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WO1996039504A2 - Procede permettant la distinction entre cancers metastatiques et cancers non metastatiques, et polynucleotides et polypeptides utilises dans ce procede - Google Patents

Procede permettant la distinction entre cancers metastatiques et cancers non metastatiques, et polynucleotides et polypeptides utilises dans ce procede Download PDF

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Publication number
WO1996039504A2
WO1996039504A2 PCT/EP1996/002461 EP9602461W WO9639504A2 WO 1996039504 A2 WO1996039504 A2 WO 1996039504A2 EP 9602461 W EP9602461 W EP 9602461W WO 9639504 A2 WO9639504 A2 WO 9639504A2
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Prior art keywords
expression
cells
gene
metastatic
sequence
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WO1996039504A3 (fr
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Emilay Shtivelman
Adam Sampson-Johannes
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Novartis Pharma GmbH Austria
Novartis AG
Sandoz AG
Systemix Inc
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Sandoz Erfindungen Verwaltungs GmbH
Novartis Erfindungen Verwaltungs GmbH
Novartis AG
Sandoz AG
Systemix Inc
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Priority to AU63008/96A priority Critical patent/AU6300896A/en
Publication of WO1996039504A2 publication Critical patent/WO1996039504A2/fr
Publication of WO1996039504A3 publication Critical patent/WO1996039504A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a novel gene (CC3) that is differentially expressed in metastatic and non-metastatic cells.
  • the invention also relates to methods for diagnosis, treatment and prevention of metastatic neuroendocrine tumors, particularly small cell lung carcinoma.
  • Metastasis refers to the ability of cancer cells to detach from the initial tumor mass, seed themselves in separate host target organs, and establish additional tumor masses. Metastasis is a multi-step process which requires highly specialized interactions of the cancer cells with the target organs. Metastasis is the primary cause of mortality from solid tumors. The ability to determine the metastatic potential of a particular tumor allows tailoring of aggressiveness of intervention and the ability to deliver a more accurate prognosis.
  • tumor suppressor genes are correlated with tumor suppression as well as formation.
  • oncogenes whose activation can lead to tumor formation
  • inactlvation of tumor suppressor genes or an absence of functional tumor suppressor gene products contributes to the development of a large number of human tumors.
  • tumor suppressor genes include the retinoblastoma (RB) gene (Huang et al. (1988) Science 24:1563-1566; and Goodrich et al. (1993) Biochim. Biophys.
  • metastases suppressor genes have been identified.
  • a region from human chromosome 11 has been shown to suppress metastasis in rat prostatic carcinoma cells. Barrett et al. (1992) Cancer Res. 52:3486.
  • the KAI1 gene is expressed in non-metastatic prostate cancer and all normal cells but not in metastatic prostate cancer.
  • KAT1 suppresses metastasis when introduced into and expressed by rat prostate cancer cells.
  • the NME1 gene is thought to be a metastasis suppressor gene in ovarian carcinoma. Buller et al. (1995) Gyn.
  • NM23 HI and NM23 H2 Two human genes (NM23 HI and NM23 H2) have been identified based on reduced expression in murine melanoma cell lines of high metastatic potential. Steeg et al. (1988) J. Natl. Cancer Inst. 80:204-208; Shahl et al. (1991) Cancer Res. 48:6550-6554; and Royds et al. (1994) J. Pathol. 172:261-266.
  • the NM23 gene is described in U.S. Pat. No. 5,049,662. Loss of NM23 does not confer growth advantage, but instead confers metastatic potential.
  • SCLC Small cell lung carcinoma
  • the two SCLCs also demonstrate biochemical differences, such as different expression of a number of biochemical markers and genetic differences, such as amplification of different members of the myc family of nuclear oncogenes.
  • Biochemical differences such as different expression of a number of biochemical markers and genetic differences, such as amplification of different members of the myc family of nuclear oncogenes.
  • v-SCLC cell lines produce more tumors in SCID-hu mice with shorter latency periods than c-SCLC cell lines, indicating a significantly higher rate of metastasis in v-SCLC than c-SCLC.
  • PCT/US94/00714 and Shtivelman et al. (1995) Proc. Natl. Acad. Sci. USA ⁇ 2_:4661-4665.
  • the invention provides a gene (CC3) which is expressed in normal cells and non-metastatic cells but not in metastatic neuroendocrine cancer cells.
  • the invention provides recombinant vectors and expression systems containing the CC3 gene.
  • Polypeptides encoded by the polynucleotides are also encompassed by the invention.
  • the invention encompasses methods of distinguishing between metastatic and non-metastatic tumor cells by measuring expression of the CC3 gene. Expression of the CC3 gene correlates with a tumor being non-metastatic and lack of or decreased expression of the CC3 gene correlates with a tumor being metastatic.
  • the invention encompasses methods of diagnosing the type of SCLC by monitoring the expression of the CC3 gene in a sample of SCLC cells.
  • Expression of the CC3 gene indicates the SCLC is c-SCLC and lack of or decreased expression of the CC3 gene indicates the SCLC is v-SCLC.
  • the invention further comprises methods of use of the CC3 gene including methods of screening agents for those effective in inhibiting or decreasing metastasis.
  • the methods include i ⁇ vitro expression systems which monitor expression of the CC3 gene or a recombinant or recombinant fusion protein in the presence or absence of various agents.
  • Potential therapeutic agents are those which increase CC3 expression either directly or indirectly relative to controls.
  • the invention also encompasses screening methods using animal model systems wherein tumor cells are placed in a suitable animal model system. The animals are then administered various potential therapeutic agents and parameters such as CC3 expression, tumor growth and metastases are measured relative to a control, untreated animal.
  • the animal model system may be used alone or in conjunction with the in vitro system.
  • the invention further encompasses methods of inhibiting metastasis comprising administering to a v-SCLC or neuroblastoma patient agents which increase expression or decrease mRNA turnover of CC3.
  • Figure 1 is a schematic diagram of the cloning of CC3.
  • the position of the original cDNA clone CC3 relative to the cDNAs cloned subsequently is shown, along with primers used to derive the cDNA extension clones pC5, 13, 16 and 15.
  • the point of divergence in the sequence of pCC(2Kbp) is indicated upstream of the Pst I site.
  • the location of the open reading frame (ORF) is shown at the bottom.
  • Figure 2 depicts the CC3 cDNA (both coding and non-coding strands) and the protein encoded thereby.
  • CC3 a gene designated CC3 is differentially expressed between metastatic and non- metastatic cells. This differential expression allows differential diagnosis of the two variants of SCLC.
  • the expression of the CC3 gene in non-metastatic "classic" SCLC (c-SCLC) but not in metastatic "variant” SCLC (v-SCLC) indicates that it may be a metastasis suppression gene. Described herein is the identification and expression patterns of CC3 and methods of use thereof.
  • CC3 refers to nucleotides or protein as appropriate from context
  • CC3 gene or “CC3 polynucleotides” refers to the nucleic acid molecules (nucleotides) and fragments thereof described herein
  • CC3 protein refers to the protein or polypeptides encoded thereby.
  • naturally occurring refers to the endogenous, genomic CC3 gene and the CC3 protein expressed thereby.
  • non-naturally occurring refers to all other CC3 polynucleotides and polypeptides.
  • the invention encompasses CC3 polynucleotide sequences including full length, coding, non-coding or portions thereof.
  • polynucleotides, both coding and noncoding strands include, but are not limited to, the CC3 cDNA, isolated genome-derived DNA, mRNA and synthetic or semi-synthetic polynucleotides such as DNA, and RNA.
  • Polynucleotide fragments are encompassed by the invention. Suitable fragments are those which hybridize specifically to CC3 DNA or RNA such that they are effective as primers or probes, e.g., fragments having at least 10, preferably at least 14, nucleotides corresponding or complementary to a characteristic sequence of CC3 DNA or RNA.
  • the primers are particularly useful in the polymerase chain reaction (PCR) .
  • the polynucleotides include the human sequences as well as sequences from other sources which are the same as, or homologous to, the human sequences disclosed herein.
  • the polynucleotides may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases.
  • the polynucleotides may be complementary to the mRNA for at least a fragment of the CC3 gene and include other polynucleotides which can bind to either the DNA or mRNA encoding CC3.
  • These complementary polynucleotides include, but are not limited to, polynucleotides capable of forming triple helices and antisense polynucleotides.
  • the polynucleotide sequences of both the coding and noncoding strands of the CC3 cDNA are shown in Figure 2. As described in the examples herein, CC3 mRNA has been detected in a variety of normal human organs and tissues by Northern blot analysis.
  • CC3 is expressed in many tumor cells, including c-SCLC, but is not expressed in v-SCLC or neuroblastoma, two highly metastatic tumors of neuroendocrine origin.
  • polynucleotide sequences encoding CC3 variants including, e.g., other alternatively processed sequences or sequences encoding CC3 fusion and deletion proteins.
  • Alternatively processed polynucleotide sequence variants are defined as polynucleotide sequences corresponding to mRNAs that differ in sequence from one another but are derived from the same genomic region, for example, mRNAs that result from: 1) the use of alternative promoters; 2) the use of alternative polyadenylation sites; or 3) the use of alternative splice sites.
  • Recombinant polynucleotides comprising sequences otherwise not naturally occurring are also provided by this invention, as are alterations of wild type protein sequences, including, but not limited to, those due to deletion, insertion, substitution of one or more nucleotides or by fusion to other polynucleotide sequences.
  • the invention includes modifications to CC3 nucleotide sequences such as deletions, substitutions and additions particularly in the non-coding regions of the DNA. Such changes are useful to facilitate cloning and modifying gene expression.
  • the invention encompasses functionally equivalent variants and derivatives of the CC3 gene which may enhance, decrease or not significantly affect the properties of the proteins encoded thereby. For instance, changes in the DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, or which result in one or a few amino acid deletions or additions, or substitution of amino acid residues by amino acid analogs are those which will not significantly affect its properties. Nucleotide substitutions that do not alter the amino acid residues encoded are useful for optimizing gene expression in different systems. Suitable substitutions are known to those of skill in the art and are made, for instance, to reflect preferred codon usage in the particular expression systems.
  • Amino acid residues which can be conservatively substituted for one another include but are not limited to: glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine; lysine/arginine; and phenylalanine/tyrosine. Any conservative amino acid substitution which does not significantly affect the properties of CC3 is encompassed by the present invention.
  • nucleotides homologous to sequences encoding CC3 are provided.
  • a polynucleotide or fragment thereof is “substantially homologous” (or “substantially similar”) to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another polynucleotide (or its complementary strand) , there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotide bases. Also included are those polynucleotides which encode CC3 or functional portions thereof, but which have nucleotide substitutions that accommodate the degeneracy of the genetic code.
  • substantial homology or (similarity) exists when a polynucleotide or fragment thereof will hybridize to another polynucleotide (or a complementary strand thereof) under selective hybridization conditions.
  • Selectivity of hybridization exists under hybridization conditions which allow one to distinguish the target polynucleotide of interest from other polynucleotides.
  • selective hybridization will occur when there is at least about 55% similarity over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See Kanehisa (1984) Nuc. Acids Res. ⁇ 2:203-213.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 17 to 20 nucleotides, and preferably at least about 36 or more nucleotides.
  • hybridization of polynucleotides is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing polynucleotides, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C.
  • Stringent salt conditions will ordinarily be less than 1 M, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.
  • the invention further encompasses polynucleotide sequences which encode the amino acid sequence or portions thereof depicted in Figure 2.
  • the polynucleotide sequence may be similar to that depicted in Figure 2 with minor changes designed to optimize codon usage or stability or may vary significantly. It is within the skill of one in the art, given the amino acid sequence in Figure 2, to design such polynucleotides.
  • An "isolated” or “substantially pure” polynucleotide is substantially separated from other polynucleotides which naturally accompany a native polynucleotide sequence. The term particularly refers to genomic DNA isolated from other, native DNA sequences.
  • the term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof.
  • the anti-sense strand of such a polynucleotide is also said to encode the sequence.
  • Methods for constructing appropriate cDNA and genomic libraries, for screening libraries for sequences of interest, for preparing suitable probes or primers (e.g., PCR primers) , for polynucleotide purification, amplification and subcloning, and for host cell transformation and other techniques of recombinant DNA technology appropriate to the practice of the present invention are provided, inter alia, in Sambrook, et al., (1989); Ausubel et al. (1987 and periodic updates); and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press: San Diego (1990) .
  • Reagents useful in applying such techniques, such as restriction enzymes, expression vectors, labels, etc. are known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U. S. Biochemicals, New England Nuclear, and a number of other commercial sources.
  • the invention further embodies a variety of DNA vectors having cloned therein the CC3 polynucleotide sequences.
  • Suitable vectors include any known in the art including, but not limited to, those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors are known in the art and need not be described in detail herein.
  • the vectors may also provide inducible promoters for expression of the CC3 polynucleotide sequences.
  • Inducible promoters are those which do not allow substantial constitutive expression of the gene but rather, permit expression only under certain circumstances. Such promoters may be induced by a variety of stimuli including, but not limited to, exposure of a cell containing the vector to a ligand, metal ion, other chemical or change in temperature.
  • the promoters may also be cell-specific, that is, inducible only in a particular cell type and often only during a specific period of time.
  • the promoter may further be cell cycle specific, that is, induced or inducible only during a particular stage in the cell cycle.
  • the promoter may be both cell type specific and cell cycle specific. Any inducible or noninducible promoter known in the art is suitable for use in the present invention.
  • a polynucleotide sequence is operably linked when it is placed into a functional relationship with another polynucleotide sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • certain genetic elements such as enhancers, may be operably linked even at a distance, i.e., even if not contiguous.
  • Polynucleotide probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to identify and isolate other polynucleotide or protein sequences. Probes comprising synthetic oligonucleotides or other polynucleotides may be derived from naturally occurring or recombinant single or double stranded nucleic acids or may be chemically synthesized. Polynucleotide probes may be labelled by any of the methods known in the art, e.g., random hexamer labeling, nick translation, or the Klenow fill-in reaction.
  • CC3 polynucleotides or polypeptides encoded thereby may be produced by replication in a suitable host cell.
  • Natural or synthetic DNA fragments coding for a protein(s) or a fragment thereof are incorporated into recombinant polynucleotide constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell.
  • the construct will be suitable for replication in a unicellular host, such as yeast or bacteria, but a multicellular eukaryotic host may also be appropriate, with or without integration within the genome of the host cells.
  • prokaryotic hosts include strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
  • Mammalian or other eukaryotic host cells include yeast, filamentous fungi, plant, insect, amphibian or avian species. Such factors as ease of manipulation, ability to appropriately glycosylate expressed protein(s), degree and control of protein expression, ease of purification of expressed protein(s) away from cellular contaminants or other factors may determine the choice of the host cell.
  • the polynucleotides may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862 or the triester method according to Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185, and may be performed on commercial automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • the invention further includes a variety of expression vectors containing the CC3 gene.
  • Expression vectors are defined as polynucleotides which, when transfected into an appropriate host cell, can express the CC3 protein or fragment thereof.
  • the polynucleotides possess a nucleotide sequence that is substantially similar to a natural protein- encoding polynucleotide or a fragment thereof.
  • Suitable expression systems include, but are not limited to, those for use in bacterial, mammalian, yeast and insect cell line expression systems. Specific expression vectors and the use thereof are known in the art and are not described in detail herein. Many useful vectors for expression may be obtained from such vendors as Stratagene, New England Biolabs, Promega Biotech, and others.
  • Expression vectors prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment.
  • Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences.
  • ARS origin of replication or autonomously replicating sequence
  • DNA encoding signal peptides may also be included, where appropriate, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
  • the appropriate promoter and other necessary vector sequences will be selected so as to be functional in the expression system. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., 1989; Ausubel et al., 1987; and Metzger et al. (1988) Nature 334:31-36.
  • the expression vector may also contain an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made.
  • DHFR e.g., DHFR
  • Enhancers and Eukaryotic Gene Expression Cold Spring Harbor Press, N.Y. (1983) . While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.
  • Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector, although such a selectable gene may be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which the selectable gene has been introduced will survive and/or grow under selective conditions.
  • Typical selection genes encode protein(s) that
  • the vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome) .
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances
  • microprojectile bombardment lipid-diluent phosphate
  • lipofection lipofection
  • infection where the vector is an infectious agent, such as a retroviral genome
  • polynucleotides and polypeptides of the present invention may be prepared by transforming suitable prokaryotic or eukaryotic host cells with protein(s)-encoding polynucleotides of the present invention in compatible vectors or other expression vehicles and culturing such transformed host cells under conditions suitable to attain expression of the protein(s)-encoding gene.
  • the protein(s) may then be recovered from the host cell and purified using standard techniques.
  • the endogenous gene may also be used to produce protein.
  • the native expression may be suitable for small amounts of protein and expression levels can be increased by upstream activation of expression. Methods of upstream activation are known and include insertion of activation sequences upstream of the endogenous CC3 gene by homologous recombination.
  • the invention encompasses cells transfected with CC3 polynucleotide sequences or expression systems containing the CC3 gene.
  • Cells transfected with DNA or vectors encoding the CC3 gene include expression systems for producing vector DNA, CC3 mRNA and/or the CC3 protein.
  • the cells may be the product of ex vivo genetic modification in which the exogenous DNA is introduced into cells removed from animals including man.
  • the cells may be immediately reintroduced into animals or expanded and cultured outside the host animal.
  • Suitable cells for modification include individual cells or cells contained within whole tissues.
  • ex vivo genetic modification can include the transfection of cells derived from an animal other than the animal or human subject into which the cells are ultimately introduced. Such cells include, but are not limited to, allografts, xenografts, and fetal tissue.
  • the invention encompasses in vivo genetic modification by, e.g., pulmonary administration of suitable vectors. Such treatment schemes are.described for instance by Roth et al. (1994) Eur. J. Cancer 30A:2032-2037. These are particularly suitable for use in the animal model described herein.
  • Transgenic animals containing the recombinant DNA vectors containing CC3 polynucleotide sequences are also encompassed by the invention. Methods of making transgenic animals are known in the art and need not be described in detail herein. For a review of methods used to make transgenic animals, see, e.g., PCT publication no. WO 93/04169. Preferably, such animals express recombinant CC3 under control of a cell-specific or a cell cycle-specific promoter.
  • the invention also includes compositions containing the substantially purified CC3 protein or fragments thereof having the amino acid residue sequences depicted in Figure 2.
  • the proteins can be isolated from recombinant expression systems, native sources or manufactured synthetically.
  • the proteins are at least partially purified from other cellular constituents.
  • the proteins are at least 50% pure. More preferably, the proteins are 50-75% pure. More highly purified proteins may also be obtained and are encompassed by the present invention.
  • the invention encompasses functionally equivalent variants of CC3 protein and variants which retain the same overall amino acid sequence but which have enhanced or decreased activity.
  • variants having at least 80%, preferably at least 90% amino acid sequence homology with the protein depicted in Figure 2, and preferably retaining most, e.g., at least 50%, of the CC3 properties (e.g., anti-metastatic activity) of the protein depicted in Figure 2, are within the scope of the invention.
  • Variants having additions or deletions, especially terminal additions or deletions, of amino acid sequences which do not compromise the function of the CC3 protein are considered to be funtionally equivalent to CC3.
  • conservative substitutions of amino acid residues and substitution of amino acid residues by amino acid analogs are within the scope of the invention. Any conservative amino acid substitution which does not significantly affect the properties of the CC3 protein is encompassed by the present invention.
  • CC3 protein As used herein, the term "CC3 protein”, “CC3 polypeptide” or “CC3 peptide” thus also includes any such functionally active polypeptides or portions of the CC3 protein.
  • the invention also includes those portions of the protein sequence provided having anti-metastatic activity.
  • Suitable methods of protein purification include, but are not limited to, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, HPLC and FPLC. Any purification scheme that does not result in substantial degradation of the protein or the function thereof is suitable for use in the present invention.
  • polyclonal and/or monoclonal antibodies capable of specifically binding to the CC3 protein and any and all use variants or fragments thereof are provided.
  • the term antibody is used to refer both to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Monoclonal or polyclonal antibodies reacting with the CC3 protein(s) may be made by methods known in the art. See, e.g., Harlow and Lane, (1988) Antibodies: A Laboratory Manual, CSH Laboratories; Goding (1986) Monoclonal Anti ⁇ bodies: Principles and Practice, 2d ed. Academic Press, New York; and Ausubel et al. (1987) .
  • recombinant immunoglobulins may be produced by method known in the art, including, but not limited to, the methods described in U.S. Patent No. 4,816,567.
  • Suitable antibodies are generated by using the CC3 proteins or antigenic portions thereof as an antigen. Methods of detecting proteins using antibodies and of generating antibodies using proteins or synthetic polypeptides are known in the art and are not described in detail herein.
  • the antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal.
  • Suitable labels include but are not limited to, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like.
  • United States Patents describing the use of such labels include but are not limited to, Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • diagnostic methods are provided to distinguish metastatic from non-metastatic cancers.
  • the methods include detecting the expression of CC3 either at the protein or mRNA level and comparing to the level of CC3 expression in non-metastatic cells.
  • Suitable non-metastatic cells for comparison include, but are not limited to, known non-metastatic cancers, normal tissue (preferably adjacent to the tumor) and/or cell lines of non-metastatic tumors.
  • Diagnosis of c-SCLC rather than v-SCLC is made by monitoring expression of the CC3 gene either at the protein level or nucleic acid level wherein decreased or absent CC3 expression is indicative of v-SCLC. Detection methods are also useful for monitoring the success of cancer therapies. Any antibody that specifically recognizes CC3 proteins is suitable for diagnostic use. Methods of detecting proteins using antibodies and of generating antibodies using proteins or synthetic polypeptides are known in the art and are not described in detail herein.
  • CC3 gene expression can also be monitored by measuring the level of CC3 mRNA. Any method for detecting specific mRNA species is suitable for use in this method. This is easily accomplished using the polymerase chain reaction (PCR) using primers corresponding to regions of the CC3 gene. Alternatively, Northern blots can be utilized to detect CC3 mRNA by using probes specific to CC3. Methods of utilizing PCR and Northern blots are known in the art and are not described in detail herein.
  • kits for use in the above methods to measure CC3 gene expression comprising monoclonal antibodies to CC3 or CC3-specific primers or probes as described above.
  • Methods of treatment of metastatic neuroendocrine tumors include increasing levels of CC3 mRNA or protein.
  • Suitable methods of increasing cellular expression of CC3 include, but are not limited to, increasing endogenous expression with therapeutic agents and transfecting the tumor cells with vectors encoding CC3 nucleotides or treatment with functional CC3 protein.
  • Increasing endogenous expression of CC3 can be accomplished by exposing the cells to potential therapeutic agents that directly or indirectly increase levels of CC3 either by increasing expression of the CC3 gene, by decreasing degradation of CC3 mRNA or by increasing translation efficiency.
  • Suitable potential therapeutic agents include, but are not limited to, chemotherapeutic drugs, cytokines, small molecules, hormones, combinations of interleukins, lectins and other stimulating agents, e.g., bispecific antibodies and other agents which modify cellular functions or protein expression.
  • Cells are exposed to such potential therapeutic agents at physiologically effective concentrations, and the expression of CC3 is measured relative to a control not exposed to the agents. Those agents which increase expression of CC3 relative to the control are selected for further study. Particularly suitable cell lines are those described in the examples presented herein.
  • the invention further encompasses screening for potential therapeutically effective agents by exposing cells expressing either endogenous or recombinant CC3 to agents which may directly or indirectly increase levels of CC3 either by changing expression of the CC3 gene, by altering the half-life of CC3 mRNA, or by altering CC3 protein degradation.
  • Cells are grown under conditions known to elicit expression of CC3, exposed to such agents at physiologically effective concentrations, and the expression of the CC3 gene is measured relative to a control not exposed to the agents. Those agents which increase the expression of CC3 relative to a control are selected for further study.
  • the levels of endogenous mRNA or protein expression may be measured or the levels of recombinant fusion proteins under control of CC3-specific promoter sequences may be measured.
  • the fusion proteins encode reporter genes which are easily measured. Reporter genes are known in the art and include, but are not limited to, chloramphenicol acetyl transferase (CAT) and ⁇ -galactosidase. Expression of the CC3 gene can be monitored as described above either by protein or mRNA levels. Expression of the reporter genes can be monitored by enzymatic assays, or antibody-based assays, like ELISAs and RIAs, also known in the art.
  • Potential pharmaceutical agents can be any therapeutic agent or chemical known to the art, or any uncharacterized compounds derived from natural sources such as fungal broths and plant extracts. Preferably, suitable pharmaceutical agents are those lacking substantial cytotoxicity and carcinogenicity.
  • Methods of screening potential therapeutic agents also include animal model systems. Metastasis of v-SCLC and c- SCLC cell lines in SCID-hu mice is described in commonly owned Patent application No. PCT/US94/00714 and is described by Shtivelman et al. (1995) Proc. Natl. Acad. Sci. USA _92:4661-4665. Briefly, v-SCLC lines produce tumors in the human fetal lung grafts of SCID-hu mice much more efficiently than c-SCLC lines, i.e., in a higher proportion of mice and grafts and with significantly shorter latency periods.
  • tumor cells are placed in a suitable animal, such as SCID-hu mice. The animals are then administered various potential therapeutic agents and parameters such as CC3 expression, tumor growth and metastases are measured relative to a control, untreated animal.
  • the animal model system may be used alone or in conjunction with the in vitro systems.
  • c-SCLC or v-SCLC or neuroblastoma tumor cells are obtained from the patient and used fresh or frozen within about 12 hours of removal from the patient and stored at below about -70°C or cultured.
  • Tumor samples are administered to the animal as a solid mass or as dissociated cells.
  • the animal is then grown and administered potential therapeutic agents suspended or dissolved in a physiologically acceptable diluent in therapeutically effective amounts.
  • Control animals receive diluent alone. After a suitable time, donor cells are analyzed for parameters of tumor progression. Diminished tumor progression in the experimental animals when compared to the controls indicates a potentially therapeutic agent.
  • the invention also encompasses therapeutic methods and compositions for use therein involving treatment of patients with therapeutic agents identified by the in vitro and/or in vivo screens to increase expression of CC3.
  • Effective concentrations and dosage regimens may be empirically derived. Such derivations are within the skill of those in the art and depend on, for instance, age, weight and gender of the patient and type and severity of the disease.
  • a therapeutically effective amount of therapeutic agent is suspended in a physiologically accepted buffer including, but not limited to, saline and phosphate buffered saline (PBS) and administered to the patient.
  • a physiologically accepted buffer including, but not limited to, saline and phosphate buffered saline (PBS)
  • PBS phosphate buffered saline
  • administration is intravenous.
  • Other methods of administration include but are not limited to, subcutaneous, intraperitoneal and gastrointestinal.
  • Suitable buffers and methods of administration are known in the art.
  • the effective concentration of a therapeutic agent will need to be determined empirically and will depend on the type and severity of the disease, disease progression and health of the patient. Such determinations are within the skill of one in the art.
  • SCLC cell lines N417, NCI-H82, NCI-H446, NCI-H146, NCI- H345 and NCI-H69 are obtained from the American Type Culture Collection. NCI-H82, NCI-H446, NCI-H146, NCI-H345 and NCI- H69 are available under accession numbers HTB 120, HTB 171, HTB 173, HTB 180 and HTB 119, respectively. The cells are cultured in RPMI medium supplemented with 10% fetal calf serum (FCS) and glutamine (2 mM) .
  • FCS fetal calf serum
  • glutamine (2 mM
  • RNA Total RNA is isolated from cultured cells according to the method described by Auffrey (1980) Eur. J. Biochem. 107:303-314. Polyadenylated RNA is purified on oligo(dT) cellulose according to the method described by Aviv et al. (1972) Proc. Natl. Acad. Sci. USA _69:1408-1412.
  • RNAmapTM Kit a kit according to the manufacturer's instructions (GeneHunter Corporation, Brookline, MA).
  • 0.1 ⁇ g of polyadenylated RNA from SCLC cell lines are reverse transcribed in the presence of 1 ⁇ M T12MA (twelve Ts; M is any base, and A) (or C, G, T) primers and 20 ⁇ M dNTPs in a total reaction volume of 20 ⁇ l.
  • reaction 2 ⁇ l of reaction is added into 18 ⁇ l of PCR reaction containing 1 ⁇ M T12MA (or C, G, T) , 0.2 ⁇ M of a random decamer primer, 2.5 ⁇ M of dNTPs (final concentration) and 10 ⁇ Ci of 35 S-dATP.
  • PCR parameters are 94°C for 30 sec, 42°C for 2 min., and 72°C for 30 sec. with 40 cycles followed by 5 min. elongation at 72°C. Reaction products are resolved on 5% polyacrylamide DNA sequencing gels.
  • Double-stranded cDNA is synthesized from mRNA of cell line HI46 and cloned into phage lambda ZAP II according to the manufacturer's instructions (Stratagene, La Jolla, CA) .
  • the library is plated on Escherichia coli strain XL-1 blue (Stratagene) and screened according to the method described by Sambrook et al. (1989).
  • differential display of RNA is performed with mRNA from two v-SCLC cell lines N417 and NCI-H82 and two c-SCLC cell lines NCI-H146 and NCI-H345.
  • a differentially displayed band of 350 nucleotides is consistently noticed when primers T12MC (twelve Ts; M is any base, and C) and AP-3 (AGGTGACCGT) were used.
  • This cDNA band designated CC3, is present in the c-SCLC cell lines but not in the v-SCLC cell lines.
  • the band is eluted from the gel, PCR-reamplified with primers AP-3 and T12MC and subcloned into the pCRII plasmid vector using the TA cloning kit (InVitrogen Corporation, San Diego, CA) .
  • the insert of this clone, pCC3 is hybridized to Northern blots containing 2 ⁇ g of polyadenylated RNA from the following sources: (i) v-SCLC cell lines NCI-H446, N417, NCI-H82 and c-SCLC NCI-H146, NCI-H345 and NCI-H69; (ii) normal human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas; and (iii) cell lines neuroblastoma NGP, colon carcinoma COLO320DM, neuroblastomas NLF, NMB and SKNSH, neuroepithelioma SKNMC, melanomas HT144 and C32r, Burkitt lymphoma Daudi.
  • This probe identifies a 1.6 Kb RNA in c-SCLC cell lines NCI-H146, NCI-H345 and NCI-H69, but not in v-SCLC N417, NCI-H446, NCI-H82.
  • a hybridizing mRNA species of 1.6 Kb is detected in all normal tissues analyzed.
  • the CC3 probe is hybridized to the Northern blot containing mRNAs isolated from a variety of human tumor cell lines of different origins, it detects the same size mRNA in all cell lines examined with the exception of neuroblastoma and v-SCLC. Cloninq of the full length cDNA for CC3
  • a cDNA library using polyadenylated RNA from cell line NCI-H146 is constructed.
  • pCC(2Kb) is isolated.
  • This cDNA clone is longer then the expected length (1.6 Kb) based on the Northern blot analyses.
  • the location of the original cDNA clone CC3 relative to the pCC(2Kb) is determined by sequence analysis ( Figure 1).
  • Clone pCC(2Kb) contains a poly(A) tail at its 3' end.
  • the RACE (Rapid Amplification of cDNA ends) procedure is performed using the AmpliFinder kit according to the manufacturer's instructions (Clontech, Palo Alto, CA) .
  • the sequence of the original clone pCC3 is used to generate the specific nested antisense oligonucleotide primers: ccpl: 5' GCCATGCGCTTTCCCCAGGTCATGGATGG 3' ccp2: 5' CTCGCTCGCCCATTGTCTCTTGGTCTCACCACATTGTTCAGC 3'
  • primer ccp2 The sequence corresponding to the primer ccp2 was located just upstream of the sequence represented by primer ccpl ( Figure 1) .
  • Primer ccp2 corresponds to the positions 812-783; primer ccp2 corresponds to the positions 757-728 in sequence presented in Figure 2.
  • the first 12 nucleotides in the primer ccp2 are sequences used for subsequent cloning into vector pDirect (see below) .
  • the primer ccpl is used to prime cDNA synthesis of CC3 cDNA from the RNA obtained from cell line NCI-H146.
  • a linker nucleotide is ligated to the 3' end of resulting cDNA which is then amplified in PCR reactions with two oligonucleotides: one complementary to the linker sequence and the other primer, ccp2.
  • the resulting double-stranded cDNAs are subcloned into vector pDirect (Clontech) and their authenticity is confirmed by specific hybridization to the CC3 probe.
  • cDNA clones containing 5' end extensions of CC3 are analyzed and further confirmed to represent true 5' extensions of CC3 by RNase protection analysis and DNA sequence analysis. Several of them are represented in Figure 1.
  • the longest two identical clones, p5 and pl3, are thought to contain the authentic 5' end of CC3 mRNA since the combined length of these cDNAs and the corresponding remaining portion of pCC(2Kbp) is 1.6 Kbp, i.e., the size of CC3 mRNA.
  • the point of divergence in the sequences of pCC(2Kb) and the cDNA extension clones is localized 45 basepairs upstream of the first PstI site ( Figure 1) .
  • a total of 1050 nucleotides from the 5' end of cDNAs representing CC3 are sequenced.
  • the nucleotide sequence obtained and amino acid sequence encoded is shown in Figure 2. It contains a 99 bp untranslated sequence at the 5' end followed by an open reading frame of 726 bp (242 aa) and an untranslated 3' sequence of about 800 bp, only the first 225 bp of which are fully sequenced.
  • Comparison of the nucleotide sequence with the Genebank database does not reveal any significant similarities to already known genes.
  • Comparison of the deduced amino acid sequence to the protein database does not reveal any significant similarities with known proteins.

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Abstract

Cette invention concerne des séquences nucléotidiques codant un nouvel ADN complémentaire classique spécifique du carcinome à petites cellules du poumon, la séquence d'acides aminés de la protéine correspondante, des procédés permettant l'expression et la purification de cette protéine, des anticorps dirigés contre cette protéine ainsi que leurs procédés d'utilisation.
PCT/EP1996/002461 1995-06-06 1996-06-06 Procede permettant la distinction entre cancers metastatiques et cancers non metastatiques, et polynucleotides et polypeptides utilises dans ce procede Ceased WO1996039504A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999060116A1 (fr) * 1998-05-18 1999-11-25 Roche Diagnostics Gmbh Acide nucleique a regulation negative de la metastase (drim) codant pour une proteine, et utilisation diagnostique et therapeutique de cet acide nucleique
WO2005007692A1 (fr) * 2003-07-11 2005-01-27 Uab Research Foundation Anticorps qui reconnaissent la proteine brms1 et leurs utilisations

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992000757A1 (fr) * 1990-07-09 1992-01-23 Research Corporation Technologies, Inc. Diagnostic du cancer metastatique a l'aide du gene mts-1
US5382521A (en) * 1992-07-14 1995-01-17 Michigan Cancer Foundation Method of determining metastatic potential of bladder tumor cells
KR950701230A (ko) * 1993-03-04 1995-03-23 죤제이 쉬바르츠 작은 동물 전이모델(small animal metastasis model)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999060116A1 (fr) * 1998-05-18 1999-11-25 Roche Diagnostics Gmbh Acide nucleique a regulation negative de la metastase (drim) codant pour une proteine, et utilisation diagnostique et therapeutique de cet acide nucleique
WO2005007692A1 (fr) * 2003-07-11 2005-01-27 Uab Research Foundation Anticorps qui reconnaissent la proteine brms1 et leurs utilisations

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