WO1999019487A1 - Homologue de la mammoglobine - Google Patents

Homologue de la mammoglobine Download PDF

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Publication number
WO1999019487A1
WO1999019487A1 PCT/US1998/021729 US9821729W WO9919487A1 WO 1999019487 A1 WO1999019487 A1 WO 1999019487A1 US 9821729 W US9821729 W US 9821729W WO 9919487 A1 WO9919487 A1 WO 9919487A1
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WIPO (PCT)
Prior art keywords
hmh
sequence
sequences
mammoglobin
human
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PCT/US1998/021729
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English (en)
Inventor
Jennifer L. Hillman
Purvi Shah
Lynn E. Murry
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Incyte Pharmaceuticals, Inc.
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Application filed by Incyte Pharmaceuticals, Inc. filed Critical Incyte Pharmaceuticals, Inc.
Priority to EP98952309A priority Critical patent/EP1023443A1/fr
Priority to JP2000516039A priority patent/JP2001520015A/ja
Priority to AU98041/98A priority patent/AU9804198A/en
Publication of WO1999019487A1 publication Critical patent/WO1999019487A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TECHNICAL FIELD This invention relates to nucleic acid and amino acid sequences of a human mammoglobin homolog and to the use of these sequences in the diagnosis, prevention, and treatment of neoplastic disorders and endometriosis.
  • BACKGROUND OF THE INVENTION Cellular functions are controlled by the timing and amount of expression attributable to thousands of individual genes. The regulation of such expression is metabolically vital in that it conserves energy and prevents the synthesis and accumulation of intermediates when a protein is not needed. Disregulation of gene expression is also known to result in disease development or progression and has been linked to neoplastic disorders. Such disregulation may change in place, time, or amount of gene expression, and these changes may be monitored and used diagnostically. For example, different types of breast cancer (invasive, noninvasive. heritable, ductal, lymphatic, metastatic. etc.) can be differentiated by monitoring the expression of genes associated with the type and stage of the neoplasm.
  • Nucleic acids are especially useful in predicting predisposition for breast cancer while antibodies to marker proteins may be used to test for the presence or stage of the disease state.
  • Changes in the expression pattern of BRC A markers have been associated with metastatic breast tumors and linked to familial susceptibility for developing breast cancer.
  • the widely known marker, BRCA1 has been mapped to 17q 21 (Hall, J.M. et al. (1990) Science 250:1584-9), and the more recently characterized marker.
  • BRCA2 has been mapped to 13q 12-13 (Fischer, S.G. et al. (1996) Proc. Natl. Acad. Sci. 93:690-694).
  • mammoglobin a mammary-specific steroid binding protein of the uteroglobin gene family.
  • the cDNA encoding mammoglobin was initially cloned from a primary human breast adenocarcinoma. and expression has been reported to be restricted to adult mammary tissue. Analyses of breast tumor biopsies showed that mRNA levels were 10-fold greater in tumor than in normal breast tissue (Watson. M.A. et al. (1996) Cancer Res. 56:860-65).
  • Another disease condition known to be linked to steroid-driven cell proliferation is endometriosis.
  • Endometrial tissue proliferation is a normal part of the monthly menstrual cycle; however, when endometrial cells become dislodged from the uterus, move into the abdomen and proliferate, they cause endometriosis. This condition may go undetected for months to years and cause considerable systemic distress by causing fibrosis throughout the pelvic cavity, sterility due to closure of the fallopian tubes, and monthly internal bleeding of variable volume and severity.
  • gonadotropin inhibitors such as danazol is effective in mild cases or in older women, surgery is often necessary to remove endometrial cells from the abdominal cavity or around the ovaries/mouth of the fallopian tube(s) prior to administration of pharmacological remedies.
  • the invention features a substantially purified polypeptide, human mammoglobin homolog (HMH), having the amino acid sequence shown in SEQ ID NO:l, or fragments thereof.
  • HMH human mammoglobin homolog
  • the invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:l or fragments thereof and a composition comprising said polynucleotide sequence.
  • the invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:l, or fragments of said polynucleotide sequence.
  • the invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:l, or fragments or variants of said polynucleotide sequence.
  • the invention also provides an isolated and purified sequence comprising SEQ ID NO:
  • the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
  • the invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2. or fragments or variants thereof.
  • the present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences.
  • the expression vector containing the polynucleotide sequence is contained within a host cell.
  • the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:l or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding HMH under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a substantially purified HMH having the amino acid sequence of SEQ ID NOT in conjunction with a suitable pharmaceutical carrier.
  • the invention also provides a purified antagonist of the polypeptide of SEQ ID NOT .
  • the invention also provides a method for diagnosing endometriosis comprising the steps of: a) obtaining abdominal fluid from a subject; b) combining an antibody to HMH with the abdominal fluid under conditions suitable for antibody binding; and c) detecting bound human mammoglobin homolog, wherein the presence of the bound molecule indicates the presence of endometriosis. Still further, the invention provides a purified agonist of the polypeptide of SEQ ID
  • the invention also provides a method for treating or preventing a neoplastic disorder comprising administering to a subject in need of such treatment an effective amount of an antagonist of HMH.
  • the invention also provides a method for treating or preventing endometriosis comprising administering to a subject in need of such treatment an effective amount of an antagonist of HMH.
  • the invention also provides a method for detecting a polynucleotide which encodes HMH in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NOT to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding HMH in the biological sample.
  • the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.
  • Figures 1 A and IB show the amino acid sequence (SEQ ID NOT) and nucleic acid sequence (SEQ ID NO:2) of human mammoglobin homolog, HMH. The alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co. Ltd. San Bruno, CA). 1. Figure 2 shows the amino acid sequence alignments between
  • HMH (2295453; SEQ ID NO NOT), and human mammoglobin (GI 1 199596; SEQ ID NO:
  • FIG 3 shows the northern analysis for HMH. produced electronically using LIFESEQTM database (Incyte Pharmaceuticals. Inc., Palo Alto. CA).
  • HMH refers to the amino acid sequences of substantially purified HMH obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which, when bound to HMH, increases or prolongs the duration of the effect of HMH.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of HMH.
  • Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none. one. or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding HMH as used herein include those with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent HMH. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HMH, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HMH.
  • the encoded protein may also be "altered” and contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HMH.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of HMH is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine. and valine, glycine and alanine, asparagine and glutamine, serine and threonine. and phenylalanine and tyrosine.
  • amino acid sequence refers to an oligopeptide, peptide. polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Fragments of HMH are preferably about 5 to about 15 amino acids in length and retain the biological activity or the immunological activity of HMH. Where "amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which, when bound to HMH, decreases the amount or the duration of the effect of the biological or immunological activity of HMH. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies or any other molecules which decrease the effect of HMH.
  • the term "antibody” refers to intact molecules as well as fragments thereof, such as Fa, F(ab') 2 , and Fv, which are capable of binding the epitopic determinant.
  • Antibodies that bind HMH polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • antigenic determinant refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
  • Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation “'negative” is sometimes used in reference to the antisense strand, and “positive " is sometimes used in reference to the sense strand.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant. or synthetic HMH. or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A”.
  • Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • composition comprising a given polynucleotide sequence refers broadly to any composition containing the given polynucleotide sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding HMH (SEQ ID NOT) or fragments thereof (e.g., SEQ ID NO:2 and fragments thereof) may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS) and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA. etc.).
  • salts e.g., NaCl
  • detergents e.g., SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA. etc.
  • Consensus refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, has been extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' direction and resequenced, or has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly (e.g., GEL VIEWTM Fragment Assembly system, GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence .
  • correlates with expression of a polynucleotide indicates that the detection of the presence of ribonucleic acid that is similar to SEQ ID NO:2 by northern analysis is indicative of the presence of mRNA encoding HMH in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.
  • a “deletion”, as used herein. refers to a change in the amino acid or nucleotide sequence and results in the absence of one or more amino acid residues or nucleotides.
  • the term “derivative”, as used herein. refers to the chemical modification of a nucleic acid encoding or complementary to HMH or the encoded HMH. Such modifications include. for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a nucleic acid derivative encodes a polypeptide which retains the biological or immunological function of the natural molecule.
  • a derivative polypeptide is one which is modified by glycosylation, pegylation. or any similar process which retains the biological or immunological function of the polypeptide from which it was derived.
  • ' refers to a degree of complementarity. There may be partial homology or complete homology (i.e.. identity). A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid 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 hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of low stringency.
  • HACs Human artificial chromosomes
  • humanized antibody refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.
  • hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases: these hydrogen bonds may be further stabilized by base stacking interactions.
  • the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R ⁇ t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion * ' or “addition" refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.
  • Microarray refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • modulate refers to a change in the activity of HMH. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional or immunological properties of HMH.
  • 'Nucleic acid sequence ' as used herein refers to an oligonucleotide. nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • “Fragments” are those nucleic acid sequences which are greater than 60 nucleotides than in length, and most preferably includes fragments that are at least 100 nucleotides or at least 1000 nucleotides, and at least 10,000 nucleotides in length.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides. and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or a hybridization assay, or a microarray. As used herein. oligonucleotide is substantially equivalent to the terms “amplimers”, “primers”, “oligomers”, and “probes”, as commonly defined in the art.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues which ends in lysine. The terminal lysine confers solubility to the composition.
  • PNAs may be pegvlated to extend their lifespan in the ceil where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
  • portion refers to fragments of that protein.
  • the fragments may range in size from five amino acid residues to the entire amino acid sequence minus one amino acid.
  • a protein "comprising at least a portion of the amino acid sequence of SEQ ID NOT" encompasses the full-length HMH and fragments thereof.
  • sample is used in its broadest sense.
  • a biological sample suspected of containing nucleic acid encoding HMH. or fragments thereof, or HMH itself may comprise a bodily fluid, extract from a cell, chromosome, organelle. or membrane isolated from a cell, a cell, genomic DNA, RNA. or cDNA(in solution or bound to a solid support, a tissue, a tissue print, and the like.
  • 'specific binding * ' or “specifically binding”, as used herein, refers to that interaction between a protein or peptide and an agonist, an antibody and an antagonist.
  • the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) of the protein recognized by the binding molecule. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A” and the antibody will reduce the amount of labeled A bound to the antibody.
  • a particular structure i.e., the antigenic determinant or epitope
  • stringent conditions refer to the conditions for hybridization as defined by the nucleic acid, salt, and temperature. These conditions are well known in the art and may be altered in order to identify or detect identical or related polynucleotide sequences. Numerous equivalent conditions comprising either low or high stringency depend on factors such as the length and nature of the sequence (DNA, RNA, base composition), nature of the target (DNA. RNA, base composition), milieu (in solution or immobilized on a solid substrate), concentration of salts and other components (e.g., formamide.
  • substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • Transformation describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation. heat shock, lipofection, and particle bombardment. Such "transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
  • a “variant" of HMH refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
  • the invention is based on the discovery of a new human mammoglobin homolog
  • ⁇ MH ⁇ -MH
  • polynucleotides encoding HMH the polynucleotides encoding HMH. and the use of these compositions for the diagnosis, prevention, or treatment of neoplastic disorders and endometriosis.
  • Nucleic acids encoding the HMH of the present invention were first identified in Incyte Clone 2295453 from a breast cDNA library (BRSTNOT05) using a computer search for amino acid sequence alignments. A consensus sequence. SEQ ID NO:2. was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2295453 (BRSTNOT05), 2381443 (ISLTNOTOl), and 2600564 (UTRSNOT10).
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NOT, as shown in Figs. 1A and IB.
  • HMH is 95 amino acids in length and has one potential N-glycosylation site at N 68 QSH and four potential phosphorylation sites at S 38 IPE, S 70 HR, T 73 LK, and T 83 VYD.
  • HMH also displays the D 51 or D 53 which acts as the "cap” for calcium binding in uteroglobins.
  • HMH has chemical and structural homology with human mammoglobin (GI 1199596; SEQ ID NO:3).
  • HMH and human mammoglobin share 57% identity.
  • Northern analysis of HMH ( Figure 3) shows expression in various libraries; 86% of which were from breast tissues and 52% of the which are from breast tumors. Of particular note was the expression of HMH in endometrial cells of the uterus
  • the invention also encompasses HMH variants.
  • a preferred HMH variant is one having at least 80%, and more preferably at least 90%, amino acid sequence identity to the HMH amino acid sequence (SEQ ID NOT) and which retains at least one biological, immunological or other functional characteristic or activity of HMH.
  • a most preferred HMH variant is one having at least 95% amino acid sequence identity to SEQ ID NOT.
  • the invention also encompasses polynucleotides which encode HMH. Accordingly, any nucleic acid sequence which encodes the amino acid sequence of HMH can be used to produce recombinant molecules which express HMH. In a particular embodiment, the invention encompasses the polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 as shown in Figs. 1A and IB.
  • nucleotide sequences encoding HMH may be produced.
  • the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring HMH, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences which encode HMH and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HMH under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HMH or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences, or fragments thereof, which encode HMH and its derivatives, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HMH or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those shown in SEQ ID NO:2, under various conditions of stringency as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel. A.R. (1987; Methods Enzymol. 152:507- 511).
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham. Chicago, IL). or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE Amplification System marketed by GIBCO/BRL (Gaithersburg, MD).
  • the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton. Reno. NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown. MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • the nucleic acid sequences encoding HMH 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.
  • various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • one method which may be employed "restriction-site "" PCR. uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
  • genomic DNA is first amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
  • the primers may be designed using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison. MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1 : 1 1 1-1 19).
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1 : 1 1 1-1 19).
  • multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Another method which may be used to retrieve unknown sequences is that of Parker,
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g. GenotyperTM and Sequence NavigatorTM. Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode HMH may be used in recombinant DNA molecules to direct expression of HMH. fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced, and these sequences may be used to clone and express HMH.
  • HMH-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HMH 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 oligonucleotides may be used to engineer the nucleotide sequences.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • nucleic acid sequences encoding HMH may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence For example, to screen peptide libraries for inhibitors of HMH activity, it may be useful to encode a chimeric HMH protein that can be recognized by a commercially available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the HMH encoding sequence and the heterologous protein sequence, so that HMH may be cleaved and purified away from the heterologous moiety.
  • sequences encoding HMH may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M.H.
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of HMH. or a fragment thereof.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge, J.Y. et al. (1995) Science
  • the newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins. Structures and Molecular Principles. WH Freeman and Co., New York, NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton. supra).
  • the amino acid sequence of HMH, or any part thereof may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • nucleotide sequences encoding HMH 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.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • the invention is not limited by the host cell employed.
  • control elements are those non-translated regions of the vector— enhancers, promoters, 5' and 3' untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla. CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla. CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock. RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding HMH, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock. RUBISCO; and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • a number of expression vectors may be selected depending upon the use intended for HMH.
  • vectors which direct high level expression of fusion proteins that are readily purified may be used.
  • Such vectors include. but are not limited to. the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene). in which the sequence encoding HMH may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH
  • PGH palidylcholine
  • the expression of sequences encoding HMH may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680: Broglie, R. et al. (1984) Science 224:838-843; and Winter. J. et al. (1991) Results Probl. Cell Differ. 17:85-105).
  • These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill. New York, NY; pp. 191-196.
  • An insect system may also be used to express HMH.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding HMH may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of HMH will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example.
  • S_. frugiperda cells or Trichoplusia larvae in which HMH may be expressed Engelhard, E.K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227).
  • a number of viral-based expression systems may be utilized.
  • sequences encoding HMH may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing HMH in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81 :3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6 to 10M are constructed and delivered via conventional delivery methods (liposomes. polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HMH. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding HMH. its initiation codon. and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including the ATG initiation codon should be provided.
  • the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation. phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro " ' form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293. and WI38), are available from the American Type Culture Collection (ATCC; Bethesda. MD) and may be chosen to ensure the correct modification and processing of the foreign protein. For long-term, high-yield production of recombinant proteins, stable expression is preferred.
  • cell lines which stably express HMH may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The pu ⁇ ose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines.
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, t ⁇ B, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci.
  • host cells which contain the nucleic acid sequence encoding HMH and express HMH may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
  • polynucleotide sequences encoding HMH can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding HMH.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding HMH to detect transformants containing DNA or RNA encoding HMH.
  • a variety of protocols for detecting and measuring the expression of HMH. using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HMH is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual. APS Press. St Paul, MN) and Maddox, D.E. et al. (1983; J. Exp. Med. 158:1211-1216).
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HMH include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the sequences encoding HMH. or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP.
  • reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent. or chromogenic agents as well as substrates, cofactors. inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding HMH may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode HMH may be designed to contain signal sequences which direct secretion of HMH through a prokaryotic or eukaryotic cell membrane.
  • Other constructions may be used to join sequences encoding HMH to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • purification facilitating domains include. but are not limited to.
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin. and the domain utilized in the FLAGS extension/affinity purification system (Immunex Co ⁇ ., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and HMH may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing HMH and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography as described in Porath, J. et al. (1992. Prot. Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides a means for purifying HMH from the fusion protein.
  • IMAC immobilized metal ion affinity chromatography as described in Porath, J. et al. (1992. Prot. Exp. Purif. 3: 263-281
  • enterokinase cleavage site provides a means for purifying HMH from the fusion protein.
  • fragments of HMH may be produced by direct peptide synthesis using solid-phase techniques Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of HMH may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • HMH is expressed in breast tumors and in endometrial cells of the uterus, and appears to play a role in steroid- responsive cell proliferation, particularly neoplastic disorders and endometriosis.
  • an antagonist of HMH may be administered to a subject to prevent or treat neoplastic disorders.
  • disorders may include. but are not limited to, adenocarcinoma, leukemia, lymphoma. melanoma, myeloma, sarcoma, and teratocarcinoma.
  • cancers include. but are not limited to, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus. thyroid, and uterus.
  • an antibody which specifically binds HMH may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express
  • a vector expressing the complement of the polynucleotide encoding HMH may be administered to a subject to treat or prevent neoplastic disorders including, but not limited to, those described above.
  • an antagonist of HMH may be administered to a subject to prevent or treat endometriosis.
  • an antibody which specifically binds HMH may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HMH.
  • a vector expressing the complement of the polynucleotide encoding HMH may be administered to a subject to treat or prevent endometriosis.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of HMH may be produced using methods which are generally known in the art.
  • purified HMH may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HMH.
  • Antibodies to HMH may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with HMH or any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides. oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corvnebacterium parvum are especially preferable.
  • the oligopeptides, peptides. or fragments used to induce antibodies to HMH have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HMH amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Monoclonal antibodies to HMH may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for HMH may also be generated.
  • fragments include, but are not limited to. the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 254:1275-1281).
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between HMH and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HMH epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra).
  • the polynucleotides encoding HMH, or any fragment or complement thereof may be used for therapeutic pu ⁇ oses.
  • the complement of the polynucleotide encoding HMH may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding HMH.
  • complementary molecules or fragments may be used to modulate HMH activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HMH.
  • Expression vectors derived from retro viruses, adenovirus, he ⁇ es or vaccinia viruses. or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence which is complementary to the polynucleotides of the gene encoding HMH. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
  • Genes encoding HMH can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes HMH. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA. RNA, or PNA) to the control, 5' or regulatory regions of the gene encoding HMH (signal sequence, promoters, enhancers, and introns). Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al.
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA. followed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HMH.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC.
  • 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 oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HMH. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP..
  • these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to. the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections or polycationic amino polymers (Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-66; inco ⁇ orated herein by reference) may be achieved using methods which are well known in the art.
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of HMH, antibodies to HMH, mimetics, agonists, antagonists, or inhibitors of HMH.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal. intraventricular. transdermal. subcutaneous, intraperitoneal, intranasal, enteral. topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone. agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e.. dosage.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate. and. optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol. or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to. hydrochloric, sulfuric, acetic, lactic, tartaric. malic, succinic. etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7%) mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g.. of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio. LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age. weight, and gender of the subject, diet, time and frequency of administration, drug combination(s). reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100.000 micrograms. up to a total dose of about 1 g. depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind HMH may be used for the diagnosis of conditions or diseases characterized by expression of HMH, or in assays to monitor patients being treated with HMH, agonists, antagonists or inhibitors.
  • the antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for HMH include methods which utilize the antibody and a label to detect HMH in human body fluids or extracts of cells or tissues. In particular, adominal fluid from a subject is combined with the antibody under conditions suitable for binding; and detection of bound human mammoglobin homolog determines the presence of endometriosis in the subject.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used, several of which are described above.
  • HMH human-derived mammalian alian alian adsorbent
  • ELISA RIA
  • FACS fluorescence-activated cell sorting
  • the polynucleotides encoding HMH may be used for diagnostic pu ⁇ oses.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HMH may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of HMH, and to monitor regulation of HMH levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HMH or closely related molecules, may be used to identify nucleic acid sequences which encode HMH.
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory region, or a less specific region, e.g., especially in the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding HMH, alleles. or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the HMH encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO:2 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring HMH.
  • Means for producing specific hybridization probes for DNAs encoding HMH include the cloning of nucleic acid sequences encoding HMH or HMH derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding HMH may be used for the diagnosis of conditions or disorders which are associated with expression of HMH.
  • conditions or disorders include neoplastic disorders such as adenocarcinoma. leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma.
  • cancers include, but are not limited to, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus.
  • HMH thyroid, and uterus and disorders of cell proliferation which are responsive to steroids such as endometriosis.
  • the polynucleotide sequences encoding HMH may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dipstick, pin, ELISA assays or microarrays utilizing fluids or tissues from patient biopsies to detect altered HMH expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding HMH may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above.
  • the nucleotide sequences encoding HMH 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 HMH 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 established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes HMH, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
  • hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • oligonucleotides designed from the sequences encoding HMH may involve the use of PCR.
  • Such oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'->3') and another with antisense (3' ⁇ -5'), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers. nested sets of oligomers. or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of HMH include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are inte ⁇ olated (Melby, P.C. et al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).
  • the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants, mutations and polymo ⁇ hisms. This information may be used to determine gene function, understanding the genetic basis of disease, diagnosing disease, and in developing and in monitoring the activities of therapeutic agents.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants, mutations and polymo ⁇ hisms. This information may be used to determine gene function, understanding the genetic basis of disease, diagnosing disease, and in developing and in monitoring the activities of therapeutic agents.
  • the microarray is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614- 10619), all of which are inco ⁇ orated herein in their entirety by reference.
  • the microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support.
  • the oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray, it may be preferable to use oligonucleotides which are only 7-10 nucleotides in length.
  • the microarray may contain oligonucleotides which cover the known 5', or 3', sequence, or contain sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence.
  • Polynucleotides used in the microarray may be oligonucleotides that are specific to a gene or genes of interest in which at least a fragment of the sequence is known or that are specific to one or more unidentified cDNAs which are common to a particular cell or tissue type or to a normal, developmental, or disease state. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray.
  • the "pairs '* will be identical, except for one nucleotide which preferably is located in the center of the sequence.
  • the second oligonucleotide in the pair serves as a control.
  • the number of oligonucleotide pairs may range from 2 to one million.
  • the gene of interest is examined using a computer algorithm which starts at the 5' or more preferably at the 3' end of the nucleotide sequence.
  • the algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • the oligonucleotides may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is inco ⁇ orated herein in its entirety by reference.
  • a "gridded" array analogous to a dot or slot blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • an array may be produced by hand or using available devices, materials, and machines (including Brinkmann® multichannel pipettors or robotic instruments) and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other multiple from 2 to one million which lends itself to the efficient use of commercially available instrumentation.
  • polynucleotides are extracted from a biological sample.
  • the biological samples may be obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • the polynucleotides extracted from the sample are used to produce nucleic acid sequences which are complementary to the nucleic acids on the microarray.
  • the microarray consists of cDNAs
  • antisense RNAs aRNA
  • mRNA is used to produce cDNA which, in turn and in the presence of fluorescent nucleotides, is used to produce fragment or oligonucleotide aRNA probes.
  • fluorescently labeled probes are incubated with the microarray so that the probe sequences hybridize to the cDNA oligonucleotides of the microarray.
  • complementary nucleic acid sequences are used as probes and can also include polynucleotides. fragments, complementary, or antisense sequences produced using restriction enzymes. PCR technologies, and Oligolabeling or TransProbe kits (Pharmacia) well known in the area of hybridization technology.
  • Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity.
  • a scanner is used to determine the levels and patterns of fluorescence.
  • the scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray.
  • a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies or functional analysis of the sequences, mutations, variants, or polymo ⁇ hisms among samples (Heller. R.A. et al., (1997) Proc. Natl. Acad. Sci. 94:2150-55).
  • the nucleic acid sequences which encode HMH may also be used to generate hybridization probes which are useful for mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome or to artificial chromosome constructions, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial chromosome cDNA libraries as reviewed in Price, CM. (1993) Blood Rev. 7:127-134, and Trask, B.J. (1991) Trends Genet. 7:149-154.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries as reviewed in Price, CM. (1993) Blood Rev. 7:127-134, and Trask, B.J. (1991) Trends Genet.
  • Fluorescent in situ hybridization FISH as described in Verma et al. (1988) Human Chromosomes: A. Manual of Basic Techniques. Pergamon Press, New York, NY
  • FISH Fluorescent in situ hybridization
  • OMIM Online Mendelian Inheritance in Man
  • Correlation between the location of the gene encoding HMH on a physical chromosomal map and a specific disease , or predisposition to a specific disease may help delimit the region of DNA associated with that genetic disease.
  • the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 lq22-23 (Gatti. R.A. et al.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • HMH its catalytic or immunogenic fragments or oligopeptides thereof
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between HMH and the agent being tested, may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • HMH large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with HMH. or fragments thereof, and washed.
  • Bound HMH is then detected by methods well known in the art.
  • Purified HMH can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • nucleotide sequences which encode HMH may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • the BRSTNOT05 cDNA library was constructed from microscopically normal breast tissue obtained from a 58-year-old Caucasian female who had undergone an unilateral extended simple mastectomy following diagnosis of multicentric invasive grade 4 mammary lobular carcinoma. .
  • the nontumorous breast tissue, the skin, nipple, and fascia were uninvolved. No evidence of vascular invasion was found. Eight mid low and two high left axillary lymph nodes were negative for tumor. Prior to surgery, the patient was prescribed tamoxifen to inhibit the induction of mammary carcinoma.
  • patient medications included Zantac® (ranitidine hydrochloride; Glaxo Wellcome, Inc., Research Triangle Park, NC), aspirin, extra-strength Tylenol® (acetaminophen; McNeil Consumer Products Company. Fort Washington, PA), and vitamin C.
  • Zantac® ranitidine hydrochloride; Glaxo Wellcome, Inc., Research Triangle Park, NC
  • aspirin extra-strength Tylenol® (acetaminophen; McNeil Consumer Products Company. Fort Washington, PA)
  • the patient history showed previous diagnoses of skin cancer, cerebrovascular disease, atherosclerosis, rheumatic heart disease, and osteoarthritis.
  • Family history included breast cancer in the mother and prostate cancer in the brother.
  • the frozen tissue was homogenized and lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments. Westbury. NY) in guanidinium isothiocyanate solution.
  • the lysate was centrifiiged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 ⁇ m at ambient temperature.
  • the RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol. resuspended in RNAse-free water, and treated with DNase at 37°C. The RNA extraction and precipitation were repeated.
  • the mRNA was isolated with the Qiagen Oligotex kit (QIAGEN, Inc.; Chatsworth, CA) and used to construct the cDNA library.
  • the mRNA was handled according to the recommended protocols in the Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Catalog #18248-013, Gibco/BRL).
  • the cDNAs were fractionated on a Sepharose CL4B column (Catalog #275105-01 , Pharmacia), and those cDNAs exceeding 400 bp were ligated into pSport 1.
  • the plasmid pSport 1 was subsequently transformed into DH5aTM competent cells (Catalog #18258-012, Gibco/BRL).
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Catalog #26173; QIAGEN. Inc.). This kit enabled the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers.
  • the recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #2271 1, GIBCO BRLTM) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4° C. The cDNAs were sequenced by the method of Sanger et al. (1975. J. Mol. Biol.
  • nucleotide sequences of the Sequence Listing or amino acid sequences deduced from them were used as query sequences against databases such as GenBank, SwissProt, BLOCKS, and Pima II. These databases which contain previously identified and annotated sequences were searched for regions of homology (similarity) using BLAST, which stands for Basic Local Alignment Search Tool (Altschul SF (1993) J Mol Evol 36:290-300; Altschul. SF et al (1990) J Mol Biol 215:403-10). BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity.
  • BLAST is especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal or plant) origin.
  • Other algorithms such as the one described in Smith, R.F. and T.F. Smith (1992; Protein Engineering 5:35-51), inco ⁇ orated herein by reference, can be used when dealing with primary sequence patterns and secondary structure gap penalties.
  • sequences have lengths of at least 49 nucleotides and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
  • threshold is set at 10-25 for nucleotides and 10-14 for peptides.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook et al., supra).
  • Analogous computer techniques using BLAST Altschul, S.F. (1993) J.Mol.Evol. 36:290-300; Altschul, S.F. et al. (1990) J.Mol.Evol. 215:403-410) are used to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQTM database (Incyte Pharmaceuticals). This analysis is much faster than multiple, membrane-
  • SUBST ⁇ TUTE SHEET (RULE 26) based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • the basis of the search is the product score which is defined as: % sequence identity x % maximum BLAST score 100
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70. the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • the nucleic acid sequence of the Incyte Clone 2295453 was used to design oligonucleotide primers for extending a partial nucleotide sequence to full length.
  • One primer was synthesized to initiate extension in the antisense direction, and the other was synthesized to extend sequence in the sense direction.
  • Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest.
  • the initial primers were designed from the cDNA using OLIGO 4.06 (National Biosciences), or another appropriate program, to be about 22 to about 30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures of about 68°to about 72° C. Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 5 65° C for 1 min
  • Step 6 68° C for 7 min
  • Step 7 Repeat step 4-6 for 15 additional cycles
  • Step 8 94° C for 15 sec
  • Step 10 68° C for 7:15 min
  • Step 11 Repeat step 8-10 for 12 cycles
  • reaction mixture A 5-10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQuickTM (QIAGEN Inc., Chatsworth, CA), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
  • QIAQuickTM QIAGEN Inc., Chatsworth, CA
  • SUBSTrrUTE SHEET (RULE 26) of each overnight culture was transferred into a non-sterile 96-well plate and after dilution 1:10 with water, 5 ⁇ l of each sample was transferred into a PCR array.
  • PCR amplification For PCR amplification, 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase. a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:
  • Step 1 94° C for 60 sec
  • 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
  • nucleotide sequence of SEQ ID NO:2 is used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
  • Hybridization probes derived from SEQ ID NO:2 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides. consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of each oligomer and 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA).
  • the labeled oligonucleotides are substantially purified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco 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 transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH). Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester. NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale, CA) for several hours, hybridization patterns are compared visually.
  • SEQ ID NO:2 is examined using a computer algorithm which starts at the 3' end of the nucleotide sequence.
  • the algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that would interfere with hybridization.
  • the algorithm identifies-specific oligonucleotides of 20 nucleotides in length (20-mers). A matched set of oligonucleotides is created in which one nucleotide in the center of each sequence is altered.
  • a chemical coupling procedure and an ink jet device is used to synthesize oligomers on the surface of a substrate (Baldeschweiler, J.D. et al., PCT/WO95/25116, inco ⁇ orated herein by reference).
  • a "gridded" array analogous to a dot (or slot) blot is used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • a typical array may be produced by hand or using available materials and machines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.
  • the microarray is washed to remove nonhybridized probes, and a detection device as simple as X-ray film or complicated as a light scanner is used to determine the levels and patterns of radioactivity or fluorescence. Scanned fluorescent images are examined to determine degree of complementarity and the relative abundance/expression level of each oligonucleotide sequence in the microarray.
  • HMH-encoding sequence Sequence complementary to the HMH-encoding sequence, or any part thereof, is used to decrease or inhibit expression of naturally occurring HMH. Although use of oligonucleotides comprising from about 15 to about 30 base-pairs is described, essentially the same procedure is used with smaller or larger sequence fragments. Appropriate oligonucleotides are designed using Oligo 4.06 software and the coding sequence of HMH, SEQ ID NO . To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the HMH-encoding transcript.
  • HMH HMH-containing cDNAs
  • the cloning vector is also used to express HMH in E. coli. Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the amino-terminal Met. and the subsequent seven residues of ⁇ -galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
  • Induction of an isolated, transformed bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first eight residues of ⁇ -galactosidase, about 5 to 15 residues of linker, and the full length protein.
  • the signal residues direct the secretion of HMH into the bacterial growth media which can be used directly in the following assay for activity.
  • HMH that is substantially purified using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • the amino acid sequence deduced from SEQ ID NO:2 is analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high immunogenicity and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions, is described by Ausubel et al. (supra), and others.
  • the oligopeptides are 15 residues in length, synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma, St. Louis. MO) by reaction with N-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS; Ausubel et al., supra).
  • KLH keyhole limpet hemocyanin
  • MBS N-maleimidobenzoyl-N- hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
  • the resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio iodinated, goat anti- rabbit IgG.
  • Naturally occurring or recombinant HMH is substantially purified by immunoaffinity chromatography using antibodies specific for HMH.
  • An immunoaffinity column is constructed by covalently coupling HMH antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • HMH Media containing HMH is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HMH (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/HMH binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HMH is collected.
  • a chaotrope such as urea or thiocyanate ion

Abstract

L'invention porte sur un homologue de la mammoglobine humaine (HMH), sur des polynucléotides l'identifiant et codant pour elle, sur des vecteurs d'expression, des cellules hôtes, des agonistes, des anticorps, et des antagonistes. Elle porte également sur des procédés permettant de diagnostiquer, traiter, ou prévenir des troubles associés à l'expression de la HMH.
PCT/US1998/021729 1997-10-16 1998-10-14 Homologue de la mammoglobine WO1999019487A1 (fr)

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AU9804198A (en) 1999-05-03
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