WO2003004532A2 - Variants d'epissures de q9ul33/q9nzz4 et q9h5p3 - Google Patents

Variants d'epissures de q9ul33/q9nzz4 et q9h5p3 Download PDF

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Publication number
WO2003004532A2
WO2003004532A2 PCT/EP2002/007541 EP0207541W WO03004532A2 WO 2003004532 A2 WO2003004532 A2 WO 2003004532A2 EP 0207541 W EP0207541 W EP 0207541W WO 03004532 A2 WO03004532 A2 WO 03004532A2
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WIPO (PCT)
Prior art keywords
polypeptide
seq
q9ul33
q9nzz4
q9h5p3
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PCT/EP2002/007541
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English (en)
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WO2003004532A3 (fr
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Lydie Bougueleret
Anne Niknejad
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Geneprot, Inc.
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Publication of WO2003004532A2 publication Critical patent/WO2003004532A2/fr
Publication of WO2003004532A3 publication Critical patent/WO2003004532A3/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates generally to human proteins that are expressed in variant forms, and more particularly, to the use of such alternatively expressed proteins as tissue and disease markers.
  • splice variants of selected genes within a tissue or organ may be indicative of its functional state or condition. Accordingly, the availability of tools to measure the relative amounts of splice variants in tissues and organs of interest will lead to improvements in diagnosis and treatment.
  • the present invention is directed to methods and compositions for measuring ratios of Q9H5P3 splice variants or Q9UL33/Q9NZZ4 splice variants in biological samples, especially human tissues.
  • the invention further includes methods and kits for monitoring the levels of Q9H5P3 splice variants and Q9UL33/Q9NZZ4 splice variants in an individual.
  • Q9H5P3 splice variants refers to the polypeptides having amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.
  • Q9UL33/Q9NZZ4 splice variants refers to the polypeptides having amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 35. Also encompassed are polypeptides encoded by a nucleic acid comprising the sequence of SEQ ID NO 31.
  • SEQ ID NO: 4 SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 35.
  • Figure 1 shows the Q9H5P3 genomic and transcript structure of SEQ ID NO: 1,SEQ ID NO: 3, and SEQ ID NO: 4.
  • FIG. 2 shows the Q9UL33/Q9NZZ4 genomic and transcript structure of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 31.
  • SEQ ID NO: 20 uses exons 1, 2,
  • SEQ ID NO: 21 uses exons 1, 2, 3, 4 and 5bis causing one S (serine) to disappear from the translated sequence.
  • SEQ ID NO: 22 uses exons 1, 2bis, 4 and 5bis.
  • SEQ ID NO: 31 shows a longer form of the final exon:exon 5ter.
  • the amino acid sequence of the isoform using exon 5ter is shown in SEQ ID NO 35
  • the upper line represents the chromosome fragment with boxes indicating the exons. Exons are numbered as in the sequence listing.
  • the lower line represents the transcript with the building exons .
  • the arrows at each end of the CDS (coding sequence) line indicate the CDS boundaries (from start codon to stop codon).
  • Figure 3 shows the variation in sequence arises at the boundary of exon4 to exon5 in the Q9UL33/Q9NZZ4 gene.
  • Two different acceptor sites can be used in the 5' region of exon5.
  • the reconstructed codon can be either S (serine) or R (arginine).
  • polypeptide or “peptide” or “peptide fragment” as used herein refers to a compound made up of a single unbranched chain of amino acid residues linked by peptide bonds.
  • the number of amino acid residues in such compounds varies widely; however, preferably, peptides referred to herein usually have from six to forty amino acid residues.
  • Polypeptides and peptide fragments referred to herein usually have from a few tens of amino acid residues, e.g. 20, to up to a few hundred amino acid residues, e.g. 100, or more.
  • polypeptides are manufactured more conveniently by recombinant DNA methods.
  • protein as used herein may be used synonymously with the term “polypeptide” or may refer to, in addition, a complex of two or more polypeptides which may be linked by bonds other than peptide bonds, for example, such polypeptides making up the protein may be linked by disulfide bonds.
  • the tenn “protein” may also comprehend a family of polypeptides having identical amino acid sequences but different post-translational modifications, such as phosphorylations, acylations, glycosylations, and the like, particularly as may be added when such proteins are expressed in eukaryotic hosts.
  • Amino acid residues are referred to herein by their standard single-letter or three-letter notations: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, Isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.
  • Perfectly matched in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one other such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand.
  • the term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed.
  • the term means that the triplex consists of a perfectly matched duplex and a third strand in which every nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a basepair of the perfectly matched duplex.
  • a "mismatch" in a duplex between a tag and an oligonucleotide means that a pair or triplet of nucleotides in the duplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/or reverse Hoogsteen bonding.
  • percent identical or like term, used in respect of the comparison of a reference sequence and another sequence (i.e. a "candidate" sequence) means that in an optimal alignment between the two sequences, the candidate sequence is identical to the reference sequence in a number of subunit positions equivalent to the indicated percentage, the subunits being nucleotides for polynucleotide comparisons or amino acids for polypeptide comparisons.
  • an "optimal alignment" of sequences being compared is one that maximizes matches between subunits and minimizes the number of gaps employed in constructing an alignment. Percent identities may be determined with commercially available implementations of algorithms described by Needleman and Wunsch, J. Mol.
  • a polypeptide having an amino acid sequence at least 95 percent identical to a reference amino acid sequence up to five percent of the amino acid residues in the reference sequence many be deleted or substituted with another amino acid, or a number of amino acids up to five percent of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence of in one or more contiguous groups with in the references sequence.
  • candidate sequence may be a component or segment of a larger polypeptide or polynucleotide and that such comparisons for the purpose computing percentage identity is to be carried out with respect to the relevant component or segment.
  • an isolated polypeptide or polynucleotide of the invention means substantially separated from the components of its natural environment.
  • an isolated polypeptide or polynucleotide is a composition that consists of at least eighty percent of the polypeptide or polynucleotide identified by sequence on a weight basis as compared to components of its natural environment; more preferably, such composition consists of at least ninety-five percent of the polypeptide or polynucleotide identified by sequence on a weight basis as compared to components of its natural environment; and still more preferably, such composition consists of at least ninety-nine percent of the polypeptide or polynucleotide identified by sequence on a weight basis as compared to components of its natural environment.
  • an isolated polypeptide or polynucleotide is a homogeneous composition that can be resolved as a single spot after conventional separation by two-dimensional gel electrophoresis based on molecular weight and isoelectric point. Protocols for such analysis by conventional two- dimensional gel electrophoresis are well known to one of ordinary skill in the art, e.g. Hames and Rickwood, Editors, Gel Electrophoresis of Proteins: A Practical Approach (IRL Press, Oxford, 1981); Scopes, Protein Purification (Springer-Verlag, New York, 1982); Rabilloud, Editor, Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods (Springer-Verlag, Berlin, 2000).
  • oligonucleotide as used herein means linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a polynucleotide by way of a regular pattem of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • monomers are linked by phosphodiester bonds, or analogs thereof, to form oligonucleotides ranging in size from a few monomeric units, e.g.
  • oligonucleotide or polynucleotide is represented by a sequence of letters, such as "ATGCCTG,” or the lower case equivalent, it will be understood that the nucleotides are in 5'- 3' order from left to right and that "A” denotes deoxy adenosine, “C” denotes deoxy cytidine, “G” denotes deoxyguanosine, “T” denotes thymidine, and “U” denotes uridine, unless otherwise noted or understood for their context.
  • oligonucleotides of the invention comprise the four natural nucleotides, and they are joined to one another by natural phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs and may also contain non-natural inter- nucleosidic linkages, particularly when employed as antisense or diagnostic compositions. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed in accordance with the invention, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.
  • nucleoside includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
  • "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, witii the only proviso that they are capable of specific hybridization.
  • Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, and the like.
  • detecttable duplex in reference to a hybridization assay means that for any type of signal generating means used the signal-to-noise ratio is at least two. Preferably, such signal-to-noise ratio is at least three, and more preferably, such signal-to- noise ratio is at least five.
  • alternative splicing in reference to a gene means different exon combinations are included in transcripts from the same gene during RNA processing.
  • splice variant refers to a polypeptide or protein encoded by a transcript which resulted from alternative splicing.
  • the invention relates to methods and compositions for measuring the relative amounts of the Q9H5P3 or Q9UL33/Q9NZZ4 splice variants in a biological sample.
  • the invention includes kits for performing eitiier quantitative immunoassays or PCRs to detect the relative abundances of either the Q9H5P3 or Q9UL33/Q9NZZ4 splice variants or their mRNA transcripts, respectively.
  • monoclonal antibody kits of the invention include at least two monoclonal antibodies, each being specific for one of the Q9H5P3 or Q9UL33/Q9NZZ4 splice variants and not cross-reactive with the others. Kits of the invention further include labeling means to detect the quantity of each monoclonal antibody bound to its target protein.
  • quantitative measurements are made using a conventional ELISA format, as described more fully below.
  • primers are selected so that the mRNAs coding the Q9H5P3 or the four Q9UL33/Q9NZZ4 splice variants are readily distinguished. Accordingly, with respect to the Q9UL33/Q9NZZ4 splice variants, to detect the mRNA encoding SEQ ID NO: 20, at least one primer is selected from exon 5 (SEQ ID NO: 29); to detect the mRNA encoding SEQ ID NO: 21, at least one primer is selected from exon 5 bIS (SEQ ID NO: 30) and at least one primer is selected from exon 2 (SEQ ID NO: 25); to detect the mRNA encoding SEQ ID NO: 22, at least one primer is selected from exon 2 bis (SEQ ID NO: 26); and to detect the mRNA encoding SEQ ID NO: 35, at least one primer is selected from exon 5 ter (SEQ ID NO: 31).
  • the antibodies of the present invention find use in diagnostic assays for the determination of Q9H5P3 or Q9UL33/Q9NZZ4 splice variants.
  • the antibodies of the invention may be used in most assays involving antigen-antibody reactions.
  • the assays may be homogeneous or heterogeneous.
  • the sample can be a biological sample or fluid such as serum, urine, whole blood, lymphatic fluid, plasma, saliva, and the like, cells, tissue, and material secreted by cells or tissues cultured in vitro.
  • the sample can be pretreated if necessary to remove unwanted materials.
  • the immunological reaction usually involves the specific antibody, labeled analyte, and the sample suspected of containing the analyte.
  • the analyte can be directly labeled with the label or indirectly labeled with a means for incorporating the label such as conjugation of the analyte to biotin and having labeled avidin or anti-biotin.
  • the signal from the label is modified, directly or indirectly, upon the binding of the antibody of the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Labels which may be employed as part of a signal producing system capable of producing a signal in relation to the amount of analyte in the sample include free radicals, chromogens, such as fluorescent dyes, chemiluminescers, enzymes, bacteriophages, coenzymes particulate labels and so forth.
  • the reagents are usually the sample, the specific antibody, and means for producing a detectable signal.
  • the specimen is generally placed on a support, such as a plate or a slide, and contacted with the antibody in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal or signal producing system.
  • the signal is related to the presence of the analyte in the sample.
  • Means for producing a detectable signal includes the use of radioactive labels, fluorescers, enzymes, and so forth.
  • Exempary of heterogeneous immunoassays are the radioimmunoassay, immunofiuorescence methods, enzyme-linked immunoassays, and the like.
  • an assay employing an antibody of the present invention involves the use of a surface to which the monoclonal antibody of the invention is attached.
  • the underlying structure of the surface may take different forms, have different compositions and may be a mixture of compositions or laminates or comthereof.
  • the surface may assume a variety of shapes and -forms and may have varied dimensions, depending on the manner of use and measurement.
  • Illustrative surfaces may be pads, beads, discs, or strips which may be flat, concave or convex. Thickness is not critical, generally being from about 0.1 to 2 mm thick and of any convenient diameter or other dimensions, surface typically will be supported on a rod, tube, capillary, fiber, strip, disc, plate, cuvette and the like.
  • the surface will typically be porous and polyfunctional or capable of being polyfunctionalized so as to permit covalent binding of the monoclonal antibody of the invention as well as to permit bonding of other compounds which form a part of a means for producing a detectable signal.
  • a wide variety of organic and inorganic polymers, both natural and synthetic, and combinations thereof, may be employed as the material for the solid surface.
  • Illustrative polymers include polyethylene, polypropylene, ⁇ oly(4- methylbutene), polystyrene, polymethracrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, latex, etc.
  • the sample is mixed with aqueous medium and the medium is contacted with the surface having a monoclonal antibody of the invention bound thereto.
  • a signal producing system and any ancillary materials may also be included in the aqueous medium, either concurrently or added subsequently so as to provide a detectable signal associated with the surface.
  • the means for producing the detectable signal can involve the incorporation in the of a labeled analyte or it may involve the use of a second monoclonal antibody having a label conjugated thereto. Separation and washing steps will be carried out as needed.
  • the signal detected is related to the presence of Q9H5P3 or Q9UL33/Q9NZZ4 splice variant in the sample. It is within the of the present invention to include a calibration as the measurement surface on the same support.
  • a particular embodiment of an assay in accordance with the present invention involves the use of a support such as a slide or a well of a petri dish. The technique involves the sample to be analyzed on the support with an appropriate fixing material such as acetone and incubating the sample on the slide with a monoclonal antibody of the invention.
  • the support After washing with an appropriate buffer such as, for example, phosphate buffered saline, the support is contacted with a labeled specific binding partner for the analyte in the sample. After incubation as desired, the slide is washed a second time with an aqueous buffer and the determination is made of the binding of the labeled monoclonal antibody to the analyte. If the label is fluorescent, the slide may be covered with a fluorescent antibody mounting fluid on a cover slip and then examined with a fluorescent microscope to determine the extent of binding.
  • an appropriate buffer such as, for example, phosphate buffered saline
  • the label can be an enzyme conjugated to the monoclonal antibody of the invention and the extent of binding can be determined by examining the slide for the presence of enzyme activity, which may be indicated by the formation of a precipitate, a color, or the like.
  • a particular example of an assay utilizing the present antibodies is a double determinant ELISA assay.
  • a support such as, e.g., a glass or vinyl plate, is coated with antibody specific for Q9H5P3 or Q9UL33/Q9NZZ4 splice variant by conventional techniques. The support is contacted with the sample suspected of containing Q9H5P3 or Q9UL33/Q9NZZ4 splice variants, usually in a aqueous medium.
  • the support is separated from the medium, washed to remove unbound Q9H5P3 or Q9UL33/Q9NZZ4 splice variants with, for example, water or an aqueous buffered medium, and contacted with an antibody specific for Q9H5P3 or Q9UL33/Q9NZZ4 splice variants, again usually in an aqueous medium.
  • the antibody is labeled wi ⁇ i an enzyme directly or indirectly such as, e.g., horseradish peroxidase or alkaline phosphatase.
  • the support is separated from the medium, and washed as above. The enzyme activity of the support or the aqueous medium is determined. This enzyme activity is related to the amount of Q9H5P3 or Q9UL33/Q9NZZ4 splice variant in the sample.
  • kits for carrying out the methods disclosed above.
  • the kit comprises in packaged combination (a) a monoclonal antibody more specifically defined above and (b) a conjugate of a specific binding partner for the above monoclonal antibody and a label capable of producing a detectable signal.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the kit may further include, where necessary, other members of the signal producing system of which system the label is a member, agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • the diagnostic kit comprises a conjugate of monoclonal antibody of the invention and a label capable of producing a detectable signal. Ancillary agents as mentioned above may also be present.
  • polynucleotide sequences described herein can be used in recombinant DNA molecules that direct the expression of the corresponding polypeptides in appropriate host cells. Because.of the degeneracy in the genetic code, other DNA sequences may encode the equivalent amino acid sequence, and may be used to clone and express the Q9H5P3 and Q9UL33/Q9NZZ4 splice variants. Codons preferred by a particular host cell may be selected and substituted into the naturally occurring nucleotide sequences, to increase the rate and/or efficiency of expression.
  • the nucleic acid e.g., cDNA or genomic DNA
  • the polypeptide may be expressed recombinantly in any of a number of expression systems according to methods known in the art (Ausubel, et al., editors, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1990).
  • Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells, for example primary cells, including stem cells, including, but not limited to bone marrow stem cells.
  • these include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, and yeast transformed with yeast expression vectors. Also included, are insect cells infected with a recombinant insect virus (such as baculovirus), and mammalian expression systems.
  • the nucleic acid sequence to be expressed may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • the Q9H5P3 and Q9UL33/Q9NZZ4 splice variants of the present invention are produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding a Q9H5P3 and Q9UL33/Q9NZZ4, under the appropriate conditions to induce or cause expression of the protein.
  • the conditions appropriate for Q9H5P3 and Q9UL33/Q9NZZ4 expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.
  • the timing of the harvest is important.
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein 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 important for correct insertion, folding and/or function.
  • host cells such as CHO, HeLa, BHK, MDCK, 293, W138, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • Drosophila melangastev cells Sacchavoinyces cevevisiae and other yeasts
  • E. coli Bacillus subtilis
  • SF9 cells C129 cells, 293 cells
  • Neurospora BHK, CHO, COS, and HeLa cells
  • fibroblasts Schwanoma cell lines
  • immortalized mammalian myeloid and lymphoid cell lines Jukat cells, human cells and other primary cells.
  • the nucleic acid encoding a Q9H5P3 and Q9UL33/Q9NZZ4 polypeptide must be "operably linked" hy placing it into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding' sequence if it is positioned so as to facilitate translation.
  • operably linked DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enliancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2: plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably, two homologous sequences which flank the expression construct.
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in die art.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
  • Selection genes are well known in the art and will vary with the host cell used.
  • Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available for from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • Host cells transformed with a nucleotide sequence encoding a Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides encoding the Q9H5P3 or Q9UL33/Q9NZZ4 can be designed with signal sequences which direct secretion of the Q9H5P3 or Q9UL33/Q9NZZ4 through a prokaryotic or eukaryotic cell membrane.
  • the desired Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus ' of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the Q9H5P3 or Q9UL33/Q9NZZ4-encoding DNA that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a- factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C.
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • the coding sequence is inserted into an appropriate vector, which in turn may require the presence of certain characteristic "control elements” or "regulatory sequences.”
  • Appropriate constructs are known generally in the art (Ausubel, et al., 1990) and, in many cases, are available from commercial suppliers such as Invitrogen (San Diego, Calif), Stratagene (La Jolla, Calif.), Gibco BRL (Rockville, Md.) or Clontech (Palo Alto, Calif). Expression in Bacterial Systems. Transformation of bacterial cells may be achieved using an inducible promoter such as the hybrid lacZ promoter of the "BLUESCRIPT" Phagemid (Stratagene) or "pSPORTl” (Gibco BRL).
  • a number of expression vectors may be selected for use in bacterial cells to produce cleavable fusion proteins that can be easily detected and/or purified, including, but not limited to "BLUESCRIPT” (a- galactosidase; Stratagene) or pGEX (glutathione S-transferase; Promega, Madison, Wis.).
  • a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of the Q9H5P3 or Q9UL33/Q9NZZ4 gene into mRNA.
  • a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence.
  • This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tat promoter is a hybrid of the trp and lac promoter sequences.
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. An efficient ribosome binding site is also desirable.
  • the expression vector may also include a signal peptide sequence that provides for secretion of the Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide in bacteria.
  • the signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art.
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include drug resistance genes such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. When large quantities of Q9H5P3 or Q9UL33/Q9NZZ4 splice variants are needed, e.g., for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, multifunctional E.
  • coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta- galactosidase so that a hybrid protein is produced; PIN vectors (Van Heeke & Schuster JBiol Chem 264:5503-5509 (1989)); PET vectors (Novagen, Madison Wis.); and the like.
  • Expression vectors for bacteria include the various components set forth above, and are well known in the art. Examples include vectors for Bacillus subtilis, E. coli,
  • Streptococcus cvemovis and Streptococcus lividans, among others.
  • Bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride mediated transfection, electroporation, and others.
  • Yeast expression systems are well known in the art, and include expression vectors for Sacchavomyces cevevisiae, Candida albicans and C. maltosa, Hansenula polymovpha, Kluyvevomyces fvagilis and A', lactis, Pichia guillevimondii and P pastoris, Schizosaccha-vomyces pombe, and Yawowia lipolytica.
  • suitable promoters for use in yeast hosts include the promoters for 3- phosphoglycerate kinase (Hitzeman et al., /. Biol. Chem.
  • glycolytic enzymes such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, tri osephosphate isomerase, phosphoglucose isomerase, alpha factor, the ADH2IGAPDH promoter, glucokinase alcohol oxidase, and PGH.
  • enolase such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, tri osephosphate isome
  • yeast promoters which are inducible have the additional advantage of transcription controlled by growth conditions, include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP 73,657. Yeast selectable markers include ADE2. HIS4. LEU2. TRP1.
  • Yeast expression vectors can be constructed for intracellular production or secretion of a Q9H5P3 or Q9UL33/Q9NZZ4 from the DNA encoding the Q9H5P3 or Q9UL33/Q9NZZ4 of interest.
  • a selected signal peptide and the appropriate constitutive or inducible promoter may be inserted into suitable restriction sites in the selected plasmid for direct intracellular expression of the Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide.
  • DNA encoding the respective Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide can be cloned into the selected plasmid, together with DNA encoding the promoter, the yeast alpha-factor secretory signal/leader sequence, and linker sequences (as needed), for expression of the Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide.
  • Yeast cells can then be transformed with the expression plasmids described above, and cultured in an appropriate fermentation media.
  • the protein produced by such transformed yeast can then be concentrated by precipitation with 10% trichloroacetic acid and analyzed following separation by SDS-PAGE and staining of the gels with Coomassie Blue stain.
  • the recombinant Q9H5P3 or Q9UL33/Q9NZZ4 can subsequently be isolated and purified from the fermentation medium by techniques known to those of skill in the art.
  • Expression in Mammalian Systems The Q9H5P3 and Q9UL33/Q9NZZ4 splice variants may be expressed in mammalian cells. Mammalian expression systems are known in the art, and include retroviral vector mediated expression systems.
  • Mammalian host cells may be transformed with any of a number of different viral-based expression systems, such as adenovirus, where the coding region can be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome results in a viable virus capable of expression of the polypeptide of interest in infected host cells.
  • a preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/101048.
  • Suitable mammalian expression vectors contain a mammalian promoter which is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for Q9H5P3 or Q9UL33/Q9NZZ4 protein into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211, 504 published Jul.
  • adenovirus such as Adenovirus 2
  • bovine papilloma virus such as Adenovirus 2
  • bovine papilloma virus such as avian sarcoma virus
  • cytomegalovirus a retrovirus
  • hepatitis-B virus and Simian Virus 40 SV40
  • heterologous mammalian promoters e.g., the actin promoter or an immunoglobulin promoter
  • heat-shock promoters provided such promoters are compatible with die host cell systems.
  • Transcription of a DNA encoding a Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into die vector.
  • Enliancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin).
  • an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enliancers.
  • the enhancer is preferably located at a site 5' from the promoter.
  • the transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank die coding sequence.
  • the 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
  • transcription terminator and polyadenylation signals include those derived from SV40. Long term, high-yield production of recombinant proteins can be effected in a stable expression system. Expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene may be used for this purpose.
  • selectable markers for use in mammalian cells are readily available commercially and are known to persons skilled in the art.
  • selectable markers include, but are not limited to herpes simplex virus thymi-dine kinase and adenine phosphoribosyltransferase for use in tk- or hprt-cells, respectively.
  • the methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used.
  • Techniques include dextran- mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of die polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Q9H5P3 and Q9UL33/Q9NZZ4 splice variants may also be produced in insect cells.
  • Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
  • the Q9H5P3 or Q9UL33/Q9NZZ4-encoding DNA is fused upstream of an epitope tag contained within a baculovirus expression vector.
  • Autographa califovnica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptevu fmgipevdu Sf9 cells or in Trichoplusia larvae.
  • the Q9H5P3 -encoding or Q9UL33/Q9NZZ4-encoding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of a Q9H5P3 or Q9UL33/Q9NZZ4-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. fingipevdu cells or Trichoplusia larvae in which the Q9H5P3 or Q9UL33/Q9NZZ4 is expressed (Smith et al., J. Wol.
  • Suitable epitope tags for fusion to the Q9H5P3 or Q9UL33/Q9NZZ4-encoding DNA include poly- his tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including commercially available plasmids such as pVL1393 (Novagen).
  • the Q9H5P3 or Q9UL33/Q9NZZ4-encoding DNA or the desired portion of die Q9H5P3 or Q9UL33/Q9NZZ4-encoding DNA is amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may incorporate flanking restriction sites.
  • the PCR product is then digested with the selected restriction enzymes and subcloned into an expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodopteva fvugipevda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL), or other methods known to those of skill in the art.
  • Virus is produced by day 4-5 of culture in Sf9 cells at 28°C, and used for further amplifications. Procedures are performed as further described in O'Reilley et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, Oxford University Press (1994).
  • Extracts may be prepared from recombinant virus-infected Sf9 cells as described in Rupert et al., Nature 362:175-179 (1993).
  • expressed epitope-tagged Q9H5P3 or Q9UL33/Q9NZZ4 splice variants can be purified by affinity chromatography, or for example, purification of an IgG tagged (or Fc tagged) Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide can be performed using chromatography techniques, including Protein A or protein G column chromatography. Evaluation of Gene Expression.
  • Gene expression may be evaluated in a sample directly, for example, by standard techniques known to those of skill in the art, e.g., Southern blotting for DNA detection, Northern blotting to determine the transcription of mRNA, dot blotting (DNA or RNA), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies may be used in assays for detection of nucleic acids, such as specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • Such antibodies may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression alternatively, may be measured by immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to directly evaluate the expression of Q9H5P3 or Q9UL33/Q9NZZ4s.
  • Antibodies useful for such immunological assays may be either monoclonal or polyclonal, and may be prepared against a native sequence Q9H5P3 or Q9UL33/Q9NZZ4 based on the DNA sequences provided herein.
  • Expressed Q9H5P3 or Q9UL33/Q9NZZ4 splice variants may be purified or isolated after expression, using any of a variety of methods known to those skilled in the art. The appropriate technique will vary depending upon what otiier components are present in the sample. Contaminant components that are removed by isolation or purification are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other solutes. The purification step(s) selected will depend, for example, on the nature of the production process used and the particular Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide produced.
  • An Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide or protein may be recovered from cultare medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage.
  • a suitable detergent solution e.g. Triton-X 100
  • cells employed in expression of Q9H5P3 or Q9UL33/Q9NZZ4 splice variants can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or by use of cell lysing agents.
  • Exemplary purification methods include, but are not limited to, ion- exchange column chromatography; chromatography using silica gel or a cation-exchange resin such as DEAE; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; chromatography using metal chelating columns to bind epitope-tagged forms of the Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide; ethanol precipitation; reverse phase HPLC; chromatofocusing; SDS-PAGE; and ammonium sulfate precipitation.
  • an isolated Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide will be prepared by at least one purification step.
  • the respective Q9H5P3 or Q9UL33/Q9NZZ4 protein may be purified using a standard anti- Q9H5P3 or anti-Q9UL33/Q9NZZ4 antibody column.
  • Ultrafiltration and dialysis techniques, in conjunction with protein concentration, are also useful (see, for example, Scopes, R., PROTEIN PURIFICATION, Springer-Verlag, New York, N.Y., 1982).
  • the degree of purification necessary will vary depending on the use of the Q9H5P3 or Q9UL33/Q9NZZ4. In some instances no purification will be necessary.
  • the Q9H5P3 or Q9UL33/Q9NZZ4 splice variants and nucleic acids of the present invention are useful in a number of applications, as detailed below.
  • the nucleic acids, proteins and antibodies of the invention may be labeled.
  • labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound.
  • labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes.
  • the labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound which is being detected.
  • Q9H5P3 and Q9UL33/Q9NZZ4 Fusion Proteins may also be modified in a way to form chimeric molecules comprising a Q9H5P3 or Q9UL33/Q9NZZ4 fused to another, heterologous polypeptide or amino acid sequence.
  • fusion protein used herein refers to a chimeric polypeptide comprising a Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide, or domain sequence thereof, fused to a "targeting polypeptide" .
  • the targeting polypeptide has enough residues to facilitate targeting to a particular cell type or receptor, yet is short enough such that it does not interfere with the biological function of the Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide.
  • the targeting polypeptide preferably is also fairly unique so that the fusion protein does not substantially cross-react with other cell types or receptors.
  • Suitable targeting polypeptides generally have at least about 10 amino acid residues and usually between from about 10 to about 500 amino acid residues.
  • Preferred targeting polypeptides have from about 20 to about 200 amino acid residues.
  • the fusion protein may also comprises a fusion of a Q9H5P3 or Q9UL33/Q9NZZ4 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generally placed at the amino-or carboxyl-terminus of the Q9H5P3 or Q9UL33/Q9NZZ4.
  • Such epitope-tagged forms of a Q9H5P3 or Q9UL33/Q9NZZ4 can be detected using an antibody against the tag polypeptide.
  • the epitope tag enables the Q9H5P3 or Q9UL33/Q9NZZ4 to be readily purified by using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • the fusion protein may comprise a fusion of a Q9H5P3 or Q9UL33/Q9NZZ4 with an immunoglobulin or a particular region of an immunoglobulin.
  • such a fusion could be to the Fc region of an IgG molecule or, for example, GM-CSF.
  • Preferred fusion proteins include, but are not limited to, molecules that facilitate immune targeting of the Q9H5P3 or Q9UL33/Q9NZZ4.
  • the Q9H5P3 or Q9UL33/Q9NZZ4 fusion protein may be made for various other purposes using techniques well known in the art. For example, for the creation of antibodies, if the desired epitope is small, a partial or complete Q9H5P3 or Q9UL33/Q9NZZ4 protein may be fused to a carrier protein to form an immunogen.
  • the Q9H5P3 or Q9UL33/Q9NZZ4 protein may be made as a fusion protein to increase the ability of the antigen to stimulate cellular and/or humoral (antibody-based) immune responses, or for other reasons.
  • Peptides of the invention are synthesized by standard techniques, e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed. (Pierce Chemical Company, Rockford, IL, 1984).
  • a commercial peptide synthesizer is used, e.g. Applied Biosystems, Inc. (Foster City, CA) model 430A, and polypeptides of the invention may be assembled from multiple, separately synthesized and purified, peptide in a convergent synthesis approach, e.g. Kent et al, U.S. patent 6,184,344 and Dawson and Kent, Annu. Rev.
  • Peptides of the invention may be assembled by solid phase synthesis on a cross-linked polystyrene support starting from the carboxyl terminal residue and adding amino acids in a step wise fashion until the entire peptide has been formed.
  • the following references are guides to the chemistry employed during synthesis: Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992); Merrifield, J. Amer. Chem. Soc, Vol. 85, pg.- 2149 (1963); Kent et al., pg 185, in Peptides 1984, Ragnarsson, Ed.
  • chemical synthesis of polypeptides of the invention is carried out by the assembly of oligopeptides by native chemical ligation, as described by Dawson et al, Science, 266: 776-779 (1994) and Kent el al, U.S. patent 6,184,344. Briefly, in the approach a first oligopeptide is provided with an N-terminal cysteine having an unoxidized sulfhydryl side chain, and a second oligopeptide is provided with a C-terminal thioester.
  • the unoxidized sulfhydryl side chain of the N-terminal cysteine is then condensed with the C-terminal thioester to produce an intermediate oligopeptide which links the first and second oligopeptides with a ⁇ -aminothioester bond.
  • the ⁇ -aminothioester bond of the intermediate oligopeptide then undergoes an intramolecular rearrangement to produce the oligopeptide product which links the first and second oligopeptides with an amide bond.
  • the N-terminal cysteine of internal fragments are protected from undesired cyclization and/ro concatenation reactions by a cyclic thiazolidine protecting group as described below.
  • such cyclic thiazolidine protecting group is a thioprolinyl group.
  • Oligopeptides having a C-terminal thioester may be produced as described in the following references, which are incorporated by reference: Kent et al, U.S. patent 6,184,344; Tarn et al, Proc. Natl. Acad. Sci., 92: 12485-12489 (1995); Blake, Int. J. Peptide Protein Res., 17: 273 (1981); Canne et al, Tetrahedron Letters, 36: 1217-1220 (1995); hackeng et al, Proc. Natl. Acad. Sci., 94: 7845-7850 (1997); or Hackeng et al, Proc. Natl. Acad. Sci., 96: 10068-10073 (1999).
  • oligopeptides are synthesized on a solid phase support (described below) typically on a 0.25 mmol scale by using the in situ neutralization/HBTU activation procedure for Boc chemistry dislosed by Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992), which reference is incorporated herein by reference.
  • HBTU is 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate and Boc is tert-butoxycarbonyl).
  • Each synthetic cycle consists of N ⁇ - Boc removal by a 1- to 2- minute treatment with neat TFA, a 1-minute DMF flow wash, a 10- to 20-minute coupling time with 1.0 mmol of preactivated Boc-amino acid in the presence of DIEA, and a second DMF flow wash.
  • N,N-dimethylformamide, and DIEA is N,N-diisopropylethylamine.
  • N ⁇ -Boc-amino acids (1.1 mmol) are preactivated for 3 minutes with 1.0 mmol of HBTU (0.5 M in DMF) in the presence of excess DIEA (3 mmol).
  • yields are determined by measuring residual free amine with a conventional quantitative ninhydrin assay, e.g. as disclosed in Sarin et al, Anal. Biochem., 117: 147-157 (1981).
  • a DCM flow wash is used before and after deprotection by using TFA, to prevent possible high-temperature (TFA/DMF)-catalyzed pyrrolidone formation.
  • TFA high-temperature
  • the oligopeptides are deprotected and cleaved from the resin by treatment with anhydrous HF for 1 hour at 0°C with 4% /?-cresol as a scavenger.
  • the imidazole side-chain 2,4-dinitrophenyl (dnp) protecting groups remain on the His residues because the dnp-removal procedure is incompatible with C-terminal thioester groups.
  • oligopeptides are precipitated with ice-cold diethylether, dissolved in aqueous acetonitrile, and lyophilized.
  • Thioester oligopeptides described above are preferably synthesized on a trityl- associated mercaptopropionic acid-leucine (TAMPAL) resin, made as disclosed by Hackeng et al (1999), or comparable protocol. Briefly, N ⁇ -Boc-Leu (4 mmol) is activated with 3.6 mmol of HBTU in the presence of 6 mmol of DIEA and coupled for 16 minutes to 2 mmol of p-methylbenzhydrylamine (MBHA) resin, or the equivalent.
  • TAMPAL trityl- associated mercaptopropionic acid-leucine
  • TAMPAL resin can be used as a starting resin for polypeptide-chain assembly after removal of the trityl protecting group with two 1-minute treatments with 3.5% triisopropylsilane and 2.5% H 2 0 in TFA.
  • the thioester bond can be formed with any desired amino acid by using standard in situ-neutralization peptide coupling protocols for 1 hour, as disclosed in Schnolzer et al (cited above). Treatment of the final oligopeptide with anhydrous HF yields the C-terminal activated mercaptopropionic acid-leucine (MPAL) thioester oligopeptides.
  • MPAL C-terminal activated mercaptopropionic acid-leucine
  • thiazolidine-protected thioester oligopeptide intermediates are used in native chemical ligation under conditions as described by Hackeng et al (1999), or like conditions. Briefly, 0.1 M phosphate buffer (pH 8.5) containing 6 M guanidine, 4% (vol/vol) benzylmercaptan, and 4% (vol/vol) thiophenol is added to dry peptides to be ligated, to give a final peptide concentration of 1-3 mM at about pH-7, lowered because of the addition of thiols and TFA from the lyophilized peptide.
  • the ligation reaction is performed in a heating block at 37°C and is periodically vortexed to equilibrate the thiol additives.
  • the reaction may be monitored for degree of completion by MALDI- MS or HPLC and electrospray ionization MS.
  • the N-terminal thiazolidine ring of the product is opened by treatment with a cysteine deprotecting agent, such as O-methylhydroxylamine (0.5 M) at pH 3.5-4.5 for 2 hours at 37°C , after which a 10-fold excess of Tris-(2-carboxyethyl)-phosphine is added to the reaction mixture to completely reduce any oxidizing reaction constituents prior to purification of the product by conventional preparative HPLC.
  • a cysteine deprotecting agent such as O-methylhydroxylamine (0.5 M) at pH 3.5-4.5 for 2 hours at 37°C
  • Tris-(2-carboxyethyl)-phosphine is added to the reaction mixture to completely reduce any oxidizing reaction constituents prior to purification of the product by conventional preparative HPLC.
  • fractions containing the ligation product are identified by electrospray MS, are pooled, and lyophilized.
  • the final polypeptide product may be refolded by conventional techniques, e.g. Creighton, Meth. Enzymol., 107: 305-329 (1984); White, Meth. Enzymol, 11: 481-484 (1967); Wetlaufer, Meth. EnzymoL, 107: 301-304 (1984); and the like.
  • a final product is refolded by air oxidation by the following, or like:
  • the reduced lyophilized product is dissolved (at about 0.1 mg/mL) in 1 M guanidine hydrochloride (or like chaotropic agent) with 100 mM Tris, 10 mM methionine, at pH 8.6. After gentle overnight stirring, the re-folded product is isolated by reverse phase HPLC with conventional protocols.
  • the present invention further provides anti- Q9H5P3 and anti-Q9UL33/Q9NZZ4 antibodies.
  • the antibodies of the present invention include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
  • the anti- Q9H5P3 or anti-Q9UL33/Q9NZZ4 antibodies of the present invention may be polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Such polyclonal antibodies can be produced in a mammal, for example, following one or more injections of an immunizing agent, and preferably, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected into the mammal by a series of subcutaneous or intraperitoneal injections.
  • the immunizing agent may include a Q9H5P3 or Q9UL33/Q9NZZ4 protein, or a fusion protein thereof.
  • immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • Adjuvants include, for example, Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicoryno-mycolate).
  • the immunization protocol may be determined by one skilled in the art based on standard protocols or by routine experimentation.
  • the anti- Q9H5P3 and anti- Q9UL33/Q9NZZ4 antibodies may be monoclonal antibodies.
  • Monoclonal antibodies may be produced by hybridomas, wherein a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lympho-cytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent [Kohler and Milstein, Nature 256:495 (1975)].
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the Q9H5P3 or Q9UL33/Q9NZZ4 or a fusion protein thereof.
  • lymph node cells are used if non-human mammalian sources are desired, or peripheral blood lymphocytes ("PBLs") are used if cells of human origin.
  • the lymphocytes are fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to produce a hybridoma cell [Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, pp. 59- 103 (1986)].
  • immortalized cell lines are transformed mammalian cells, for example, myeloma cells of rat, mouse, bovine or human origin.
  • the hybridoma cells are cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT), substances which prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level production of antibody, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine or human myeloma lines, which can be obtained, for example, from the American Type Culture Collection (ATCC), Rockville, MD. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Zmmunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)].
  • the culture medium (supernatant) in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against a Q9H5P3 or Q9UL33/Q9NZZ4.
  • the binding specificity of monoclonal antibodies present in the hybridoma supernatant is determined by immunoprecipitation or by an in vitro binding assay, such as radio- immunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Appropriate techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of
  • the cells may be cloned by limiting dilution procedures and grown by standard methods [Goding, 1986]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by selected clones may be isolated or purified from the culture medium or ascites fluid by immunoglobulin purification procedures routinely used by those of skill in the art such as, for example, protein A-Sepharose, hydroxyl-apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be isolated from the Q9H5P3 or Q9UL33/Q9NZZ4-specific hybridoma cells and sequenced, e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies.
  • the DNA may be inserted into an expression vector, which is then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for the human heavy and light chain constant domains for the homologous murine sequences [Morrison et al., Proc. Nat. Acad. Sci. 81:6851-6855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)], or by covalently joining to the immunoglobulin coding sequence all * or part of the coding sequence for a non-immunoglobulin polypeptide.
  • the non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of die invention to create a chimeric bivalent antibody.
  • the antibodies may also be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, in vitro methods are suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • Antibodies and antibody fragments characteristic of hybridomas of the invention can also be produced by recombinant means by extracting messenger RNA, constructing a cDNA library, and selecting clones which encode segments of the antibody molecule, e.g. Wall et al., Nucleic Acids Research, Vol. 5, pgs. 3113-3128 (1978); Zakut et al., Nucleic Acids Research, Vol. 8, pgs. 3591-3601 (1980); Cabilly et al., Proc. Natl. Acad. Sci., Vol. 81, pgs. 3273-3277 (1984); Boss et al., Nucleic Acids Research, Vol. 12, pgs.
  • Both polyclonal and monoclonal antibodies can be screened by ELISA.
  • the test is based on the tendency of macromolecules to adsorb nonspecifically to plastic. The irreversibility of this reaction, without loss of immunological activity, allows the formation of antigen-antibody complexes with a simple separation of such complexes from unbound material.
  • peptide conjugated to a carrier different from that used in immunization is adsorbed to the wells of a 96-well microtiter plate. The adsorbed antigen is then allowed to react in the wells with dilutions of anti-peptide serum.
  • the invention includes peptides derived from Q9H5P3 or Q9UL33/Q9NZZ4, and immunogens comprising conjugates between carriers and peptides of the invention.
  • immunogen refers, to a substance which is capable of causing an immune response.
  • carrier refers to any substance which when chemically conjugated to a peptide of the invention permits a host organism immunized with the resulting conjugate to generate antibodies specific for the conjugated peptide.
  • Carriers include red blood cells, bacteriophages, proteins, or synthetic particles such as agarose beads.
  • carriers are proteins, such as serum albumin, gamma-globulin, keyhole limpet hemocyanin, thyroglobulin, ovalbumin, fibrinogen, or the like.
  • a general rule for -selecting an appropriate method for coupling a given peptide to a protein carrier can be stated as follows: the group involved in attachment should occur only once in the sequence, preferably at the appropriate end of the segment.
  • BDB should not be used if a tyrosine residue occurs in the main part of a sequence chosen for its potentially antigenic character.
  • centrally located lysines rule out the glutaraldehyde method, and the occurrences of aspartic and glutamic acids frequently exclude the carbodiimide approach.
  • suitable residues can be positioned at either end of chosen sequence segment as attachment sites, whether or not they occur in the "native" protein sequence. Internal segments, unlike the amino and carboxy termini, will differ significantly at the "unattached end" from the same sequence as it is found in the native protein where the polypeptide backbone is continuous.
  • the problem can be remedied, to a degree, by acetylating the ⁇ -amino group and then attaching the peptide by way of its carboxy terminus.
  • the coupling efficiency to the carrier protein is conveniently measured by using a radioactively labeled peptide, prepared either by using a radioactive amino acid for one step of the synthesis or by labeling the completed peptide by the iodination of a tyrosine residue.
  • the presence of tyrosine in the peptide also allows one to set up a sensitive radioimmune assay, if desirable. Therefore, tyrosine can be introduced as a terminal residue if it is not part of die peptide sequence defined by the native polypeptide.
  • Preferred carriers are proteins, and preferred protein carriers include bovine serum albumin, myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH), or the like.
  • Peptides can be linked to KLH through cysteines by MBS as disclosed by Liu et al., Biochemistry, Vol. 18, pgs. 690-697 (1979).
  • MBS phosphate- buffered saline
  • pH 9.0 0.1 M sodium borate buffer
  • 1.0 M sodium acetate buffer pH for the dissolution of the peptide is chosen to optimize peptide solubility.
  • the content of free cysteine for soluble peptides is determined by Ellman's method, Ellman, Arch. Biochem. Biophys., Vol. 82, pg. 7077 (1959).
  • 4 mg KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg MBS (dissolved in dimethyl formamide) and stirred for 30 min at room temperature.
  • the MBS is added dropwise to ensure that the local concentration of formamide is not too high, as KLH is insoluble in >30% formamide.
  • the reaction product, KLH-MBS is then passed through Sephadex G-25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0) to remove free MBS, KLH recovery from peak fractions of the column eluate (monitored by OD280) is estimated to be approximately 80% .
  • KLH-MBS is then reacted with 5 mg peptide dissolved 25 in 1 ml of the chosen buffer.
  • the pH is adjusted to 7-7.5 and the reaction is stirred for 3 hr at room temperature. Coupling efficiency is monitored with radioactive peptide by dialysis of a sample of the conjugate against phosphate-buffered saline, and ranged from 8% to 60%.
  • polyclonal or monoclonal antibodies are produced by standard techniques, e.g. as disclosed by Campbell, Monoclonal Antibody Technology (Elsevier, New York, 1984); Hurrell, ed. Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982); Schreier et al. Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); U.S. Patent 4,562,003; or the like. In particular, U.S. Patent 4,562,003 is incorporated by reference.
  • the anti-Q9H5P3 and anti-Q9UL33/Q9NZZ4 antibodies of the invention may further comprise humanized antibodies or human antibodies.
  • humanized antibody refers to humanized forms of non-human (e.g., murine) antibodies that are chimeric antibodies, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding partial sequences of antibodies) which contain some portion of the sequence derived from non-human antibody.
  • Humanized antibodies include human immunoglobulins in which residues from a complementary determining region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non-human species such as mouse, rat or rabbit having the desired binding specificity, affinity and capacity.
  • CDR complementary determining region
  • the humanized antibody will comprise substantially all of at least one, and generally two, variable domains, in which all or substantially all of the CDR regions ' correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature 321:522-525 (1986) and Presta, Cuvv. Op. Stvuct. Biol. 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • Methods for humanizing non-human antibodies are well known in the art.
  • a humanized antibody has one or more amino acids introduced into it from a source which is non-human in order to more closely resemble a human antibody, while still retaining the original binding activity of the antibody.
  • Heteroconjugate Antibodies Heteroconjugate antibodies which comprise two covalently joined antibodies, are also within the scope of the present invention. Heteroconjugate antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be prepared using a disulfide exchange reaction or by forming a thioether bond.
  • Bispecific Antibodies have binding specificities for at least two different antigens. Such antibodies are monoclonal, and preferably human or humanized. One of the binding specificities of a bispecific antibody of the present invention is for a Q9H5P3 or Q9UL33/Q9NZZ4, and the other one is preferably for a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art, and in general, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs in hybridoma cells, where the two heavy chains have different specificities [Milstein and Cuello, Nature 305:537-539 (1983)]. Given that the random assortment of immunoglobulin heavy and light chains results in production of potentially ten different antibody molecules by the hybridomas, purification of the correct molecule usually requires some sort of affinity purification, e.g. affinity chromatography.
  • Antibody antagonists are derived from antibodies specific for Q9H5P3 or Q9UL33/Q9NZZ4. More preferably, die antagonists of the invention comprise fragments or binding compositions specific for Q9H5P3 or Q9UL33/Q9NZZ4. Antibodies comprise an assembly of polypeptide chains linked together by disulfide bridges. Two major polypeptide chains, referred to as die light chain and the heavy chain, make up all major structural classes (isotypes) of antibody. Botii heavy chains and light chains are further divided into subregions referred to as variable regions and constant regions.
  • Heavy chains comprise a single variable region and three different constant regions
  • light chains comprise a single variable region (different from that " of the heavy chain) and a single constant region (different from those of the heavy chain).
  • the variable regions of the heavy chain and light chain are responsible for the antibody's binding specificity.
  • the term "heavy chain variable region” means a polypeptide (1) which is from 110 to 125 amino acids in length, and (2) whose amino acid sequence corresponds to that of a heavy chain of a monoclonal antibody of the invention, starting from the heavy chain's N-terminal amino acid.
  • the term "light chain variable region” means a polypeptide (1) which is from 95 to 115 amino acids in length, and (2) whose amino acid sequence corresponds to that of a light chain of a monoclonal antibody of the invention, starting from the light chain's N-terminal amino acid.
  • the term “monoclonal antibody” refers to homogeneous populations of immunoglobulins which are capable of specifically binding to Q9H5P3 or Q9UL33/Q9NZZ4.
  • binding composition means a composition comprising two polypeptide chains (1) which, when operationally associated, assume a conformation having high binding affinity for Q9H5P3 or Q9UL33/Q9NZZ4, and (2) which are derived from a hybridoma producing monoclonal antibodies specific for Q9H5P3 or Q9UL33/Q9NZZ4.
  • operationalally associated is meant to indicate that die two polypeptide chains can be positioned relative to one another for binding by a variety of means, including by association in a native antibody fragment, such as Fab or Fv, or by way of genetically engineered cysteine-containing peptide linkers at the carboxyl termini.
  • the two polypeptide chains correspond to the light chain variable region and heavy chain variable region of a monoclonal antibody specific for Q9H5P3 or Q9UL33/Q9NZZ4.
  • antagonists of the invention are derived from monoclonal antibodies specific for Q9H5P3 or Q9UL33/Q9NZZ4.
  • Monoclonal antibodies capable of blocking, or neutralizing, Q9H5P3 or Q9UL33/Q9NZZ4 are selected by their ability to inhibit Q9H5P3 or Q9UL33/Q9NZZ4-induced effects.
  • Polynucleotide sequences (or the complements thereof) which encode Q9H5P3 and Q9UL33/Q9NZZ4 splice variants have various applications, including uses as hybridization probes, in chromosome and gene mapping, and in the generation of antisense RNA and DNA.
  • Q9H5P3 and Q9UL33/Q9NZZ4-encoding nucleic acids are useful as targets for pharmaceutical intervention, e.g. for the development of DNA vaccines, and for the preparation of Q9H5P3 and Q9UL33/Q9NZZ4 splice variants by recombinant techniques, as described herein.
  • polynucleotides described herein can be used in diagnostic assays. Accordingly, diagnostic methods based on detecting the presence of such polynucleotides in body fluids or tissue samples are a feature of the present invention.
  • nucleic acid based diagnostic assays in accordance with the present invention include, but are not limited to, hybridization assays, e.g., in situ hybridization, and PCR-based assays.
  • Polynucleotides, including extended length polynucleotides, sequence variants and fragments thereof, as described herein, may be used to generate hybridization probes or PCR primers for use in such assays.
  • Such probes and primers will be capable of detecting polynucleotide sequences, including genomic sequences that are similar, or complementary to, the Q9H5P3 or Q9UL33/Q9NZZ4 polynucleotides described herein.
  • the invention includes primer pairs for carrying out a PCR to amplify a segment of a polynucleotide of the invention.
  • Each primer of a pair is an oligonucleotide having a length of between 15 and 30 nucleotides such that i) one primer of the pair forms a perfectly matched duplex with one strand of a polynucleotide of the invention and the other primer of the pair form a perfectly match duplex with the complementary strand of the same polynucleotide, and ii) the primers of a pair form such perfectly matched duplexes at sites on the polynucleotide that separated by a distance of between 10 and 2500 nucleotides.
  • the annealing temperature of each primer of a pair to its respective complementary sequence is substantially the same.
  • Q9UL33/Q9NZZ4-related polypeptides have been described as tumor markers, thereby having use in the diagnosis and treatment of cancer (International Patent Publication no. WO 00/55350).
  • DbEST entries show that the mRNA encoding the Q9UL33/Q9NZZ4 protein of SEQ ID NO 20 is expressed in adrenal gland (AV703452), pheochromocytoma (AV733899), cervix (BI091920), prostate (adenocarcinoma, cell line) (BG285280), human skeletal muscle (adult) (AL712099), brain (BM926792).
  • the mRNA encoding the Q9UL33/Q9NZZ4 protein of SEQ ID NO 21 is expressed (dbEST) in eye (adult, retina foveal and macular) (BM690783), bone (osteosarcoma, cell line) (BG025979), bone marrow (CD34+ hematopoietic stem/progenitor cell) (AV761880), cartilage (BG900037), kidney
  • Database entries show that the mRNA encoding the Q9H5P3 protein of SEQ ID NO 1 is expressed in Lung (Q8WWF (TrEmbl)) and brain (anaplastic oligodendroglioma) (AW005929).
  • DbEST entries show that mRNA encoding the Q9H5P3 protein of SEQ ID NO 3 can be found in testis (AA824370), fetal lung (19 weeks) (AI192974), lung (metastatic chondrosarcoma) (BQ000937), lung (BF512797), prostate (adult) (AI401045).
  • the mRNA encoding the Q9H5P3 protein of SEQ ID NOS 5 has been found in pooled germ cell tumors (AI342055).
  • Hybridization probes derived from polynucleotides of the invention can be used, for example, in performing in situ hybridization on tissue samples, such as fixed or frozen tissue sections prepared on microscopic slides or suspended cells. Briefly, a labeled DNA or RNA probe is allowed to bind its DNA or RNA target sample in the tissue section on a prepared microscopic, under controlled conditions. Generally, dsDNA probes consisting of the DNA of interest cloned into a plasmid or bacteriophage DNA vector are used for this purpose, although ssDNA or ssRNA probes may also be used. Probes are generally oligonucleotides between about 15 and 40 nucleotides in length.
  • the probes can be polynucleotide probes generated by PCR random pruning primer extension or in vitro transcription of RNA from plasmids (riboprobes). These latter probes are typically several hundred base pairs in lengtii.
  • the probes can be labeled by any of a number of methods, including fluorescent tags, enzymes or radioactive moieties, according to methods well known in the art. The particular detection method will correspond to the type of label utilized on the probe (e.g. , autoradiography, X-ray detection, fluorescent or visual microscopic analysis, as appropriate).
  • the reaction can be further amplified in situ using immunocytochemical techniques directed against the label of the detector molecule used, such antibodies directed to a fluorescein moiety present on a fluorescently labeled probe, or against avidin, or marker enzymes (peroxidase, alkaline phosphatase).
  • immunocytochemical techniques directed against the label of the detector molecule used, such antibodies directed to a fluorescein moiety present on a fluorescently labeled probe, or against avidin, or marker enzymes (peroxidase, alkaline phosphatase).
  • specific labeling and in situ detection methods can be found, for example, in Howard, G. C, Ed., Methods in Nonradioactive Detection, Appleton & Lange, Norwalk, Conn., (1993), herein incorporated by reference.
  • nucleic acids present in a test tissue or cell sample are amplified by polymerase chain reaction (PCR) using two primers consisting of at least 15 nucleotides derived from one or more of a group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 23, 24, 25, 26, 27, 28, 29, 30 and 31, including primers derived from variants and/or extensions of such sequences, as described herein.
  • PCR polymerase chain reaction
  • Amplification products are detected in the sample by a method that is appropriate to the particular label used to label the amplification products, according to methods as described in U.S. Pat. No. 4,683,195.
  • PCR primers are selected to be at least 15 nucleotides in length, and preferably between about 15 and 30 nucleotides in length, and are selected from the DNA molecule of interest, according to methods known in the art. While such primers can be selected from within the sequences identified as SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 23, 24, 25, 26, 27, 28, 29, 30 and 31, herein, it may also be desirable to select sequences that encompass the longer nucleotide sequences.
  • the probes are selected such that the two hybridization sites are separated by between about 10 to 1,000 nucleotides (occasionally up to about 10,000 nucleotides).
  • PCR in situ hybridization of tissue sections and/or cell samples provides a highly sensitive detection method for rare cell types in fixed cell or tissue samples.
  • the PCR in situ hybridization detection method is carried out in accordance with methods that are known in the art, e.g., Nuovo, G. J., PCR IN SITU HYBRIDIZATION: PROTOCOLS AND APPLICATIONS, Raven Press, N.Y., 1992; U.S. Pat. No. 5,538,871, both of which are incorporated herein by reference.
  • a cell sample tissue on microscopic slide, pelleted cell suspension
  • a common fixative preparation such as buffered formalin, formaldehyde or the like.
  • Proteinase or detergent treatment is favored following fixation, to increase cell permeability to reagents.
  • the PCR reaction is carried out in situ by polymerase chain reaction (PCR) using two primers.
  • PCR polymerase chain reaction
  • the primers are designed to selectively amplify one or more of the nucleotide sequences described herein, and particularly sequences described as SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 23, 24, 25, 26, 27, 28, 29, 30 and 31.
  • the amplification reaction mixture contains, in addition to the target nucleotide sample and the primers, a thermostable DNA polymerase, such as a polymerase derived from Themus aquaticus (Taq polymerase, U.S. Pat. 4,889,818), and a sufficient quantity of the four standard deoxyribonucleotides (dNTPs), one or more of which may be labeled to facilitate detection.
  • a thermostable DNA polymerase such as a polymerase derived from Themus aquaticus (Taq polymerase, U.S. Pat. 4,889,818)
  • dNTPs deoxyribonucleotides
  • the reaction mixture is subjected to several rounds of thermocycling to produce multiple copies (amplification products) of the target nucleotide sequence.
  • Amplification products are tiien detected in the sample, for example by detecting radioactively labeled amplification products.
  • Hybridization probes and PCR primers may also be selected from the genomic sequences corresponding to the full-length proteins identified in accordance with the present invention, including promoter, enhancer elements and introns of the gene encoding the naturally occurring polypeptide.
  • Nucleotide sequences encoding a Q9H5P3 or Q9UL33/Q9NZZ4 polypeptide can also be used to construct hybridization probes for mapping the gene which encodes the respective Q9H5P3 or Q9UL33/Q9NZZ4 and for the genetic analysis of individuals.
  • sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening witii libraries. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the Q9H5P3 or Q9UL33/Q9NZZ4 cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, which would complicate the amplification process.
  • Nucleic acids used for diagnosis may be obtained from a patient's cells, including, for example, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR [Saiki, et al. Nature 324:163-166 (1986)] prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention.
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of die invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method [e.g. Cotton, et al., Pvoc. Natl. Acad. Sci. USA 85:4397-4401 (1985)], or by differences in melting temperatures. "Molecular beacons" [Kostrikis L. G.
  • hairpin-shaped, single-stranded synthetic oligonucleotides containing probe sequences which are complementary to the nucleic acid of the present invention may also be used to detect point mutations or other sequence changes as well as monitor expression levels of Q9H5P3 or Q9UL33/Q9NZZ4.
  • Oligonucleotides of the invention are synthesized by conventional means on a commercially available automated DNA synthesizer, e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer, or like instrument.
  • a commercially available automated DNA synthesizer e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer, or like instrument.
  • phosphoramidite chemistry is employed, e.g. as disclosed in the following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. patent 4,980,460; Koster et al, U.S. patent 4,725,677; Caruthers et al, U.S.
  • nuclease resistant backbones are preferred.
  • modified oligonucleotides are available that confer nuclease resistance, e.g. phosphorothioate, phosphorodithioate, phosphoramidate, or the like, described in many references, e.g. phosphor othioates: Stec et al, U.S. patent 5,151,510; Hirschbein, U.S. patent 5,166,387; Bergot, U.S.
  • Antisense compounds of the invention are preferably selected so that they are complementary to a contiguous subsequence of SEQ ID NO: 8 to 16 or 24 to 30.
  • the length of the antisense oligonucleotides, i.e. such contiguous subsequence, is sufficiently large to ensure that specific binding will take place only at the desired target polynucleotide and not at other fortuitous sites.
  • the upper range of the length is determined by several factors, including the inconvenience and expense of synthesizing and purifying oligomers greater than about 30-40 nucleotides in length, the greater tolerance of longer oligonucleotides for mismatches than shorter oligonucleotides, and the like.
  • the antisense oligonucleotides of the invention have lengths in the range of about 15 to 40 nucleotides. More preferably, the oligonucleotide moieties have lengths in the range of about 18 to 25 nucleotides.
  • polypeptides of the present invention When polypeptides of the present invention are expressed in soluble form, for example as a secreted product of transformed yeast or mammalian cells, they can be purified according to standard procedures of the art, including steps of ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and/or the like, e.g. "Enzyme Purification and Related Techniques," Methods in Enzymology, 22:233-577 (1977), and Scopes, R., Protein Purification: Principles and Practice (Springer-Verlag, New York, 1982) provide guidance in such purifications.
  • polypeptides of the invention when expressed in insoluble form, for example as aggregates, inclusion bodies, or the like, they can be purified by standard procedures in the art, including separating the inclusion bodies from disrupted host cells by centrifugation, solublizing the inclusion bodies with chaotropic and reducing agents, diluting the solubilized mixture, and lowering the concentration of chaotropic agent and reducing agent so that the polypeptide takes on a biologically active conformation.
  • the latter procedures are disclosed in the following references, which are incorporated by reference: Winkler et al, Biochemistry, 25: 4041-4045 (1986); Winkler et al, Biotechnology, 3: 992-998 (1985); Koths et al, U.S. patent 4,569,790; and European patent applications 86306917.5 and 86306353.3.
  • Q9H5P3 and Q9UL33/Q9NZZ4 polypeptides can be purified from culture supernatants of mammalian cells transiently transfected or stably transformed by an expression vector carrying a Q9H5P3 or Q9UL33/Q9NZZ4 gene.
  • the Q9H5P3 or Q9UL33/Q9NZZ4 is purified from culture supernatants of COS 7 cells transiently transfected by the pcD expression vector.
  • Transfection of COS 7 cells with pcD proceeds as follows: One day prior to transfection, approximately 10 ⁇ COS 7 monkey cells are seeded onto individual 100 mm plates in Dulbecco's modified Eagle medium (DME) containing 10% fetal calf serum and 2 mM glutamine. To perform the transfection, the medium is aspirated from each plate and replaced with 4 ml of DME containing 50 mM Tris.HCl pH 7.4, 400 mg/ml DEAE-Dextran and 50 ⁇ g of plasmid DNA. The plates are incubated for four hours at 37°C, then the DNA-containing medium is removed, and the plates are washed twice with 5 ml of serum-free DME.
  • DME Dulbecco's modified Eagle medium
  • DME is added back to the plates which are then incubated for an additional 3 hrs at 37°C.
  • the plates are washed once with DME, after which DME containing 4% fetal calf serum, 2 mM glutamine, penicillin (100 U/L) and streptomycin (100 ⁇ g/L) at standard concentrations is added.
  • the cells are then incubated for 72 hrs at 37°C, after which the growth medium is collected for purification of Q9H5P3 or Q9UL33/Q9NZZ4.
  • transfection can be accomplished by electroporation as described in the examples.
  • Plasmid DNA for the transfections is obtained by growing pcD(SR ⁇ ), or like expression vector, containing the Q9H5P3 or Q9UL33/Q9NZZ4 cDNA insert in E. coli MC1061, described by Casadaban and Cohen, "J. Mol. Biol., Vol. 138, pgs. 179-207 (1980), or like organism.
  • the plasmid DNA is isolated from the cultures by standard techniques, e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, New York, 1989) or Ausubel et al (1990, cited above).
  • Example 1 Monoclonal Antibodies Specific for Q9H5P3 or Q9UL33/Q9NZZ4
  • a male Lewis rat is immunized with semi-purified preparations of COS 7-cell expressed Q9H5P3 or Q9UL33/Q9NZZ4.
  • the rat is first immunized with approximately 50 ⁇ g of Q9H5P3 or Q9UL33/Q9NZZ4 in Freund's Complete Adjuvant, and boosted twice with the same amount of material in Freund's Incomplete Adjuvant. Test bleeds are taken. The animal is given a final boost of 25 ⁇ g in phosphate-buffered saline, and four days later the spleen is obtained for fusion.
  • Approximately 3 x 10 ⁇ rat splenocytes are fused with an equal number of P3X63- AG8.653 mouse myeloma cells (available from the ATCC under accession number CRL 1580). 3840 microtiter plate wells are seeded at 5.7 x 10 ⁇ parental myeloma cells per well. Standard protocols for the fusion and subsequent culturing of hybrids are followed, e.g. as described by Chretien et al, J. Immunol. Meth., Vol. 117, pgs. 67-81 (1989). 12 days after fusion supernatants are harvested and screened by indirect ELISA on PVC plates coated with COS 7-produced Q9H5P3 or Q9UL33/Q9NZZ4.
  • Example 2 Antibodies Specific for the RSTPTENELK-Peptide 50 mg of ovalbumin (OVA) and 50 mg of myoglobulin (MYO) (e.g. available from Sigma) are each dissolved in 10 ml of 0.1 M sodium bicarbonate, and reacted with 1 ml of 0.12 iodoacetamide solution (88 mg of iodoacetamide dissolved in 4 ml 0.1 M sodium bicarbonate) for 1 hour at room temperature in a 15 ml Falcon tube (Falcon Plastics, Oxnard, CA), or the like. Each reaction mixture is dialyzed overnight against 4 liters of 0.1 M sodium bicarbonate at 4RC.
  • OVA ovalbumin
  • MYO myoglobulin
  • RSTPTENELK 10 mg is dissolved in 2 ml of 0.1 M DTT (dithiotheitol) solution (containing 50 mM Tris and 2.5 mM EDTA at pH8) in a 4 ml tube, incubated at 37°C overnight; and then applied to a GF05 gel-filtration column (1.5 x 26.5 cm) (LKB, Bromma, Sweden) and eluted with a peptide elution buffer consisting of 0.015 M acetic acid and 0.005 M beta-mercaptoethanol.
  • DTT dithiotheitol
  • the dialyzed iodoacetylated OVA and MYO are recovered, separately mixed with equal volumes (preferably 2 ml) of borate reduction buffer containing the peptide, and incubated overnight at room temperature.
  • the resulting conjugates are analyzed by SDS-PAGE (12.5% gel).
  • the conjugate containing solution is diluted with PBS to 1 mg/ml, sterile filtered, and aliquotted to convenient volumes (e.g. 500 microliters) for immunizations, and/or stored at 4°C.
  • Polyclonal anti-sera against the MYO conjugate is produced in both rats and rabbits (New Zealand White).
  • the immunization schedule for rabbits is as follows: Initially (week 0) a 10 ml sample of serum is extracted as a control. One week later (week 1) 0.5 ml of peptide-carrier conjugate is mixed with 0.5 ml Freund's Complete Adjuvant and injected I. P. Three weeks later (week 4) a booster is given consisting of 0.5 ml peptide-carrier conjugate mixed with 0.5 ml Freund's Incomplete Adjuvant. The following week (week 5) an additional booster is given, again consisting of 0.5 ml peptide-carrier conjugate mixed with 0.5 ml Freund's Incomplete Adjuvant, followed by yet another identical booster the next week (week 6).

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Abstract

L'invention concerne des méthodes et des compositions de mesure des rapports des variants d'épissures de Q9UL33/Q9NZZ4 ainsi que les rapports d'épissures de Q9H5P3 dans des échantillons biologiques, particulièrement dans des tissus humains. L'invention concerne par ailleurs des méthodes et des kits de surveillance des taux d'épissures de Q9UL33/Q9NZZ4 et des taux d'épissures de Q9H5P3 chez un individu.
PCT/EP2002/007541 2001-07-05 2002-07-05 Variants d'epissures de q9ul33/q9nzz4 et q9h5p3 WO2003004532A2 (fr)

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US30318701P 2001-07-05 2001-07-05
US30319701P 2001-07-05 2001-07-05
US60/303,197 2001-07-05
US60/303,187 2001-07-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020026167A1 (fr) * 2018-08-02 2020-02-06 Novartis Ag Nouvelles myokines et leurs utilisations

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0791660A1 (fr) * 1996-02-22 1997-08-27 Smithkline Beecham Corporation Nouveau marqueur diagnostique pour variations d'épissage de gènes en association avec fonctions neurologiques
WO1999055902A1 (fr) * 1998-04-29 1999-11-04 University Of South Florida Marqueurs diagnostiques de l'infertilite chez la femme

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0791660A1 (fr) * 1996-02-22 1997-08-27 Smithkline Beecham Corporation Nouveau marqueur diagnostique pour variations d'épissage de gènes en association avec fonctions neurologiques
WO1999055902A1 (fr) * 1998-04-29 1999-11-04 University Of South Florida Marqueurs diagnostiques de l'infertilite chez la femme

Non-Patent Citations (3)

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Title
DATABASE SWALL [Online] 1 March 2001 (2001-03-01), KAWAKAMI, T: "Hypothetical protein FLJ23221" XP002248568 accession no. EBI Database accession no. Q9H5P3 *
DATABASE SWALL [Online] 1 May 2000 (2000-05-01), ZHU, X: "Hypothetical protein" XP002248569 accession no. EBI Database accession no. Q9UL33 *
DATABASE SWALL [Online] 1 October 2000 (2000-10-01), ZHANG, Q: "HSPC176 (hypothetical protein)" XP002248570 accession no. EBI Database accession no. Q9NZZ4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020026167A1 (fr) * 2018-08-02 2020-02-06 Novartis Ag Nouvelles myokines et leurs utilisations
CN112512553A (zh) * 2018-08-02 2021-03-16 诺华股份有限公司 新的肌肉因子及其用途
JP2021533113A (ja) * 2018-08-02 2021-12-02 ノバルティス アーゲー 新規なマイオカイン及びその使用

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