Classifications

• • 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

Definitions

• This invention relates to nucleic acid and amino acid sequences of a growth factor-like protein and to the use of these sequences in the diagnosis, prevention, and treatment of cancer and connective tissue disorders.
• PDGF Human platelet-derived growth factor
• the growth factors such as PDGF.
• transforming growth factor- ⁇ , and epidermal growth factor have a variety of roles in cellular growth and development including pattern formation, organogenesis. and tissue-specific transcription. Growth factor binding to specific receptors induces expression of many immediate-early genes, including transcription factors, secreted proteins, protein kinases. and protein phosphatases.
• PDGF regulates cell proliferation and differentiation, extracellular matrix formation, migration, adhesion, and other cellular functions important for development, maturation, and homeostasis. It is a member of a family of proteins that includes human connective tissue growth factor, its mouse homologue. fisp-12. and the human and chicken nov proteins. These proteins share a common structural motif which is a consensus sequence found in the insulin growth factor-binding protein. All members of this family are believed to be involved in cell proliferation.
• PDGF is a two chain (A and B) secreted protein that acts as a chemoattractant at low concentrations, and as a mitogen at higher concentrations.
• the B-chain sequence is very similar to a segment of the transforming protein of simian sarcoma virus and, without the A chain, is sufficient for mitogenesis. Stimulation of cell proliferation through the PDGF receptor has been implicated in atherogenesis and in cell transformation by simian sarcoma virus oncogene.
• PDGF-action such as PDGF antibodies
• PDGF antibodies are inhibitors of simian sarcoma virus-transformation and can reverse the phenotype of the transformed cells.
• PDGF is secreted by activated platelets and macrophages, and by vascular cells at sites of inflammation and vascular damage. It is also expressed in human sarcoma cell lines and smooth muscle cells within atherosclerotic lesions (Snaith, et al. (1996) Genomics 38: 425-428; Westermark, B. and Heldin, C. H. (1986) Med. Oncol. Tumor Pharmacother. 3: 177-183; and Durante. W. et al (1996) J. Biol. Chem. 271 : 1 1838-1 1843).
• Connective tissue growth factor is a peptide that exhibits PDGF-like activities and is produced by fibroblasts after activation with transforming growth factor-beta. It was first isolated from the medium of human umbilical vein endothelial cell cultures through its ability to bind to anti-PDGF antibodies. Like PDGF, it is a member of the family of proteins that contain the insulin growth factor-binding consensus sequence. CTGF is antigenically and functionally related to PDGF and has been shown to induce connective tissue cell proliferation and extracellular matrix synthesis. It is expressed in wound repair, cancer stroma. and fibroproliferative disorders (Bradham. D. M. et al.(1991 ) J. Cell Biol. 1 14: 1285-1294; Frazier, K. et al. (1996) J. Invest. Dermatol. 107:404-41 1 )
• the invention features a substantially purified polypeptide, growth factor-like protein (GRFLP), having the amino acid sequence shown in SEQ ID NO: l , or fragments thereof.
• the invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: l or fragments thereof and a composition comprising said polynucleotide sequence.
• the invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO: l, or fragments of said polynucleotide sequence.
• the invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:l, or fragments or variants of said polynucleotide sequence.
• the invention also provides an isolated and purified sequence comprising SEQ ID NO.2 or variants thereof.
• the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
• the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
• the invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2.
• the present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences.
• the expression vector containing the polynucleotide sequence is contained within a host cell.
• the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: l or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding GRFLP under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
• the invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising a substantially purified GRFLP having the amino acid sequence of SEQ ID NO: l in conjunction with a suitable pharmaceutical carrier.
• the invention also provides a purified antagonist which decreases the activity of a polypeptide of SEQ ID NO: 1.
• the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO:l .
• the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO: 1.
• the invention also provides a method for treating or preventing cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist which decreases the activity of GRFLP.
• the invention also provides a method for treating or preventing connective tissue disorders comprising administering to a subject in need of such treatment an effective amount of an antagonist which decreases the activity of GRFLP.
• the invention also provides a method for detecting a polynucleotide which encodes GRFLP in a biological sample comprising the steps of: a) hybridizing a polynucleotide sequence complementary to the polynucleotide which encodes GRFLP (SEQ ID NO: l) to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding GRFLP in the biological sample.
• the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
• Figures 1A, IB, 1C, and ID show the amino acid sequence (SEQ ID NO: l) and nucleic acid sequence (SEQ ID NO:2) of GRFLP.
• the alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co. Ltd. San Bruno, CA).
• Figures 2A and 2B show the amino acid sequence alignments among GRFLP (SEQ ID NO: l), human connective tissue growth factor (GI 180924; SEQ ID NO:3), and mouse fisp-12 (GI 193314; SEQ ID NO:4) produced using the multisequence alignment program of DNASTARTM software (DNASTAR Inc. Madison WI).
• GRFLP refers to the amino acid sequences of substantially purified GRFLP obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
• agonist refers to a molecule which, when bound to GRFLP, increases or prolongs the duration of the effect of GRFLP.
• Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of GRFLP.
• Alleles are an alternative form of the gene encoding GRFLP. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none. one. or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
• altered nucleic acid sequences encoding GRFLP as used herein include those with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent GRFLP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular ohgonucleotide probe of the polynucleotide encoding GRFLP, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding GRFLP.
• the encoded protein may also be "altered” and contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent GRFLP.
• Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of GRFLP is retained.
• negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophihcity values may include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.
• Amino acid sequence refers to an ohgopeptide, peptide. polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Fragments of GRFLP are preferably about 5 to about 15 amino acids in length and retain the biological activity or the immunological activity of GRFLP.
• amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
• PCR polymerase chain reaction
• Antagonist refers to a molecule which, when bound to GRFLP, decreases the amount or the duration of the effect of the biological or immunological activity of GRFLP.
• Antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules which decrease the effect of GRFLP.
• the term " antibody” refers to intact molecules as well as fragments thereof, such as Fa, F(ab ' )-,, and Fv, which are capable of binding the epitopic determinant.
• Antibodies that bind GRFLP polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or ohgopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
• antigenic determinant refers to that fragment of a molecule
• an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
• antisense refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence.
• the term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
• Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand.
• biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
• immunologically active refers to the capability of the natural, recombinant. or synthetic GRFLP, or any ohgopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
• complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
• sequence "A-G-T” binds to the complementary sequence "T-C-A”.
• Complementarity between two single-stranded molecules may be " partial " , in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules.
• the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands and in the design and use of PNA molecules.
• composition comprising a given polynucleotide sequence refers broadly to any composition containing the given polynucleotide sequence.
• the composition may comprise a dry formulation or an aqueous solution.
• Compositions comprising polynucleotide sequences encoding GRFLP (SEQ ID NO:l) or fragments thereof (e.g., SEQ ID NO:2 and fragments thereof) may be employed as hybridization probes.
• the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
• the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS) and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
• salts e.g., NaCl
• detergents e.g., SDS
• other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
• Consensus refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, has been extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' direction and resequenced. or has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly (e.g., GEL VIEWTM Fragment Assembly system, GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence .
• ribonucleic acid that is similar to SEQ ID NO:2 by northern analysis is indicative of the presence of mRNA encoding GRFLP in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.
• a “deletion” refers to a change in the amino acid or nucleotide sequence and results in the absence of one or more amino acid residues or nucleotides.
• derivative refers to the chemical modification of a nucleic acid encoding or complementary to GRFLP or the encoded GRFLP. Such modifications include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
• a nucleic acid derivative encodes a polypeptide which retains the biological or immunological function of the natural molecule.
• a derivative polypeptide is one which is modified by glycosylation, pegylation. or any similar process which retains the biological or immunological function of the polypeptide from which it was derived.
• low stringency conditions are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
• the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g.. less than about 30% identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.
• HACs Human artificial chromosomes
• HACs are linear microchromosomes which may contain DNA sequences of 10K to 10M in size and contain all of the elements required for stable mitotic chromosome segregation and maintenance (Harrington, J.J. et al. (1997) Nat Genet. 15:345-355).
• humanized antibody refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.
• hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
• hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
• the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
• a hybridization complex may be formed in solution (e.g., C 0 t or Rgt analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g.. paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
• insertion refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.
• “Microarray” refers to an array of distinct polynucleotides or ohgonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
• modulate refers to a change in the activity of GRFLP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional or immunological properties of GRFLP.
• Nucleic acid sequence refers to an ohgonucleotide, nucleotide, or polynucleotide. and fragments thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
• “Fragments” are those nucleic acid sequences which are greater than 60 nucleotides than in length, and most preferably includes fragments that are at least 100 nucleotides or at least 1000 nucleotides, and at least 10,000 nucleotides in length.
• ohgonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides. and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or hybridization assays. As used herein, ohgonucleotide is substantially equivalent to the terms “amplimers”. “primers”, “oligomers”, and “probes”, as commonly defined in the art.
• PNA protein nucleic acid
• PNA refers to an antisense molecule or anti-gene agent which comprises an ohgonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues which ends in lysine. The terminal lysine confers solubility to the composition.
• PNAs may be pegylated to extend their lifespan in the cell where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen. P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
• portion refers to fragments of that protein.
• the fragments may range in size from five amino acid residues to the entire amino acid sequence minus one amino acid.
• a protein "comprising at least a portion of the amino acid sequence of SEQ ID NO: l " encompasses the full-length GRFLP and fragments thereof.
• sample as used herein, is used in its broadest sense.
• a biological sample suspected of containing nucleic acid encoding GRFLP, or fragments thereof, or GRFLP itself may comprise a bodily fluid, extract from a cell, chromosome, organelle. or membrane isolated from a cell, a cell, genomic DNA. RNA. or cDNA(in solution or bound to a solid support, a tissue, a tissue print, and the like.
• binding refers to that interaction between a protein or peptide and an agonist, an antibody and an antagonist. The interaction is dependent upon the presence of a particular structure (i.e.. the antigenic determinant or epitope) of the protein recognized by the binding molecule. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.
• stringent conditions refer to the conditions for hybridization as defined by the nucleic acid, salt, and temperature. These conditions are well known in the art and may be altered in order to identify or detect identical or related polynucleotide sequences. Numerous equivalent conditions comprising either low or high stringency depend on factors such as the length and nature of the sequence (DNA, RNA, base composition), nature of the target (DNA.
• RNA, base composition RNA, base composition
• milieu in solution or immobilized on a solid substrate
• concentration of salts and other components e.g., formamide, dextran sulfate and/or polyethylene glycol
• temperature of the reactions within a range from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature.
• salts and other components e.g., formamide, dextran sulfate and/or polyethylene glycol
• temperature of the reactions within a range from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature.
• concentration of salts and other components e.g., formamide, dextran sulfate and/or polyethylene glycol
• temperature of the reactions within a range from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature.
• One or more factors be may be varied to generate conditions of either low or high stringency different from
• substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
• substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
• Transformation describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation. heat shock, lipofection. and particle bombardment. Such "transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome.
• They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
• a “variant" of GRFLP refers to an amino acid sequence that is altered by one or more amino acids.
• the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
• Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software. THE INVENTION
• the invention is based on the discovery of a new human growth factor-like protein (hereinafter referred to as "GRFLP”), the polynucleotides encoding GRFLP, and the use of these compositions for the diagnosis, prevention, or treatment of cancer and connective tissue disorders.
• GRFLP human growth factor-like protein
• Nucleic acids encoding the GRFLP of the present invention were first identified in Incyte Clone 69291 1 from the lung tumor cDNA library (LUNGTUT02) using a computer search for amino acid sequence alignments. A consensus sequence. SEQ ID NO:2, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 69291 1 (LUNGTUT02), 393590 (TMLR2DT01 ). 360014 (SYNORABOl), 17321 14 (BRSTTUT08). 643423 (BRSTTUT02).
• the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: l , as shown in Figures 1A, IB, 1C, and I D.
• GRFLP is 250 amino acids in length and has a potential insulin-like growth factor binding signature from residues G 9 through C 64 .
• GRFLP has chemical and structural homology with human connective tissue growth factor (GI 180924; SEQ ID NO:3) and mouse fisp-12 (GI 193314; SEQ ID NO:4).
• GRFLP and human connective tissue growth factor share 44% identity
• GRFLP and mouse fisp-12 share 39% identity.
• Northern analysis shows the expression of this sequence in various libraries, at least 63% of which are immortalized or cancerous.
• the invention also encompasses GRFLP variants.
• a preferred GRFLP variant is one having at least 80%, and more preferably 90%. amino acid sequence identity to the GRFLP amino acid sequence (SEQ ID NO: 1).
• a most preferred GRFLP variant is one having at least 95% ⁇ amino acid sequence identity to SEQ ID NO: l .
• the invention also encompasses polynucleotides which encode GRFLP. Accordingly, any nucleic acid sequence which encodes the amino acid sequence of GRFLP can be used to produce recombinant molecules which express GRFLP. In a particular embodiment, the invention encompasses the polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 as shown in Figure 1.
• nucleotide sequences encoding GRFLP may be produced.
• the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring GRFLP, and all such variations are to be considered as being specifically disclosed.
• nucleotide sequences which encode GRFLP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring GRFLP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding GRFLP or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
• RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
• the invention also encompasses production of DNA sequences, or fragments thereof, which encode GRFLP and its derivatives, entirely by synthetic chemistry.
• the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.
• synthetic chemistry may be used to introduce mutations into a sequence encoding GRFLP or any fragment thereof.
• polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those shown in SEQ ID NO:2, under various conditions of stringency as taught in Wahl, G.M. and S.L. Berger ( 1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-51 1). Methods for DNA sequencing which are well known and generally available in the art and may be used to practice any of the embodiments of the invention.
• the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD).
• the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton. Reno. NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown. MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
• the nucleic acid sequences encoding GRFLP may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
• one method which may be employed, "restriction-site" PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
• genomic DNA is first amplified in the presence of primer to a linker sequence and a primer specific to the known region.
• the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
• Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
• Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al. ( 1988) Nucleic Acids Res. 16:8186).
• the primers may be designed using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C.
• the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
• Another method which may be used is capture PCR which involves PCR amplification of
• Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991 ; Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions. When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
• Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
• capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
• Output/light intensity may be converted to electrical signal using appropriate software (e.g. GenotyperTM and Sequence NavigatorTM, Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
• Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
• polynucleotide sequences or fragments thereof which encode GRFLP may be used in recombinant DNA molecules to direct expression of GRFLP, fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced, and these sequences may be used to clone and express GRFLP. As will be understood by those of skill in the art. it may be advantageous to produce
• GRFLP-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
• nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter GRFLP encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
• DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic ohgonucleotides may be used to engineer the nucleotide sequences.
• site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
• natural, modified, or recombinant nucleic acid sequences encoding GRFLP may be ligated to a heterologous sequence to encode a fusion protein.
• a heterologous sequence to encode a fusion protein.
• a fusion protein may also be engineered to contain a cleavage site located between the GRFLP encoding sequence and the heterologous protein sequence, so that GRFLP may be cleaved and purified away from the heterologous moiety.
• sequences encoding GRFLP may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223. Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).
• the protein itself may be produced using chemical methods to synthesize the amino acid sequence of GRFLP, or a fragment thereof.
• peptide synthesis can be performed using various solid-phase techniques (Roberge, J.Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer).
• the newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins. Structures and Molecular Principles. WH Freeman and Co., New York, NY).
• the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton. supra).
• the amino acid sequence of GRFLP. or any part thereof may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
• nucleotide sequences encoding GRFLP or functional equivalents may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
• a variety of expression vector/host systems may be utilized to contain and express sequences encoding GRFLP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus. TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
• the invention is not limited by the host cell employed.
• control elements are those non-translated regions of the vector—enhancers, promoters, 5' and 3" untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla. CA) or pSport 1TM plasmid (Gibco BRL) and the like may be used.
• inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla. CA) or pSport 1TM plasmid (Gibco BRL) and the like may be used.
• the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO: and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding GRFLP. vectors based on SV40 or EBV may be used with an appropriate selectable marker.
• a number of expression vectors may be selected depending upon the use intended for GRFLP.
• vectors which direct high level expression of fusion proteins that are readily purified may be used.
• Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding GRFLP may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke. G. and S.M. Schuster (1989) J. Biol. Chem.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
• Proteins made in such systems may be designed to include heparin, thrombin. or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
• Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
• constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
• sequences encoding GRFLP may be driven by any of a number of promoters.
• viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu. N. ( 1987) EMBO J. 3:131 1).
• plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3: 1671 - 1680; Broglie. R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991 ) Results Probl.
• Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
• the sequences encoding GRFLP may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of GRFLP will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
• the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which GRFLP may be expressed (Engelhard, E.K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227).
• a number of viral-based expression systems may be utilized.
• sequences encoding GRFLP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing GRFLP in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81 :3655-3659).
• transcription enhancers such as the Rous sarcoma virus (RSV) enhancer
• RSV Rous sarcoma virus
• HACs Human artificial chromosomes
• 6 to 10M are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
• Specific initiation signals may also be used to achieve more efficient translation of sequences encoding GRFLP. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding GRFLP, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf. D. et al. ( 1994) Results Probl. Cell Differ. 20: 125-162).
• a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
• modifications of the polypeptide include, but are not limited to. acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
• Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function.
• Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities are available from the American Type Culture Collection (ATCC; Bethesda, MD) and may be chosen to ensure the correct modification and processing of the foreign protein.
• ATCC American Type Culture Collection
• cell lines which stably express GRFLP may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
• any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 1 1 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk " or aprt cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
• npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150: 1 -14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase. respectively (Murry, supra). Additional selectable genes have been described, for example. trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci.
• marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
• sequence encoding GRFLP is inserted within a marker gene sequence
• transformed cells containing sequences encoding GRFLP can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with a sequence encoding GRFLP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
• host cells which contain the nucleic acid sequence encoding GRFLP and express GRFLP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
• polynucleotide sequences encoding GRFLP can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding GRFLP.
• Nucleic acid amplification based assays involve the use of ohgonucleotides or ohgomers based on the sequences encoding GRFLP to detect transformants containing DNA or RNA encoding GRFLP.
• a variety of protocols for detecting and measuring the expression of GRFLP. using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA). and fluorescence activated cell sorting (FACS).
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• FACS fluorescence activated cell sorting
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on GRFLP is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton. R. et al. (1990: Serological Methods, a Laboratory Manual. APS Press, St Paul, MN) and Maddox, D.E. et al. (1983; J. Exp. Med. 158: 121 1 - 1216).
• Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding GRFLP include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
• the sequences encoding GRFLP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
• RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
• T7, T3, or SP6 RNA polymerase
• Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors. inhibitors, magnetic particles, and the like.
• Host cells transformed with nucleotide sequences encoding GRFLP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
• the protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
• expression vectors containing polynucleotides which encode GRFLP may be designed to contain signal sequences which direct secretion of GRFLP through a prokaryotic or eukaryotic cell membrane.
• Other constructions may be used to join sequences encoding GRFLP to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
• Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
• metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
• protein A domains that allow purification on immobilized immunoglobulin
• the domain utilized in the FLAGS extension/affinity purification system Immunex Corp., Seattle, WA.
• cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and GRFLP may be used to facilitate purification.
• One such expression vector provides for expression of a fusion protein containing GRFLP and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
• the histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides a means for purifying GRFLP from the fusion protein.
• IMAC immobilized metal ion affinity chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281
• the enterokinase cleavage site provides a means for purifying GRFLP from the fusion protein.
• fragments of GRFLP may be produced by direct peptide synthesis using solid-phase techniques Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of GRFLP may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
• GRFLP human connective tissue growth factor
• mouse fisp-12 GI 193314
• GRFLP is expressed in various libraries derived from cancerous tissues. Therefore, GRFLP appears to play a role in cancer and connective tissue disorders, particularly disorders in which GRFLP is overexpressed.
• an antagonist of GRFLP may be administered to a subject to prevent or treat cancer.
• cancer may include, but is not limited to, adenocarcinoma.
• antibodies which specifically bind GRFLP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express GRFLP.
• a vector expressing the complement of the polynucleotide encoding GRFLP may be administered to a subject to treat or prevent the cancer, including, but not limited to, the cancers described above.
• an antagonist of GRFLP may be administered to a subject to prevent or treat a connective tissue disorder.
• connective tissue disorder may include, but are not limited to, ankylosing spondylitis, atherosclerosis, Dupuytren's contracture. eosinophilic fasciitis, Felty syndrome, Goodpasture's disease. Hunter syndrome, Hurler syndrome, keloids. Marfan syndrome, nodular fasciitis, osteogenesis imperfecta, polyarthritis nodosa. rheumatoid arthritis, scleroderma, systemic lupus erythematosus, and restenosis following angioplasty.
• antibodies which specifically bind GRFLP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express GRFLP.
• a vector expressing the complement of the polynucleotide encoding GRFLP may be administered to a subject to treat or prevent a connective tissue disorder, including, but not limited to. the connective tissue disorders described above.
• any of the therapeutic proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
• the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
• Antagonists or inhibitors of GRFLP may be produced using methods which are generally known in the art.
• purified GRFLP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind GRFLP.
• Antibodies to GRFLP may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal. monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
• various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with GRFLP or any fragment or ohgopeptide thereof which has immunogenic properties.
• various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to.
• Freund's mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides. oil emulsions, keyhole limpet hemocyanin. and dinitrophenol.
• BCG Bacilli Calmette-Guerin
• Corynebacterium parvum are especially preferable.
• the oligopeptides, peptides. or fragments used to induce antibodies to GRFLP have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of GRFLP amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.
• Monoclonal antibodies to GRFLP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 :31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S.P. et al. (1984) Mol. Cell Biol. 62: 109-120).
• chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. 81 :6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
• techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce GRFLP-specific single chain antibodies.
• Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton D.R. (1991) Proc. Natl. Acad. Sci. 88: 1 1 120-3).
• Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
• Antibody fragments which contain specific binding sites for GRFLP may also be generated.
• fragments include, but are not limited to. the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
• Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse. W.D. et al. (1989) Science 254: 1275-1281).
• immunoassays may be used for screening to identify antibodies having the desired specificity.
• Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
• Such immunoassays typically involve the measurement of complex formation between GRFLP and its specific antibody.
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering GRFLP epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra).
• the polynucleotides encoding GRFLP. or any fragment or complement thereof may be used for therapeutic purposes.
• the complement of the polynucleotide encoding GRFLP may be used in situations in which it would be desirable to block the transcription of the mRNA.
• cells may be transformed with sequences complementary to polynucleotides encoding GRFLP.
• complementary molecules or fragments may be used to modulate GRFLP activity, or to achieve regulation of gene function.
• sense or antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding GRFLP.
• Expression vectors derived from retro viruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence which is complementary to the polynucleotides of the gene encoding GRFLP. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
• Genes encoding GRFLP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes
• GRFLP GRFLP
• Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
• modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA. or PNA) to the control. 5' or regulatory regions of the gene encoding GRFLP (signal sequence, promoters, enhancers, and introns). Ohgonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases. transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee. J.E. et al.
• the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
• Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
• the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding GRFLP.
• ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the ohgonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides using ribonuclease protection assays.
• RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing ohgonucleotides such as solid phase phosphoramidite chemical synthesis.
• RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding GRFLP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
• these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
• RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine. and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine. cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
• vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections or polycationic amino polymers (Goldman. C.K. et al. (1997) Nature Biotechnology 15:462-66; incorporated herein by reference) may be achieved using methods which are well known in the art. Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
• compositions may consist of GRFLP, antibodies to GRFLP, mimetic, agonists, antagonists, or inhibitors of GRFLP.
• the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
• the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
• compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
• these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co.. Easton, PA).
• Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees. capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
• compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol. or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
• disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
• Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol. and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol. and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e.. dosage.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
• Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
• compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
• Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• Non-lipid polycationic amino polymers may also be used for delivery.
• the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• penetrants appropriate to the particular barrier to be permeated are used in the formulation.
• penetrants are generally known in the art.
• compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
• the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric. acetic, lactic, tartaric. malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
• the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol. at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
• the determination of an effective dose is well within the capability of those skilled in the art.
• the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
• a therapeutically effective dose refers to that amount of active ingredient, for example
• GRFLP or fragments thereof, antibodies of GRFLP, agonists, antagonists or inhibitors of GRFLP. which ameliorates the symptoms or condition.
• Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
• Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
• Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age. weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
• Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
• antibodies which specifically bind GRFLP may be used for the diagnosis of conditions or diseases characterized by expression of GRFLP, or in assays to monitor patients being treated with GRFLP, agonists, antagonists or inhibitors.
• the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for GRFLP include methods which utilize the antibody and a label to detect GRFLP in human body fluids or extracts of cells or tissues.
• the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
• a wide variety of reporter molecules which are known in the art may be used, several of which are described above.
• GRFLP expression A variety of protocols including ELISA, RIA, and FACS for measuring GRFLP are known in the art and provide a basis for diagnosing altered or abnormal levels of GRFLP expression.
• Normal or standard values for GRFLP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to GRFLP under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, but preferably by photometric, means.
• the polynucleotides encoding GRFLP may be used for diagnostic purposes.
• the polynucleotides which may be used include ohgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
• the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of GRFLP may be correlated with disease.
• the diagnostic assay may be used to distinguish between absence, presence, and excess expression of GRFLP, and to monitor regulation of GRFLP levels during therapeutic intervention.
• hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding GRFLP or closely related molecules, may be used to identify nucleic acid sequences which encode GRFLP.
• the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory region, or a less specific region, e.g., especially in the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding GRFLP, alleles, or related sequences.
• Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the GRFLP encoding sequences.
• the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO:2 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring GRFLP.
• Means for producing specific hybridization probes for DNAs encoding GRFLP include the cloning of nucleic acid sequences encoding GRFLP or GRFLP derivatives into vectors for the production of mRNA probes.
• Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
• Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
• Polynucleotide sequences encoding GRFLP may be used for the diagnosis of conditions, disorders, or diseases which are associated with expression of GRFLP. Examples of such conditions or diseases include cancers such as adenocarcinoma, leukemia, lymphoma.
• melanoma myeloma, sarcoma, and teratocarcinoma and particularly cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus, and ankylosing spondylitis, atherosclerosis, Dupuytren's contracture, eosinophilic fasciitis, Felty syndorme, Goodpasture's disease, Hunter syndrome, Hurler syndrome, keloids, Marfan syndrome, nodular fasciitis, osteogenesis imperfecta, polyarthritis nodosa, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, and restenosis following angioplasty.
• the polynucleotide sequences encoding GRFLP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dipstick, pin, ELISA assays or microarrays utilizing fluids or tissues from patient biopsies to detect altered GRFLP expression. Such qualitative or quantitative methods are well known in the art.
• the nucleotide sequences encoding GRFLP may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above.
• the nucleotide sequences encoding GRFLP may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
• nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding GRFLP in the sample indicates the presence of the associated disease.
• assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. In order to provide a basis for the diagnosis of disease associated with expression of
• GRFLP a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes GRFLP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
• hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
• the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
• a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
• ohgonucleotides designed from the sequences encoding GRFLP may involve the use of PCR.
• Such ohgomers may be chemically synthesized, generated enzymatically, or produced in vitro.
• Ohgomers will preferably consist of two nucleotide sequences, one with sense orientation (5'->3') and another with antisense (3' ⁇ -5'), employed under optimized conditions for identification of a specific gene or condition.
• the same two ohgomers, nested sets of ohgomers, or even a degenerate pool of ohgomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
• Methods which may also be used to quantitate the expression of GRFLP include radiolabeling or biotinylating nucleotides. coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby, P.C. et al. (1993) J. Immunol. Methods. 159:235-244; Duplaa. C. et al. (1993) Anal. Biochem. 229-236).
• the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
• ohgonucleotides derived from any of the polynucleotide sequences described herein may be used as probes in microarrays.
• the microarrays can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants, mutations and polymorphisms. This information will be useful in determining gene function, understanding the genetic basis of disease, diagnosing disease, and in developing and monitoring the activity of therapeutic agents.
• the microarray is prepared and used according to the methods described in PCT application W095/1 1995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference.
• the microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense ohgonucleotides or fragments of cDNAs fixed to a solid support.
• Microarrays may contain ohgonucleotides which cover the known 5', or 3', sequence, or contain sequential ohgonucleotides which cover the full length sequence; or unique ohgonucleotides selected from particular areas along the length of the sequence.
• Polynucleotides used in the microarray may be ohgonucleotides that are specific to a gene or genes of interest in which at least a fragment of the sequence is known or that are specific to one or more unidentified cDNAs which are common to a particular cell type, developmental or disease state.
• the gene of interest is examined using a computer algorithm which starts at the 5' or more preferably at the 3' end of the nucleotide sequence.
• the algorithm identifies ohgomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization.
• the ohgomers are synthesized at designated areas on a substrate using a light-directed chemical process.
• the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
• the ohgomers may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251 1 16 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference.
• a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or ohgonucleotides to the surface of a substrate using a vacuum system, thermal. UV, mechanical or chemical bonding procedures.
• An array may be produced by hand or using available devises (slot blot or dot blot apparatus) materials and machines (including robotic instruments) and contain grids of 8 dots. 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots, or any other multiple which lends itself to the efficient use of commercially available instrumentation.
• RNA or DNA from a biological sample is made into hybridization probes.
• the mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA).
• aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray so that the probe sequences hybridize to complementary ohgonucleotides of the microarray. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence.
• the scanned images are examined to determine degree of complementarity and the relative abundance of each ohgonucleotide sequence on the microarray.
• the biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
• a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies on the sequences, mutations, variants, or polymorphisms among samples.
• the nucleic acid sequences which encode GRFLP may also be used to generate hybridization probes which are useful for mapping the naturally occurring genomic sequence.
• the sequences may be mapped to a particular chromosome, to a specific region of a chromosome or to artificial chromosome constructions, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions or single chromosome cDNA libraries as reviewed in Price, CM. (1993) Blood Rev. 7: 127-134, and Trask, B.J. (1991) Trends Genet. 7: 149-154.
• FISH Fluorescent in situ hybridization
• Chromosomes A Manual of Basic Techniques. Pergamon Press, New York, NY
• CMOS complementary metal-oxide-semiconductor
• Examples of genetic map data can be found in various scientific journals or at Online Mendelian Inheritance in Man (OMIM).
• OMIM Online Mendelian Inheritance in Man
• Correlation between the location of the gene encoding GRFLP on a physical chromosomal map and a specific disease , or predisposition to a specific disease may help delimit the region of DNA associated with that genetic disease.
• the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.
• In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 lq22-23 (Gatti, R.A. et al.
• any sequences mapping to that area may represent associated or regulatory genes for further investigation.
• the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
• GRFLP its catalytic or immunogenic fragments or oligopeptides thereof
• the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between GRFLP and the agent being tested, may be measured.
• Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
• GRFLP small test compounds
• a solid substrate such as plastic pins or some other surface.
• the test compounds are reacted with GRFLP, or fragments thereof, and washed.
• Bound GRFLP is then detected by methods well known in the art.
• Purified GRFLP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
• non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
• nucleotide sequences which encode GRFLP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
• the LUNGTUT02 cDNA library was constructed from lung tumor tissue removed from a 79 year old Caucasian male who was diagnosed as having a malignant neoplasm in the upper lobe of left lung.
• the pathology report revealed a grade four carcinoma with H ⁇ rthle cells that had metastasized from thyroid cancer.
• the frozen tissue was homogenized and lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments. Westbury NJ). The reagents and extraction procedures were used as supplied in the Stratagene RNA Isolation Kit (Cat. # 200345; Stratagene). The lysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 ⁇ m at ambient temperature. The RNA was extracted twice with phenol chloroform pH 8.0, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in water and DNase treated for 15 min at 37°C. The RNA was isolated using the Qiagen Oligotex kit (QIAGEN Inc. Chatsworth CA) and used to construct the cDNA library.
• Qiagen Oligotex kit QIAGEN Inc. Chatsworth CA
• RNA was handled according to the recommended protocols in the Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat. #18248-013; Gibco/BRL). cDNAs were fractionated on a Sepharose CL4B column (Cat. #275105, Pharmacia), and those cDNAs exceeding 400 bp were ligated into pSport I. The plasmid pSport I was subsequently transformed into DH5aTM competent cells (Cat. #18258-012, Gibco/BRL).
• Plasmid DNA was released from the cells and purified using the Miniprep Kit (Catalog #77468; Advanced Genetic Technologies Co ⁇ oration, Gaithersburg MD). This kit consists of a 96-well block with reagents for 960 purifications. The recommended protocol was employed except for the following changes: 1 ) the 96 wells were each filled with only 1 ml of sterile Terrific Broth (Catalog #2271 1.
• the cDNAs were sequenced by the method of Sanger F and AR Coulson (1975; J Mol Biol 94:44 If), using a Hamilton Micro Lab 2200 (Hamilton. Reno NV) in combination with four Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown MA) and Applied Biosystems 377 or 373 DNA Sequencing Systems, and the reading frame was determined. III Homology Searching of cDNA Clones and Their Deduced Proteins
• nucleotide sequences of the Sequence Listing or amino acid sequences deduced from them were used as query sequences against databases such as GenBank, SwissProt, BLOCKS, and Pima II. These databases which contain previously identified and annotated sequences were searched for regions of homology (similarity) using BLAST, which stands for Basic Local Alignment Search Tool (Altschul (1993) supra. Altschul (1990) supra).
• BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal or plant) origin. Other algorithms such as the one described in Smith RF and TF Smith (1992 Protein Engineering 5:35-51 ), inco ⁇ orated herein by reference, can be used when dealing with primary sequence patterns and secondary structure gap penalties. As disclosed in this application, the sequences have lengths of at least 49 nucleotides, and no more than 12%) uncalled bases (where N is recorded rather than A, C, G, or T).
• threshold was set at 10 "25 for nucleotides and 10 "14 for peptides.
• Incyte nucleotide sequence were searched against the GenBank databases for primate (pri), rodent (rod), and mammalian sequences (mam), and deduced amino acid sequences from the same clones are searched against GenBank functional protein databases, mammalian (mamp), vertebrate (vrtp) and eukaryote (eukp), for homology.
• GenBank functional protein databases mammalian (mamp), vertebrate (vrtp) and eukaryote (eukp), for homology.
• the product score is calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the %> maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. Where an Incyte Clone was homologous to several sequences, up to five matches were provided with their relevant scores. In an analogy to the hybridization procedures used in the laboratory, the electronic stringency for an exact match was set at 70, and the conservative lower limit for an exact match was set at approximately 40 (with 1-2%) error due to uncalled bases).
• Column 4 provides the log-likelihood where the value reflects the log of (probability divided by threshold); column 5. the relevant GenBank release; and column 6, a GenBank description of the protein, or an edited version thereof.
• Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook et al.. supra).
• the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
• the results of northern analysis are reported as a list of libraries in which the transcript encoding GRFLP'occurs. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library.
• the nucleic acid sequence of the Incyte Clone 69291 1 was used to design ohgonucleotide primers for extending a partial nucleotide sequence to full length.
• One primer was synthesized to initiate extension in the antisense direction, and the other was synthesized to extend sequence in the sense direction.
• Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest.
• the initial primers were designed from the cDNA using OLIGO 4.06 (National
• Biosciences or another appropriate program, to be about 22 to about 30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures of about 68°to about 72° C. Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
• Selected human cDNA libraries (Gibco/BRL) were used to extend the sequence If more than one extension is necessary or desired, additional sets of primers are designed to further extend the known region.
• High fidelity amplification was obtained by following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. Beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit. PCR was performed using the Peltier Thermal Cycler (PTC200; M.J. Research. Watertown. MA) and the following parameters:
• Step 1 94° C for 1 min (initial denaturation)
• Step 2 65 ° C for 1 min
• Step 3 68° C for 6 min
• Step 4 94° C for 15 sec
• Step 5 65 ° C for 1 min
• Step 7 Repeat step 4-6 for 15 additional cycles
• Step 8 94° C for 15 sec
• Step 9 65 ° C for 1 min
• Step 1 Repeat step 8-10 for 12 cycles
• Step 12 72° C for 8 min Step 13 - 4° C (and holding)
• reaction mixture A 5-10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQuickTM (QIAGEN Inc., Chatsworth, CA), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
• QIAQuickTM QIAGEN Inc., Chatsworth, CA
• Step 2 94° C for 20 sec
• Step 3 55 ° C for 30 sec
• Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72° C for 180 sec
• nucleotide sequence of SEQ ID NO:2 is used to obtain 5' regulatory sequences using the procedure above, ohgonucleotides designed for 5' extension, and an appropriate genomic library.
• Hybridization probes derived from SEQ ID NO:2 are employed to screen cDNAs, genomic DNAs. or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments.
• Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of each oligomer and 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN ® . Boston, MA).
• the labeled ohgonucleotides are substantially purified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco R Pst I, Xba 1 , or Pvu II; DuPont NEN ® ).
• the DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% > sodium dodecyl sulfate. After XOMAT ARTM film (Kodak. Rochester. NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics. Sunnyvale. CA) for several hours, hybridization patterns are compared visually.
• ohgonucleotides for a microarray.
• the nucleotide sequence described herein is examined using a computer algorithm which starts at the 3' end of the nucleotide sequence.
• the algorithm identifies ohgomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that would interfere with hybridization.
• the algorithm identifies 20 sequence-specific ohgonucleotides of 20 nucleotides in length (20-mers).
• a matched set of ohgonucleotides is created in which one nucleotide in the center of each sequence is altered. This process is repeated for each gene in the microarray. and double sets of twenty 20 mers are synthesized and arranged on the surface of the silicon chip using a light-directed chemical process (Chee, M. et al., PCT/W095/1 1995, inco ⁇ orated herein by reference).
• a chemical coupling procedure and an ink jet device are used to synthesize ohgomers on the surface of a substrate (Baldeschweiler, J.D. et al., PCT/W095/251 16, inco ⁇ orated herein by reference).
• a "gridded" array analogous to a dot (or slot) blot is used to arrange and link cDNA fragments or ohgonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
• An array may be produced by hand or using available materials and machines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.
• the microarray is washed to remove nonhybridized probes, and a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each ohgonucleotide sequence on the micro-array.
• Sequence complementary to the GRFLP-encoding sequence, or any part thereof, is used to decrease or inhibit expression of naturally occurring GRFLP. Although use of ohgonucleotides comprising from about 15 to about 30 base-pairs is described, essentially the same procedure is used with smaller or larger sequence fragments. Appropriate ohgonucleotides are designed using Oligo 4.06 software and the coding sequence of GRFLP. SEQ ID NO: l . To inhibit transcription, a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary ohgonucleotide is designed to prevent ribosomal binding to the GRFLP-encoding transcript.
• GRFLP GRFLP-induced GRFLP
• the cloning vector is also used to express GRFLP in E. coli. Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase. followed by sequence containing the amino-terminal Met. and the subsequent seven residues of ⁇ -galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
• Induction of an isolated, transformed bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first eight residues of ⁇ -galactosidase, about 5 to 15 residues of linker, and the full length protein.
• the signal residues direct the secretion of GRFLP into the bacterial growth media which can be used directly in the following assay for activity.
• Cell proliferation may be assayed by evaluation of changes in cell number and 3 H leucine uptake.
• Confluent NIH 3T3 cells are treated with different concentrations of GRFLP added directly to the culture medium. After incubation for 72 hours, the cultures are washed twice with 0.02%) EDTA in Dulbecco's phosphate-buffered saline, detatched with 0.25% trypsin in Tris- buffered saline and counted to determine the cell number in each sample.
• the cells may be washed three times with Dulbecco's phosphate-buffered saline, placed in 96-well microtiter plates (2 * 10 4 cells/well) in RPMI 1640 medium containing 10% FCS. 0.5 ⁇ Ci/ml 3 H leucine, and different concentrations of GRFLP. After incubation for 24 hours, the medium is aspirated and the cells are incubated for 30 minutes in cold 5% trichloroacetic acid. The wells are then washed once with trichloroacetic acid and three times with water. The 3 H leucine inco ⁇ orated into trichloroacetic acid-precipitable material is measured by liquid scintillation counting after solubilization in 0.1 M NaOH and compared to controls.
• GRFLP that is substantially purified using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
• the amino acid sequence deduced from SEQ ID NO:2 is analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high immunogenicity and a corresponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Selection of appropriate epitopes. such as those near the C-terminus or in hydrophilic regions, is described by Ausubel et al. (supra), and others.
• the oligopeptides are 15 residues in length, synthesized using an Applied Protocol
• KLH keyhole limpet hemocyanin
• MBS N-maleimidobenzoyl-N- hydroxysuccinimide ester
• Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
• the resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio iodinated, goat anti-rabbit IgG.
• Naturally occurring or recombinant GRFLP is substantially purified by immunoaffinity chromatography using antibodies specific for GRFLP.
• An immunoaffinity column is constructed by covalently coupling GRFLP antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
• Media containing GRFLP is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of GRFLP (e.g., high ionic strength buffers in the presence of detergent).
• the column is eluted under conditions that disrupt antibody/GRFLP binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope. such as urea or thiocyanate ion), and GRFLP is collected.
• GRFLP or biologically active fragments thereof are labeled with I25 I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).
• Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled GRFLP, washed and any wells with labeled GRFLP complex are assayed. Data obtained using different concentrations of GRFLP are used to calculate values for the number, affinity, and association of GRFLP with the candidate molecules.
• TGCCTCCTCT CAAAGGTGCG TACCCAGCTG TGCCCGACAC CATGTACCTG CCCCTGGCCA 360
• GCCCAAGGAC CCCAGTTTTC TGGCCTTGTC TCTTCCCTGC CCCCTGGTGT CCCCTGCCCA 840 GAATGGAGCA CGGCCTGGGG ACCCTGCTCG ACCACCTGTG GGCTGGGCAT GGCCACCCGG 900
• CCCTGCCCAC CCTCCAGGGG TCGCAGTCCA CAAAACAGTG CCTTCTAGAG CCGGGCTGGG 1020
• Lys Cys Pro Asp Gly Glu lie Met Lys Lys Asn Met Met Phe lie Lys 305 310 315 320

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