds, and the like, generally being in the range of about 1 ⁇ g to 100 ⁇ g .
- the subject polypeptide may be employed with a physiologically acceptable carrier, such as saline, phosphate- buffered saline, or the like.
- the amount of compound employed will be determined empirically, based on the response of cells in vitro and response of experimental animals to the subject polypeptides or formulations containing the subject polypeptides.
- the EEGF or soluble fragment thereof may be employed in the modulation c ' angiogenesis, bone resorption, immune response, and synaptic and neuronal effector functions. EEGF may also be used in the modulation of the arachidonic acid cascade.
- Certain disease pathologies may be partially or completely ameliorated by the systemic clinical administration of the EEGF growth factor.
- This administration can be in the form of gene therapy (see below) ; or through the administration of peptides or proteins synthesized from recombinant constructs of EEGF DNA or from peptide chemical synthesis (Woo, et al., Protein Engineering 3:29-37 (1989) .
- This invention provides a method for identification of EEGF receptors.
- the gene encoding a receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al. , Current Protocols in Immun., 1(2), Chapter 5, (1991)).
- expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to EEGF, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to EEGF.
- EEGF vascular endothelial growth factor
- labeled EEGF can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to auto- radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
- labeled ligand can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film.
- the labeled complex containing the ligand- receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
- the amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
- Another assay for identifying potential antagonists specific to the receptors to the polypeptide of the present invention is a competition assay which comprises isolating plasma membranes which over-express a receptor to the polypeptide of the present invention, for example, human A431 carcinoma cells.
- Serially diluted test sample in a medium (volume is approximately 10 microliters) containing 10 nM 125 I-EEGF is added to five micrograms of the plasma membrane in the presence of the potential antagonist compound and incubated for 4 hours at 4°C.
- the reaction mixtures 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 EEGF is then measured.
- a control assay is also performed in the absence of the compound to determine if the antagonists reduce the amount of bound EEGF.
- Potential antagonist compounds include an antibody, or in some cases, an oligopeptide, which binds to the polypeptide.
- a potential antagonist may be a closely related protein which binds to the receptor which is an inactive forms of the polypeptide and thereby prevent the action of the polypeptide of the present invention.
- Another antagonist compound is an antisense construct prepared using antisense technology.
- Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
- the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
- a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl.
- the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide of the present invention (Antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
- the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the polypeptide of the present invention.
- Antagonist compounds include a small molecule which binds to the polypeptide of the present invention and blocks its action at the receptor such that normal biological activity is prevented.
- the small molecules 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 molecules.
- the antagonists may be employed to treat neoplasia, for example, cancers and tumors. It is known that inhibition of secretion or production of members of the EGF family by tumor cells in mice causes regression of tumors, since these proteins stimulate induction of DNA synthesis in all cells including neoplastic cells.
- the antagonists to the polypeptides of the present invention may also be used therapeutically for the treatment of certain skin disorders, for example, psoriasis. Elevated levels of expression of members of this family of growth factors in skin biopsies taken from diseases such as psoriatic lesions have been found to be elevated (Cook, et al., Cancer Research. 52:3224-3227 (1992)).
- the antagonists may be employed in a compositicn with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
- compositions comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient.
- a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
- the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
- the polypeptides or compounds of the present invention may be employed in conjunction with other therapeutic compounds.
- the pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
- the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
- cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
- a packaging cell is transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
- These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
- Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
- the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
- the vector includes one or more promoters.
- Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter,- and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and 3-actin promoters) .
- Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
- Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters,- viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter,- retroviral LTRs (including the modified retroviral LTRs hereinabove described) ,- the ⁇ -actin promoter,- and human growth hormone promoters.
- the promoter also may be the native promoter which controls the gene en
- the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
- packaging cells which may be transfected include, but are not limited to, the PE501, PA317, -2 , -RM, PA12, T19-14X, VT-19-17- H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
- the vector may transduce the packaging cells through any means known in the art.
- retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
- the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
- retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
- the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
- Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
- This invention is also related to the use of the gene of the present invention as a diagnostic. Detection of a mutated form of the gene of the present invention will allow a diagnosis of a disease or a susceptibility to a disease which results from underexpression of the polypeptide of the present invention, for example, improper wound healing, improper neurological functioning, ocular disorders, kidney and liver disorders, hair follicular development, angiogenesis and embryogenesis.
- deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
- Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
- Sequence differences between the reference gene and genes having mutations 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 sequencing procedures with fluorescent-tags.
- DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985)).
- Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and 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 methods such as hybridization, RNase 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)
- mutations can also be detected by in si tu analysis.
- the present invention also relates to diagnostic assays for detecting altered levels of the polypeptide of the present invention in various tissues since an over-expression of the proteins compared to normal control tissue samples can detect the presence of certain disease conditions such as neoplasia, skin disorders, ocular disorders and inflammation.
- Assays used to detect levels of the polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and include radioimmunoas ⁇ ays, competitive-binding assays, Western Blot analysis and preferably an ELISA assay.
- An ELISA assay initially comprises preparing an antibody specific to an antigen of the polypeptide of the present invention, preferably a monoclonal antibody.
- a reporter antibody is prepared against the monoclonal antibody.
- a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme.
- a sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin.
- the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any polypeptides of the present invention attached to the polystyrene dish. All 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 present invention. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of protein present in a given volume of patient sample when compared against a standard curve.
- a competition assay may also be employed to determine levels of the polypeptide of the present invention in a sample derived from the hosts.
- Such an assay comprises isolating plasma membranes which over-express the receptor for the polypeptide of the present invention.
- a test sample containing the polypeptides of the present invention which have been labeled are then added to the plasma membranes and then incubated for a set period of time.
- Also added to the reaction mixture is a sample derived from a host which is suspected of containing the polypeptide of the present invention.
- the reaction mixtures are then passed through a filter which is rapidly washed and the bound 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.
- Antibodies specific to EEGF may be used for cancer diagnosis and therapy, since many types of cancer cells up-regulate various members of the TGF ⁇ family during the process of neoplasia or hyperplasia. These antibodies bind to and inactivate EEGF. Monoclonal antibodies against EEGF (and/or its family members) are in clinical use for both the diagnosis and therapy of certain disorders including (but not limited to) hyperplastic and neoplastic growth abnormalities. Upregulation of growth factor expression by neoplastic tissues forms the basis for a variety of serum assays which detect increases in growth factor in the blood of affected patients. These assays are typically applied not only in diagnostic settings, but are applied in prognostic settings as well (to detect the presence of occult tumor cells following surgery, chemotherapy, etc) .
- malignant cells expressing the EEGF receptor may be detected by using labeled EEGF in a receptor binding assay, or by the use of antibodies to the EEGF receptor itself.
- Cells may be distinguished in accordance with the presence and density of receptors for EEGF, thereby providing a means for predicting the susceptibility of such cells to the biological activities of EEGF.
- sequences of the present invention are also valuable for chromosome identification.
- the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
- Few chromosome 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 is an important first step in correlating those sequences with genes associated with disease.
- PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
- sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
- Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
- Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
- This technique can be used with cDNA as short as 50 or 60 bases.
- Verma et al. Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
- a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes l megabase mapping resolution and one gene per 20 kb) .
- Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
- any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
- “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
- the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
- typically l ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
- typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume.
- Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37"C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction i ⁇ electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
- Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).
- Ligase refers to the proces ⁇ of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T. , et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
- ligase T4 DNA ligase
- the DNA sequence encoding EEGF is initially amplified using PCR oligonucleotide primers corresponding to the 5' sequences of the EEGF protein and the sequences 3' of the EEGF gene.
- the 5' oligonucleotide primer has the sequence 5' GACTTCATGATGTGTGTTAACCAAAATGG 3' (SEQ ID NO:3) contains a Bsp HI restriction enzyme site followed by 19 nucleotides of EEGF coding sequence starting from the presumed terminal amino acid.
- the 3' sequence 5' GACTGGATCCGAATGGGTACTGCGACACATATATC 3' contains complementary sequences to a BamHI site and is followed by 25 nucleotides of EEGF.
- the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, CA) .
- pQE-60 encodes antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG- regulatable promoter operator (P/O) , a riboso e binding site (RBS) , a 6-His tag and restriction enzyme sites.
- pQE-60 is then digested with BamHI.
- O.D. 600 optical density 600
- EEGF is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.
- the DNA sequence encoding the EEGF protein, ATCC 8 97285, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
- the primer sequences are as follows: 5' CAGTGGATCCGCCATC ATGATGTGTGTTAACCAAAATGGCG 3' (SEQ ID NO:5) , has a BamHI re ⁇ triction enzyme site (in bold) followed by 6 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol. Biol., 196:947-950 (1987) (the initiation codon for translation is "ATG”) .
- the 3' primer 5' GCATGGTACCCGTCGGAGGCTCCAGCCCGAGG 3' contains the cleavage site for the restriction endonuclease A ⁇ n718 (bold) and 22 nucleotides complementary to the 3' end of the EEGF gene.
- the amplified sequences are isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with the endonucleases BamHI and Asp7l8 and then purified again on a 1% agarose gel. This fragment is designated F2.
- the vector pA2 is used (modification of pVL941 vector, discussed below) for the expression of the EEGF protein using the baculovirus expression system (for review see: Summer? M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
- the polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation.
- the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene.
- the polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co ⁇ transfected wild-type viral DNA.
- Many other baculovirus vectors could be used such as pAc373, pRGl, pVL94l and pAcIMl (Luckow, V.A. and Summers, M.D. , Virology, 170:31-39).
- the plasmid is digested with the restriction enzymes BamHI and Asp718 and then dephosphorylated using calf intestinal phosphatase by procedures known in the art.
- the DNA is then isolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This vector DNA is designated V2.
- Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase.
- E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid (pBacEEGF) with the EEGF gene using the restriction enzymes BamHI and Asp7l8. The sequence of the cloned fragment is confirmed by DNA sequencing.
- plasmid pBacEEGF 5 ⁇ g of the plasmid pBacEEGF is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417
- the plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and l ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation continued at 27°C for four days.
- plaque assay After four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra) . As a modification an agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A detailed description of a "plaque assay” can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc. , Gaithersburg, page 9-10) .
- the virus is added to the cells and blue stained plaques are picked with the tip of an Eppendorf pipette.
- the agar containing the recombinant viruses is then resu ⁇ pended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
- the agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
- the supematants of these culture dishes are harvested and then stored at 4°C.
- Sf9 cells are grown in Grace's medium supplemented with 1 0% heat-inactivated FBS.
- the cells are infected with the recombinant baculovirus V-EEGF at a multiplicity of infection (MOD of 2.
- MOD multiplicity of infection
- the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Ine. , Gaithersburg) .
- the cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
- EEGF HA The expression of plasmid, EEGF HA is 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 EEGF precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
- the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, R.
- HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
- the plasmid construction strategy is described as follows:
- the DNA sequence encoding EEGF, ATCC # 97285, is constructed by PCR using two primers: the 5' primer 5' GACTGGATCCGCCACCATGATGTGTGTTAACCAAAATG 3' (SEQ ID NO:7) contains a BamHI site (in bold) followed by 6 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells and 22 nucleotides of EEGF coding sequence starting from the initiation codon ,- the 3 ' sequence 5 ' GACTTCTAGAGAATGGGTACTGCGACACATAT 3' (SEQ ID NO: 8) contains complementary sequences to an Xbal site, 22 nucleotides of the EEGF gene followed by sequences encoding the HA tag.
- the pcDNA3/Amp vector contains BamHI/Xbal cloning sites which bring the PCR insert in frame with the 3' HA tag followed by a stop codon.
- the PCR amplified DNA fragment and the vector, pcDNA3/Amp are digested with BamHI and Xbal restriction enzyme and ligated.
- the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
- COS cells are transfected with the expres ⁇ ion vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)).
- the expression of the EEGF HA protein is detected by radiolabelUng and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with 3i S-cysteine two days post transfection.
- Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 ⁇ M Tris, pH 7.5) (Wilson, I. et al. , Id. 37:767 (1984)) . Both cell lysate and culture media are precipitated with an HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.
- RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 ⁇ M Tris, pH 7.5
- HA specific monoclonal antibody Proteins precipitated are analyzed on 15% SDS-PAGE gels.
- Fibroblasts are obtained from a subject by skin biopsy.
- the resulting tissue 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 pieces are placed in each flask.
- the flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37°C for approximately one week. At this time, fresh media is added and subsequently changed every several days.
- fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin
- pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and Hindlll and subsequently treated with calf intestinal phosphatase.
- the linear vector is fractionate d on agarose gel and purified, using glass beads.
- the cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3 ' end ⁇ equences respectively.
- the 5' primer containing an EcoRI site and the 3' primer further includes a Hindlll site.
- Equal quantities of the Moloney murine sarcoma 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 is used to transform bacteria HB101, 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 Eagles Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin.
- DMEM Dulbecco's Modified Eagles Medium
- CS calf serum
- 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 infectious viral particles containing the gene (the packaging cells are now referred to as producer cells) .
- Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells.
- the spent media containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells.
- Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblast ⁇ 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 fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcamer beads.
- the fibroblasts now produce the protein product.
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US1996/005247 WO1997038002A1 (en) | 1996-04-10 | 1996-04-10 | Extracellular/epidermal growth factor-like protein |
CA002249213A CA2249213A1 (en) | 1996-04-10 | 1996-04-10 | Extracellular/epidermal growth factor-like protein |
AU55492/96A AU5549296A (en) | 1996-04-10 | 1996-04-10 | Extracellular/epidermal growth factor-like protein |
JP09536143A JP2000508531A (en) | 1996-04-10 | 1996-04-10 | Extracellular / epidermal growth factor-like protein |
EP96912802A EP0910569A4 (en) | 1996-04-10 | 1996-04-10 | Extracellular/epidermal growth factor-like protein |
US08/839,525 US6852506B1 (en) | 1996-04-10 | 1997-04-11 | Extracellular/epidermal growth factor-like protein |
US09/275,805 US20010051358A1 (en) | 1996-04-10 | 1999-03-25 | Extracellular/epidermal growth factor-like protein |
US11/017,065 US7393832B2 (en) | 1996-04-10 | 2004-12-21 | Extracellular/epidermal growth factor like protein |
US12/144,864 US20090104622A1 (en) | 1996-04-10 | 2008-06-24 | Extracellular/Epidermal Growth Factor Like Protein |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US1996/005247 WO1997038002A1 (en) | 1996-04-10 | 1996-04-10 | Extracellular/epidermal growth factor-like protein |
CA002249213A CA2249213A1 (en) | 1996-04-10 | 1996-04-10 | Extracellular/epidermal growth factor-like protein |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
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US08/839,525 Continuation-In-Part US6852506B1 (en) | 1996-04-10 | 1997-04-11 | Extracellular/epidermal growth factor-like protein |
Publications (1)
Publication Number | Publication Date |
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WO1997038002A1 true WO1997038002A1 (en) | 1997-10-16 |
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ID=25680533
Family Applications (1)
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PCT/US1996/005247 WO1997038002A1 (en) | 1996-04-10 | 1996-04-10 | Extracellular/epidermal growth factor-like protein |
Country Status (5)
Country | Link |
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EP (1) | EP0910569A4 (en) |
JP (1) | JP2000508531A (en) |
AU (1) | AU5549296A (en) |
CA (1) | CA2249213A1 (en) |
WO (1) | WO1997038002A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999000410A2 (en) * | 1997-06-27 | 1999-01-07 | Incyte Pharmaceuticals, Inc. | Human extracellular matrix proteins |
WO1999055863A1 (en) * | 1998-04-28 | 1999-11-04 | Ono Pharmaceutical Co., Ltd. | NOVEL POLYPEPTIDE, cDNA ENCODING THE SAME AND UTILIZATION THEREOF |
WO2000006188A1 (en) * | 1998-07-28 | 2000-02-10 | Mediko Pty. Ltd. | Hair growth/maintenance compositions and methods involving the same |
US6300311B1 (en) | 1998-04-14 | 2001-10-09 | Texas Tech University | Regulation of vertebrate ovarian maturation and function using growth factors |
AU752206B2 (en) * | 1997-04-11 | 2002-09-12 | Human Genome Sciences, Inc. | Extracellular/epidermal growth factor like protein |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS60501737A (en) * | 1983-07-05 | 1985-10-17 | チロン コ−ポレイシヨン | Epidermal growth factor hybrid DNA synthesis |
IL89673A0 (en) * | 1988-03-24 | 1989-09-28 | Oncogen | Novel polypeptides having growth factor activity and nucleic acid sequences encoding the polypeptides |
-
1996
- 1996-04-10 CA CA002249213A patent/CA2249213A1/en not_active Abandoned
- 1996-04-10 EP EP96912802A patent/EP0910569A4/en not_active Withdrawn
- 1996-04-10 JP JP09536143A patent/JP2000508531A/en not_active Ceased
- 1996-04-10 WO PCT/US1996/005247 patent/WO1997038002A1/en not_active Application Discontinuation
- 1996-04-10 AU AU55492/96A patent/AU5549296A/en not_active Abandoned
Non-Patent Citations (4)
Title |
---|
CURRENT OPINIONS IN STRUCTURAL BIOLOGY, 1993, Vol. 3, CAMPBELL et al., "Epidermal Growth Factor-Like Modules", pages 385-392. * |
FEBS LETTERS, April 1988, Vol. 231, No. 1, APPELLA et al., "Structure and Function of Epidermal Growth Factor-Like Regions in Proteins", pages 1-4. * |
FEBS LETTERS, July 1989, Vol. 251, No. 1, ENGEL et al., "EGF-Like Domains in Extracellular Matrix Proteins; Localized Signals for Growth and Differentiation?", pages 1-7. * |
See also references of EP0910569A4 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU752206B2 (en) * | 1997-04-11 | 2002-09-12 | Human Genome Sciences, Inc. | Extracellular/epidermal growth factor like protein |
WO1999000410A2 (en) * | 1997-06-27 | 1999-01-07 | Incyte Pharmaceuticals, Inc. | Human extracellular matrix proteins |
WO1999000410A3 (en) * | 1997-06-27 | 1999-03-18 | Incyte Pharma Inc | Human extracellular matrix proteins |
US6303765B1 (en) | 1997-06-27 | 2001-10-16 | Incyte Genomics, Inc. | Human extracellular matrix proteins |
US6300311B1 (en) | 1998-04-14 | 2001-10-09 | Texas Tech University | Regulation of vertebrate ovarian maturation and function using growth factors |
WO1999055863A1 (en) * | 1998-04-28 | 1999-11-04 | Ono Pharmaceutical Co., Ltd. | NOVEL POLYPEPTIDE, cDNA ENCODING THE SAME AND UTILIZATION THEREOF |
WO1999055864A1 (en) * | 1998-04-28 | 1999-11-04 | Ono Pharmaceutical Co., Ltd. | NOVEL POLYPEPTIDE, cDNA ENCODING THE SAME AND UTILIZATION THEREOF |
US6846647B1 (en) | 1998-04-28 | 2005-01-25 | Ono Pharmaceutical Co., Ltd. | Polypeptides suppressing smooth muscle cell proliferation, the encoding cDNA, and related methods |
WO2000006188A1 (en) * | 1998-07-28 | 2000-02-10 | Mediko Pty. Ltd. | Hair growth/maintenance compositions and methods involving the same |
Also Published As
Publication number | Publication date |
---|---|
CA2249213A1 (en) | 1997-10-16 |
EP0910569A4 (en) | 2002-05-08 |
JP2000508531A (en) | 2000-07-11 |
EP0910569A1 (en) | 1999-04-28 |
AU5549296A (en) | 1997-10-29 |
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