CN1182431A - Human Criptin Growth Factor - Google Patents

Abstract

A human Criptin Growth Factor polypeptide (CGF) and DNA(RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for wound healing or tissue generation, stimulating implant fixation and angiogenesis. Antagonist against such polypeptides and their use as a therapeutic to treat and/or prevent neoplasia such as tumors is also disclosed. Diagnostic assays for idenfifying mutations in CGF nucleic acid sequences and altered levels of the CGF for the detection of cancer are also disclosed.

Description

Human Criptin Growth Factor

The present invention relates to newly identified polynucleotides, polypeptides encoded by the polynucleotides, uses of the polynucleotides and polypeptides and production of the polynucleotides and polypeptides. More specifically, the polypeptides of the present invention have been putatively identified as human Criptin growth factor, sometimes referred to hereinafter as "CGF". The invention also relates to inhibiting the action of the polypeptide.

Growth factors and other mitogens, including transforming oncogenes, can rapidly induce the expression of a range of genes by specific cells (Lau, L.F. and Nathans, D., Molecular Aspects of Cellular Regulation, 6:165-202 (1991)). These genes, designated immediate early or early response genes, are transcriptionally activated within minutes of exposure to growth factors or mitogens, independent of de novo protein synthesis. A group of such immediate early genes encode secreted extracellular proteins that are necessary for the coordination of complex biological processes such as differentiation and proliferation, regeneration and wound healing (Ryseck, R.P. et al., Cell Growth Differentiation, 2:235-233( 1991)).

Expression of these immediate early genes acts as "third messengers" in a cascade of events triggered by growth factors. They are also thought to be necessary for the integration and coordination of complex biological processes such as differentiation and wound healing, where cell proliferation is a common event.

The criptin growth factor is overexpressed and secreted by certain types of cancer cells such as pancreatic cancer.

The CGF of the present invention shows amino acid sequence homology to the cripto growth factor disclosed in U.S. Pat. Upregulated and expressed in pancreatic cancer.

According to one aspect of the present invention, there are provided a novel mature polypeptide and its biologically active and useful fragments, analogs and derivatives in diagnostics or therapeutics.

According to another aspect of the present invention, there is provided an isolated nucleic acid molecule encoding the polypeptide of the present invention, which nucleic acid molecule includes mRNA, DNA, cDNA, genomic DNA, and analogs thereof and which have biological activity and are useful in diagnostics or therapy. Scientifically useful fragments and derivatives.

According to another aspect of the present invention, there is provided a method for producing such a polypeptide by recombinant technology, the method comprising culturing a plant containing a nucleic acid sequence encoding a polypeptide of the present invention under conditions that promote expression of the protein and subsequent recovery of the protein. Recombinant prokaryotic and/or eukaryotic host cells.

According to yet another aspect of the present invention, there is provided a method of using the polypeptide or a polynucleotide encoding the polypeptide for therapeutic purposes, such as treating sarcoidosis, osteoporosis, assisting graft fixation, stimulating wound healing and tissue Regenerates, promotes angiogenesis, and stimulates vascular smooth muscle proliferation and endothelial cell production.

According to yet another aspect of the invention, antibodies against such polypeptides are provided.

According to another aspect of the invention, antagonists against such polypeptides are provided, which are useful for inhibiting the effects of such polypeptides, for example limiting the production of excess connective tissue during wound healing and pulmonary fibrosis.

According to another aspect of the present invention, there is provided a nucleic acid probe comprising a nucleic acid molecule of sufficient length that can specifically hybridize to the polynucleotide sequence encoding the present invention.

According to yet another aspect of the present invention, there is provided a diagnostic assay for the detection of diseases associated with overexpression of the polypeptide of the present invention and mutations in the nucleic acid sequence encoding the polypeptide.

According to yet another aspect of the present invention, there is provided a method of using such a polypeptide or a polynucleotide encoding such a polypeptide for in vitro purposes related to scientific research, DNA synthesis and production of DNA vectors.

These and other aspects of the invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are only meant to illustrate specific embodiments of the present invention, and are not meant to limit the present invention in any way.

Figure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the polypeptide of the present invention, using standard one-letter amino acid abbreviations. Sequencing was performed with a 373 automatic DNA sequencer (Applied Biosystems, Inc.).

Figure 2 shows the amino acid sequence homology between polypeptides of the invention (upper row) and cripto growth factors (lower row).

According to one aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) encoding a mature polypeptide having the deduced amino acid sequence (SEQ ID NO: 2) of FIG. , the mature polypeptide encoded by the cloned cDNA with the deposit number of No.97142.

The polynucleotide encoding the polypeptide of the present invention was originally discovered from a cDNA library obtained from human pancreatic cancer tissue. It is structurally related to the human cripto growth factor and contains an open reading frame encoding a protein of 230 amino acid residues, of which the initial 23 amino acid residues of the polypeptide are putative signal sequences, whereby the mature polypeptide contains 207 amino acids. As shown in Figure 2, the polypeptide of the present invention has the same conserved cysteine residues as cripto growth factor.

In addition, the polypeptide of the present invention has a putative soluble portion comprising amino acids 45-128 of SEQ ID NO: 2, so amino acids 129-207 are putative transmembrane portions.

Initial Northern blot analysis showed very high expression in pancreatic cancer cells.

The polynucleotides of the present invention can be in the form of RNA or DNA, wherein DNA includes cDNA, genomic DNA and synthetic DNA, the DNA can be double-stranded or single-stranded, and if single-stranded it can be coding and non-coding (antisense) strand. The coding sequence encoding the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO: 1) or the coding sequence of the deposited clone, or the sequence may be a different coding sequence, but due to the redundancy of the genetic code or degeneracy it encodes the same mature polypeptide as the DNA of Figure 1 (SEQ ID NO: 1) or the deposited cDNA.

The polynucleotide encoding the mature polypeptide of Figure 1 (SEQ ID NO: 2) or encoding the mature polypeptide encoded by the deposited cDNA may include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide together with additional coding sequences, such as A leader or secretory sequence or a proprotein sequence; the coding sequence of the mature polypeptide (and optionally additional coding sequences) and non-coding sequences, such as introns or 5' and/or 3' non-coding sequences of the mature polypeptide coding sequence sequence.

Thus, the term "polynucleotide encoding a polypeptide" includes polynucleotides comprising only the coding sequence for the polypeptide as well as polynucleotides comprising additional coding and/or non-coding sequences.

The present invention further relates to variants of polynucleotides as described above, which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence (SEQ ID NO: 2) of Figure 1 or encoded by the cDNA of the deposited clone Fragments, analogs and derivatives of polypeptides. A variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide, or a non-naturally occurring variant of the polynucleotide.

Therefore, the present invention includes polynucleotides encoding the same mature polypeptide (SEQ ID NO: 2) as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone, and variants thereof, wherein the variant encodes the same mature polypeptide as shown in Figure 1 The polypeptide (SEQ ID NO: 2) or a fragment, derivative or analogue of the polypeptide encoded by the cDNA of the deposited clone. The nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As indicated above, the polynucleotide may have a coding sequence that is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID NO: 1) or the coding sequence of the deposited clone. As known in the art, an allelic variant is a variable form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, but which is substantially unchanged Function of the encoded polypeptide.

The invention also includes polynucleotides in which the coding sequence for the mature polypeptide is fused in the same reading frame to a polynucleotide sequence that facilitates expression and secretion of the polypeptide from a host cell, e.g., as a control A leader sequence that functions as a secretory sequence for the polypeptide to be transported from the cell. A polypeptide having a leader sequence is a preprotein that can be cleaved by a host cell to form the mature form of the polypeptide. The polynucleotide may also encode a proprotein with additional 5' amino acid residues added to the mature protein. A mature protein with a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved, an active mature protein remains.

Thus, for example, a polynucleotide of the invention may encode a mature protein, or encode a protein with a prosequence, or encode a protein with a prosequence and a presequence (leader sequence).

A polynucleotide of the invention may also have a coding sequence fused in frame to a marker sequence which facilitates purification of the polypeptide of the invention. In the case of bacterial hosts the marker sequence may be the hexahistidine tag provided by the pQE-9 vector to ensure purification of the mature polypeptide fused to the tag, or for example when using mammalian hosts such as COS-7 cells. When , the marker sequence may be a hemagglutinin (HA) marker. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" refers to the segment of DNA involved in the production of a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences between individual coding segments (exons) (intron).

Fragments of the full-length CGF gene can be used as hybridization probes for a cDNA library in order to isolate the full-length gene and isolate other genes with high sequence similarity or similar biological activity to the gene. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe can also be used to identify cDNA clones corresponding to a full-length transcript as well as a genomic clone or multiple genomic clones containing the complete CGF gene including regulatory and promoter regions, exons and introns. An example of a screen involves isolating the coding region of the CGF gene by synthesizing oligonucleotide probes using known DNA sequences. Labeled oligonucleotides having a sequence complementary to the gene sequence of the invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which library members hybridize to the probe.

The invention further relates to polynucleotides which hybridize to the above sequences, provided that 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 to the above polynucleotides under stringent conditions. The term "stringent conditions" as used herein means that hybridization occurs only when there is at least 95% and preferably at least 97% identity between the sequences. In a preferred embodiment, the polypeptide encoded by the polynucleotide hybridized with the above-mentioned polynucleotide substantially retains the mature polypeptide encoded by the cDNAs (SEQ ID NO: 1) or the deposited cDNA(s) of Fig. 1 have the same biological function or activity.

On the other hand, the polynucleotide may contain at least 20 bases, preferably 30 bases and more preferably at least 50 bases, hybridizes to the polynucleotide of the present invention and has identity as described above, and may retain or No activity retained. For example such a polynucleotide can be used as a probe for the polynucleotide of SEQ ID NO: 1, for example for the recovery of the polynucleotide or as a diagnostic probe or PCR primer.

Accordingly, the present invention relates to polynucleotides having at least 70% identity, preferably at least 90% identity and more preferably at least 95% identity to a polynucleotide encoding a polypeptide of SEQ ID NO: 2 and fragments thereof acid, and the polypeptide encoded by the polynucleotide, wherein said fragment has at least 30 bases and preferably at least 50 bases.

The deposit referred to herein will be deposited in accordance with the Budapest Treaty Concerning the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits are provided solely for the convenience of those skilled in the art, and are not an acknowledgment of the deposits required under 35 U.S.C. §112. The sequences of the polynucleotides contained in the deposited materials and the amino acid sequences of the polypeptides they encode are incorporated herein by reference, and in the event of any conflict with the sequence description herein, control. A license is required to make, use, or sell the deposited material, and no such license is granted here.

The present invention further relates to a polypeptide having the deduced amino acid sequence (SEQ ID NO: 2) of Figure 1 or having the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of the polypeptide.

When referring to the polypeptide of Figure 1 (SEQ ID NO: 2) or the polypeptide encoded by the deposited cDNA, the terms "fragment", "derivative" and "analog" refer to substantially the same biological Functional or active polypeptide. Thus, an analog includes a proprotein that 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 polypeptide of Figure 1 (SEQ ID NO: 2) or a fragment, derivative or analog of the polypeptide encoded by the deposited cDNA may be: (i) a polypeptide in which one or more amino acid residues are replaced by a conserved or A non-conserved amino acid residue (preferably a conservative amino acid residue) is substituted and the substituted amino acid residue may or may not be encoded by the genetic code, or (ii) a polypeptide in which one or more amino acids The residue contains a substituent, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g. polyethylene glycol), or (iv) a A polypeptide wherein the mature polypeptide is fused to additional amino acids, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a proprotein sequence, or (v) a splice variant of the mature polypeptide which lacks Certain amino acid residues but still retain biological activity. Such fragments, derivatives and analogs are considered to be within the purview of those skilled in the art from the teachings herein.

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

The term "isolated" means that the material is separated from its original environment (eg, the natural environment if the material occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide isolated from some or all of the coexisting materials in the natural system is isolated . Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition, and if the vector or composition is not part of its natural environment, it is still isolated of.

Polypeptides of the present invention include polypeptides of SEQ ID NO: 2 (especially mature polypeptides) and polypeptides having at least 70% similarity (preferably at least 70% identity) with the polypeptide of SEQ ID NO: 2, more preferably with SEQ ID NO: 2 The polypeptide of NO: 2 has at least 90% similarity (more preferably at least 90% identity) polypeptide, and particularly preferably has at least 95% similarity (particularly preferably at least 95% identity) with the polypeptide of SEQ ID NO: 2 identity), the polypeptides of the present invention also include portions of the polypeptide, wherein these portions of the polypeptide generally contain at least 30 amino acids and more preferably at least 50 amino acids.

As is known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutions to the sequence of a second polypeptide.

Fragments or portions of polypeptides of the invention can be used to produce corresponding full-length polypeptides by peptide synthesis; thus, the fragments can be used as intermediates in the production of full-length polypeptides. Fragments or parts of polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention.

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention and the production of polypeptides of the present invention by recombinant techniques.

The host cell can be genetically engineered (transduced or transformed or transfected) with the vector of the present invention, wherein the vector can be, for example, a cloning vector or an expression vector, and the vector can be in the form of a plasmid, virus particle, phage, etc., for example. The engineered host cells can be cultured on conventional nutrient media modified for activation of promoters, selection of transformants, or amplification of the CGF gene. Culture conditions (such as temperature, pH, and others) are those previously employed by the host cell selected for expression and will be apparent to those of ordinary skill in the art.

The polynucleotides of the invention can be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide sequence may be contained in any of a variety of expression vectors, such as in particular vectors or plasmids, that express polypeptides. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example: SV40 derivatives; bacterial plasmids; phage DNA; baculoviruses; yeast plasmids; virus, fowl pox virus and pseudorabies). However, any other vector or plasmid can be used as long as it can replicate and survive in the host.

The appropriate DNA sequence can be inserted into a vector by a number of methods. In general, the DNA sequence can be inserted into an appropriate restriction enzyme site by methods known in the art. This and other methods are considered to be within the purview of those skilled in the art.

The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct the synthesis of mRNA. As representative examples of the aforementioned promoters there may be mentioned: LTR or SV40 promoters, Escherichia coli lac or trp, bacteriophage _λPL promoters and others known to regulate gene expression in prokaryotic or eukaryotic cells or their viruses. Promoter. Expression vectors also contain a ribosome binding site for translation initiation and a transcription terminator. The vector may also include suitable sequences for amplifying expression.

In addition, the expression vector preferably contains a gene that confers a phenotypic trait for selection in transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as in E. coli Tetracycline or ampicillin resistance in

A vector containing an appropriate DNA sequence as described above and an appropriate promoter or regulatory sequence can be used to transform an appropriate host to allow expression of the protein by the host. As representative examples of suitable hosts there may be mentioned: bacterial cells such as Escherichia coli, Streptomyces sp, Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 and Autographa californica Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; plant cells, etc. Selection of an appropriate host is considered to be within the purview of those skilled in the art in light of the teachings herein.

More specifically, the invention also includes recombinant constructs comprising one or more sequences as broadly defined above. Constructs include vectors, such as plasmid or viral vectors, into which sequences of the invention are inserted in forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence, such as a promoter, operably linked to the sequence. Many suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors are provided by way of example: Bacteria: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223- 3. pKK233-3, pDR540, pRIT5 (Pharmacia); eukaryotic cells: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used as long as it can replicate and survive in the host.

The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vectors with a selection marker. Two suitable vectors are pKK232-8 and pCM7. Bacterial promoters of particular note include lacI, lacZ, T3, T7, gpt, _λPR , _PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruses and mouse metallothionein-I. Selection of appropriate vectors and promoters is within the ordinary skill level of the art.

In a still further embodiment, the present invention relates to host cells comprising the above constructs. The host cell can be a higher eukaryotic cell (such 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 this construct into host cells can be achieved by calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (Davies, L., Dibner, M., Battey, I., Fundamental Methods in Molecular Biology , (1986)).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. On the other hand, the polypeptide of the present invention can also be synthesized by a conventional peptide synthesizer.

The mature protein can be expressed in mammalian cells, yeast, bacteria or other cells under the control of a suitable promoter. The protein can also be produced by cell-free translation systems using RNAs derived from the DNA constructs of the invention. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, NY, 1989), the disclosure of which is incorporated herein by reference .

Transcription of DNA encoding a polypeptide of the invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, generally about 10 to 300 bp in length, that act on promoters to increase their transcription. Examples include the SV40 enhancer (bp100 to 270) on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

In general, recombinant expression vectors include an origin of replication with selectable markers that allow transformation of host cells (such as the ampicillin resistance gene in E. coli and the TRP1 gene in Saccharomyces cerevisiae) and transcription of downstream structural sequences from a highly expressed gene. promoter. These promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins. The heterologous structural sequence is suitably assembled with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or into the extracellular medium. The heterologous sequence may optionally encode a fusion protein containing an N-terminal identification peptide, wherein the identification peptide confers desired characteristics on the expressed recombinant product, such as stability or ease of purification.

Expression vectors useful for use in bacteria can be constructed by inserting a structural DNA sequence encoding a desired protein in operable reading with appropriate translation initiation and termination signals and a functional promoter. The vector will include one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if necessary, amplification in the host. Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium and many species belonging to the genera Pseudomonas, Streptomyces and Staphylococcus, although other species may also be used.

As a representative but non-limiting example, expression vectors useful for bacterial use may include a selectable marker and bacterial origin of replication from a commercially available plasmid containing genetic elements from the well known cloning vector pBR322 (ATCC 37017) . These commercially available vectors include, for example, pKK223-3 (Phamacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" portions are combined with an appropriate promoter and structural sequence to be expressed.

After transformation of a suitable host strain and growth of the host strain to a suitable cell density, the selected promoter is induced by a suitable method (eg temperature shift or chemical induction) and the cells are cultured for an additional period of time.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells used in protein expression may be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents.

Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 cell line of monkey kidney fibroblasts described by Gluzman (Cell, 23:175 (1981)) and other cell lines that can express compatible vectors, such as C127, 3T3, CHO , HeLa and BHK cell lines. Mammalian expression vectors include an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non- Transcribed sequence. DNA sequences obtained from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splicing and polyadenylation sites, can be used to provide the required non-transcriptional genetic elements.

The polypeptides of the present invention can be recovered and purified from recombinant cell cultures by methods hitherto used, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction Chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be employed, if desired, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for the final purification step.

The polypeptide of the present invention may be a natural purified product, or the product of a chemical synthesis method, or obtained from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cell culture) Prepared by recombinant technology. Depending on the host used in the recombinant production method, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptide may also contain an initial methionine residue.

The CGF gene of the present invention can be labeled and used as a probe in Southern blot analysis of DNA preparations containing endonuclease digestion to determine the presence of amplification, rearrangement, Deletion or restriction fragment length polymorphism.

The tagged criptin gene can be used to analyze Northern blots containing RNA to determine the relative levels of mRNA expression in various normal and pathological tissues.

CGF can be used to generate probes suitable for in situ RNA:RNA hybridization for histological localization in normal or pathological cells expressing CGF mRNA.

CGF oligonucleotides (sense strand) can be used to detect CGF mRNA levels in various tissues.

CGF polypeptides are overexpressed and secreted by certain types of cancer cells, such as pancreatic cancer, therefore, detection of CGF gene transcription or excess of CGF protein enables the diagnosis of pancreatic cancer. Therefore, anti-CGF antibodies can be used in the diagnosis of neovascularization associated with tumor formation, as altered levels of this polypeptide can be indicative of this disease.

A competition assay can be employed in which an antibody specific for CGF is bound to a solid support, and labeled CGF and a sample obtained from the host are passed over the solid support, and the detected protein is then adsorbed to the solid support. The amount of label on the substance can be correlated with the amount of CGF in the sample.

A "sandwich" assay is similar to an ELISA. In a "sandwich" assay, a CGF polypeptide is passed over a solid support and bound to an antibody already attached to the solid support, and a second antibody is then bound to the CGF polypeptide. A third antibody, labeled and specific for the second antibody, is then introduced to the solid support and bound to the second antibody, after which its amount can be quantified.

The polypeptides of the present invention can be used in wound healing and related treatments related to tissue regeneration, such as connective tissue, skin, bone, cartilage, muscle, lung or kidney.

The polypeptides of the invention can also be used to stimulate angiogenesis, eg, to promote the growth of vascular smooth muscle cells and endothelial cells. Increased angiogenesis favors ischemic tissue and favors bypass coronary artery development following coronary stenosis.

Polypeptides of the invention may also be employed in graft fixation to stimulate cell growth around the graft and thus promote attachment of the graft to its intended site.

According to a further aspect of the present invention, the present invention provides a method of using such polypeptides or polynucleotides encoding such polypeptides as in vitro purposes related to scientific research, synthesis of DNA, production of DNA vectors research reagents for the development of therapeutics and diagnostics to treat human disease.

The invention provides a method of identifying a receptor for a polypeptide of the invention. Genes encoding receptors can be identified by a number of methods known to those of ordinary skill in the art, such as ligand panning and FACS sorting (Coligan, et al., General Protocols in Immunology, 1(2), Chapter 5, ( 1991)). Expression cloning is preferably employed, wherein polyadenylated RNA is prepared from a cell responsive to a CGF polypeptide, and the cDNA library generated from this RNA is split into pools and used to transfect COS cells or Other cells that do not respond to CGF. Transfected cells grown on glass slides are contacted with labeled CGF, which can be labeled by a number of methods including iodination or introduction of a recognition site for a site-specific protein kinase. After fixation and incubation, slides were subjected to autoradiographic analysis. Positive pools were identified and subpools were prepared and retransfected using an iterative subpooling and rescreening approach, resulting in a single clone encoding a putative receptor.

As an alternative method for receptor identification, labeled CGF can be photoaffinity linked to cell membranes or preparations of extracts expressing the receptor molecule. Cross-linked material was resolved by PAGE analysis and exposed to X-ray film. The tagged complex containing the CGF receptor can be excised, resolved into peptide fragments and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing can be used to design a degenerate set of oligonucleotide probes for screening cDNA libraries to identify the gene encoding the putative receptor.

The present invention also devises a method for screening compounds in order to identify those substances that bind to and activate CGF receptors. An example of such an assay involves stimulation of proliferation of endothelial cells in the presence of comitogen Con A. Human umbilical vein endothelial cells were cultured on a 96-well flat-bottom culture plate (Costar, Cambridge, MA) and supplemented with a reaction mixture suitable for stimulating cell proliferation, which contained Con-A (Calbiochem, La Jolla, CA). Con-A and compounds to be screened were added, cultures were pulsed with3[H]thymidine after incubation at 37°C and harvested onto glass fiber filters (PhD; Cambridge Technology, Watertown, MA). The ^average3 [H]thymidine incorporation (cpm) of triplicate cultures was determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA). Significant ³ [H]thymidine incorporation indicates stimulation of endothelial cell proliferation.

To determine antagonist compounds, the analytical assay described above can be performed, however in this assay CGF is added with the compound to be screened, the ability of the compound to inhibit ³ [H]thymidine incorporation in the presence of CGF indicates that the compound is a member of CGF. an antagonist.

Alternatively, CGF antagonists can be detected by combining labeled CGF and a potential antagonist compound with membrane bound CGF receptor or recombinant receptor under conditions suitable for competitive inhibition assays. CGF can be radiolabeled so that the number of CGF molecules bound to the receptor can be used to determine the effect of potential antagonists.

Alternatively, the response of known second messenger systems can be measured following interaction of a potential antagonist compound with a receptor. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels, or phosphoinositide hydrolysis. Compounds can be labeled to detect binding, and a compound that binds but does not stimulate a second messenger response is a potent antagonist compound.

Examples of potential CGF antagonist compounds include antibodies, or in some cases oligonucleotides, which bind to the polypeptide itself or to a polypeptide receptor. Alternatively, a potential antagonist could be a closely related protein, such as a mutant form of CGF, which recognizes but has no effect on the CGF receptor, thus competitively inhibiting the action of CGF.

Another potential CGF antagonist 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 are based on the binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of a polynucleotide sequence encoding a mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide that is from about 10 to 40 base pairs in length. A DNA oligonucleotide was designed to be complementary to the region of the gene involved in transcription (triple helix - see Lee et al., Nucleic Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); with Dervan et al. , Science, 251:1360 (1991)), thereby preventing transcription and production of CGF. Antisense RNA oligonucleotides hybridize to mRNA in vivo and prevent translation of the mRNA molecule into CGF (antisense-Okano, Journal of Neurochemistry, 56:560 (1991); oligodeoxynucleosides as antisense inhibitors of gene expression Acids, CRC Press, Boca Raton, FL (1988)). The above oligonucleotides can also be delivered to cells so that the antisense RNA or DNA can be expressed in vivo to inhibit the production of CGF.

Potential CGF antagonists include small molecules that bind to the active site, receptor binding site, or other growth factor binding site of the polypeptide to prevent the normal biological activity of CGF. Examples of small molecules include, but are not limited to, small peptides or peptoid molecules.

Antagonists can be used directly or indirectly to inhibit tumor growth, for example by antagonizing CGF activity and/or antagonizing neovascularization and smooth muscle cell neointimal proliferation common in atherosclerosis and restenosis following balloon angioplasty. inhibition.

Antagonists may be used in a composition together with a pharmaceutically acceptable carrier as described below.

The CGF polypeptides and antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier, such compositions include a therapeutically effective amount of the polypeptide or antagonist compound and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to, saline, buffered saline. Glucose, water, glycerin, ethanol and mixtures 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 composition of the invention. Accompanying the container may be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. In addition, the pharmaceutical compositions can be used in combination with other therapeutic compounds.

The pharmaceutical composition can be administered in a conventional manner, such as orally, topically, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally or intradermally. The pharmaceutical composition is administered in an amount effective for the treatment and/or prevention of the particular condition. Generally, the composition will be administered in an amount of at least about 10 μg/kg body weight, and in most cases the composition will be administered in an amount not exceeding about 8 mg/kg body weight/day. The dose ranges from about 10 µg/kg body weight to about 1 mg/kg body weight per day in most cases in consideration of the route of administration, symptoms and the like.

Physiological responses as seen in wound healing can be accelerated by CGF in combination with other growth factors including, but not limited to, PDGF, IGF, FGF, EGF or TGF-beta.

According to the present invention, CGF polypeptides, as well as agonists and antagonists of the same polypeptides, can also be used by expressing said polypeptides in vivo, which is commonly referred to as "gene therapy",

Thus, for example, cells from a patient can be engineered in vitro with a polynucleotide (DNA or RNA) encoding a polypeptide, and the engineered cells can then be provided to a patient to be treated with the polypeptide. The above methods are well known in the art. For example, cells can be engineered by methods known in the art using a retroviral particle containing RNA encoding a polypeptide of the invention.

Similarly, cells can be engineered in vivo for expression of a polypeptide in vivo, eg, by methods known in the art. As is known in the art, to engineer cells in vivo and express a polypeptide in vivo, a patient may be administered a producer cell that produces retroviral particles containing RNA encoding a polypeptide of the invention. These and other methods of administering a polypeptide of the invention by the methods described above will be apparent to those of ordinary skill in the art from the teachings of the present invention. For example, in addition to retroviral particles, the expression vector for engineered cells can be, for example, an adenovirus, which can be used to engineer cells in vivo in combination with a suitable delivery vehicle.

Retroviruses from which the above retroviral plasmid vectors can be obtained include but are not limited to Moloney murine leukemia virus, spleen necrosis virus, retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus, human immunodeficiency virus , adenovirus, myeloproliferative sarcoma virus and mammary tumor virus. In a specific embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus.

The vector contains one or more promoters. Suitable promoters that may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human The cytomegalovirus (CMV) promoter, or any other promoter (eg, a cellular promoter, such as a eukaryotic promoter, including but not limited to histone, pol III, and β-actin promoters). Other viral promoters that can be used include, but are not limited to, the adenovirus promoter, the thymidine kinase (TK) promoter, and the B19 parvovirus promoter. Selection of an appropriate promoter will be apparent to one of ordinary skill in the art from the teachings herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that may be employed include, but are not limited to, adenovirus promoters, such as the adenovirus major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; Inducible promoters, such as MMT promoter, metallothionein promoter; thermogram promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter, such as herpes simplex thymidine Kinase promoters; retroviral LTRs (including the modified retroviral LTRs described above); beta-actin promoter; and human growth hormone promoter. The promoter may also be the native promoter controlling the gene encoding the polypeptide.

Retroviral plasmid vectors are used to transduce packaging cell lines to form production cell lines. Examples of packaging cells that can be transduced include, but are not limited to, PE501, PA3 17, φ-2, φ-AM, PA12, T19-14X, VT-19-17-H2, φCRE, φCRIP, GP+E-86, GP+envAm12 and the DAN cell line described in Miller, Human Gene Therapy, Vol. 1, pp. 5-14 (1990), which is incorporated herein by reference in its entirety. Vectors can be used to transduce packaging cells by any method known in the art, including but not limited to electroporation, using liposomes, and _CaPO4 precipitation. In another approach, the retroviral plasmid vector can be embedded in liposomes, or conjugated to lipids, before the vector is introduced into the host.

Producer cell lines produce infectious retroviral vector particles containing the nucleic acid sequence encoding the polypeptide, which can then be used to transduce eukaryotic cells either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinomatous cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets and hybridizes to a particular location on a single human chromosome. Furthermore, there is now a need to identify specific loci on chromosomes. Currently, there are only a few chromosomal labeling reagents based on actual sequence data (repeated polymorphisms) that can be used to mark chromosomal locations. The DNA chromosome mapping of the present invention is an important first step in correlating these sequences with disease-associated genes.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 10-25 bp) from cDNA. In silico analysis of 3' untranslated regions allows rapid selection of primers that should not span more than one exon on the genomic DNA or complicate the amplification method. These primers were then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will produce amplified fragments.

PCR mapping of somatic cell hybrids is a rapid procedure for mapping a specific DNA to a specific chromosome. Using the same oligonucleotide primers according to the invention, sublocalization can be achieved in a similar manner with a set of fragments from a specific chromosome or from a collection of large genome clones. Other mapping strategies that can similarly be used to map chromosomes include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of cDNA clones and a metaphase smear can be used to achieve accurate chromosome mapping in one step. The technique can use cDNAs as short as 50 or 60 bases in length. For a review of this technique see Verma et al., The Human Chromosome: A Handbook of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to an accurate chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library). Linkage analysis (co-inheritance of physically adjacent genes) is then used to identify relationships between genes and diseases that have mapped to the same chromosomal region.

Differences in cDNA or genome sequences between affected and unaffected individuals then need to be determined. If a mutation is observed in some or all affected individuals, but not in any normal individual, then the mutation is likely causative of the disease.

At the current resolution of physical and genetic mapping techniques, a cDNA pinpointed to a disease-associated chromosomal region could be one of 50-500 potentially causative genes (of which a hypothetical 1 trillion Base mapping resolution and every 20kb is a gene).

Polypeptides, fragments thereof, or other derivatives or analogs of the polypeptides, or cells expressing the above substances can be used as immunogens for producing antibodies thereof. These antibodies may be, for example, polyclonal or monoclonal antibodies. The invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments or products of Fab expression libraries. Various methods known in the art can be used to produce these antibodies and fragments.

Antibodies raised against the polypeptides corresponding to the sequences of the present invention can be obtained by directly injecting the polypeptides into human animals or by administering the polypeptides to animals, wherein the animals are preferably non-human. The antibody thus obtained will then bind to the polypeptide. In this way, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies that bind the entire native polypeptide. The antibody can then be used to isolate the polypeptide from tissue expressing the polypeptide.

For the preparation of monoclonal antibodies, any technique for producing antibodies by continuous cell line culture can be used. Examples include hybridoma technology (Kohler and Milstein, 1975, Nature, 256:495-497), trisomy hybridoma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72) and production of human EBV-hybridoma technology for monoclonal antibodies (Cole, et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques for the production of single chain antibodies (US Patent 4,946,778) can be adapted to produce single chain antibodies directed against the immunogenic polypeptide preparations of the invention. Transgenic mice can also be used to express humanized antibodies to the immunogenic polypeptide preparations of the invention.

The present invention will be further illustrated with reference to the following examples; however, it should be understood that the invention is not limited to these examples. All parts or amounts are by weight unless otherwise stated.

In order to facilitate the understanding of the following embodiments, some frequently occurring methods and/or terms are now described.

"Plasmids" are designated by a preceding lowercase p and/or followed by several uppercase letters and/or numbers. The starting plasmids herein are either commercially available or publicly available on an unrestricted basis, or can be constructed from available plasmids according to published methods. Furthermore, equivalent plasmids to those described are known in the art and will be apparent to those of ordinary skill in the art.

"Digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme that acts only on certain sequences on the DNA. A variety of restriction enzymes employed herein are commercially available, and the reaction conditions, cofactors, and other requirements for their use are known to those of ordinary skill in the art. For analytical purposes, typically 1 µg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 µl of buffer solution. To isolate DNA fragments for plasmid construction, 5 to 50 μg of DNA is usually digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffer solutions and amounts of substrate for a particular restriction enzyme are specified by the manufacturer. An incubation time of about 1 hour at 37°C is typically employed, but this time may vary according to the product supplier's instructions. After digestion, the reaction mixture was run directly on a polyacrylamide gel to isolate the desired fragments.

Size separation of cleaved fragments was performed using an 8% polyacrylamide gel as described by Goeddel, D. et al., Nucleic Acids Res. 8:4057 (1980).

"Oligonucleotide" refers to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which can be chemically synthesized. These synthetic oligonucleotides do not have a 5' phosphate, so unless a phosphate is added with ATP in the presence of a kinase, the oligonucleotide will not ligate to another oligonucleotide. Synthetic oligonucleotides will be ligated to the non-dephosphorylated fragments.

"Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis, T., et al., supra, p. 146). Unless otherwise provided, ligation was achieved using known buffers and conditions with approximately equimolar amounts of 10 units of T4 DNA ligase ("ligase") per 0.5 μg of the DNA fragments to be ligated.

Unless otherwise stated, transformations were performed as described by Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Example 1

Bacterial expression and purification of CGF

The DNA sequence encoding CGF, ATCC #97142 was initially amplified using PCR oligonucleotide primers corresponding to the 5' sequence of the processed CGF protein (minus the signal peptide sequence) and the vector sequence 3' of the CGF gene. Additional nucleotides corresponding to the CGF gene were added to the 5' and 3' sequences, respectively. The 5' oligonucleotide primer has the sequence 5'ACTCTTGGATCCAATTTGGGAAACAGCTATCAAGA 3' (SEQ ID NO: 3) and contains a BamHI restriction enzyme site (in bold) followed by a sequence from the putative terminal amino acid codon of the processed protein. CGF coding sequence. The 3' oligonucleotide primer sequence is 5'TACAACTCTAGACTATTATTTACAACATAGAAAATTAAAGGC3' (SEQ ID NO: 4), which contains an Xbal restriction site (in bold) followed by the nucleoside corresponding to the carboxy-terminal 5' amino acid and the translation stop codon Acid reverse complement sequence. The restriction sites corresponded to those on the bacterial expression vector pQE (Qiagen, Inc. Chatsworth, CA 91311). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulated promoter operator (P/O), a ribosome binding site (RBS), a 6-histidine markers and restriction enzyme sites. pQE-9 was then digested with HindIII and XbaI, the amplified sequence was ligated to pQE-9, and inserted in frame containing the sequence encoding the histidine tag and the ribosome binding site (RBS). Then, by the method described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1989), the E. coli strain M15/rep 4 (Qiagen, Inc. .). M15/rep 4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and confers kanamycin resistance (Kan ^r ). Transformants were identified by their ability to grow on LB plates, and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired construct were cultured overnight (O/N) on LB medium supplemented with Amp (100 μg/ml) and Kan (25 μg/ml). O/N cultures were used to inoculate larger cultures at a ratio of 1:100 to 1:250. Cells were grown to an optical density 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6, and then IPTG (“Isopropyl-BD-thiogalactopyranoside”) was added to a final concentration of 1 mM. IPTG induces increased gene expression by inactivating the lacI repressor and initiating P/O induction. Cells were incubated for an additional 2.5 hours to produce an exponentially growing culture and harvested by centrifugation. M15[pREP4] cells containing CGF/6-histidine were lysed in 6 M guanidine hydrochloride, 50 mM NaPO ₄ pH 8.0. The lysate was loaded onto a nickel chelate column and the flow-through was collected. The column was washed with 6 molar guanidine hydrochloride, 50 mM _NaPO4 , pH 8.0, 6.0 and 5.0. The CGF fusion protein was eluted at pH 2.0 (purity >90%). For renaturation, the pH 2.0 eluent was adjusted to 3 M guanidine hydrochloride, 100 mM sodium phosphate, 10 mmol glutathione (reduced form) and 2 mmol glutathione (oxidized form). After 12 hours of incubation in this solution the protein was dialyzed against 10 mM sodium phosphate. For gel electrophoresis, the pellet was resuspended in SDS/NaOH and 2×SDS-PAGE loading buffer, heat denatured, and run on a 4-20% SDS-PAGE gel. Proteins were visualized by Coomassie Brilliant Blue R-250 staining.

Example 2

    Cloning and expressing CGF using baculovirus expression system

The DNA sequence encoding the full-length CGF protein was amplified using PCR oligonucleotide primers containing 5'BamHI and 3'XbaI, ATCC #97142. The primer sequences were 5'ACTCCTGGATCCGCCATCATGACCTGGAGGCACCT 3' (SEQ ID NO: 5) and 5' TACAACTCTAGACTATTATTTACAACATAGAAAATTAAAGGC 3' (SEQ ID NO: 6). The BamHI-XbaI fragment contains the entire CGF coding region including the signal sequence for secretion. This fragment, designated F2, was isolated from a 1% agarose gel using a commercially available kit ("Geneclean", BIO 101 Inc., La Jolla, Ca.).

The CGF protein was expressed by the baculovirus expression system using the vector pA2 (for review, refer to Summers, M.D. and Smith, G.E. 1987, Method Manual for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of Autographa californica polynuclear polyhedrosis virus (AcMNPV), followed by recognition sites for the restriction endonucleases BamHI and XbaI. The polyadenylation site of Simian Virus (SV) 40 is used for efficient polyadenylation. For easy selection of recombinant viruses, the β-galactosidase gene from E. coli was inserted in the same orientation as the polyhedrin promoter, followed by the polyadenylation signal for the polyhedrin gene. The polyhedrin sequence is flanked by viral sequences for cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors, such as pRG1, pAc373, pVL941 and pAcIM1 can be used in place of pA2 (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).

Plasmid pA2 was digested with restriction enzymes BamHI and XbaI and dephosphorylated with bovine intestinal phosphatase by methods known in the art, followed by a commercially available kit ("Geneclean", BIO 101 Inc., La Jolla, Ca.) isolated DNA from a 1% agarose gel, and the vector DNA was named V2.

BamHI-XbaI cleavage fragment F2 and dephosphorylated plasmid V2 were ligated by T4 DNA ligase. Escherichia coli XL1 Blue (Stratagene Cloning Systems, 11011 North Torrey Pines Road La Jolla, Ca. 92037) was then transformed and the bacteria containing the plasmid (pBacCGF) with the CGF cDNA were identified using the enzymes BamHI and XbaI. The sequence of the cloned fragment was confirmed by DNA sequencing.

5 μg of plasmid pBacCGF and 1.0 μg of commercially available linearized baculovirus ("BaculoGold ^™ Baculovirus DNA", Pharmingen, San Diego, CA.) for co-transfection.

1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid pBacCGF were mixed in a well of a sterile microtiter plate containing 50 μl of serum-free Grace medium (Life Technologies, Gaithersburg, MD). Then 10 μl Lipofectin and 90 μl Grace medium were added, mixed and incubated at room temperature for 15 minutes. This transfection mixture was then added dropwise to Sf9 insect cells (ATCC CRL 1711 ) seeded on 35 mm tissue culture plates containing 1 ml of serum-free Grace medium. Shake the plate back and forth to mix the newly added solution. The plates were then incubated at 27°C for 5 hours. After 5 hours, the transfection solution was removed from the plates and 1 ml of Grace insect medium supplemented with 10% fetal bovine serum was added. The plate was returned to the incubator and incubation was continued for 4 days at 27°C.

Supernatants were collected after 4 days and plaque assays were performed similarly as described by Summers and Smith (supra). As a modification, an agarose gel containing "Blue Gal" (Life Technologies, Gaithersburg, MD), which allows easy separation of blue-stained plaques, was used. (A detailed description of the "plaque assay" can also be found on pages 9-10 of the User's Guide for Insect Cell Culture and Baculovirology, distributed by Life Technologies, Gaithersburg).

Virus was added to the cells 4 days after serial dilution and blue-stained plaques were picked with the tip of an Eppendorf pipette. The agar containing the recombinant virus was then resuspended in an Eppendorf tube containing 200 μl Grace medium. The agar was removed by brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells seeded on 35mm dishes. The supernatants from these culture plates were collected after 4 days and stored at 4°C.

Sf9 cells were grown on Grace medium supplemented with 10% heat-inactivated FBS. Cells were infected with recombinant baculovirus V-CGF at a multiplicity of infection (MOI) of 2. After 6 hours the medium was removed and replaced with SF900 II medium (Life Technologies, Gaithersburg) without methionine and cysteine. 5 μCi of ³⁵ S-methionine and 5 μCi of ³⁵ S-cysteine (Amersham) were added after 42 hours. Cells were harvested by centrifugation after further incubation for 16 hours, and labeled proteins were visually visualized by SDS-PAGE and autoradiography.

Example 3

       Expression of Recombinant CGF in COS Cells

The CGF protein was expressed using vector pN346, which is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids contain the mouse dhfr gene under the control of the SV40 early promoter. Chinese hamster ovary cells transfected with these plasmids, or other cells lacking dihydrofolate activity, can be selected by culturing the cells in selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. Amplification of the DHFR gene in methotrexate (MTX) resistant cells is known (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Biochem. 253:1357-1370, Hamlin, J.L. and Ma, C. 1990, Annals of Biochemistry and Biophysics 1097: 107-143, Page, M.J. and Sydenham, M.A. 1991, Bioengineering, Vol. 9, 64-68). Cells cultured in increasing concentrations of MTX developed resistance to the drug, which was brought about by overproduction of the target enzyme DHFR by amplification of the DHFR gene. If the second gene is linked to the dhfr gene, it is usually coamplified and overexpressed. Thus, when methotrexate is removed, the cell line contains the amplified gene integrated into the chromosome.

Plasmid pN346 contains a strong promoter for the long terminal repeat (LTR) of Rouse sarcoma virus (Cullen et al., Molecular and Cellular Biology, March 1985, 438-447) as well as from human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985) for the expression of a gene of interest. Downstream of the promoter are the following single restriction enzyme cleavage sites for gene integration: BamHI, PvuII and Nrul. Following these cloning sites, the plasmid contains translation stop codons in all 3 reading frames, followed by the 3' intron of the rat preproinsulin gene and a polyadenylation site. Other highly efficient promoters can also be used for expression, such as the human β-actin promoter, the SV40 early or late promoter, or the long terminal repeats from other retroviruses such as HIV and HTLVI. For polyadenylation of mRNA, other signals may also be used, such as those from the human growth hormone or globin genes.

Stable cell lines carrying the gene of interest integrated into the chromosome can also be selected after co-transfection with selectable markers such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker at the outset, eg G418 plus methotrexate.

Plasmid pN346 was digested with the restriction enzyme BamHI and then dephosphorylated with bovine intestinal phosphatase according to known techniques, and the vector was then isolated from a 1% agarose gel.

The DNA sequence encoding the full-length CGF protein, ATCC #97142, was amplified with PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.

The 5' primer sequence is 5'ACTCTTGGATCCGCCATCATGACCTGGAGGCACCAT3' (SEQ ID NO: 7), which contains a BamHI restriction enzyme site (bold), followed by 6 nucleosides representing an efficient signal for translation initiation in eukaryotic cells acid (Kozak, M., Molecular Biology 196:947-950, (1987)), and the remaining nucleotides correspond to the amino-terminal 6 amino acids (including the translation initiation codon). The sequence of the 3' primer is 5'TACAACCAGCTGCTATTATTTACAACATAG 3' (SEQ ID NO: 8), which contains a PvuII restriction site and 18 nucleotides from the 3' CGF DNA originating from the translation stop codon reverse complement. The PCR product was digested with BamHI-PvuII and purified on a 1% agarose gel using a commercially available kit ("Geneclean", BIO 100 Inc., LaJolla, Ca.). This fragment was then ligated into BamHI-PvuII digested phosphatized pN346 plasmid using T4 DNA ligase. Escherichia coli strain XllBlue (Stratagene) was transformed and plated on 50 μg/ml ampicillin LB plates, and colonies with the desired recombinants in the appropriate orientation were screened by PCR using 5' primers corresponding to the Rous sarcoma virus promoter and corresponding to 3' primer for the reverse complement of CGF codons 73-79. The sequence of the cloned fragment was confirmed by DNA sequencing. Transfection of CHO-dhfr cells

Chinese hamster ovary cells lacking active DHFR enzyme were used for transfection, and 5 μg of expression plasmid pN346CGF and 0.5 μg of plasmid pSVneo were co-transfected by lipofection (Felgner et al., supra). Plasmid pSV2-neo contains a primary selectable marker, the neo gene from Tn5 encoding enzymes that confer resistance to a panel of antibiotics including G418. The cells were seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells were trypsinized and seeded on a hybridoma cloning plate (Greiner, Germany), and cultured for 10-14 days. After this period, single clones were trypsinized and seeded in 6-well dishes with different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones grown in the highest concentration of methotrexate were then transferred to new 6-well dishes containing higher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). This procedure was repeated until clones grew at a concentration of 100 μM.

Expression of the desired gene product was analyzed by Western blot analysis and SDS-PAGE.

Example 4

Expression through gene therapy

Fibroblasts were obtained from a study subject by skin biopsy. The resulting tissue was placed on tissue culture medium and dissected into small pieces. Small pieces of tissue were placed on the wet surface of tissue culture flasks, with approximately 10 pieces of tissue placed in each bottle. The bottle was placed upside down, capped tightly and left overnight at room temperature. After 24 hours at room temperature, invert the bottle, fix the tissue pieces at the bottom of the bottle, add fresh medium (such as Ham's F12 medium containing 10% FBS, penicillin, and streptomycin), and incubate at 37°C for about 1 hour. week. Fresh medium was added at this point, followed by medium changes every few days. After an additional 2 weeks of culture, a monolayer of fibroblasts appeared. The monolayer was trypsinized and placed into larger flasks.

pMV-7 flanked by Moloney murine sarcoma virus long terminal repeats (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)) was digested with EcoRI and Hind III, followed by treatment with bovine intestinal phosphatase. Linear vectors were fractionated on agarose gels and purified using glass beads.

The cDNA encoding the polypeptide of the present invention is amplified using PCR primers corresponding to the 5' and 3' end sequences, respectively. The 5' primer contains an EcoRI site and the 3' primer contains a Hind III site. Equal amounts of the linearized backbone of Moloney murine sarcoma virus are added to the amplified EcoRI and Hind III fragments in the presence of T4 DNA ligase, and the resulting mixture is maintained under conditions suitable for ligation of the two fragments. The ligation mixture was used to transform bacteria HB101, which were then plated on agar containing kanamycin to confirm that the vector had inserted the correct gene of interest.

Amphotropic pA317 or GP+am12 packaging cells were grown on tissue culture plates in Dulbecco's Modified Eagles Medium (DMEM) containing 10% calf serum (CS), penicillin, and streptomycin until reaching confluence density. The MSV vector containing the gene is then added to the medium and packaging cells are transduced with the vector. The packaging cells then produce infectious virus particles containing the gene (packaging cells are now called producer cells).

Fresh medium was added to the transduced producer cells, followed by harvesting the medium from a 10 cm plate confluent with the producer cells. The medium containing infectious viral particles is filtered through a millipore filter to remove detached producer cells, and then used to infect fibroblasts. Medium was removed from sub-confluent plates of fibroblasts and quickly replaced with medium from producer cells. The medium was removed and replaced with fresh medium. If the titer of the virus is high, then virtually all fibroblasts are infected and no selection is required. If the titer is very low, then a retrovirus with a selectable marker such as neo or his will need to be used.

The engineered fibroblasts are then injected into the host, either alone or after they have been grown to confluence on a cytodex 3 microcarrier bead.

Many modifications and variations of the present invention are possible in light of the above teaching, so that the invention may be otherwise embodied within the scope of the appended claims.

Sequence Listing (1) General Information: (i) Applicant: Meissner et al. (ii) Invention Name: Human criptin Growth Factor (iii) Sequence Number: 10 (iv) Contact Address:

(A)Address: CARELLA, BYRNE, BAIN, GILFILLLAN, CECCHI,

STEWART&OLSTEIN

(B) Street: 6 BECKERFARMROAD

(C) City: ROSELAND

(D) State: New Jersey

(E) Country: United States

(F) Zip code (ZIP): 07068

(v) in computer readable form:

(A) Storage media type: 3.5-inch floppy disk

(B) Computer: IBM PS/2

(C) Operating system: MS-DOS

(D) Software: WORD PERFECT 5.1

(vi) Current application data:

(A) Application number:

(B) Filing date: At the same time

(C) Classification:

(vii) Prior application data:

(A) Application number: None

(B) Filing date: None

(viii) Lawyer/Representative Information:

(A) Name: FERRARO, GREGORYD.

(B) Registration number: 36,134

(C) Case number/document number: 325800-

(ix) Telecommunication information:

(A) Tel: 201-994-1700

(B) Fax: 201-994-1744 (2) SEQ ID NO: 1 Information:

(i) Sequential features:

(A) Length: base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 1: (2) SEQ ID NO: 2 information:

(i) Sequential features:

(A) Length: 205 amino acids

(B) Type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecule type: protein

(xi) Sequence description: SEQ ID NO: 2: (2) SEQ ID NO: 3 Information:

(i) Sequential features:

(A) Length: base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 3: (2) SEQ ID NO: 4 Information:

(i) Sequential features:

(A) Length: base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 4: (2) SEQ ID NO: 5 information:

(i) Sequential features:

(A) Length: base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 5: (2) SEQ ID NO: 6 information:

(i) Sequential features:

(A) Length: base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 6:

Claims (20)

1, a kind of isolating polynucleotide, it comprises a member who is selected from as in next group:

(a) a kind of coding comprises the-26 polynucleotide to the polypeptide of the 207th amino acids as shown in Figure 1;

(b) a kind of coding comprises the 1st polynucleotide to the polypeptide of the 207th amino acids as shown in Figure 1;

(c) a kind of coding comprises the 1st polynucleotide to the polypeptide of the 150th amino acids as shown in Figure 1;

(d) a kind of coding comprises the 45th polynucleotide to the polypeptide of the 150th amino acids as shown in Figure 1;

(e) a kind of can and (a) and (b), (c) or multi-nucleotide hybrid (d) and and (a) and (b), (c) or polynucleotide (d) between have the polynucleotide of 70% homogeny at least; And

(f) (a), (b), (c), (d) or a polynucleotide passage of polynucleotide (e).

2, the polynucleotide of claim 1, wherein these polynucleotide are DNA.

3, a kind of isolating polynucleotide, it comprises a member who is selected from as in next group:

(a) a kind of coding is by the polynucleotide of the mature polypeptide that is included in the DNA expression in the ATCC preserving number 97142;

(b) a kind of coding is by the polynucleotide that are included in the DNA polypeptide expressed in the ATCC preserving number 97142;

(c) a kind of can and (a) or multi-nucleotide hybrid (b) and and (a) or have the polynucleotide of 70% homogeny between the polynucleotide (b) at least; And

(d) (a), (b) or a polynucleotide passage of polynucleotide (c).

4, the polynucleotide of claim 2 comprise 771 the sequence from Nucleotide 1 to Nucleotide as shown in Figure 1.

5, the polynucleotide of claim 2 comprise 771 the sequence from Nucleotide 62 to Nucleotide as shown in Figure 1.

6, the polynucleotide of claim 2 comprise 771 the sequence from Nucleotide 151 to Nucleotide as shown in Figure 1.

7, the polynucleotide of claim 2 comprise 600 the sequence from Nucleotide 283 to Nucleotide as shown in Figure 1.

8, a kind of carrier that contains the DNA of claim 2.

9, a kind of genetically engineered host cell of carrier with claim 8.

10, a kind of method of producing polypeptide, this method comprises: express the polypeptide by said dna encoding in the host cell of Accessory Right requirement 9.

11, the method for the cell that a kind of production can express polypeptide, this method comprises the genetically engineered cell of carrier with claim 8.

12, a kind of polypeptide that is selected from as a member in next group that comprises: (i) polypeptide of the deduced amino acid of a kind of Fig. 1 of having and fragment thereof, analogue and derivative; And (ii) a kind of cDNA encoded polypeptides, and the fragment of said polypeptide, analogue and derivative by ATCC preserving number 97142.

13, a kind of compound of acceptor of the polypeptide that activates claim 12.A kind of antibody of polypeptide of anti-claim 10.

14, a kind of compound that suppresses the polypeptide of claim 12.

15, a kind of antibody of polypeptide of anti-claim 12.

16, a kind of method of identifying the activatory compound of the polypeptide that suppresses claim 12 comprises:

To under being suitable for the condition of part and described receptors bind, the CGF of the cell of its surface expression CGF acceptor and mark and compound to be screened contact; And

The labelled amount that is attached to acceptor by mensuration is determined the CGF of mark and the combination degree of acceptor.

17, a kind of method of identifying the activatory compound of the polypeptide that suppresses claim 12 comprises:

To under being suitable for the condition of part and described receptors bind, the cell of its surface expression CGF acceptor and compound to be screened contact; And

Determine that compound is with the combination degree of acceptor and by the forfeiture that combines the signal that produces.

18, a kind of method of compound of the acceptor of identifying the polypeptide that activates claim 12 comprises:

To under being suitable for the condition of part and described receptors bind, the cell of its surface expression CGF acceptor and compound to be screened contact; And

Determine that compound is with the combination degree of acceptor and by the existence that combines the signal that produces.

19, the sudden change diseases associated in the polynucleotide of a kind of diagnosis and claim 1 and the method for disease susceptibility comprise:

Sudden change in the polynucleotide of mensuration claim 1.

20, a kind of diagnostic method comprises:

Analysis is from the polypeptide that whether has claim 12 in host's the sample.

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