WO2001025250A1 - Differentially expressed genes associated with her-2/neu overexpression - Google Patents
Differentially expressed genes associated with her-2/neu overexpression Download PDFInfo
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- WO2001025250A1 WO2001025250A1 PCT/US2000/027649 US0027649W WO0125250A1 WO 2001025250 A1 WO2001025250 A1 WO 2001025250A1 US 0027649 W US0027649 W US 0027649W WO 0125250 A1 WO0125250 A1 WO 0125250A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
Definitions
- This invention relates to nucleic acid and amino acid sequences of new genes that are differentially expressed in cells that overexpress the Her-2/neu oncogene.
- Cancers of the breast and ovary are among the leading causes of death among women.
- the cumulative lifetime risk of a woman developing breast cancer estimated to be 1 in 9. Consequently, understanding the origins of these malignancies as well as models for the identification of new diagnostic and therapeutic modalities is of significant interest to health care professionals.
- cancer cells have been shown to exhibit unique gene expression, and dozens of cancer-specific genetic markers, tumor antigens, have been identified.
- the human HER-2//zr ⁇ (c-erbB-2) proto-oncogene encodes a transmembrane receptor tyrosine kinase with extensive sequence homology to the epidermal growth factor receptor (EGFR) (Bargmann, C. I., Hung, M. C. and Weinberg, R. A. (1986) Cell, 45(5), 649-57).
- EGFR epidermal growth factor receptor
- Amplification and/or overexpression of ⁇ R-2/ ne has been found in one-third of human breast and one-fifth of ovarian cancers ( Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A. and McGuire, W. L.
- XXEK-2/neu overexpression may be an epiphenomenon serving merely as a marker of aggressive breast cancers. Conversely, the alteration may be causal for the aggressive phenotype.
- differential hybridization also known as plus/minus screening (St John, T. P. and Davis, R. W. (1979) Cell, 16(2), 443-52)
- differential hybridization also known as plus/minus screening
- This approach has the advantage of comparing two human breast cancer cell lines which are identical except for HER-2/»r ⁇ overexpression allowing for a direct comparison of cD ⁇ As derived from the two cell populations.
- the present invention provides new Her-2/neu overexpression modulated proteins (including proteins having both new and known amino acid sequences such as novel splice variants of known proteins) hereinafter designated HOMPS.
- a first HOMPS protein is designated HI 7. The expression of HI 7 increases in cells which overexpress Her-2/neu.
- a second HOMPS protein is designated C40. The expression of C40 decreases in cells which overexpress Her-2/neu.
- a third HOMPS protein is designated H41.
- the expression of H41 increases in cells which overexpress Her-2/neu.
- a fourth HOMPS protein is designated H13.
- the expression of H13 increases in cells which overexpress Her-2/neu.
- a fifth HOMPS protein is designated H14.
- the expression of H14 increases in cells which overexpress Her-2/neu.
- the present invention discloses a HOMPS related polynucleotide sequence designated H63. The expression of H63 increases in cells which overexpress Her-2/neu.
- the invention provides polynucleotides corresponding or complementary to all or part of the HOMPS genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding HOMPS proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oUgonucleotides complementary to the HOMPS genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the HOMPS genes, mRNAs, or to HOMPS-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding HOMPS.
- HOMPS proteins and polypeptide fragments thereof are also provided.
- the invention further provides HOMPS proteins and polypeptide fragments thereof.
- the invention further provides antibodies that bind to HOMPS proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker.
- the invention provides a substantially purified HOMPS having the amino acid sequence shown in Figure 2, Figure 4, Figure 6, Figure 9 or Figure 11.
- a typical embodiment of the invention provides an isolated and substantially purified polynucleotide that encodes HOMPS.
- the polynucleotide is the nucleotide sequence shown in Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10.
- the invention also provides a polynucleotide sequence comprising the complement of the nucleotide sequences shown in Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10 or variants thereof.
- the invention provides polynucleotide sequences which hybridize under stringent conditions to the nucleotide sequences shown in Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10.
- the invention further provides nucleic acid sequences encoding fragments or the complement of the polynucleotide sequences, as well as expression vectors and host cells comprising polynucleotides that encode HOMPS.
- the invention further provides methods for detecting the presence and status of HOMPS polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express HOMPS.
- a typical embodiment of this invention provides methods for monitoring HOMPS gene products in a tissue sample having or suspected of having some form of growth disregulation such as cancer.
- FIGURES Figure 1 shows a nucleic acid sequence of HI 7 (SEQ ID NO: 1).
- Figure 2 shows an amino acid sequence of HI 7 (SEQ ID NO: 2).
- Figure 3 shows a nucleic acid sequence of C40 (SEQ ID NO: 3).
- Figure 4 shows an amino acid sequence of C40 (SEQ ID NO: 4).
- Figure 5 shows a nucleic acid sequence of H41 (SEQ ID NO: 5).
- Figure 6 shows an amino acid sequence of H41 (SEQ ID NO: 6).
- Figure 7 shows a nucleic acid sequence of H63 (SEQ ID NO: 7).
- Figure 8 shows an nucleic acid sequence of HI 3 (SEQ ID NO: 8).
- Figure 9 shows an amino acid sequence of H13 (SEQ ID NO: 9).
- Figure 10 shows a nucleic acid sequence of H14 (SEQ ID NO: 10).
- Figure 11 shows an amino acid sequence of H14 (SEQ ID NO: 11).
- the transcription-coupled translation reaction was performed using T3 RNA polymerase, rabbit reticulocyte lysate and [ 35 S]methionine labeling.
- the first lane represents the 61 kDa luciferase protein product which was used as a positive control.
- the C40, HI 3, HI 7, and H37 protein products are seen as distinct bands at 55, 30, 50, and 90 kDa, respectively, whereas the H41 cDNA produced two faint bands at 30 kDa and a lower molecular weight.
- the protein molecular weight marker is shown on the left.
- Figure 14 shows a schematic representation of the three differentially expressed novel genes.
- the thin line indicates a stretch of nucleotide sequences ending at poly A tail, denoted by (A) N , at the base pair number written next.
- the filled box illustrates location of the most probable open reading frame, with the numbers below indicating the base pair positions of start and stop codons respectively.
- A Map of the C40 cDNA, the leucine zipper motif is denoted by LZ and shown above is the corresponding amino acid positions and sequences.
- B Map of the HI 7 cDNA, the asterisks above poly A tail indicate the presence of polyadenylation signals.
- the hatched box illustrates the amino acid region of shared homology and the sequence alignments are shown below the gene.
- Figure 16 shows that the upregulation of the H37 and H41 transcripts correlates with HER-2/ neu overexpression in human breast tumors (p ⁇ 0.005 and p ⁇ 0.075 respectively).
- A Northern blot analysis was performed to compare expression levels of the HER-2 vs. H37 cDNAs in 15 individual breast tumor samples. Ten ⁇ g of total RNA was loaded in each lane, and the same blot was stripped for rehybridization with the second probe.
- B The expression levels of the HER-2 vs. H41 cDNAs were analyzed in a separate Northern blot experiment. The same set of breast tumor samples were used as in panel A except that the #16 tumor was substituted for the #14 due to depletion of the sample.
- RNA loading control Fifteen ⁇ g of total RNA was loaded in each lane except for tumor #15 for which only 5 ⁇ g were used because of lack of material. The blot was stripped as in A. For both A and B, ethidium bromide staining of 28 S ribosomal RNA is shown below the autoradiograms for RNA loading control.
- polynucleotide means a polymeric form of nucleotides of at least about 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA.
- polypeptide means a polymer of at least about 6 ammo acids. Throughout the specification, standard three letter or smgle letter designations for ammo acids are used.
- Nucleic acid sequence refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be smgle- or double-stranded, and represent the sense or antisense strand.
- amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protem sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules.
- amino acid sequence is recited herem to refer to an amino acid sequence of a naturally occurring protem molecule
- amino acid sequence and like terms, such as “polypeptide” or “protem” are not meant to limit the ammo acid sequence to the complete, native ammo acid sequence associated with the recited protem molecule.
- Peptide nucleic acid refers to a molecule which comprises an oligomer to which an ammo acid residue, such as lysme, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8:53-63).
- HOMPS refers to the am o acid sequences of substantially purified HOMPS obtained from any species, particularly mammalian, including bovine, ovme, porcme, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombmant.
- Consensus refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, or which has been extended usmg the XL- PCR kit. (Perkin Elmer, Norwalk, Conn.
- a “variant" of HOMPS refers to an ammo acid sequence that is altered by one or more ammo acids.
- the variant may have "conservative" changes, wherem a substituted amino acid has similar structural or chemical properties, e. g. , replacement of leucme with isoleucine. More rarely, a variant may have "nonconservative" changes, e. g. , replacement of a glycine with a tryptophan. Similar minor variations may also include ammo acid deletions or insertions, or both.
- Guidance m determining which ammo acid residues may be substituted, mserted, or deleted without abolishing biological or lmmunological activity may be found usmg computer programs well known m the art, for example, LASERGENE software.
- a “deletion”, as used herem, refers to a change m either amino acid or nucleotide sequence m which one or more ammo acid or nucleotide residues, respectively, are absent.
- An “msertion” or “addition”, as used herem, refers to a change m an ammo acid or nucleotide sequence resulting in the addition of one or more ammo acid or nucleotide residues, respectively, as compared to the naturally occurrmg molecule
- substitution refers to the replacement of one or more ammo acids or nucleotides by different amino acids or nucleotides, respectively
- biologically active refers to a protem having structural, regulatory, or biochemical functions of a naturally occurrmg molecule.
- immunologically active refers to the capability of the natural, recombmant, or synthetic HOMPS, or any oligopeptide thereof, to mduce a specific immune response m appropriate animals or cells and to bind with specific antibodies.
- agonist refers to a molecule which, when bound to HOMPS, causes a change in HOMPS which modulates the activity of HOMPS.
- Agonists may mclude proteins, nucleic acids, carbohydrates, or any other molecules which bmd to HOMPS.
- Antagonist refers to a molecule which, when bound to HOMPS, blocks or modulates the biological or lmmunological activity of HOMPS.
- Antagonists and inhibitors may mclude proteins, nucleic acids, carbohydrates, or any other molecules which bmd to HOMPS.
- modulate refers to a change or an alteration m the biological activity of HOMPS. Modulation may be an mcrease or a decrease m protem activity, a change in binding characteristics, or any other change m the biological, functional or lmmunological properties of HOMPS.
- mietic refers to a molecule, the structure of which is developed from knowledge of the structure of HOMPS or portions thereof and, as such, is able to effect some or all of the actions related to the human Her-2/neu overexpression modulated proteins.
- derivative refers to the chemical modification of a nucleic acid encoding HOMPS or the encoded HOMPS.
- nucleic acid derivative would encode a polypeptide which retains essential biological characteristics of the natural molecule.
- 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.
- PCR polymerase chain reaction
- 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.
- a hybridization complex may be formed in solution (e. g. C 0t or Rlit t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e. g. , membranes filters, chips, pins or glass slides to which cells have been fixed for in situ hybridization).
- a solid support e. g. , membranes filters, chips, pins or glass slides to which cells have been fixed for in situ hybridization.
- 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.
- a partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term "substantially homologous. " The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i. e. , the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency.
- 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 nonspecific binding, the probe will not hybridize to the second non-complementary target sequence.
- hybridize As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6XSSC/0.1% SDS/ 100 ⁇ g/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1X SSC/0.1% SDS are above 55 degrees C, and most preferably to stringent hybridization conditions.
- “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
- “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sul
- washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
- An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
- the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
- identity is used to express the percentage of amino acid residues at the same relative positions that are the same.
- homoology is used to express the percentage of amino acid residues at the same relative positions that are either identical or are similar, using the conserved amino acid criteria of BLAST analysis, as is generally understood in the art. For example, % identity values may be generated by WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology 266:460-480; ht ://blast.wusd/edu/blast/README.html). Further details regarding amino acid substitutions, which are considered conservative under such criteria, are provided below.
- the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences.
- antisense refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence.
- antisense strand is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
- Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may be generated.
- the designation “negative” is sometimes used in reference to the antisense strand, and "positive” is sometimes used in reference to the sense strand.
- portion refers to fragments of that protein.
- the fragments may range in size from four 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 Figure 2, Figure 4, Figure 6, Figure 9 or Figure 11” encompasses the full-length human HOMPS and fragments thereof.
- 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 host cell being transformed and may include, but is not limited to, viral infection, electroporation, 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.
- antigenic determinant refers to that portion of a molecule that makes contact with a particular antibody (i. e. an epitope).
- a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.
- An antigenic determinant may compete with the intact antigen (i. e. , the immunogen used to elicit the immune response) for binding to an antibody.
- telomere binding in reference to the interaction of an antibody and a protein or peptide, mean that the interaction is dependent upon the presence of a particular structure (i. e., the antigenic determinant or epitope) on the protein; in other words, the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. 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.
- a biological sample suspected of containing nucleic acid encoding HOMPS or fragments thereof may comprise a cell, chromosomes isolated from a cell (e. g. , a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), an extract from cells or a tissue, and the like.
- the term "correlates with expression of a polynucleotide”, as used herein, indicates that the detection of the presence of ribonucleic acid that is similar to Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10 by northern analysis is indicative of the presence of mRNA encoding HOMPS in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.
- "Alterations" in the polynucleotide of Figure 1, Figure 3, Figure 5, Figure 8 or Tigure 10, as used herein, comprise any alteration in the sequence of polynucleotides encoding HOMPS including deletions, insertions, and point mutations that may be detected using hybridization assays.
- HOMPS alterations to the genomic DNA sequence which encodes HOMPS
- alterations to the genomic DNA sequence which encodes HOMPS e. g. , by alterations in the pattern of restriction fragment length polymorphisms capable of hybridizing to Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10
- the inability of a selected fragment of Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10 to hybridize to a sample of genomic DNA e. g. , using allele-specific oligonucleotide probes
- improper or unexpected hybridization such as hybridization to a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HOMPS (e. g. , using fluorescent in situ hybridization [FISH] to metaphase chromosomes spreads).
- FISH fluorescent in situ hybridization
- the term "antibody” refers to intact molecules as well as fragments thereof, such as Fab, F(ab') 2 and Fv, which are capable of binding the epitopic determinant.
- Antibodies that bind HOMPS polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen.
- the polypeptide or peptide used to immunize an animal can be derived from the transition 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. The coupled peptide is then used to immunize the animal (e. g. , a mouse, a rat, or a rabbit).
- 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.
- the invention is based on the discovery of new human Her-2/neu overexpression modulated proteins (HOMPS), the polynucleotides encoding HOMPS, and the use of these compositions for the evaluation and characterization of disorders associated with abnormal cellular proliferation.
- HMPS Her-2/neu overexpression modulated proteins
- Amplification and resulting overexpression of the H ⁇ X -2/neu proto- oncogene is found in approximately 30% of human breast and 20% of human ovarian cancers.
- differential hybridization was used to identify genes whose expression levels are altered in cells overexpressing this receptor. As illustrated below, of 16,000 clones screened from an overexpression cell cDNA library, a total of 19 non-redundant clones were isolated including 7 whose expression decreases (C clones) and 12 which increase (H clones) in association with HEK-2/neu overexpression. Of these, 5 C clones and 11 H clones have been confirmed to be differentially expressed by Northern blot analysis.
- This group includes nine genes of known function, three previously sequenced genes of relatively uncharacterized function and four novel genes without match in GenBank. Examination of the previously characterized genes indicates that they represent sequences known to be frequently associated with the malignant phenotype, suggesting that the subtraction cloning strategy used identified appropriate target genes. In addition, differential expression of 12 of 16 (75%) cDNAs identified in the breast cancer cell lines are also seen in H ⁇ R.-2/neu overexpressing ovarian cancer cells, indicating that they represent generic associations with HER-2/neu overexpression. Finally, upregulation of two of the identified cDNAs, one novel and one identified but as yet uncharacterized gene, was confirmed in human breast cancer specimens in association with HEK-2/neu overexpression.
- human MCF-7 breast cancer cells which express normal levels of the receptor were transfected with a retroviral expression vector containing a full length cDNA encoding the human HER-2 gene (Chazin, V. R., Kaleko, M., Miller, A. D. and Slamon, D. J. (1992) Oncogene, 7(9), 1859-66).
- Multiple rounds of infection and sorting of the top 5% of HER-2 overexpressing, pooled transfectants generated a stably transfected cell line, MCF-7/HER-2, in which HER-2 expression levels were comparable to those observed in human HER-2 overexpressing breast cancer specimens (Pegram, M. D., Finn, R.
- MCF-7 cells were similarly transfected using an empty neomycin resistance vector including multiple infections, producing the MCF- 7/control cell line.
- Increased amounts and/or activation of this ceU surface receptor may affect either the expression or function of other molecules involved in regulation of ceU proUferation.
- Direct effects of HER-2 overexpression on other ceUular proteins can be accompUshed by changes in 1) expression at the mR ⁇ A transcript level, 2) protein production at the translational level, or 3) protein activation / modification at the post-translational level.
- the ceUular changes associated with ⁇ ER-2/ neu overexpression are likely to be induced by most or aU of these mechanisms. To identify those changes associated with differential expression of genes at the transcript level, we undertook a differential screening analysis.
- differential hybridization also known as plus/minus screening (St John, T. P. and Davis, R. W. (1979) Cell, 16(2), 443-52)
- differential hybridization also known as plus/minus screening
- This approach has the advantage of comparing two human breast cancer ceU lines which are identical except for H ⁇ R-2/ neu overexpression aUowing for a direct comparison of cD ⁇ As derived from the two ceU populations.
- a relatively conventional subtraction cloning method termed differential hybridization has been successfully used by other investigators in the cloning of genes associated with various biologic phenomenon including the galactose-inducible genes of yeast (St John, T. P. and Davis, R. W. (1979) Cell, 16(2), 443-52), human fibroblast interferon (Taniguchi, T., Fuju-Kuriyama, Y. and Muramatsu, M. (1980) Proc Natl Acad Sci U S A, 77(7), 4003-6), a variety of heat-shock proteins (Mason, I. J., Taylor, A., Williams, J. G., Sage, H. and Hogan, B. L.
- Recent data indicates that downreguiation of cytokeratin (C29, C49) gene expression may result in disorganization of the cytoskeleton leading to enhanced invasive properties (Mukhopadhyay, T. and Roth, J. A. (1996) Anticancer Res, 16(1), 105-12).
- the gamma actin (C72) transcript level is markedly decreased in saUvary gland adenocarcinoma ceUs on acquisition of metastatic abiUty (Suzuki, H., Nagata, H, Shimada, Y. and Konno, A. (1998) Int] Oncol, 12(5), 1079-84).
- ribosomal proteins L8 (HI 6) and LLrep3(H35), GAPDH (H31), and succinyl coA transferase (H45), may be merely reflective of higher proUferation in HER-2 overexpressing tumors.
- differential expression of these genes may be more specificaUy linked to HER-2 overexpression.
- An example of this could be the LLrep3 which was also identified in differential hybridization screening of a ras-transfected teratocarcinoma ceU line compared to isogenic ceU control as increased 25-fold (Chiao, P. J., Shin, D. M., Sacks, P.
- DNA fragmentation factor (DFF) (HI 3) is also overexpressed in the HER-2 overexpressing ceUs and has recently been identified as a protein which functions downstream of caspase-3 during apoptosis (Liu, X., Zou, H., Slaughter, C. and Wang, X. (1997) Cell, 89(2), 175-84). Its exact ceUular role in this process, i.e. inhibition or promotion of apoptosis, however, is as yet undefined (Mitamura, S., Ikawa, H., Mizuno, N., Kaziro, Y. and Itoh, H.
- H37 cDNA found to be overexpressed in HER-2 overexpressing ceUs in the current study and demonstrating convincing differential expression in actual tumor samples, is localized to a region of chromosome 3 ⁇ 21.3 aUeged to contain a putative lung cancer tumor suppressor gene(s) (Wei, M. H., Latif, F., Bader, S., Kashuba, V., Chen, J. Y., Duh, F. M., Sekido, Y., Lee, C. C, GeU, L., Kuzmin, I., Zabarovsky, E., Klein, G., Zbar, B., Minna, J. D. and Lerman, M. I. (1996) Cancer Res, 56(7), 1487-92). Further characterization of this gene at the functional and genomic levels should give further insight into this phenomenon.
- oncogenesis associated markers such as Her-2 and PSA provide in the monitoring of cancers in patients as weU as the need for the identification of additional oncogenesis associated markers (see e.g. Bostwick et al., J CeU Biochem Suppl 1996;25:156-64 and Morote et al., Int J Cancer 1999 Aug 20;84(4):421-5).
- oncogenesis is a multistep process and the identification of a variety of different oncogenesis associated markers can be used to identify and characterize precancerous and cancerous svndromes earUer and more efficiently (see e.g.
- HOMPS proteins described herein include them in the class of oncogenesis associated markers that can be used to evaluate and/or evaluate oncogenetic processes in cancers. Understandably, a number of the HOMPS proteins disclosed herein have been independently identified by other artisans as oncogenesis associated markers which can be used to examine growth disregulation in conditions such as cancer (see e.g.
- HOMPS gene products exhibit specific properties that are analogous to those found in a famUy of genes whose polynucleotides, polypeptides and anti-polypeptide antibodies are used in weU known diagnostic assays directed to examining conditions associated with disregulated ceU growth such as cancer.
- WeU known members of this class include Her-2 as weU as PSA, the archetypal markers that have been used by medical practitioners for years to identify and monitor the presence of cancers such as prostate cancer (see e.g. MerriU et al, J. Urol. 163(2): 503-5120 (2000); Polascik et al, J. Urol.
- HOMPS polynucleotides and polypeptides as weU as the HOMPS polynucleotide probes and anti-HOMPS antibodies used to identify the presence of these molecules
- HOMPS polynucleotide probes and anti-HOMPS antibodies used to identify the presence of these molecules
- their properties aUows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
- Typical embodiments of diagnostic methods which utilize the HOMPS polynucleotides, polypeptides and antibodies described herein are analogous to those methods from weU estabUshed diagnostic assays which employ Her-2 and PSA polynucleotides, polypeptides and antibodies.
- PSA polynucleotides are used as probes (for example in Northern analysis, see e.g. Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see e.g. Okegawa et al., J. Urol.
- the HOMPS polynucleotides described herein can be utilized in the same way to evaluate or monitor the ceUular growth disregulation that is associated with cancer.
- PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods of monitoring PSA protein overexpression (see e.g. Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate ceUs (see e.g. Alanen et al, Pathol. Res. Pract. 192(3):233-7 (1996))
- the HOMPS polypeptides described herein can be utilized to generate antibodies for use in detecting HOMPS overexpression as seen in ceUs expected of exhibiting some form of growth disregulation.
- HOMPS polynucleotide fragments and polynucleotide variants can also be used in an analogous manner.
- typical PSA polynucleotides used in methods of monitoring this molecule are probes or primers which consist of fragments of the PSA cDNA sequence. IUustrating this, primers used to PCR ampUfy a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction.
- Polynucleotide fragments and variants are typicaUy useful in this context as long as they have the common attribute or characteristic of being capable of binding to a target polynucleotide sequence (e.g. the HOMPS polynucleotide shown in Figure 2) under conditions of high stringency.
- a target polynucleotide sequence e.g. the HOMPS polynucleotide shown in Figure 2
- HOMPS polypeptide fragments and polypeptide variants can also be used in an analogous manner.
- typical PSA polypeptides used in methods of monitoring this molecule are fragments of the PSA protein which contain an epitope that can be recognized by an antibody which will specificaUy bind to the PSA protein.
- This practice of using polypeptide fragments or polypeptide variants used to generate antibodies is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see e.g. Current Protocols In Molecular Biology, Volume 2, Unit 1 (., Frederick M.
- each of the variety of epitopes in a protein of interest functions to provide the architecture upon which the antibody is generated.
- TypicaUy skilled artisans generaUy create a variety of different polypeptide fragments that can be used in order to generate antibodies specific for different portions of a polypeptide of interest (see e.g. U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533).
- Polypeptide fragments and variants are typicaUy useful in this context as long as they have the common attribute or characteristic of having an epitope capable of generating an antibody specific for a target polypeptide sequence (e.g. the HOMPS polypeptide shown in Figure 2).
- the HOMPS polynucleotides and polypeptides exhibit specific properties that can make them useful in examining cancerous ceUs or tissues.
- the described diagnostic assays that measures the presence of HOMPS gene products, in order to provide evidence of growth disregulation are particularly useful in identifying potential candidates for preventive measures or further monitoring, as has been done so successfuUy with Her-2 and PSA (see e.g. Scheurle et al, Anticancer Res. 2000 May-Jun;20(3B):2091-6; Fontana et al, Anticancer Res 1994 Sep- Oct;14(5B):2099-104 and Sahin, Adv Anat Pathol 2000 May;7(3):158-66).
- the invention is directed to Her-2 /neu overexpression modulated proteins (HOMPS) genes and HOMPS gene products as weU as HOMPS antibodies and assays for detecting these molecules.
- HOMPS Her-2 /neu overexpression modulated proteins
- the invention encompasses HOMPS proteins as weU as the polynucleotides which encode HOMPS proteins.
- any nucleic acid sequence which encodes the amino acid sequence of HOMPS can be used to generate recombinant molecules which express HOMPS.
- the invention encompasses the polynucleotide comprising the nucleic acid sequence of Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10.
- the invention encompasses HOMPS variants which retain biological or other functional activity of HOMPS.
- a preferred HOMPS variant is one having at least 80%, and more preferably 90%, amino acid sequence identity to the HOMPS amino acid sequence of Figure 2, Figure 4, Figure 6, Figure 9 or Figure 11.
- a most preferred HOMPS variant is one having at least 95% amino acid sequence identity to the amino acid sequence of Figure 2, Figure 4, Figure 6, Figure 9 or Figure 11.
- nucleotide sequences which encode HOMPS and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturaUy occurring HOMPS under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HOMPS or its derivatives possessing a substantiaUy 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.
- Other reasons for substantiaUy altering the nucleotide sequence encoding HOMPS and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-Ufe, than transcripts produced from the naturaUy occurring sequence.
- the invention also encompasses production of DNA sequences, or portions thereof, which encode HOMPS and its derivatives, entirely by synthetic chemistry.
- the synthetic sequence may be inserted into any of the many available expression vectors and ceU systems using reagents that are weU known in the art at the time of the filing of this appUcation.
- synthetic chemistry may be used to introduce mutations into a sequence encoding HOMPS or any portion thereof.
- polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those shown in Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10, under various conditions of stringency.
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Ward, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel A. R. (1987; Methods Enzymol. 152:507-511), and may be used at a defined stringency.
- Altered nucleic acid sequences encoding HOMPS which are encompassed by the mvention mclude deletions, insertions, or substitutions of different nucleotides resulting m a polynucleotide that encodes the same or a functionaUy equivalent HOMPS.
- the encoded protem may also contam deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionaUy equivalent HOMPS.
- DeUberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubiUty, hydrophobicity, hydrophiUcity, and/or the amphipathic nature of the residues as long as the biological activity of HOMPS is retamed.
- negatively charged amino acids may mclude aspartic acid and glutamic acid; positively charged ammo acids may mclude lysme and arginine; and amino acids with uncharged polar head groups having similar hydrophiUcity values may mclude leucme, isoleucme, and valine; glycme and alanme; asparagme and glutamine; serme and threonine; phenylalanine and tyrosine.
- the mvention is directed to Her-2 /neu overexpression modulated proteins (HOMPS).
- HMPS Her-2 /neu overexpression modulated proteins
- the typical embodiments of the mvention discussed herem e.g. polynucleotides, polypeptides, antibodies and assays for HOMPS gene products etc.
- aU of the HOMPS genes and gene products l e. H13, H14, H17, H41, H63 and C40.
- embodiments of a smgle HOMPS gene are used (for example the H41 gene) to lUustrate typical embodiments of the mvention that apply to aU of the HOMPS molecules provided herem.
- a ⁇ usans understand that the use of a smgle HOMPS molecule m lUustrative typical embodiments common to aU of the HOMPS molecules eliminates unnecessary redundancy m the description of the mvention
- polynucleotides correspondmg or complementary to aU or part of a HOMPS gene, mRNA, and/or coding sequence, preferably m isolated form, mcludmg polynucleotides encoding a HOMPS protem and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oUgonucleotides complementary to a HOMPS gene or mRNA sequence or a part thereof, and polynucleotides or oUgonucleotides that hybridize to a HOMPS gene, mRNA, or to a HOMPS encoding polynucleotide (coUectively, "HOMPS polynucleotides").
- HOMPS gene and protein is meant to include the HOMPS genes and proteins specificaUy described herein.
- a H41 polynucleotide may comprise a polynucleotide having the nucleotide sequence of human H41 as shown in FIG. 5 (SEQ ID NO: 5), wherein T can also be U; a polynucleotide that encodes aU or part of the H41 protein; a sequence complementary to the foregoing; or a polynucleotide fragment of any of the foregoing.
- Another embodiment comprises a polynucleotide that is capable of hybridizing under stringent hybridization conditions to the human H41 cDNA shown in FIG. 5 (SEQ ID NO: 5) or to a polynucleotide fragment thereof.
- Typical embodiments of the invention disclosed herein include H41 polynucleotides containing specific portions of the H41 mRNA sequence (and those which are complementary to such sequences) such as those that encode the protein and fragments thereof.
- representative embodiments of the invention disclosed herein include: polynucleotides encoding about amino acid 1 to about amino acid 10 of the H41 protein shown in FIG. 5 (SEQ ID NO: 5), polynucleotides encoding about amino acid 20 to about amino acid 30 of the H41 protein shown in FIG. 5 (SEQ ID NO: 5), polynucleotides encoding about amino acid 30 to about amino acid 40 of the H41 protein shown in FIG.
- polynucleotides encoding about amino acid 40 to about amino acid 50 of the H41 protein shown in FIG. 5 polynucleotides encoding about amino acid 50 to about amino acid 60 of the H41 protein shown in FIG. 5 (SEQ ID NO: 5), polynucleotides encoding about amino acid 60 to about amino acid 70 of the H41 protein shown in FIG. 5 (SEQ ID NO: 5), polynucleotides encoding about amino acid 70 to about amino acid 80 of the H41 protein shown in FIG. 5 (SEQ ID NO: 5), polynucleotides encoding about amino acid 80 to about amino acid 90 of the H41 protein shown in FIG.
- polynucleotides encoding about amino acid 90 to about amino acid 100 of the H41 protein shown in FIG. 5 may be generated by a variety of techniques weU known in the art.
- Additional iUustrative embodiments of H41 polynucleotides include embodiments consisting of a polynucleotide having the sequence as shown in FIG. 5 (SEQ ID NO: 5) from about nucleotide residue number 1 through about nucleotide residue number 500, from about nucleotide residue number 500 through about nucleotide residue number 1000 and from about nucleotide residue number 500 through about nucleotide residue number 1000 and from about nucleotide residue number 1000 through about nucleotide residue number 1500 and from about nucleotide residue number 1500 through about nucleotide residue number 2000 and from about nucleotide residue number 2000 through about nucleotide residue number 2500 and from about nucleotide residue number 2500 through about nucleotide residue number 3000 and from about nucleotide residue number 3000 through about nucleotide residue number 3346.
- polynucleotide fragments can be of any size an include any portion of the H41 sequence as shown in FIG. 5 (SEQ ID NO: 5), for example a polynucleotide having the sequence as shown in FIG. 5 (SEQ ID NO: 5) from about nucleotide residue number 324 through about nucleotide residue number 2248.
- H41 proteins and polypeptide fragments thereof are provided.
- the H41 proteins of the invention include those specificaUy identified herein, as weU as aUeUc variants, conservative substitution variants and homologs that can be isolated/generated and characterized without undue experimentation foUowing the methods outlined below. Fusion proteins that combine parts of different H41 proteins or fragments thereof, as weU as fusion proteins of a H41 protein and a heterologous polypeptide are also included.
- H41 proteins wiU be coUectively referred to as the H41 proteins, the proteins of the invention, or H41.
- H41 polypeptide refers to a polypeptide fragment or a H41 protein of at least 6 amino acids, preferably at least 15 amino acids.
- Specific embodiments of H41 proteins comprise a polypeptide having the amino acid sequence of human H41 as shown in FIG. 6 (SEQ ID NO: 6).
- embodiments of H41 proteins comprise variant polypeptides having alterations in the amino acid sequence of human H41 as shown in FIG. 6 (SEQ ID NO: 6).
- aUeUc variants of human HOMPS such as H41 wiU share a high degree of structural identity and homology (e.g., 90% or more identity).
- aUeUc variants of the HOMPS proteins wiU contain conservative amino acid substitutions within the HOMPS sequences described herein or wiU contain a substitution of an amino acid from a corresponding position in a HOMPS homologue.
- HOMPS aUeUc variants wiU be proteins that share a high degree of homology with at least a smaU region of a particular HOMPS amino acid sequence, but wiU further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. Conservative amino acid substitutions can frequendy be made in a protein without altering either the conformation or the function of the protein.
- Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
- Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V).
- Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequendy interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. StiU other changes can be considered "conservative" in particular environments.
- Embodiments of the invention disclosed herein include a wide variety of art accepted variants of HOMPS proteins such as polypeptides having amino acid insertions, deletions and substitutions. HOMPS variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis.
- Such amino acids include alanine, glycine, serine, and cysteine.
- Alanine is typicaUy a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typicaUy preferred because it is the most common amino acid. Further, it is frequendy found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, 1976, J. Mol. Biol, 150:1). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
- HOMPS variants have the distinguishing attribute of having at least one epitope in common with a HOMPS protein (such as the H41 protein having the amino acid sequence of FIG. 6 (SEQ ID NO: 6)), such that an antibody that specificaUy binds to a HOMPS variant wiU also specificaUy bind to the HOMPS protein (such as the HOMPS protein having the amino acid sequence of FIG. 6 (SEQ ID NO: 6)).
- a polypeptide ceases to be a variant of the H41 protein shown in FIG.
- embodiments of the claimed invention include polypeptides containing less than the 258 amino acid sequence of the H41 protein shown in FIG. 6 (SEQ ID NO: 6) (and the polynucleotides encoding such polypeptides).
- representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of the H41 protein shown in FIG.
- polypeptides consisting of about amino acid 20 to about amino acid 30 of the H41 protein shown in FIG. 6 polypeptides consisting of about amino acid 30 to about amino acid 40 of the H41 protein shown in FIG. 6 (SEQ ID NO: 6), polypeptides consisting of about amino acid 40 to about amino acid 50 of the H41 protein shown in FIG. 6 (SEQ ID NO: 6), polypeptides consisting of about amino acid 50 to about amino acid 60 of the H41 protein shown in FIG. 6 (SEQ ID NO: 6), polypeptides consisting of about amino acid 60 to about amino acid 70 of the H41 protein shown in FIG.
- polypeptides consisting of about amino acid 70 to about amino acid 80 of the H41 protein shown in FIG. 6 polypeptides consisting of about amino acid 80 to about amino acid 90 of the H41 protein shown in FIG. 6 (SEQ ID NO: 6) and polypeptides consisting of about amino acid 90 to about amino acid 100 of the H41 protein shown in FIG. 6 (SEQ ID NO: 6), etc.
- polypeptides consisting of portions of the amino acid sequence of amino acids 100-258 of the H41 protein are typical embodiments of the invention. Polypeptides consisting of larger portions of the H41 protein are also contemplated.
- polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the H41 protein shown in FIG. 6 (SEQ ID NO: 6) may be generated by a variety of techniques weU known in the art.
- aUeles of the genes encoding HOMPS are also included within the scope of the present invention.
- an "aUele” or “aUeUc sequence” is an alternative form of the gene which may result from at least one mutation in the nucleic acid sequence.
- AUeles may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many aUeUc forms.
- Common mutational changes which give rise to aUeles are generaUy 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.
- Methods for DNA sequencing which are weU known and generaUy available in the art may be used to practice any embodiments of the invention.
- the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase Amersham Pharmacia Biotech (Pisctaway N. J. ), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE AmpUfication System marketed by Life Technologies (Gaithersburg, Md. ).
- the process is automated with machines such as the HamUton Micro Lab 2200 (HamUton, Reno, Nev. ), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass. ) and the ABI 377 DNA sequencers (Perkin Elmer).
- the nucleic acid sequences encoding HOMPS 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. For example, 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 AppUc. 2:318-322).
- genomic DNA is first ampUfied in the presence of primer to linker sequence and a primer specific to the known region.
- the ampUfied 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 ampUfy or extend sequences using divergent primers based on a known region (TrigUa, T. et al. (1988) Nucleic Acids Res. 16:8186).
- the primers may be designed using OLIGO 4. 06 Primer Analysis software (National Biosciences Inc. , Plymouth, Minn.
- 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 Ugation and used as a PCR template.
- Another method which may be used is capture PCR which involves PCR ampUfication of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods AppUc. 1:111-119). In this method, multiple restriction enzyme digestions and Ugations may also be used to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before performing PCR.
- Genomic Ubraries may be useful for extension of sequence into the 5' and 3' non- transcribed regulatory regions.
- CapiUary electrophoresis systems which are commerciaUy avaUable may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
- capiUary 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 device camera.
- Output/Ught intensity may be converted to electrical signal using appropriate software (e. g.
- CapiUary electrophoresis is especiaUy preferable for the sequencing of smaU pieces of DNA which might be present in limited amounts in a particular sample.
- polynucleotide sequences or fragments thereof which encode HOMPS, or fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules to direct expression of HOMPS in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantiaUy the same or a functionaUy equivalent amino acid sequence may be produced and these sequences may be used to clone and express HOMPS.
- codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-Ufe which is longer than that of a transcript generated from the naturaUy occurring sequence.
- nucleotide sequences of the present invention can be engineered using methods generaUy known in the art in order to alter HOMPS 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 oUgonucleotides 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 spUce variants, or introduce mutations, and so forth.
- natural, modified, or recombinant nucleic acid sequences encoding HOMPS may be Ugated 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 HOMPS encoding sequence and the heterologous protein sequence, so that HOMPS may be cleaved and purified away from the heterologous moiety.
- sequences encoding HOMPS may be synthesized, in whole or in part, using chemical methods weU 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 HOMPS, or a portion thereof.
- peptide synthesis can be performed using various soUd-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer).
- the newly synthesized peptide may be substantiaUy purified by preparative high performance Uquid chromatography (e. g. , Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co. , New York, N. Y. ).
- the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e. g. , the Edman degradation procedure; Creighton, supra).
- AdditionaUy the amino acid sequence of HOMPS, 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.
- the nucleotide sequences encoding HOMPS 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.
- appropriate expression vector i. e. , a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- Methods which are weU known to those skiUed in the art may be used to construct expression vectors containing sequences encoding HOMPS and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N. Y. , and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N. Y.
- a variety of expression vector/host systems may be utilized to contain and express sequences encoding HOMPS. 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 ceU systems infected with virus expression vectors (e. g. , baculovirus); plant ceU systems transformed with virus expression vectors (e. g. , cauUflower mosaic virus, CaMV; tobacco mosaic virus, TMN) or with bacterial expression vectors (e. g. , Ti or ⁇ BR322 plasmids); or animal ceU systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
- yeast transformed with yeast expression vectors insect ceU systems infected with virus expression vectors (e. g. , baculovirus)
- control elements or "regulatory sequences” are those non- translated regions of the vector— enhancers, promoters, 5' and 3' untranslated regions—which interact with host ceUular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.
- any number of suitable transcription and translation elements may be used.
- inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, LaJoUa, CaUf. ) or PSPORT1 plasmid (Gibco BRL) and the like may be used.
- the baculovirus polyhedrin promoter may be used in insect ceUs. Promoters or enhancers derived from the genomes of plant ceUs (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.
- promoters from mammalian genes or from mammaUan viruses are preferable.
- 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 HOMPS. For example, when large quantities of HOMPS are needed for the induction of antibodies, 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.
- coU cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding HOMPS may be Ugated into the vector in frame with sequences for the ammo-te ⁇ ninal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced; pI ⁇ vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like.
- pGEX vectors Promega, Madison, Wis.
- GST glutathione S-transferase
- such fusion proteins are soluble and can easUy be purified from lysed ceUs by adsorption to glutathione-agarose beads foUowed by elution in the presence of free glutathione.
- Proteins made in such systems may be designed to include heparin, thrombm, or factor XA protease cleavage sites so that the cloned polypeptide of mterest can be released from the GST moiety at wiU.
- yeast 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.
- the expression of sequences encoding HOMPS 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 m combmation with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311.
- plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; and Wmter, J. et al.
- HOMPS Autographa caUfornica nuclear polyhedrosis virus
- AcNPV Autographa caUfornica nuclear polyhedrosis virus
- the sequences encoding HOMPS may be cloned into a non- essential region of the virus, such as the polyhedrm gene, and placed under control of the polyhedrm promoter. Successful insertion of HOMPS wiU render the polyhedrm gene inactive and produce recombmant virus lacking coat protem.
- the recombmant viruses may then be used to infect, for example, S frugiperda ceUs or Trichoplusia larvae m which HOMPS may be expressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227).
- HOMPS may be expressed in mammaUan host ceUs.
- sequences encoding HOMPS may be Ugated mto an adenovirus transcription/ translation complex consisting of the late promoter and tripartite leader sequence.
- Insertion m a non-essential El or E3 region of the viral genome may be used to obta a viable virus which is capable of expressing HOMPS m mfected host ceUs (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655- 3659).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to mcrease expression in mammaUan host ceUs.
- RSV Rous sarcoma virus
- Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HOMPS. Such signals mclude the ATG initiation codon and ad j acent sequences.
- Enhancers which are appropriate for the particular ceU system which is used, such as those described m the Uterature (Scharf, D. et al. (1994) Results Probl CeU Differ. 20-125-162).
- a host ceU strain may be chosen for its abiUty to modulate the expression of the mserted 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, Upidation, and acylation.
- Post-translational processing which cleaves a "prepro" form of the protem may also be used to faciUtate correct msertion, folding and/or function.
- Different host ceUs such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific ceUular machinery and characteristic mechanisms for such post- translational activities, may be chosen to ensure the correct modification and processmg of the foreign protem.
- ceU Unes which stably express HOMPS may be transformed usmg expression vectors which may contam viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
- ceUs may be aUowed to grow for 1-2 days m 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 aUows growth and recovery of ceUs which successfuUy express the introduced sequences.
- Resistant clones of stably transformed ceUs may be proUferated using tissue culture techniques appropriate to the ceU type.
- ceU Unes Any number of selection systems may be used to recover transformed ceU Unes. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) CeU 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) CeU 22:817-23) genes which can be employed in tk “ or aprt " ceUs, respectively. Also, antimetaboUte, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methoteexate (Wigler, M. et al. (1980) Proc. Nad.
- npt which confers resistance to the arninoglycosides, 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 aUows ceUs to utilize indole in place of tryptophan, or hisD, which aUows ceUs to utilize histinol in place of histidine (Hartman, S. C.
- marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
- a marker gene can be placed in tandem with a sequence encoding HOMPS under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.
- host ceUs which contain the nucleic acid sequence encoding HOMPS and express HOMPS may be identified by a variety of procedures known to those of skiU 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 HOMPS can be detected by DNA-DNA or DNA-RNA hybridization or ampUfication using probes or portions or fragments of polynucleotides encoding HOMPS.
- Nucleic acid ampUfication based assays involve the use of oUgonucleotides or oUgomers based on the sequences encoding HOMPS to detect transformants containing DNA or RNA encoding HOMPS.
- oUgonucleotides or “oUgomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.
- a variety of protocols for detecting and measuring the expression of HOMPS, using eidier 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 ceU sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescence activated ceU sorting
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HOMPS 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. Minn. ) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HOMPS include oUgolabeUng, nick translation, end-labeling or PCR ampUfication using a labeled nucleotide.
- the sequences encoding HOMPS, or any portions thereof may be cloned into a vector for the production of an mRNA probe.
- a vector for the production of an mRNA probe Such vectors are known in the art, are commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commerciaUy avaUable kits Amersham Pharmacia Biotech, Promega, and US Biochemical Corp. Suitable reporter molecules or labels, which may be used, include radionucUdes, enzymes, fluorescent, chem uminescent, or chromogenic agents as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host ceUs transformed with nucleotide sequences encoding HOMPS may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
- the protein produced by a recombinant ceU may be secreted or contained inttaceUularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode HOMPS may be designed to contain signal sequences which direct secretion of HOMPS through a prokaryotic or eukaryotic ceU membrane.
- purification faciUtating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that aUow purification on immobiUzed metals, protein A domains that aUow purification on immobilized immunoglobulin, and the domain utiUzed in the FLAGS extension/affinity purification system (Immunex Corp. , Seattle, Wash. ).
- cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, CaUf. ) between the purification domain and HOMPS may be used to faciUtate purification.
- One such expression vector provides for expression of a fusion protein containing HOMPS and a nucleic acid encoding 6 histidine residues preceding a fhioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobiUzed metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif.
- HOMPS In addition to recombinant production fragments of HOMPS may be produced by direct peptide synthesis using soUd-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 AppUed Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments of HOMPS may be chemicaUy synthesized separately and combined using chemical methods to produce the fuU length molecule.
- antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specificaUy bind DNA or RNA in a base pair-dependent manner.
- PNAs peptide nucleic acids
- non-nucleic acid molecules such as phosphorothioate derivatives
- Antisense technology entaUs the administration of exogenous oUgonucleotides that bind to a target polynucleotide located within the ceUs.
- the term "antisense” refers to the fact that such oUgonucleotides are complementary to their inttaceUular targets, e.g., HOMPS. See for example, Jack Cohen, 1988, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitors of Gene Expression, CRC Press; and Synthesis 1:1-5 (1988).
- the HOMPS antisense oUgonucleotides of the present invention include derivatives such as S-oUgonucleotides (phosphorothioate derivatives or S-oUgos, see, Jack Cohen, supra), which exhibit enhanced cancer ceU growth inhibitory action.
- S-oUgos are isoelecttonic analogs of an oUgonucleotide (O-oUgo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom.
- the S-oUgos of the present invention may be prepared by treatment of the corresponding O-oUgos with 3H-l,2-benzodithiol-3-one-l,l-dioxide, which is a sulfur transfer reagent. See Iyer, R. P. et al, 1990, J. Org. Chem. 55:4693-4698; and Iyer, R. P. et al, 1990, J. Am. Chem. Soc. 112:1253-1254, the disclosures of which are fuUy incorporated by reference herein. Additional HOMPS antisense oUgonucleotides of the present invention include morphoUno antisense oUgonucleotides known in the art (see e.g.
- the HOMPS antisense oUgonucleotides of the present invention typicaUy may be RNA or DNA that is complementary to and stably hybridizes with the first 100 N-terminal codons or last 100 C-terminal codons of the HOMPS genomic sequence or the corresponding mRNA. WhUe absolute complementarity is not required, high degrees of complementarity are preferred. Use of an oUgonucleotide complementary to this region aUows for the selective hybridization to HOMPS mRNA and not to mRNA specifying other regulatory subunits of protein kinase.
- the HOMPS antisense oUgonucleotides of the present invention are a 15 to 30-mer fragment of the antisense DNA molecule having a sequence that hybridizes to HOMPS mRNA.
- HOMPS antisense oUgonucleotide is a 30-mer oUgonucleotide that is complementary to a region in the first 10 N-terminal codons and last 10 C- terminal codons of HOMPS.
- the antisense molecules are modified to employ ribozymes in the inhibition of HOMPS expression (L. A. Couture & D. T. Stinchcomb, 1996, Trends Genet. 12: 510-515).
- probes and primer pairs which aUow the specific ampUfication of the polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specificaUy hybridize to nucleic acid molecules of the invention or to any part thereof.
- Probes may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemUuminescent compound, metal chelator or enzyme.
- a detectable marker such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemUuminescent compound, metal chelator or enzyme.
- Such probes and primers can be used to detect the presence of a HOMPS polynucleotide in a sample and as a means for detecting a ceU expressing a HOMPS protein.
- probes include polypeptides comprising aU or part of the human HOMPS H41 cDNA sequences shown in FIG. 5.
- primer pairs capable of specificaUy ampUfying HOMPS mRNAs are also described in the Examples that foUow. As wiU be understood by the skiUed artisan, a great many different primers and probes may be prepared based on the sequences provided herein and used effectively to ampUfy and/ or detect a HOMPS mRNA.
- a polynucleotide is said to be "isolated” when it is substantiaUy separated from contaminant polynucleotides that correspond or are complementary to genes other than the HOMPS gene or that encode polypeptides other than HOMPS gene product or fragments thereof.
- a skiUed artisan can reacUly employ nucleic acid isolation procedures to obtain an isolated HOMPS polynucleotide.
- the HOMPS polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the ampUfication and/or detection of the HOMPS gene(s), mRNA(s), or fragments thereof; as reagents for the evaluation and/or diagnosis and/or prognosis of cancers; as coding sequences capable of directing the expression of HOMPS polypeptides; as tools for modulating or inhibiting the expression of the HOMPS gene(s) and/or translation of the HOMPS ttanscript(s); and as therapeutic agents.
- Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for deUvery of nucleotide sequences to the targeted organ, tissue or ceU population. Methods which are weU known to those skiUed in the art can be used to construct recombinant vectors which wiU express antisense molecules complementary to the polynucleotides of the gene encoding HOMPS. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
- HOMPS Genes encoding HOMPS can be turned off by ttansforming a ceU or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes HOMPS. Such constructs may be used to inttoduce untranslatable sense or antisense sequences into a ceU. 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-repUcating vector and even longer if appropriate repUcation elements are part of the vector system.
- modifications of gene expression can be obtained by designing antisense molecules, DNA, RNA, or PNA, to the control regions of the gene encoding HOMPS, i. e. the promoters, enhancers, and inteons.
- OUgonucleotides derived from the transcription initiation site e. g. , between positions -10 and +10 from the start site, are preferred.
- inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the abiUty of the double helix to open sufficiendy for the binding of polymerases, transcription factors, or regulatory molecules.
- the antisense molecules 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, foUowed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specificaUy and efficiendy catalyze endonucleolytic cleavage of sequences encoding HOMPS.
- Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites which include the foUowing sequences: GUA, GUU, and GUC.
- 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 oUgonucleotide inoperable.
- the suitabiUty of candidate targets may also be evaluated by testing accessibiUty to hybridization with complementary oUgonucleotides using ribonuclease protection assays.
- Antisense 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 chemicaUy synthesizing oUgonucleotides such as soUd phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HOMPS. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into ceU Unes, ceUs, or tissues.
- RNA molecules may be modified to increase inttaceUular stabiUty and half-Ufe.
- 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 aU of these molecules by the inclusion of nonttaditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easUy recognized by endogenous endonucleases.
- vectors into ceUs or tissues are avaUable and equaUy suitable for use in vivo, in vitro, and ex vivo.
- vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. DeUvery by transfection and by Uposome injections may be achieved using methods which are weU known in the art. Where appropriate, the methods described above may be appUed to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
- antibodies which specificaUy bind HOMPS may be used for the evaluation and characterization of conditions or diseases characterized by expression of HOMPS, or in assays to monitor patients being treated with HOMPS, 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 HOMPS include methods which utilize the antibody and a label to detect HOMPS in human body fluids or exttacts of ceUs or tissues.
- the antibodies may be used with or without modification, and may be labeled by joining them, either covalendy or non-covalendy, 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.
- HOMPS human immunosorbent assays
- normal or standard values for HOMPS expression are estabUshed by combining body fluids or ceU exttacts taken from normal mammaUan subjects, preferably human, with antibody to HOMPS under conditions suitable for complex formation.
- the amount of standard complex formation may be quantified by various methods, but preferably by photometric, means.
- Quantities of HOMPS expressed in subject samples, control and diseases from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
- the polynucleotides encoding HOMPS may be used for diagnostic purposes.
- the polynucleotides which may be used include oUgonucleotide sequences, antisense RNA and DNA molecules, and PNAs.
- the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HOMPS may be correlated with disease.
- the diagnostic assay may be used to distinguish between absence, presence, and excess expression of HOMPS, and to monitor regulation of HOMPS levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HOMPS or closely related molecules, may be used to identify nucleic acid sequences which encode HOMPS.
- 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. , especiaUy in the 3' coding region, and the stringency of the hybridization or ampUfication (maximal, high, intermediate, or low) wiU determine whether the probe identifies only naturaUy occurring sequences encoding HOMPS, aUeles, 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 HOMPS encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10 or from genomic sequence including promoter, enhancer elements, and inttons of the naturaUy occurring HOMPS.
- Means for producing specific hybridization probes for DNAs encoding HOMPS include the cloning of nucleic acid sequences encoding HOMPS or HOMPS derivatives into vectors for the production of mRNA probes.
- Such vectors are known in the art, commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate
- Hybridization probes may be labeled by a variety of reporter groups, for example, radionucUdes such as
- Polynucleotide sequences encoding HOMPS may be used for the evaluation and characterization of disorders associated with the expression of HOMPS. Examples of such disorders include: various types of cancer such as breast cancer.
- the polynucleotide sequences encoding HOMPS may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered HOMPS expression. Such quaUtative or quantitative methods are weU known in the art.
- the nucleotide sequences encoding HOMPS may be useful in assays that detect activation or induction of various cancers such as breast cancer, particularly those mentioned above.
- the nucleotide sequences encoding HOMPS 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 HOMPS 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.
- a normal or standard profile for expression is estabUshed. This may be accompUshed by combining body fluids or ceU exttacts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes HOMPS, under conditions suitable for hybridization or ampUfication. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantiaUy 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 estabUsh the presence of disease. Once disease is estabUshed and a treatment protocol is initiated, 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 evaluation and characterization of this type may aUow health professionals to employ preventative measures or aggressive treatment earUer thereby preventing the development or further progression of the cancer.
- oUgonucleotides designed from the sequences encoding HOMPS may involve the use of PCR.
- Such oUgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced from a recombinant source.
- OUgomers wiU preferably consist of two nucleotide sequences, one with sense orientation (5'— ?>') and another with antisense (3' ⁇ — 5'), employed under optimized conditions for identification of a specific gene or condition.
- the same two oUgomers, nested sets of oUgomers, or even a degenerate pool of oUgomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
- Another aspect of the present invention relates to methods for detecting HOMPS polynucleotides and HOMPS proteins and variants thereof, as weU as methods for identifying a ceU that expresses HOMPS.
- the expression profile of HOMPS makes it a potential diagnostic marker for local and/or metastasized disease.
- the status of HOMPS gene products may provide information useful for predicting a variety of factors including susceptibiUty to advanced stage disease, rate of progression, and/or tumor aggressiveness.
- the status of HOMPS gene products in patient samples may be analyzed by a variety protocols that are weU known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), western blot analysis and tissue array analysis. More particularly, the invention provides assays for the detection of HOMPS polynucleotides in a biological sample, such as breast or uterine tissue, serum, bone, prostate, and other tissues, urine, semen, ceU preparations, and the Uke.
- a biological sample such as breast or uterine tissue, serum, bone, prostate, and other tissues, urine, semen, ceU preparations, and the Uke.
- Detectable HOMPS polynucleotides include, for example, a HOMPS gene or fragments thereof, HOMPS mRNA, alternative spUce variant HOMPS mRNAs, and recombinant DNA or RNA molecules containing a HOMPS polynucleotide.
- a number of methods for ampUfying and/ or detecting the presence of HOMPS polynucleotides are weU known in the art and may be employed in the practice of this aspect of the invention.
- a method for detecting a HOMPS mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; ampUfying the cDNA so produced using a HOMPS polynucleotides as sense and antisense primers to ampUfy HOMPS cDNAs therein; and detecting the presence of the ampUfied HOMPS cDNA.
- the sequence of the ampUfied HOMPS cDNA can be determined.
- a method of detecting a HOMPS gene in a biological sample comprises first isolating genomic DNA from the sample; ampUfying the isolated genomic DNA using HOMPS polynucleotides as sense and antisense primers to ampUfy the HOMPS gene therein; and detecting the presence of the ampUfied HOMPS gene.
- HOMPS polynucleotides as sense and antisense primers to ampUfy the HOMPS gene therein
- Any number of appropriate sense and antisense probe combinations may be designed from the nucleotide sequences provided for the HOMPS (e.g. H41 as shown in FIG. 6) and used for this purpose.
- the invention also provides assays for detecting the presence of a HOMPS protein in a tissue of other biological sample such as breast or uterine tissue, serum, bone, prostate, and other tissues, urine, semen, ceU preparations, and the Uke.
- Methods for detecting a HOMPS protein are also weU known and include, for example, immunoprecipitation, immunohistochemical analysis, Western Blot analysis, molecular binding assays, ELISA, ELIFA and the Uke.
- a method of detecting the presence of a HOMPS protein in a biological sample comprises first contacting the sample with a HOMPS antibody, a HOMPS-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a HOMPS antibody; and then detecting the binding of HOMPS protein in the sample thereto.
- Methods for identifying a ceU that expresses HOMPS are also provided.
- an assay for identifying a ceU that expresses a HOMPS gene comprises detecting the presence of HOMPS mRNA in the ceU.
- Methods for the detection of particular mRNAs in ceUs include, for example, h) bridization assays using complementary DNA probes (such as in situ hybridization using labeled HOMPS riboprobes, Northern blot and related techniques) and various nucleic acid ampUfication assays (such as RT-PCR using complementary primers specific for HOMPS, and other ampUfication type detection methods, such as, for example, branched DNA, SISBA, TMA and the Uke).
- an assay for identifying a ceU that expresses a HOMPS gene comprises detecting the presence of HOMPS protein in the ceU or secreted by the ceU.
- HOMPS expression analysis may also be useful as a tool for identifying and evaluating agents that modulate HOMPS gene expression. Identification of a molecule or biological agent that could inhibit HOMPS expression or over- expression in cancer ceUs may be of therapeutic value. Such an agent may be identified by using a screen that quantifies HOMPS expression by RT-PCR, nucleic acid hybridization or antibody binding.
- Assays that evaluate the status of the HOMPS gene and HOMPS gene products in an individual may provide information on the growth or oncogenic potential of a biological sample from this individual. For example, because HOMPS are modulated by Her-2, an oncogene associated with a number of cancers, assays that evaluate the relative levels of HOMPS mRNA transcripts or proteins in a biological sample may be used to evaluate diseases associated with HOMPS disregulation such as cancer and may provide prognostic information useful in defining appropriate therapeutic options.
- HOMPS expression is modulated, for example, in ceUs which cv ⁇ rexpress the Her-2 oncogene
- the expression status of HOMPS can provide information useful for deterniining information including the presence, stage and location of displasic, precancerous and cancerous ceUs, predicting susceptibiUty to various stages of disease, and/or for gauging tumor aggressiveness.
- an important aspect of the mvention is directed to the various molecular methods for examining the status of HOMPS m biological samples such as those from individuals suffermg from, or suspected of suffermg from a pathology characterized by disregulated ceUular growth such as cancer.
- Oncogenesis is known to be a multistep process where ceUular growth becomes progressively disregulated and ceUs progress from a normal physiological state to precancerous and then cancerous states (see e.g. Alers et al, Lab Invest. 77(5): 437-438 (1997) and Isaacs et al, Cancer Surv. 23: 19-32 (1995))
- examining a biological sample for evidence of disregulated ceU growth can aUow the early detection of such aberrant ceUular physiology before a pathology such as cancer has progressed to a stage at which therapeutic options are more limited.
- the status of HOMPS m a biological sample of mterest can be compared, for example, to the status of HOMPS m a corresponding normal sample (e.g. a sample from that mdividual (or alternatively another mdividual) that is not effected by a pathology, for example one not suspected of havmg disregulated ceU growth) with alterations m the status of HOMPS in the biological sample of mterest (as compared to the normal sample) providing evidence of disregulated ceUular growth.
- a corresponding normal sample e.g. a sample from that mdividual (or alternatively another mdividual) that is not effected by a pathology, for example one not suspected of havmg disregulated ceU growth
- a predetermined normative value such as a predetermined normal level of mRNA expression (see e.g. Grever et al, J. Comp. Neurol. 1996 Dec 9,376(2):306-14 and U.S. patent No. 5,837,501) to compare HOMPS m normal versus suspect samples
- a predetermined normative value such as a predetermined normal level of mRNA expression
- the term "status" m this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. As specificaUy described herem, the status of HOMPS can be evaluated by a number of parameters known m the art.
- HOMPS an alteration m the status of HOMPS comprises a change m the location of HOMPS expressmg ceUs (as occurs in metastases) and/or an mcrease m HOMPS mRNA and/or protem expression.
- TypicaUy skiUed artisans use a number of parameters to evaluate the condition or state of a gene and its products. These mclude, but are not limited to the location of expressed gene products (mcludmg the location of HOMPS expressmg ceUs) as weU as the, level, and biological activity of expressed gene products (such as HOMPS mRNA polynucleotides and polypeptides). Alterations in the status of HOMPS can be evaluated by a wide variety of methodologies weU known in the art, typicaUy those discussed below. TypicaUy an alteration in the status of HOMPS comprises a change in the location of HOMPS and/or HOMPS expressing ceUs and/or an increase in HOMPS mRNA and/or protein expression.
- the status of HOMPS in a biological sample may be evaluated by a number of methods utilized by skilled artisans including, but not Limited to genomic Southern analysis (to examine, for example perturbations in the HOMPS gene), northerns and/or PCR analysis of HOMPS mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of HOMPS mRNAs), and western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of HOMPS proteins and/or associations of HOMPS proteins with polypeptide binding partners).
- genomic Southern analysis to examine, for example perturbations in the HOMPS gene
- northerns and/or PCR analysis of HOMPS mRNA to examine, for example alterations in the polynucleotide sequences or expression levels of HOMPS mRNAs
- western and/or immunohistochemical analysis to examine, for example alterations in polypeptide sequences, alterations in polypeptide local
- Detectable HOMPS polynucleotides include, for example, a HOMPS gene or fragments thereof, HOMPS mRNA, alternative spUce variants HOMPS mRNAs, and recombinant DNA or RNA molecules containing a HOMPS polynucleotide.
- HOMPS The expression profile of HOMPS makes them potential markers for disregulated ceU growth.
- the status of HOMPS may provide information useful for predicting susceptibiUty to particular disease stages, progression, and/ or tumor aggressiveness.
- the invention provides methods and assays for determining HOMPS status and diagnosing cancers that express HOMPS.
- HOMPS status in patient samples may be analyzed by a number of means weU known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, western blot analysis of clinical samples and ceU Unes, and tissue array analysis. Typical protocols for evaluating the status of the HOMPS gene and gene products can be found, for example in Ausubul et al.
- the status of HOMPS in a biological sample can be examined by a number of weU known procedures in the art.
- the status of HOMPS in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of HOMPS expressing ceUs (e.g. those that express HOMPS mRNAs or proteins).
- This examination can provide evidence of disregulated ceUular growth for example, when HOMPS expressing ceUs are found in a biological sample that does not normaUy contain such ceUs (such as a lymph node). Such alterations in the status of HOMPS in a biological sample are often associated with disregulated ceUular growth.
- SpecificaUy one indicator of disregulated ceUular growth is the metastases of cancer ceUs from an organ of origin (such as the breast) to a different area of the body (such as a lymph node).
- antibody is used in the broadest sense and specificaUy covers single anti-HOMPS monoclonal antibodies (including agonist, antagonist and neuttaUzing antibodies) and anti-HOMPS antibody compositions with polyepitopic specificity.
- monoclonal antibody mAb as used herein refers to an antibody obtained from a population of substantiaUy homogeneous antibodies, i.e. the antibodies comprising the individual population are identical except for possible naturaUy-occurring mutations that may be present in minor amounts.
- Another aspect of the invention provides antibodies that bind to HOMPS proteins and polypeptides.
- the most preferred antibodies wiU specificaUy bind to a HOMPS protein and wiU not bind (or wiU bind weakly) to non-HOMPS proteins and polypeptides.
- Anti-HOMPS antibodies that are particulady contemplated include monoclonal and polyclonal antibodies as weU as fragments containing the antigen binding domain and/or one or more complementarity determining regions of these antibodies.
- an antibody fragment is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen binding region.
- HOMPS antibodies of the invention may be particularly useful in imaging methodologies.
- InttaceUularly expressed antibodies may be therapeuticaUy useful in treating cancers in which the expression of HOMPS is involved, such as for example Her-2 overexpressing cancers.
- Such antibodies may- be useful in the analysis, treatment, evaluation and characterization, and/or prognosis of other cancers, to the extent HOMPS is also expressed or overexpressed in other types of cancers.
- the invention also provides various immunological assays useful for the detection and quantification of HOMPS and mutant HOMPS proteins and polypeptides.
- Such assays generaUy comprise one or more HOMPS antibodies capable of recognizing and binding a HOMPS or mutant HOMPS protein, as appropriate, and may be performed within various immunological assay formats weU known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the Uke.
- immunological imaging methods capable of detecting cancers expressing HOMPS are also provided by the invention, including but limited to radioscintigraphic imaging methods using labeled HOMPS antibodies.
- HOMPS antibodies may also be used in methods for purifying HOMPS and mutant HOMPS proteins and polypeptides and for isolating HOMPS homologues and related molecules.
- the method of purifying a HOMPS protein comprises incubating a HOMPS antibody, which has been coupled to a soUd matrix, with a lysate or other solution containing HOMPS under conditions that permit the HOMPS antibody to bind to HOMPS; washing the soUd matrix to eliminate impurities; and elating the HOMPS from the coupled antibody.
- Other uses of the HOMPS antibodies of the invention include generating anti-idiotypic antibodies that mimic the HOMPS protein.
- antibodies may be prepared by immunizing a suitable mammaUan host using a HOMPS protein, peptide, or fragment, in isolated or immunoconjugated form (Harlow, and Lane, eds., 1988, Antibodies: A Laboratory Manual, CSH Press; Harlow, 1989, Antibodies, Cold Spring Harbor Press, NY).
- HOMPS HOMPS GST- fusion protein
- a GST fusion protein comprising aU or most of the open reading frame amino acid sequence of H41 as shown in FIG. 6 may be produced and used as an immunogen to generate appropriate antibodies.
- a HOMPS peptide may be synthesized and used as an immunogen.
- naked DNA immunization techniques known in the art may be used (with or without purified HOMPS protein or HOMPS expressing ceUs) to generate an immune response to the encoded immunogen (for review, see DonneUy et al., 1997, Ann. Rev. Immunol. 15:617-648).
- the amino acid sequence of the H41 may be used (with or without purified HOMPS protein or HOMPS expressing ceUs) to generate an immune response to the encoded immunogen (for review, see DonneUy et al., 1997, Ann. Rev. Immunol. 15:617-648).
- HOMPS protein as shown in FIG. 6 may be used to select specific regions of the HOMPS protein for generating antibodies.
- hydrophobicity and hydrophiUcity analyses of the HOMPS amino acid sequence may be used to identify hydrophiUc regions in the HOMPS structure.
- Regions of the HOMPS protein that show immunogenic structure, as weU as other regions and domains, can reacUly be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-DooUtde, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.
- Methods for preparing a protein or polypeptide for use as an immunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other carrier proteins are weU known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be used; in other instances Unking reagents such as those suppUed by Pierce Chemical Co., Rockford, IL, may be effective.
- Administration of a HOMPS immunogen is conducted generaUy by injection over a suitable time period and with use of a suitable adjuvant, as is generaUy understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
- HOMPS monoclonal antibodies are preferred and may be produced by various means weU known in the art.
- immortalized ceU Unes that secrete a desired monoclonal antibody may be prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize producing B ceUs, as is generaUy known.
- the immortalized ceU Unes secreting the desired antibodies are screened by immunoassay in which the antigen is the HOMPS protein or a HOMPS fragment.
- the ceUs may be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
- the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specificaUy to the desired regions of the HOMPS protein can also be produced in the context of chimeric or CDR grafted antibodies of multiple species origin. Humanized or human HOMPS antibodies may also be produced and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences are weU known (see for example, Jones et al, 1986, Nature 321:522-525; Riechmann et al, 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536).
- FuUy human HOMPS monoclonal antibodies may be generated using cloning technologies employing large human Ig gene combinatorial Ubraries (i.e., phage display) (Griffiths and Hoogenboom, BuUding an in vitro immune system: human antibodies from phage display Ubraries. In: Clark, M., ed., 1993, Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic AppUcations in Man, Nottingham Academic, pp 45-64; Burton and Barbas, Human Antibodies from combinatorial Ubraries. Id., pp 65-82).
- FuUy human HOMPS monoclonal antibodies may also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent AppUcation W098/24893, Kucherlapati and Jakobovits et al, pubUshed December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7 (4): 607-614). This method avoids the in vitro manipulation required with phage display technology and efficiendy produces high affinity authentic human antibodies. 3
- Reactivity of HOMPS antibodies with a HOMPS protein may be estabUshed by a number of weU known means, including western blot, iiumunoprecipitation, ELISA, and FACS analyses using, as appropriate, HOMPS proteins, peptides, HOMPS-expressing ceUs or exttacts thereof.
- a HOMPS antibody or fragment thereof of the invention may be labeled with a detectable marker or conjugated to a second molecule.
- Suitable detectable markers include, but are not Limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemUuminescent compound, a metal chelator or an enzyme.
- a second molecule for conjugation to the HOMPS antibody can be selected in accordance with the intended use. For example, for therapeutic use, the second molecule can be a toxin or therapeutic agent.
- bi-specific antibodies specific for two or more HOMPS epitopes may be generated using methods generaUy known in the art. Homodimeric antibodies may also be generated by cross-Unking techniques known in the art (e.g., Wolff et al, 1993, Cancer Res. 53: 2560-2565).
- Nucleic acids that encode HOMPS or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeuticaUy useful reagents.
- a ttansgenic animal e.g., a mouse or rat
- ceUs that contain a ttansgene, which ttansgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.
- a transgene is a DNA that is integrated into the genome of a ceU from which a ttansgenic animal develops.
- cDNA encoding HOMPS can be used to clone genomic DNA encoding HOMPS in accordance with estabUshed techniques and the genomic sequences used to generate ttansgenic animals that contain ceUs that express DNA encoding HOMPS.
- Methods for generating ttansgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009.
- TypicaUy particular ceUs would be targeted for HOMPS ttansgene incorporation with tissue-specific enhancers.
- Transgenic animals that include a copy of a ttansgene encoding HOMPS introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding HOMPS.
- Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression.
- an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the ttansgene, would indicate a potential therapeutic intervention for the pathological condition.
- non-human homologues of HOMPS can be used to construct a HOMPS "knock out" animal that has a defective or altered gene encoding HOMPS as a result of homologous recombination between the endogenous gene encoding HOMPS and altered genomic DNA encoding HOMPS introduced into an embryonic ceU of the animal.
- cDNA encoding HOMPS can be used to clone genomic DNA encoding HOMPS in accordance with estabUshed techniques.
- a portion of the genomic DNA encoding HOMPS can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration.
- Idlobases of unaltered flanking DNA are included in the vector (see e.g., Thomas and Capecchi, 1987, CeU 51:503) for a description of homologous recombination vectors].
- the vector is introduced into an embryonic stem ceU line (e.g., by electroporation) and ceUs in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see e.g., Li et al, 1992, CeU 69:915).
- ceUs are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see e.g., Bradley, in Robertson, ed., 1987, Teratocarcinomas and Embryonic Stem CeUs: A Practical Approach, (IRL, Oxford), pp. 113-152).
- a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
- Progeny harboring the homologously recombined DNA in their germ ceUs can be identified by standard techniques and used to breed animals in which aU ceUs of die animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their abiUty to defend against certain pathological conditions and for their development of pathological conditions due to absence of the HOMPS polypeptide.
- HOMPS protein sequences disclosed herein aUow the skiUed artisan to identify molecules that interact with them via any one of a variety of art accepted protocols.
- molecules that interact reconstitute a transcription factor and direct expression of a reporter gene, the expression of which is then assayed.
- Typical systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic ttanscriptional activator and are disclosed for example in U.S. Patent Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.
- peptides that bind to selected receptor molecules such as HOMPS are identified by screening Ubraries that encode a random or conttoUed coUection of amino acids.
- Peptides encoded by the Ubraries are expressed as fusion proteins of bacteriophage coat proteins, and bacteriophage particles are then screened against the receptors of interest.
- Peptides having a wide variety of uses, such as therapeutic or diagnostic reagents may thus be identified without any prior information on the structure of the expected Ugand or receptor molecule.
- Typical peptide Ubraries and screening methods that can be used to identify molecules that interact with HOMPS protein sequences are disclosed for example in U.S. Patent Nos. 5,723,286 and 5,733,731.
- ceU Unes expressing HOMPS can be used to identify protein-protein interactions mediated by HOMPS. This possibiUty can be examined using immunoprecipitation techniques as shown by others (HamUton, B.J., et al, 1999, Biochem. Biophys. Res. Commun. 261:646-51).
- TypicaUy HOMPS protein can be immunoprecipitated from HOMPS expressing cancer ceU Unes using anti-HOMPS antibodies.
- antibodies against His-tag can be used in ceU line engineered to express HOMPS (vectors mentioned above).
- the immunoprecipitated complex can be examined for protein association by procedures such as western blotting, 35 S-methionine labeling of proteins, protein microsequencing, sUver staining and two dimensional gel electrophoresis.
- Screen assays include methods for identifying smaU molecules that interact with HOMPS. Typical methods are discussed for example in U.S. Patent No. 5,928,868 and include methods for forming hybrid Ugands in which at least one Ugand is a smaU molecule.
- the hybrid Ugand is introduced into ceUs that in turn contain a first and a second expression vector.
- Each expression vector includes DNA for expressing a hybrid protein that encodes a target protein Linked to a coding sequence for a ttanscriptional module.
- the ceUs further contains a reporter gene, the expression of which is conditioned on the proximity of the first and second hybrid proteins to each other, an event that occurs only if the hybrid Ugand binds to target sites on both hybrid proteins. Those ceUs that express the reporter gene are selected and the unknown smaU molecule or the unknown hybrid protein is identified.
- Methods which may also be used to quantitate the expression of HOMPS include radiolabeling or biotinylating nucleotides, coampUfication 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 oUgomer of interest is presented in various dUutions and a specttophotomettic or colorimettic response gives rapid quantitation.
- the nucleic acid sequences which encode HOMPS may also be used to generate hybridization probes which are useful for mapping the naturaUy occurring genomic sequence.
- the sequences may be mapped to a particular chromosome or to a specific region of the chromosome using weU known techniques.
- Such techniques include FISH, FACS, or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions or single chromosome cDNA Ubraries as reviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.
- FISH as described in Verma 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 ttanslocation, inversion, etc. among normal, carrier, or affected individuals.
- HOMPS its catalytic or immunogenic fragments or oUgopeptides thereof
- the fragment employed in such screening may be free in solution, affixed to a soUd support, borne on a ceU surface, or located intraceUularly.
- the formation of binding complexes, between HOMPS and the agent being tested, may be measured.
- HOMPS soUd substrate
- HOMPS soUd substrate
- HOMPS HOMPS
- Bound HOMPS is then detected by methods weU known in the art.
- Purified HOMPS can also be coated direcdy onto plates for use in the aforementioned drug screening techniques.
- non-neutraUzing antibodies can be used to capture the peptide and immobilize it on a soUd support.
- HOMPS 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 currendy known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- kits are also provided by the invention.
- Such kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the Uke, each of the container means comprising one of the separate elements to be used in the method.
- one of the container means may comprise a probe that is or can be detectably labeled.
- probe may be an antibody or polynucleotide specific for a HOMPS protein or a HOMPS gene or message, respectively.
- the kit may also have containers containing nucleotide(s) for ampUfication of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or stteptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.
- a reporter-means such as a biotin-binding protein, such as avidin or stteptavidin
- a reporter molecule such as an enzymatic, florescent, or radioisotope label.
- the kit of the invention wiU typicaUy comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, dUuents, filters, needles, syringes, and package inserts with instructions for use.
- a label may be present on the container to indicate that the composition is used for a specific therapy or non- therapeutic app
- CeUs were grown m RPMI medium 1640, supplemented with 10 % fetal bovine serum, 2mM glutamine, and 1% penicillin G-stteptomycin-fungizone solution. CeUs were harvested at 80 % confluency for total RNA extraction.
- RNA Total ceUular RNA was purified by guanidmium / cesium chloride ulttacenttifugation (16). Messenger RNA was isolated by two passages through an ohgo dT ceUulose column (T3 - Collaborative Research) (17). The quaUty and mRNA composition of the resulting RNA population were confirmed by Northern blot analysis by probmg with ⁇ -actin and HER-2 cDNAs. Both MCF- 7/control and MCF-7/HER2 mRNA pools contain equivalent, basal expression of the endogenous HER-2 transcript whereas the transcript representing transfected HER-2 cDNA was present only m the MCF-7/HER2 ceUs.
- the MCF-7/HER2 cDNA Ubrary was plated on XLl-Blue MRF' host ceUs at a density of 2,000 pfu per each of eight 150 mm petti dishes. After plating, actin and HER-2 clones purified from the same Ubrary were each loaded onto four designated spots withm the mdividual plates to be used as hybridization controls.
- the nitroceUulose filters (MiUipore) were placed on the agar plates 1.5 mm. for the first filter, 3 mm. for the second, and 7 mm. for the third.
- the phage DNA was denatured for 3 mm. m a solution containing 0.5 M NaOH 1.5 M NaCl, neutralized for 3 mm. m a solution containing 3 M NaCl 0.5
- 1XTEN buffer 0.1 M NaCl, 10 mM Tris-pH 8.0, 1 mM EDTA.
- Hybridization solution contained 50% formamide, 25X Denhardts, sonicated salmon sperm DNA, NaPO ⁇ pH 6.8, sodium pyrophosphate, and ribo
- ATP ATP. Prehybridization was performed at 42°C for 4 h. and hybridization at 42°C for 4 days (overnight hybridization for the third set of filters).
- FUters were washed at room temperature for 5 min. (x3) in 0.2xSSC/0.1%SDS, and at 60°C for 15 min. (x7) in the same solution. The washed filters were exposed with an intensifying screen to Kodak-XAR5 films at -70°C for various time periods.
- the primary screening procedure was repeated except that each clone was separately plated onto a 100 mm petti dish at a low density of 25-50 plaques/plate .
- the pBK-CMV phagemid was in vivo excised from the lamdaphage vector according to the manufacturer's instructions (Stratagene).
- the cDNA inserts were isolated from the plasmid either by restriction enzyme digestions or by PCR ampUfication using T3 and T7 sequences as primers.
- the purified cDNA inserts were random-labeled in a 50 ⁇ l reaction mix which contained 50 ng template, [ ⁇ - 2p] dCTP, 20 ⁇ g BSA, 6 U Klenow. Incorporated counts were eluted from a G-50 Sephadex spin column (Pharmacia). Approximately 3x10" dpm counts per 1 ml hybridization solution were used. The hybridization was carried out in 50% formamide, 2xSSC, 0.1% SDS, 10 mg/ml salmon sperm DNA, and 10% dextran sulfate, at 42°C for 16 hours.
- Membranes were washed in 2xSSC/0.1%SDS at 25°C for 10 min. (x3), and in the same solution at 65°C for 5 min. (x2). The washed membranes were exposed with an intensifying screen to Kodak-XAR film at -70°C.
- the cDNA inserts were translated into polypeptides in a TNT coupled reticulocyte system (Promega) according to the manufacturer's protocol ; Approximately 1 ⁇ g of purified plasmid template was transcribed and ttanslated in the 50 ⁇ l reaction containing T3 RNA polymerase, rabbit reticulocyte lysate, [ 35 S] methionine, etc. 5 ⁇ l of the end product was aUquoted to estimate the molecular size of the in vitto ttanslated protein using 10% SDS-PAGE and prestained protein size markers (Bio-Rad).
- RNA from breast tumor samples were obtained from patients at the time of surgery as part of a core tissue procurement resource sponsored by the DOD breast cancer program. AU tumor samples were snap frozen in Uquid nitrogen and kept at - 70°C before extraction of RNA. Frozen tissues were pulverized in Uquid nitrogen prior to homogenization in cold 4 M guanidine thiocyanate buffer (7.5 ml/g of tissue). The homogenates were centtifuged for 10 min. at 4°C at 8000 g in order to remove ceU debris.
- a total of 127 differentiaUy expressed clones were isolated including 77 C clones and 50 H clones representing genes whose expression levels are decreased (C clones) or increased (H clones) respectively in association with HER-2 overexpression.
- Each clone was ranked according to degree of differential hybridization based on signal intensity ranging from more than a five fold to less than a two fold change based on visualization.
- the MCF-7/HER-2 ceUs behave significandy differendy than their isogenic conttol parental counterparts; with increases in DNA synthesis, ceU growth in vitro, soft agar cloning efficiency and tumorigenicity.
- cytokeratin 8 C29
- cytokeratin 18 C49
- Oshima R. G., MiUan, J. L. and Cecena, G.
- HI 3 Three genes (H13, H14, H37) found to be overexpressed in the HER-2 overexpressing ceUs matched cDNA sequences which were previously identified by other investigators but not fuUy characterized.
- the HI 3 clone appears to be an alternate spUce variant of DNA fragmentation factor (DFF) (GenBank accession no. U91985) (Liu, X, Zou, H, Slaughter, C. and Wang, X. (1997) Cell, 89(2), 175-84); the first 261 amino acid sequences contained in both H13 and the DFF open reading frames are identical, but HI 3 lacks 70 amino acids at the 3' end and contains 7 different amino acids in their place.
- DFF DNA fragmentation factor
- the predictive amino acid sequence for HI 3 is highly simUar to the ICAD (Inhibitor of Caspase- Activated Dnase)- S and -L proteins (Sakahira, H., Enari, M. and Nagata, S. (1998) Nature, 391(6662), 96-9; Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. and Nagata, S. (1998) Nature, 391(6662), 43-50) (73% and 69% sequence identity, respectively).
- Both the HI 4 and DRP-1 cDNAs are Likely to be partial, 3' end sequences of a larger transcript since no suitable initiating codon was found in either sequences, and the HI 4 cDNA hybridized with an additional transcript of approximately 6.5 kb on Northern blot analysis.
- the 5' end of the HI 4 cDNA sequence can be extended, and the ESTs covering the extended portion of the gene is designated THC202438 (deposited in the Tentative Human Consensus effort) (Kirkness, E. F. and Kerlavage, A. R. (1997) Methods Mol Biol, 69, 261-8).
- the H37 cDNA sequence has been previously deposited into GenBank as RNA binding motif protein 5 (RBM5) (accession no.
- AF091263, unpubUshed found within a region reported to be homozygously deleted in lung cancer and beUeved to contain (a) major tumor suppressor gene(s) involved in a majority of smaU ceU and non-smaU ceU lung cancers (Wei, M. H., Latif, F., Bader, S., Kashuba, V., Chen, J. Y., Duh, F. M., Sekido, Y., Lee, C. C, GeU, L., Kuzmin, I., Zabarovsky, E., Klein, G., Zbar, B., Minna, J. D. and Lerman, M. I. (1996) Cancer Res, 56(7), 1487-92).
- the H37 cDNA contains an open reading frame of 816 amino acid and is ttanslated in mtro into a predicted protein product of approximately 90 kDa ( Figure 13).
- Analysis of the putative H37 protein against PROSITE protein profile databases recognized the presence of two RNA binding domains, located at amino acid residues 140-147 and 274-281, which are perfect matches with the consensus eukaryotic sequence for a putative RNA-binding region RNP-1 (BandziuUs, R. J., Swanson, M. S. and Dreyfuss, G. (1989) Genes Dev, 3(4), 431-7).
- C40, HI 7, H41, H63 Four clones (C40, HI 7, H41, H63) represented as-yet unknown genes in the DNA databases, and three of these (C40, H17, H41) were found to contain probable open reading frames.
- the 1750 bp long C40 clone contains a 510 a .iino acid open reading frame ( Figure 14A).
- the coding region of this gene begins with a start codon at nucleotide position 74 and has an in-frame stop codon at position 1604 ( Figure 14A).
- the C40 clone was in mtro - ttanslated into the predicted major protein product of approximately 55 kDa ( Figure 13).
- the 1981 bp long HI 7 clone contains a consensus initiation codon (Kozak, M. (1991) J Biol Chem, 266(30), 19867-70) at nucleotide 66 foUowed by a 486 amino acid open reading frame and a 458 bp 3' untranslated region mcludmg a polyadenylation signal (AATAAA) ( Figure 14B).
- the nucleic acid sequence around the 5' proximal AUG codon is typicaUy a Kozak consensus sequence where eukaryotic nbosomes initiate translation and the general rule the eukaryotic nbosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated only under rare conditions (see e.g. Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).
- In vitro translation generated the predicted protem product of approximately 50 kDa ( Figure 13).
- the putative HI 7 protem has 39.3% identity with a C.
- the 3346 bp H41 clone contams a 323 bp 5' untranslated region foUowed by an initiation codon with a Kozak consensus (Kozak, M. (1991) / Biol Chem, 266(30), 19867-70) and an extensive, 2249 bp 3' untranslated region (Figure 14C).
- the 258 ammo acid residues encoded by its open reading frame was ttanslated in mtro into the predicted protem product of approximately 30 kDa and an additional protem of lower molecular weight ( Figure 13).
- the putative H41 protem is related to one of the "fast evolving" drosoph a genes of unknown function (AF005858) (Schmid, K. J.
- the H41 gene product is predicted to be a nuclear protem based on the presence of a nuclear locaUzation signal, 4 basic ammo acid (lysine) residues, at its N-terminus (94.1% reUabiUty by Remhardt's method) (Reinhardt, A. and Hubbard, T. (1998) Nucleic Acids Res, 26(9), 2230-6) ( Figure 14C).
- wc could not find any ESTs which would extend our sequences furdier either at the 5' or 3' ends.
- the novel H63 clone is beUeved to be a partial, 3' sequence of a longer ttanscript because this sequence did not contain any probable open reading frames, and the 2068 bp DNA hybridized with a ttanscript of approxknatcly 4.5 kb on a Northern blot.
- die 5' end of the H63 cDNA sequence can be extended and assembled as THC175350.
- Table 1 Identity of differentially expressed clones
- Prefix "C” and “I I” denotes genes whose expression level decrease and increase, respectively, in MCF-7/HER-2 cells
- the amount of HER-2/ neu protem expressed was approximately 1.62 pg/ceU for MCF-7/HER-2 ceUs and 1.14 pg/ceU for the CaOv-3/HER-2 ceUs as compared to 0.36 pg/ceU and 0.41 pg/ceU for the conttol transfected ceUs respectively (Press, M. F., Pike, M. C, Chazm, V. R., Hung, G., Udove, J A , Markowicz, M., Danyluk, J., Godolphin, W., SUwkowski, M., Akita, R. and et al. (1993) Cancer Res, 53(20), 4960-70).
- the biologic changes mduced by HER-2/neu overexpression m the human ovarian cancer ceUs were simdar to those seen and described above m the human breast cancer ceUs (Chazm, N.R. (1991).
- the biologic effects of HER-2/ neu proto-oncogene overexpression Chapter 2. Department of Microbiology and Immunology, University of CaUfornia, Los Angeles).
- oUgonucleotide primers for extending a partial nucleotide sequence to fuU length or for obtaining 5' or 3', intron or other conttol sequences from genomic Ubraries.
- One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF).
- Primers are used to faciUtate the extension of the known sequence "outward" generating ampUcons containing new, unknown nucleotide sequence for the region of mterest.
- the initial primers are designed from the cDNA usmg OLIGO 4. 06 (National Biosciences), or another appropriate program to be 22-30 nucleotides m length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68 ° -72
- the original, selected cDNA Ubraries, or a human genomic Ubrary are used to extend the sequence; the latter is most useful to obtain 5' upstream regions. If more extension is necessary or desired, additional sets of primers are designed to further extend the known region.
- Peltier Thermal Cycler (PTC200; M. J. Research, Watertown, Mass. ) and the foUowmg parameters:
- Step 1 94 °C. for 1 mm (initial denaturation)
- Step 2 65 °C. for 1 mm
- Step 4 94 °C. for 15 sec
- Step 5 65 °C. for 1 mm
- Step 6 68 °C. for 7 mm
- Step 7 Repeat step 4-6 for 15 additional cycles
- Step 8 94 °C. for 15 sec
- Step 9 65 °C. for 1 mm
- Step 10 68°C. for 7:15 mm
- Step 11 Repeat step 8-10 for 12 cycles
- Step 12 72 °C. for 8 mm
- Step 13 4°C. (and holding) A 5-10 ⁇ l aUquot of the reaction mixture is 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 are selected and removed from the gel. Further purification involves using a commercial gel exttaction method such as the QIA QUICK kit (QIAGEN Inc. , Chatsworth, CaUf). After recovery of the DNA, Klenow enzyme is used to trim single-stranded, nucleotide overhangs creating blunt ends which faciUtate reUgation and cloning.
- QIA QUICK kit QIAGEN Inc. , Chatsworth, CaUf
- PCR ampUfication For PCR ampUfication, 18 ⁇ l of concentrated PCR reaction mix (3. 3. times. ) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction are added to each weU. AmpUfication is performed using the foUowing conditions:
- Step 2 94 °C. for 20 sec
- Step 3 55 °C. for 30 sec
- Step 4 72 °C. for 90 sec
- Step 5 Repeat steps 2-4 for an additional 29 cycles
- Step 6 72 °C. for 180 sec
- Step 7 4°C. (and holding) AUquots of the PCR reactions arc run on agarose gels together with molecular weight markers. The sizes of the PCR products are compared to the original partial cDNAs, and appropriate clones are selected, Ugated into plasmid, and sequenced.
- Hybridization probes derived from Figure 1, Figure 3, Figure 5, Figure 7, Figure 8 or Figure 10 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base- pairs, is specificaUy described, essentiaUy the same procedure is used with larger cDNA fragments. OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.
- a portion containing 10 7 counts per minute of each of the sense and antisense oUgonucleotides is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1 , or Pvu II; DuPont NEN).
- the DNA from each digest is fractionated on a 0. 7 percent agarose gel and ttansferred to nylon membranes (Nyttan Plus, Schleicher & SchueU, Durham, N. H. ). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentiaUy washed at room temperature under increasingly stringent conditions up to 0. 1. times, saline sodium cittate and 0. 5% sodium dodecyl sulfate. After XOMAT AR film Eastman Kodak Rochester, N. Y ) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale, CaUf.) for several hours, hybridization patterns are compared visuaUy.
- Phosphoimager cassette Molecular Dynamics, Sunnyvale, CaUf.
- Antisense molecules or nucleic acid sequences complementary to the HOMPS-encoding sequence, or any part thereof, are used to inhibit in vivo or in vitto expression of naturaUy occurring HOMPS.
- use of antisense oUgonucleotides, comprising about 20 base-pairs, is specificaUy described, essentiaUy the same procedure is used with larger cDNA fragments.
- An oUgonucleotide based on the coding sequences of HOMPS is used to inhibit expression of naturaUy occurring HOMPS.
- the complementary oUgonucleotide is designed from the most unique 5' sequence and used either to inhibit ttanscription by preventing promoter binding to the upstteam nonttanslated sequence or translation of an HOMPS-encoding ttanscript by preventing the ribosome from binding.
- an effective antisense oUgonucleotide includes any 15-20 nucleotides spanning the region which translates into the signal or 5' coding sequence of the polypeptide.
- HOMPS expression of HOMPS is accompUshed by subcloning the cDNAs into appropriate vectors and transforming the vectors into host ceUs.
- the cloning vector, pINCYl previously used for the generation of the cDNA Ubrary is used to express HOMPS in E. coU. Upstteam of the cloning site, this vector contains a promoter for ⁇ -galactosidase, foUowed by sequence containing the ammo-terminal Met, and the subsequent seven residues of ⁇ -galactosidase. I*nmediately foUowing these eight residues is a bacteriophage promoter useful for ttanscription 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 fuU length protein.
- the signal residues direct the secretion of HOMPS into the bacterial growth media which can be used direcdy in the foUowing assay for activity.
- HOMPS that is substantiaUy 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 Figure 1, Figure 3, Figure 5, Figure 8 or Figure 10 is analyzed using LASERGENE software (DNASTAR Inc) to determine regions of high immunogenicity and a corresponding oUgopolypeptide is synthesized and used to raise antibodies by means known to those of skiU in the art. Selection of appropriate epitopes, such as those near the C-terminus or in hydrophiUc regions, is described by Ausubel et al. (supra), and others.
- oUgopeptides are 15 residues in length, synthesized using an
- Rabbits are immunized with the oUgopeptide-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 radioiodinated, goat anti-rabbit IgG.
- HOMPS Media containing HOMPS is passed over the imrnunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of HOMPS (e. g. , high ionic strength buffers in the presence of detergent).
- the column is eluted under conditions that disrupt antibody/HOMPS binding (e.g., a buffer of pH 2-3 or a high concenttation of a chaottope, such as urea or thiocyanate ion), and HOMPS is coUected.
- HOMPS or biologicaUy active fragments thereof are labeled with 125 I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).
- Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled HOMPS, washed and any weUs with labeled HOMPS complex are assayed. Data obtained using different concentrations of HOMPS are used to calculate values for the number, affinity, and association of HOMPS with the candidate molecules.
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Abstract
Description
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU11929/01A AU777096B2 (en) | 1999-10-06 | 2000-10-06 | Differentially expressed genes associated with Her-2/neu overexpression |
CA002391805A CA2391805A1 (en) | 1999-10-06 | 2000-10-06 | Differentially expressed genes associated with her-2/neu overexpression |
EP00973424A EP1218394A4 (en) | 1999-10-06 | 2000-10-06 | Differentially expressed genes associated with her-2/neu overexpression |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US15792399P | 1999-10-06 | 1999-10-06 | |
US60/157,923 | 1999-10-06 |
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WO2001025250A1 true WO2001025250A1 (en) | 2001-04-12 |
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ID=22565913
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PCT/US2000/027649 WO2001025250A1 (en) | 1999-10-06 | 2000-10-06 | Differentially expressed genes associated with her-2/neu overexpression |
Country Status (4)
Country | Link |
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EP (1) | EP1218394A4 (en) |
AU (1) | AU777096B2 (en) |
CA (1) | CA2391805A1 (en) |
WO (1) | WO2001025250A1 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7526387B2 (en) | 2003-07-10 | 2009-04-28 | Genomic Health, Inc. | Expression profile algorithm and test for cancer prognosis |
US7569345B2 (en) | 2003-01-15 | 2009-08-04 | Genomic Health, Inc. | Gene expression markers for breast cancer prognosis |
US7622251B2 (en) | 2004-11-05 | 2009-11-24 | Genomic Health, Inc. | Molecular indicators of breast cancer prognosis and prediction of treatment response |
US7723033B2 (en) | 2003-06-24 | 2010-05-25 | Genomic Health, Inc. | Prediction of likelihood of cancer recurrence |
US7767391B2 (en) | 2003-02-20 | 2010-08-03 | Genomic Health, Inc. | Use of intronic RNA to measure gene expression |
US7838224B2 (en) | 2002-03-13 | 2010-11-23 | Genomic Health, Inc. | Gene expression profiling in biopsied tumor tissues |
US7871769B2 (en) | 2004-04-09 | 2011-01-18 | Genomic Health, Inc. | Gene expression markers for predicting response to chemotherapy |
US7930104B2 (en) | 2004-11-05 | 2011-04-19 | Genomic Health, Inc. | Predicting response to chemotherapy using gene expression markers |
US8008003B2 (en) | 2002-11-15 | 2011-08-30 | Genomic Health, Inc. | Gene expression profiling of EGFR positive cancer |
US8329398B2 (en) | 2003-12-23 | 2012-12-11 | Genomic Health, Inc. | Universal amplification of fragmented RNA |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1074617A3 (en) * | 1999-07-29 | 2004-04-21 | Research Association for Biotechnology | Primers for synthesising full-length cDNA and their use |
EP1248798A2 (en) * | 1999-08-18 | 2002-10-16 | Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. | Human dna sequences |
-
2000
- 2000-10-06 CA CA002391805A patent/CA2391805A1/en not_active Abandoned
- 2000-10-06 AU AU11929/01A patent/AU777096B2/en not_active Ceased
- 2000-10-06 EP EP00973424A patent/EP1218394A4/en not_active Withdrawn
- 2000-10-06 WO PCT/US2000/027649 patent/WO2001025250A1/en not_active Application Discontinuation
Non-Patent Citations (2)
Title |
---|
OH ET AL.: "Identification of differentially expressed genes associated with HER-2/neu overexpression in human breast cancer cells", NUCLEI ACIDS RESEARCH, vol. 27, no. 20, October 1999 (1999-10-01), pages 4008 - 4017, XP002937284 * |
See also references of EP1218394A4 * |
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US7838224B2 (en) | 2002-03-13 | 2010-11-23 | Genomic Health, Inc. | Gene expression profiling in biopsied tumor tissues |
US10241114B2 (en) | 2002-03-13 | 2019-03-26 | Genomic Health, Inc. | Gene expression profiling in biopsied tumor tissues |
US8071286B2 (en) | 2002-03-13 | 2011-12-06 | Genomic Health, Inc. | Gene expression profiling in biopsied tumor tissues |
US7858304B2 (en) | 2002-03-13 | 2010-12-28 | Genomic Health, Inc. | Gene expression profiling in biopsied tumor tissues |
US8008003B2 (en) | 2002-11-15 | 2011-08-30 | Genomic Health, Inc. | Gene expression profiling of EGFR positive cancer |
US8148076B2 (en) | 2002-11-15 | 2012-04-03 | Genomic Health, Inc. | Gene expression profiling of EGFR positive cancer |
US8741605B2 (en) | 2003-01-15 | 2014-06-03 | Genomic Health, Inc. | Gene expression markers for breast cancer prognosis |
US11220715B2 (en) | 2003-01-15 | 2022-01-11 | Genomic Health, Inc. | Gene expression markers for breast cancer prognosis |
US7569345B2 (en) | 2003-01-15 | 2009-08-04 | Genomic Health, Inc. | Gene expression markers for breast cancer prognosis |
US8034565B2 (en) | 2003-01-15 | 2011-10-11 | Genomic Health, Inc. | Gene expression markers for breast cancer prognosis |
US9944990B2 (en) | 2003-01-15 | 2018-04-17 | Genomic Health, Inc. | Gene expression markers for breast cancer prognosis |
US8206919B2 (en) | 2003-01-15 | 2012-06-26 | Genomic Health, Inc. | Gene expression markers for breast cancer prognosis |
US7767391B2 (en) | 2003-02-20 | 2010-08-03 | Genomic Health, Inc. | Use of intronic RNA to measure gene expression |
US7723033B2 (en) | 2003-06-24 | 2010-05-25 | Genomic Health, Inc. | Prediction of likelihood of cancer recurrence |
US10619215B2 (en) | 2003-06-24 | 2020-04-14 | Genomic Health, Inc. | Prediction of likelihood of cancer recurrence |
US7939261B2 (en) | 2003-07-10 | 2011-05-10 | Genomic Health, Inc. | Expression profile algorithm and test for cancer prognosis |
US7526387B2 (en) | 2003-07-10 | 2009-04-28 | Genomic Health, Inc. | Expression profile algorithm and test for cancer prognosis |
US8329398B2 (en) | 2003-12-23 | 2012-12-11 | Genomic Health, Inc. | Universal amplification of fragmented RNA |
US9605318B2 (en) | 2004-04-09 | 2017-03-28 | Genomic Health, Inc. | Gene expression markers for predicting response to chemotherapy |
US7871769B2 (en) | 2004-04-09 | 2011-01-18 | Genomic Health, Inc. | Gene expression markers for predicting response to chemotherapy |
US8868352B2 (en) | 2004-11-05 | 2014-10-21 | Genomic Health, Inc. | Predicting response to chemotherapy using gene expression markers |
US7622251B2 (en) | 2004-11-05 | 2009-11-24 | Genomic Health, Inc. | Molecular indicators of breast cancer prognosis and prediction of treatment response |
US7930104B2 (en) | 2004-11-05 | 2011-04-19 | Genomic Health, Inc. | Predicting response to chemotherapy using gene expression markers |
Also Published As
Publication number | Publication date |
---|---|
AU777096B2 (en) | 2004-09-30 |
CA2391805A1 (en) | 2001-04-12 |
EP1218394A4 (en) | 2004-04-14 |
EP1218394A1 (en) | 2002-07-03 |
AU1192901A (en) | 2001-05-10 |
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