WO2000000608A2 - Molecules du systeme immunitaire - Google Patents

Molecules du systeme immunitaire Download PDF

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Publication number
WO2000000608A2
WO2000000608A2 PCT/US1999/013995 US9913995W WO0000608A2 WO 2000000608 A2 WO2000000608 A2 WO 2000000608A2 US 9913995 W US9913995 W US 9913995W WO 0000608 A2 WO0000608 A2 WO 0000608A2
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Prior art keywords
ismo
polynucleotide
sequence
polypeptide
sequences
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PCT/US1999/013995
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English (en)
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WO2000000608A3 (fr
Inventor
Preeti Lal
Y. Tom Tang
Neil C. Corley
Gina Gorgone
Karl J. Guegler
Chandra Patterson
Mariah R. Baughn
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Incyte Pharmaceuticals, Inc.
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Priority to CA002330546A priority Critical patent/CA2330546A1/fr
Priority to AU47036/99A priority patent/AU4703699A/en
Priority to EP99930508A priority patent/EP1092022A2/fr
Priority to JP2000557361A priority patent/JP2002519028A/ja
Publication of WO2000000608A2 publication Critical patent/WO2000000608A2/fr
Publication of WO2000000608A3 publication Critical patent/WO2000000608A3/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 immune system molecules and to the use of these sequences in the diagnosis, treatment, and prevention of disorders associated with the immune system and cell proliferation.
  • a key feature of the immune system is its ability to specifically discriminate foreign molecules from self molecules.
  • a foreign molecule, or antigen triggers an immune response by stimulating host cell differentiation, proliferation, and communication.
  • the immune response coordinates the progressive selection, amplification, and activation of cellular defense mechanisms which ultimately lead to the destruction or neutralization of the pathogen.
  • the immune response although indispensable for survival, may cause undesirable conditions such as inflammation, allergies, and autoimmune disorders.
  • lymphocytes include T- and B-cells, which specifically recognize and respond to foreign antigens.
  • T-cells are important for fighting viral infections and activating other leukocytes, while B-cells secrete antibodies that neutralize bacteria and other microbes.
  • Granulocytes and monocytes are primarily migratory, phagocytic cells that exit the bloodstream to fight infection in tissues.
  • Neutrophils which are the most common type of granulocyte, are particularly important for the phagocytosis and destruction of bacteria.
  • Antibodies which are the most common type of granulocyte, are particularly important for the phagocytosis and destruction of bacteria.
  • Antibodies or immunoglobulins (Ig), are secreted by B-cells into the circulation and are capable of recognizing antigens on the surface of microbial cells.
  • the prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules.
  • Antibodies are classified based on their H-chain composition.
  • the five antibody classes, IgA, IgD, IgE, IgG and IgM are defined by the ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ H-chain types, respectively.
  • L-chains There are two types of L-chains, K and ⁇ , either of which may associate as a pair with any H-chain pair.
  • IgG the most common class of antibody found in the circulation, is tetrameric, as described above, while the other classes of antibodies are generally variants or multimers of this basic structure.
  • H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region.
  • the sequence of the constant region which consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains, is nearly identical among H- or L-chains of a particular class.
  • the sequence of the variable region which consists of about 1 10 amino acids, differs among H- or L-chains of a particular class.
  • Within each H- and L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen binding site.
  • rodent antibodies directed against human disease-associated proteins can be "humanized” by replacing their constant regions with those from human antibodies.
  • the variable regions of these humanized antibodies recognize human disease-associated proteins, while the constant regions activate downstream effectors and prevent the antibodies themselves from being recognized as foreign in a human host.
  • Humanized antibodies have proved to be effective therapeutic agents for the prevention of transplant rejection in primate model systems and for their anti-proliferative activity in breast tumor cell lines. (Brown, P. S. et al. (1991) Proc. Natl. Acad. Sci. USA 88:2663-2667.) Autoimmuniry
  • SLE Systemic lupus erythematosus
  • Other symptoms of SLE include joint pain, facial rash, anemia, fatigue, fever, nausea, renal failure, pleurisy, arthritis, pericarditis, and neurological disturbances.
  • SLE is particularly common among young women of color, aged 20 to 40 years.
  • Various autoantibodies are found in the circulation of SLE patients and include antibodies to whole nuclei and nuclear components, cytoplasmic components, and specific cell populations.
  • the La antigen is a cytoplasmic protein recognized by some SLE autoantibodies.
  • La is a 408-amino acid polypeptide that associates with poly-uridine tracts at the termini of newly synthesized RNA polymerase III transcripts.
  • La contains an N-terminal ribonucleoprotein (RNP) consensus motif, a central extended alpha helical domain, and a hydrophilic C-terminal domain.
  • RNP N-terminal ribonucleoprotein
  • the C-terminal and RNP regions of La are particularly reactive with SLE autoimmune antibodies.
  • La is conserved among diverse organisms, including mosquitos, fruit flies, mice, frogs, yeasts, and humans. (Pardigon, N. (1996) J. Virol. 70:1173- 1 181.)
  • Neutrophils are phagocytic cells that contain numerous types of lysosomal and secretory granules. These granules contain digestive enzymes for the degradation of phagocytosed material.
  • One type of granule called the secondary or specific granule, contains neutrophil gelatinase- associated lipocalin (NGAL).
  • NGAL neutrophil gelatinase- associated lipocalin
  • Lipocalins in general, bind and transport small hydrophobic molecules. It is proposed that NGAL, in particular, binds to bacterial lipopolysaccharides and chemotactic peptides, such as formylpeptides. Furthermore, NGAL may function as a modulator of inflammation. NGAL is expressed in bone marrow and in tissues prone to exposure to microorganisms. A variety of immune system disorders are associated with uncontrolled cell proliferation.
  • the invention is based on the discovery of new immune system molecules (ISMO), the polynucleotides encoding ISMO, and the use of these compositions for the diagnosis, treatment, or prevention of disorders associated with the immune system and cell proliferation.
  • the invention features substantially purified polypeptides, immune system molecules, referred to collectively as "ISMO” and individually as “ISMO-1,” “ISMO-2,” “ISMO-3,” and “ISMO-4.”
  • the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof.
  • the invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequence of SEQ ID NO: 1-4, or to a fragment of any of these sequences.
  • the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof.
  • the invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof.
  • the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:5-8 and fragments thereof.
  • the invention further provides an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:5-8 and fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO:5-8 and fragments thereof.
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -4 and fragments thereof.
  • the expression vector is contained within a host cell.
  • the invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide 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 polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
  • the invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof, as well as a purified agonist and a purified antagonist to the polypeptide.
  • the invention also provides a method for treating or preventing a disorder associated with the immune system, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof.
  • the invention also provides a method for treating or preventing a disorder associated with cell proliferation, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof.
  • the invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample.
  • the method further comprises amplifying the polynucleotide prior to hybridization.
  • Figure 1 shows the amino acid sequence alignment between ISMO-1 (2192748; SEQ ID NO:l) and NGAL (GI 929657; SEQ ID NO:9). The alignments were produced using the multisequence alignment program of LASERGENETM software (DNASTAR Inc. Madison WI).
  • Figures 2A and 2B show the amino acid sequence alignments among ISMO-3 (2937262; SEQ ID NO:3), mosquito La (GI 131 1676; SEQ ID NO: 10), and human La (GI 178687; SEQ ID NO: 11).
  • Table 1 shows the programs, their descriptions, references, and threshold parameters (where appropriate) used to identify and characterize MAPOP.
  • ISMO refers to the amino acid sequences, or variant thereof, of substantially purified ISMO obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which, when bound to ISMO, increases or prolongs the duration of the effect of ISMO.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of ISMO.
  • allelic variant is an alternative form of the gene encoding ISMO. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in 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 allelic variants 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 ISMO include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as ISMO or a polypeptide with at least one functional characteristic of ISMO. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding ISMO, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding ISMO.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent ISMO.
  • 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 ISMO 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 or amino acid sequence refer to an oligopeptide. peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
  • fragments refer to fragments of ISMO which are preferably at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of ISMO.
  • 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.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art. (See, e.g., Dieffenbach, C.W. and G.S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, pp.1-5.)
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which, when bound to ISMO, decreases the amount or the duration of the effect of the biological or immunological activity of ISMO.
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of ISMO.
  • the term "antibody” refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2> and Fv fragments, which are capable of binding the epitopic determinant.
  • Antibodies that bind ISMO polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin. and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • 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 a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence. Antisense molecules 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 to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer 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 ISMO, or of 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 by base pairing.
  • sequence 5' A-G-T 3'
  • complementary sequence 3' T-C-A 5'.
  • Complementarity between two single-stranded molecules may be "partial.” such that only some of the nucleic acids bind, or it may be "complete,” such that 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 the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence,” as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding ISMO or fragments of ISMO 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.,sodium dodecyl sulfate (SDS), and other components, e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • salts e.g., NaCl
  • detergents e.g.,sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Denhardt's solution e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCRTM kit (The Perkin-Elmer Corp., Norwalk, CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence.
  • the term "correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding ISMO, by Northern analysis is indicative of the presence of nucleic acids encoding ISMO in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding ISMO.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • similarity refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.”
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
  • 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 reduced stringency.
  • a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency.
  • Percent identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MegAlignTM program (DNASTAR, Inc., Madison WI). The MegAlignTM program can create alignments between two or more sequences according to different methods, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
  • the percentage similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
  • HACs Human artificial chromosomes
  • linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance. (See, e.g., Harrington, J.J. et al. (1997) Nat Genet. 15:345-355.)
  • humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its 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 bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or formed 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,” as used herein, refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • microarray refers to an arrangement of distinct polynucleotides arrayed on a substrate, e.g., paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • element or “array element” as used herein in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
  • modulate refers to a change in the activity of ISMO. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of ISMO.
  • nucleic acid or “nucleic acid sequence,” as used herein, refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • fragments refers to those nucleic acid sequences which, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
  • operably associated refers to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
  • oligonucleotide is substantially equivalent to the terms "amplimer,” “primer,” “oligomer,” and “probe,” as these terms are 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 about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)
  • sample as used herein, is used in its broadest sense.
  • a biological sample suspected of containing nucleic acids encoding ISMO, or fragments thereof, or ISMO itself may comprise a bodily fluid; an 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; etc.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope. recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • stringent conditions refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides.
  • Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 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. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation 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.
  • transformed cells includes 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, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a “variant" of ISMO polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues.
  • 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 glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENETM software.
  • variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to ISMO. This definition may also include, for example, "allelic” (as defined above), “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • the invention is based on the discovery of new human immune system molecules (ISMO), the polynucleotides encoding ISMO. and the use of these compositions for the diagnosis, treatment, or prevention of disorders associated with the immune system and cell proliferation.
  • ISMO new human immune system molecules
  • Nucleic acids encoding the ISMO-1 of the present invention were first identified in Incyte Clone 2192748 from the thyroid tumor cDNA library (THYRTUT03) using a computer search, e.g., BLAST, for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:5 was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2192748H1 (THYRTUT03), 1384450T1 (BRAITUT08), and 1291692T1 (PGANNOT03).
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: l.
  • ISMO-1 is 153 amino acids in length and has one potential protein kinase C phosphorylation site at S52 and a predicted signal peptide from Ml to A 19.
  • BLOCKS and PRINTS analyses indicate that the regions of ISMO-1 from R2 to L14, from S33 to A49, from G109 to F124, and from C126 to P141 are similar to lipocalin sequence motifs.
  • ISMO-1 has chemical and structural similarity with human NGAL (GI 929657; SEQ ID NO:9).
  • NGAL GI 929657; SEQ ID NO:9
  • ISMO-1 and NGAL share 31% identity. Note that in Figure 1, amino acids 1 19 through 154 of NGAL are omitted from the alignment for reasons of clarity.
  • NGAL SEQ ID NO:9
  • a fragment of SEQ ID NO:5 from about nucleotide 64 to about nucleotide 96 is useful as a hybridization probe.
  • Northern analysis shows the expression of this sequence in various libraries, particularly those associated with diseased thyroid.
  • Nucleic acids encoding the ISMO-2 of the present invention were first identified in Incyte Clone 2849752 from the breast tumor cDNA library (BRSTTUT13) using a computer search, e.g., BLAST, for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:6, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
  • ISMO-2 is 470 amino acids in length and has two potential N- glycosylation sites at N 120 and N320; four potential casein kinase II phosphorylation sites at T105, T232. S290, and S377; nine potential protein kinase C phosphorylation sites at S47, S81, T92, S98, SI 42, SI 54, T322, S347, and T460; and two potential tyrosine kinase phosphorylation sites at Y69 and Y319. Protein sequence analysis using various search algorithms indicates that regions of ISMO-2 show strong homology to Ig superfamily protein domains.
  • BLOCKS analysis indicates that ISMO-2 contains two conserved Ig/MHC protein blocks from S387 to Q409 and from F446 to S463.
  • PFAM analysis indicates that ISMO-2 contains three regions with similarity to Ig superfamily protein domains from S34 to Rl 16, from G160 to V225, and from K383 to V450.
  • BLAST analyses indicate that ISMO-2 shows significant amino acid identity to vertebrate immunoglobulin ⁇ H-chain.
  • Two independent search algorithms also predict a signal peptide sequence in ISMO-2 from M 1 to S 19.
  • a fragment of SEQ ID NO:6 from about nucleotide 432 to about nucleotide 473 is useful as a hybridization probe.
  • Northern analysis shows the expression of this sequence in various libraries, at least 61% of which are associated with cancer or cell proliferation and at least 38% of which are associated with the immune response or trauma.
  • 27% of the libraries expressing ISMO-2 are derived from reproductive tissue and 26% 5 are derived from gastrointestinal tissue.
  • Nucleic acids encoding the ISMO-3 of the present invention were first identified in Incyte Clone 2937262 from the fetal thymus cDNA library (THYMFET02) using a computer search, e.g., BLAST, for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:7 was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:3.
  • ISMO-3 is 582 amino acids in length and has three potential N-
  • ISMO-3 is predominantly hydrophilic and alpha- helical.
  • Various search algorithms indicate that regions of ISMO-3 show strong homology to RNA binding motifs.
  • BLOCKS and Profilescan indicate that ISMO-3 contains an RNP-1 RNA binding signature from K166 to F173.
  • BLOCKS and PFAM analyses further indicate that the region of ISMO-3 from VI 27 to 1199 is similar to an RNA recognition motif.
  • ISMO-3 has chemical and structural similarity with mosquito La (GI 131 1676; SEQ ID NO: 10) and human La (GI 178687; SEQ ID NO: l 1). Amino acids 1 through 229 of ISMO-3 share 20% identity with amino acids 1 through
  • Northern analysis shows the expression of this sequence in various libraries, at least 46% of which are associated with cancer or cell proliferation and at least 25% of which are associated with the immune response or trauma.
  • Nucleic acids encoding the ISMO-4 of the present invention were first identified in Incyte Clone 2995108 from the ovarian tumor cDNA library (OVARTUT07) using a computer search, e.g., BLAST, for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:8, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2995108H1 (OVARTUT07), 1000491 T6 (BRSTNOT03), 132994R6 (BMARNOT02), 1348978F 1 (LATRTUT02), and shotgun sequences SARB01777F 1 , SBJA01451 F 1 , and SAEA01628F1.
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:4.
  • ISMO-4 is 497 amino acids in length and has two potential N-glycosylation sites at N288 and N484; five potential casein kinase II phosphorylation sites at Tl 10, T160, T184, S400, and S425; nine potential protein kinase C phosphorylation sites at S86, T97, S126, S149, T193, S310, T369, S400, and T469.
  • Protein sequence analysis using various search algorithms indicates that regions of ISMO-4 show strong homology to Ig superfamily protein domains.
  • BLOCKS analysis indicates that ISMO-4 contains two conserved Ig/MHC protein blocks from T391 to Q413 and from F455 to T472.
  • PFAM analysis indicates that ISMO-4 contains three regions with similarity to Ig superfamily protein domains from S35 to R121, from G284 to A350, and from N387 to V459.
  • BLAST analyses indicate that ISMO-4 shows significant amino acid identity to vertebrate immunoglobulin ⁇ H-chain.
  • Two independent search algorithms also predict a signal peptide sequence in ISMO-4 from Ml to S20. A fragment of SEQ ID NO:8 from about nucleotide 266 to about nucleotide 295 is useful as a hybridization probe.
  • Northern analysis shows the expression of this sequence in various libraries, at least 61% of which are associated with cancer or cell proliferation and at least 40% of which are associated with the immune response or trauma.
  • 28% of the libraries expressing ISMO-4 are derived from reproductive tissue and 28% are derived from gastrointestinal tissue.
  • the invention also encompasses ISMO variants.
  • a preferred ISMO variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the ISMO amino acid sequence, and which contains at least one functional or structural characteristic of ISMO.
  • the invention also encompasses polynucleotides which encode ISMO.
  • the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO:5, which encodes ISMO-1.
  • the invention encompasses the polynucleotide sequence comprising the sequence of SEQ ID NO:6, which encodes ISMO-2.
  • the invention encompasses the polynucleotide sequence comprising the sequence of SEQ ID NO:7, which encodes ISMO-3.
  • the invention encompasses the polynucleotide sequence comprising the sequence of SEQ ID NO:8, which encodes ISMO-4.
  • the invention also encompasses a variant of a polynucleotide sequence encoding ISMO.
  • a variant polynucleotide sequence will have at least about 70%, more preferably at least about 85%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding ISMO, selected from the group consisting of SEQ ID NO:5-8.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of ISMO.
  • nucleotide sequences which encode ISMO and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring ISMO under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding ISMO possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. 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 which encode ISMO and
  • ISMO derivatives, or fragments thereof, 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 well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding ISMO or any fragment thereof. Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:5-8 and fragments thereof, under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
  • additional parameters such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • SDS sodium dodecyl sulfate
  • hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM
  • wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C.
  • wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at
  • wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE ® enzyme
  • sequence preparation is automated with machines, e.g., the ABI CATALYSTTM 800 (Perkin-Elmer) or MICROLAB® 2200 (Hamilton
  • Sequencing can also be automated, such as by ABI PRISMTM 373 or 377 systems (Perkin-Elmer) or the MEGABACETM 1000 capillary electrophoresis system (Molecular Dynamics, Inc., Sunnyvale, CA). Sequences can be analyzed using computer programs and algorithms well known in the art. (See, e.g., Ausubel, supra, unit 7.7; and Meyers, R.A. ( 1995) Molecular Biology and Biotechnology, Wiley VCH, Ine, New York, NY.)
  • the nucleic acid sequences encoding ISMO may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al.
  • primers may be designed using commercially available software, such as OLIGOTM 4.06 Primer Analysis software (National Biosciences Inc., Madison, MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50%) or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • 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 nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software, 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 sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode ISMO may be cloned in recombinant DNA molecules that direct expression of ISMO, or 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 used to express ISMO.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter ISMO-encoding sequences for a variety of purposes including, but not limited to, modification of 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.
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • sequences encoding ISMO may be synthesized, in whole or in part, using chemical methods well known in the art.
  • chemical methods See. e.g., Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232.
  • ISMO itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solid-phase techniques.
  • the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392- 421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties. WH Freeman and Co., New York, NY.)
  • the nucleotide sequences encoding ISMO or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding ISMO. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding ISMO. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • 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 appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162.)
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding ISMO. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus (CaMV) or 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.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding ISMO.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding ISMO can be achieved using a multifunctional _co vector such as pBluescript® plasmid (Stratagene) or pSportlTM plasmid (Life Technologies). Ligation of sequences encoding ISMO into the vector's multiple cloning site disrupts the lacL gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of ISMO may be used.
  • vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of ISMO.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of ISMO. Transcription of sequences encoding ISMO may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 3:17-311.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., 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.
  • viral promoters e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection See, e.g., Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, NY; pp. 191-196.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding ISMO 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 infective virus which expresses ISMO in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb
  • -99- are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • sequences encoding ISMO can be transformed into cell lines 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 about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk or apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 1 1 :223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase. respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -D-glucuronoside, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. et al. (1995) Methods Mol. Biol. 55: 121-131.)
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding ISMO is inserted within a marker gene sequence
  • transformed cells containing sequences encoding ISMO can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding ISMO under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding ISMO and that express ISMO may be identified by a variety of procedures known to those of skill in the art.
  • DNA-DNA or DNA-RNA hybridizations include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, 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 sequences.
  • Immunological methods for detecting and measuring the expression of ISMO using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding ISMO include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding ISMO, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection 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 ISMO 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 retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode ISMO may be designed to contain signal sequences which direct secretion of ISMO through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate 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 specify protein targeting, folding, and/or activity.
  • 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.
  • natural, modified, or recombinant nucleic acid sequences encoding ISMO may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric ISMO protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of ISMO activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6- His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the ISMO encoding sequence and the heterologous protein sequence, so that ISMO may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel, F. M. et al. ( 1995 and periodic supplements) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, ch 10. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled ISMO may be achieved in vitro using the TNTTM rabbit reticulocyte lysate or wheat germ extract systems (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 35 S-methionine.
  • Fragments of ISMO may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton. supra pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431 A Peptide Synthesizer (Perkin- Elmer). Various fragments of ISMO may be synthesized separately and then combined to produce the full length molecule.
  • Partial chemical and structural similarity e.g., in the context of sequences and motifs, exists among regions of ISMO-1, human NGAL (GI 929657), and other lipocalin-related proteins.
  • partial chemical and structural similarity exists among regions of ISMO-2, ISMO-4, and Ig superfamily proteins.
  • partial chemical and structural similarity exists among regions of ISMO-3, RNA binding proteins, and lupus-associated La proteins, particularly mosquito La (GI 131 1676) and human La (GI 178687).
  • ISMO is expressed in tissues associated with the immune system and cell proliferation. Therefore, ISMO appears to play a role in disorders associated with the immune system and cell proliferation.
  • an antagonist of ISMO may be administered to a subject to treat or prevent a disorder associated with the immune system.
  • disorders can include, but are not limited to, autoimmune and inflammatory diseases such as Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis.
  • bronchitis cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis.
  • a vector expressing the complement of the polynucleotide encoding ISMO may be administered to a subject to treat or prevent a disorder associated with the immune system including, but not limited to, those described above.
  • an antagonist of ISMO may be administered to a subject to treat or prevent a disorder associated with cell proliferation.
  • disorders can include, but are not limited to, actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia such as multiple myeloma, lymphoma such as Hodgkin's disease, melanoma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas
  • a vector expressing the complement of the polynucleotide encoding ISMO may be administered to a subject to treat or prevent a disorder associated with cell proliferation including, but not limited to, those described above.
  • 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 ISMO may be produced using methods which are generally known in the art.
  • purified ISMO may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind ISMO.
  • Antibodies to ISMO may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, 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 ISMO or with any fragment or oligopeptide thereof which has immunogenic properties. Rats and mice are preferred hosts for downstream applications involving monoclonal antibody production.
  • 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, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides. peptides. or fragments used to induce antibodies to ISMO have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 14 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of ISMO amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to ISMO may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV- hybridoma technique.
  • the hybridoma technique See, e.g., Kohler. G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 :31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries.
  • 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.
  • Antibody fragments which contain specific binding sites for ISMO may also be generated.
  • fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments 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. (See, e.g., Huse, W.D. et al. ( 1989) Science 246: 1275- 1281.)
  • immunoassays may be used for screening to identify antibodies having the desired specificity and minimal cross-reactivity.
  • 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 ISMO and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering ISMO epitopes is preferred, but a competitive binding assay may also be employed (Maddox. supra).
  • K a is defined as the molar concentration of ISMO-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
  • K a association constant
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular ISMO epitope represents a true measure of affinity.
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the ISMO-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of ISMO, preferably in active form, from the antibody.
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is preferred for use in procedures requiring precipitation of ISMO-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
  • the polynucleotides encoding ISMO may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding ISMO 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 ISMO.
  • complementary molecules or fragments may be used to modulate ISMO 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 ISMO.
  • Expression vectors derived from retroviruses. adenoviruses. or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding ISMO. (See, e.g., Sambrook. supra; and Ausubel. supra.)
  • Genes encoding ISMO can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide. or fragment thereof, encoding ISMO. 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 may last 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 ISMO.
  • Oligonucleotides derived from the transcription initiation site e.g., between about positions -10 and +10 from the start site, are preferred.
  • inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the 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. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
  • a 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.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding ISMO.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU. and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the 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 ISMO. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half-life.
  • flanking sequences at the 5' and/or 3' ends of the molecule Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine. as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine. and uridine which are not as easily recognized by endogenous endonucleases.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman. C.K. et al. (1997) Nature Biotechnology 15:462-466.)
  • 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 or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of ISMO, antibodies to ISMO, and mimetics, agonists, antagonists, or inhibitors of ISMO.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular. transdermal. subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and 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, and 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.
  • 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 fillers 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, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. 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 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutical ly effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs.
  • An 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 ISMO or fragments thereof, antibodies of ISMO, and agonists, antagonists or inhibitors of ISMO, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED 50 /LD 50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, the 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 requiring 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, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, 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 ISMO may be used for the diagnosis of disorders characterized by expression of ISMO, or in assays to monitor patients being treated with ISMO or agonists, antagonists, or inhibitors of ISMO.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for ISMO include methods which utilize the antibody and a label to detect ISMO in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • ISMO immunosorbent assays
  • ELISAs ELISAs
  • RIAs RIAs
  • FACS fluorescence-activated cell sorting
  • normal or standard values for ISMO expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to ISMO under conditions suitable for complex formation
  • the amount of standard complex formation may be quantitated by various methods, preferably by photometric means.
  • Quantities of ISMO expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding ISMO may be used for diagnostic purposes.
  • 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 ISMO may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of ISMO, and to monitor regulation of ISMO levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding ISMO or closely related molecules may be used to identify nucleic acid sequences which encode ISMO.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding ISMO, allelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the ISMO encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or from genomic sequences including promoters, enhancers, and introns of the ISMO gene.
  • Means for producing specific hybridization probes for DNAs encoding ISMO include the cloning of polynucleotide sequences encoding ISMO or ISMO derivatives into vectors for the production of mRNA probes.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding ISMO may be used for the diagnosis of a disorder associated with expression of ISMO.
  • a disorder associated with expression of ISMO include, but are not limited to, disorders associated with the immune system including autoimmune and inflammatory diseases such as Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis.
  • anemia asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis.
  • leukemia such as multiple myeloma, lymphoma such as Hodgkin's disease, melanoma, sarcoma, teratocarcinoma, and, in particular, 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.
  • lymphoma such as Hodgkin's disease, melanoma, sarcoma, teratocarcinoma
  • the polynucleotide sequences encoding ISMO may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered ISMO expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding ISMO may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding ISMO 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.
  • the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding ISMO in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of
  • ISMO 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, encoding ISMO, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • 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 an abnormal 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. Additional diagnostic uses for oligonucleotides designed from the sequences encoding
  • ISMO may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding ISMO, or a fragment of a polynucleotide complementary to the polynucleotide encoding ISMO, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of ISMO include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
  • radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
  • 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 and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
  • nucleic acid sequences encoding ISMO may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding ISMO on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques 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 by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • 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.
  • ISMO in another embodiment, ISMO, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • 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 ISMO 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.
  • a solid substrate such as plastic pins or some other surface.
  • the test compounds are reacted with ISMO, or fragments thereof, and washed.
  • Bound ISMO is then detected by methods well known in the art.
  • Purified ISMO 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.
  • the nucleotide sequences which encode ISMO 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 THYRTUT03 library was constructed using RNA isolated from benign thyroid tumor tissue removed from a 17-year-old Caucasian male during a thyroidectomy. Pathology indicated encapsulated follicular adenoma forming a circumscribed mass.
  • the BRSTTUT13 library was constructed using RNA isolated from breast tumor tissue removed from the right breast of a 46-year-old Caucasian female during a unilateral extended simple mastectomy with breast reconstruction.
  • Pathology indicated an invasive grade 3 adenocarcinoma, ductal type with apocrine features and greater than 50% intraductal component.
  • Patient history included breast cancer.
  • the THYMFET02 library was constructed using RNA isolated from thymus tissue removed from a Caucasian female fetus, who died at 17 weeks' gestation from anencephalus.
  • the OVARTUT07 library was constructed using RNA isolated from right ovarian tumor tissue removed from a 58-year-old Caucasian female during bilateral salpingo-oophorectomy, regional lymph node excision, destruction of peritoneal tissue, cystocele repair, and skin repair.
  • Pathology indicated grade 3 adenocarcinoma, serous type, forming a mass and entirely replacing the right ovary.
  • the left pelvic sidewall revealed a microscopic focus of metastatic adenocarcinoma.
  • Patient history included hyperlipidemia, thrombophlebitis, and carcinoma in situ of the cervix uteri.
  • Family history included cerebrovascular disease, breast cancer, hyperlipidemia, atherosclerotic coronary artery disease, and heart failure.
  • frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, NY). The lysate was centrifuged over a CsCl cushion to isolate RNA. The RNA was extracted with phenol and precipitated with sodium acetate and ethanol.
  • frozen tissue was homogenized and lysed in TRIzol reagent (1 gm tissue/10 ml TRIzol; Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate.
  • RNA preparations were resuspended in RNase-free water, treated with DNase, re-extracted as necessary with acid phenol, and reprecipitated with sodium acetate and ethanol. From each RNA preparation, poly(A+) RNA was isolated using the Qiagen OLIGOTEX kit (QIAGEN Ine, Chatsworth, CA).
  • Poly(A+) RNA was used for cDNA synthesis and construction of each cDNA library according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies).
  • the cDNAs were fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia Biotech), and those cDNAs exceeding 400 bp were ligated into pINCY 1 plasmid (Incyte Pharmaceuticals, Palo Alto, CA). Recombinant plasmids were transformed into DH5 ⁇ TM competent cells (Life Technologies).
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (QIAGEN Ine). The recommended protocol was employed except for the following changes: 1 ) the bacteria were cultured in 1 ml of sterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/1 and glycerol at 0.4%; 2) after the cultures were incubated for 19 hours, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellets were each resuspended in 0.1 ml of distilled water. The DNA samples were stored at 4°C
  • the cDNAs were prepared for sequencing using either an ABI CATALYSTTM 800 (Perkin-Elmer Applied Biosystems, Foster City, CA) or a MICROLAB® 2200 (Hamilton) sequencing preparation system in combination with Peltier PTC-200 thermal cyclers (MJ Research, Inc., Watertown, MA).
  • the cDNAs were sequenced using the ABI PRISMTM 373 or 377 sequencing systems and ABI protocols, base calling software, and kits (Perkin-Elmer Applied Biosystems). Alternatively, solutions and dyes from Amersham Pharmacia Biotech, Ltd. were used in place of the ABI kits. In some cases, reading frames were determined using standard methods (Ausubel. supra).
  • the polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation, using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were re-analyzed by querying against databases such as the GenBank databases described above and SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
  • 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. (See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)
  • the product score encompasses 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 may have a possibility of a 1% to 2% error, in contrast, a product score of 70 indicates that the match will be exact. Similar molecules were identified by product scores between 15 and 40, although lower scores may identify related molecules.
  • organ/tissue categories included cardiovascular, dermatologic,
  • the disease categories included cancer, inflammation/trauma, fetal, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was divided by the total number of libraries across all categories. The results above were reported as a percentage distribution.
  • the full length nucleic acid sequences were produced by extension of the component fragments as disclosed in the invention description. Oligonucleotide primer pairs designed from at least one of the component fragments were used to initiate extension of sense and antisense
  • Step 1 94° C for 1 min (initial denaturation)
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 5 65 ° C for 1 min
  • Step 8 94° C for 15 sec
  • Step 9 65 ° C for 1 min
  • Step 10 68° C for 7: 15 min Step 1 1 Repeat steps 8 through 10 for an additional 12 cycles
  • T4-DNA ligase 15 units
  • l ⁇ l T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16° C.
  • Competent E. coli cells in 40 ⁇ l of appropriate media
  • the E. coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra. Appendix A, p. 1) containing carbenicillin (2x carb). The following day, several colonies were randomly picked from each plate and cultured in 150 ⁇ l of liquid LB/2x carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 ⁇ l of each overnight culture was transferred into a non-sterile 96-well plate and,
  • 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 2 94° C for 20 sec
  • Step 5 Repeat steps 2 through 4 for an additional 29 cycles
  • nucleotide sequences of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
  • SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 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 OLIGOTM 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA).
  • the labeled oligonucleotides are substantially purified using a SephadexTM G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
  • the DNA from each digest is fractionated on a 0.7% 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 to film for several hours, hybridization patterns are compared visually.
  • a chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate.
  • An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
  • nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
  • Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENETM software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
  • Fluorescent probes are prepared and used for hybridization to the elements on the substrate.
  • the substrate is analyzed by procedures described above.
  • Sequences complementary to the ISMO-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring ISMO. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGOTM 4.06 software (National Biosciences) and the coding sequence of ISMO. 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 ISMO-encoding transcript.
  • ISMO trp-lac
  • T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts. e.g., BL21(DE3).
  • Antibiotic resistant bacteria express ISMO upon induction with isopropyl beta- D-thiogalactopyranoside (IPTG).
  • ISMO Autographica californica nuclear polyhedrosis virus
  • AcMNPV Autographica californica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding ISMO by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
  • ISMO is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST glutathione S-transferase
  • FLAG peptide epitope tag
  • GST a 26-kilodalton enzyme from Schistosoma japonicum. enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from ISMO at specifically engineered sites.
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, ch 10, 16. Purified ISMO obtained by these methods can be used directly in the following activity assay.
  • ISMO activity is exemplified by that of immunoglobulins, which recognize and precipitate antigens from serum. The quantitative precipitin reaction measures this activity.
  • immunoglobulins which recognize and precipitate antigens from serum. The quantitative precipitin reaction measures this activity.
  • the quantitative precipitin reaction measures this activity.
  • ISMO is isotopically labeled using methods known in the art. Various serum concentrations are added to constant amounts of labeled ISMO. ISMO-antigen complexes precipitate out of solution and are collected by centrifugation. The amount of precipitable ISMO- antigen complex is proportional to the amount of radioisotope detected in the precipitate. The amount of precipitable ISMO-antigen complex is plotted against the serum concentration.
  • the amount of precipitable ISMO-antigen complex is a measure of ISMO activity which is characterized by sensitivity to both limiting and excess quantities of antigen.
  • ISMO function is assessed by expressing the sequences encoding ISMO at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORTTM plasmid (Life Technologies) and pCRTM 3.1 plasmid (Invitrogen, Carlsbad, CA), both of which contain the cytomegalovirus promoter.
  • 5- 10 ⁇ g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co- transfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down- regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies: and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod. M. G. (1994) Flow Cytometry. Oxford, New York, NY.
  • the influence of ISMO on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding ISMO and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success, NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding ISMO and other genes of interest can be analyzed by Northern analysis or microarray techniques.
  • PAGE polyacrylamide gel electrophoresis
  • the ISMO amino acid sequence is analyzed using LASERGENETM 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.
  • LASERGENETM software DNASTAR Inc.
  • Methods for selection of appropriate epitopes. such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel supra, ch. 1 1.)
  • oligopeptides 15 residues in length are synthesized using an Applied Biosystems Peptide Synthesizer Model 43 1 A using fmoc-chemistry and coupled to KLH (Sigma- Aldrich, St. Louis, MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, 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 ISMO is substantially purified by immunoaffinity chromatography using antibodies specific for ISMO.
  • An immunoaffinity column is constructed by covalently coupling anti-ISMO antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • ISMO suppression protein
  • Media containing ISMO are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of ISMO (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/ISMO binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and ISMO is collected.
  • a buffer of pH 2 to pH 3 or a high concentration of a chaotrope, such as urea or thiocyanate ion
  • ISMO or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton et al. (1973) Biochem. J. 133:529.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled ISMO, washed, and any wells with labeled ISMO complex are assayed. Data obtained using different concentrations of ISMO are used to calculate values for the number, affinity, and association of ISMO with the candidate molecules.
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing and Perkin-Elmer Applied Biosystems, Mismatch ⁇ 50'/ annotating amino acid or nucleic acid sequences Foster City, CA, Paracel Ine , Pasadena, CA
  • ABI AuloAssembler A program that assembles nucleic acid Perkin Elmer Applied Biosystems, sequences Foster City, CA
  • Phred A base-calling algorithm that examines Ewing, B et al ( 1998) Genome automated sequencer traces with high sensitivity Res 8 175- 185, Ewing, B and P and probability Green ( 1998) Genome Res 8 186 194

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Abstract

L'invention concerne des molécules du système immunitaire humain (ISMO), ainsi que les polynucléotides identifiant et codant pour ces molécules ISMO. Cette invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes, et des antagonistes. Cette invention concerne enfin des méthodes permettant de diagnostiquer, de traiter, ou de prévenir les troubles liés à l'expression desdites molécules ISMO.
PCT/US1999/013995 1998-06-30 1999-06-21 Molecules du systeme immunitaire WO2000000608A2 (fr)

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EP99930508A EP1092022A2 (fr) 1998-06-30 1999-06-21 Molecules du systeme immunitaire
JP2000557361A JP2002519028A (ja) 1998-06-30 1999-06-21 免疫系分子

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029576A1 (fr) * 1998-11-13 2000-05-25 Zymogenetics, Inc. Homologues de la lipocaline
US7943328B1 (en) 2006-03-03 2011-05-17 Prometheus Laboratories Inc. Method and system for assisting in diagnosing irritable bowel syndrome
US8463553B2 (en) 2006-08-15 2013-06-11 Nestec S.A. Methods for diagnosing irritable bowel syndrome

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ADAMS MD ET AL: "Epididymus homo spaiens cDNA 5' end." EMEST DATABASE ENTRY HSZZ41272, ACCESSION NUMBER AA336050, 18 April 1997, XP002120451 *
BUDGAARD JR: "Molecular cloning and expression of a cDNA encoding NGASL: a lipocalin expressed in human neutrophils" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 202, 1994, ORLANDO, FL US, pages 1468-1475, XP002120453 *
KJELDSEN L ET AL: "ISOLATION AND PRIMARY STRUCTURE OF NGAL, A NOVEL PROTEIN ASSOCIATED WITH HUMAN NEUTROPHIL GELATINASE" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 14, 15 May 1993, pages 10425-10432, XP000608298 *
NATIONAL CANCER INSTITUTE, CANCER GENOME ANATOMY PROJECT (CGAP): "Homo sapiens cDNA clone" EMEST DATABASE ENTRY AA916297, ACCESSION NUMBER AA916297, 16 April 1998, XP002120452 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029576A1 (fr) * 1998-11-13 2000-05-25 Zymogenetics, Inc. Homologues de la lipocaline
US7943328B1 (en) 2006-03-03 2011-05-17 Prometheus Laboratories Inc. Method and system for assisting in diagnosing irritable bowel syndrome
US8463553B2 (en) 2006-08-15 2013-06-11 Nestec S.A. Methods for diagnosing irritable bowel syndrome

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