+

WO2001023567A1 - Proteine-60 membranaire - Google Patents

Proteine-60 membranaire Download PDF

Info

Publication number
WO2001023567A1
WO2001023567A1 PCT/US2000/026664 US0026664W WO0123567A1 WO 2001023567 A1 WO2001023567 A1 WO 2001023567A1 US 0026664 W US0026664 W US 0026664W WO 0123567 A1 WO0123567 A1 WO 0123567A1
Authority
WO
WIPO (PCT)
Prior art keywords
zsigόo
amino acid
gene
polypeptide
sequence
Prior art date
Application number
PCT/US2000/026664
Other languages
English (en)
Inventor
Scott R. Presnell
Original Assignee
Zymogenetics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zymogenetics, Inc. filed Critical Zymogenetics, Inc.
Priority to AU77281/00A priority Critical patent/AU7728100A/en
Publication of WO2001023567A1 publication Critical patent/WO2001023567A1/fr

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the hypothalamus and pituitary complex is a major the most dominant portion of the endocrine system regulating the function of the thyroid, adrenal, and reproductive glands. It also regulates somatic growth, lactation, milk secretion, and water metabolism.
  • the major hormones that the pituitary is known to produce are growth hormone, (GH), prolactin (PRL), luteinizing hormone, (LH), follicle stimulating hormone, (FSH), thyroid-stimulating hormone (TSH, thyrotropin) and adrenocorticotropin hormone (ACTH).
  • GH growth hormone
  • PRL prolactin
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • TSH thyroid-stimulating hormone
  • ACTH adrenocorticotropin hormone
  • GH regulates growth. PRL is necessary for lactation. Luteinizing hormone and follicle-stimulating hormone control the gonads in both men and women.
  • ACTH controls glucocorticoid function of the adrenal gland.
  • Vasopressin and oxytocin are produced in the neurons of the hypothalamus and stored in the posterior lobe of the pituitary. The underproduction or overproduction of these hormones very often results in disease and physiological malfunctions. The ove ⁇ roduction of growth hormone can result in acromegaly or giantism. An enlarged pituitary gland may indicate that the pituitary gland is producing too much growth hormone. It would be useful to discover membrane-bound proteins that are expressed by cells of the pituitary so that antibodies to the membrane bound protein could be developed. These antibodies could be tagged with an appropriate identifying compound.
  • tagged antibodies Using the tagged antibodies, one could separate cells of the pituitary gland from other cells.using, for example a fluorescence-activated cell sorter (FACS) machine. Labeled antibodies of a membrane-bound protein expressed by the pituitary gland could be used by surgeons to determine that all of the pituitary gland has been removed during an operation. Radiolabeled antibodies that bound to a membrane-bound protein of cells of the pituitary gland could also be used to radioimage the pituitary gland in an effort to diagnose pathology of the pituitary gland. Thus, there is a need to discover new membrane bound proteins produced by the pituitary gland that can be useful to develop antibodies that can be developed to tag the cells of the pituitary gland.
  • FACS fluorescence-activated cell sorter
  • the present invention fills this need by providing for a novel membrane- bound protein, designated "Zsig ⁇ O.”
  • Zsig ⁇ O novel membrane- bound protein
  • the present invention also provides Zsig ⁇ O polypeptides and Zsig ⁇ O fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins.
  • the present invention provides isolated polypeptides having an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of the polypeptide defined by SEQ ID NO: 2; the polypeptide sequence of amino acid residues 16, an isoleucine, to and including amino acid residue 271, an isoleucine, of SEQ ID NO: 2 also defined by SEQ ID NO:3; and the polypeptide sequence of amino acid residues 19, an isoleucine, to and including amino acid residue 271 of SEQ ID NO: 2, also defined by SEQ ID NO:4.
  • the mature extracellular portion of the polypeptide extends from amino acid residue 16, an isoleucine to and including amino acid residue 178, an arginine, of SEQ ID NO:2.
  • the mature extracellular portion of the receptor is also represented by SEQ ID NO:6.
  • the portion of the polypeptide which extends through the membrane of the cell extends from amino acid residue 179, a serine, to and including amino acid residue 197, a valine of SEQ ID NO: 2.
  • the intracellular or cytoplasmic region of the polypeptide extends from amino acid residue 198, a histidine, to and including amino acid residue 271, an isoleucine of SEQ ID NO: 2.
  • An alternative mature extracellular portion of the polypeptide extends from amino acid residue 19, an isoleucine to and including amino acid residue 178, an arginine, of SEQ ID NO: 2. This sequence is also represented by SEQ ID NO: 7.
  • the present invention further provides pharmaceutical compositions that comprise such polypeptides, and a pharmaceutically acceptable carrier.
  • the present invention also includes variant human Zsig ⁇ O polypeptides, wherein the variant polypeptide shares an identity with the amino acid sequence of SEQ ID NOs: 2, 3, or 4 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NOs:2, 3 and 4 is due to one or more conservative amino acid substitutions.
  • the present invention also provides for isolated polynucleotides that encode the above-described polypeptides.
  • the present invention further provides human Zsig ⁇ O polypeptides that comprise an amino acid sequence consisting of ID NO:2.
  • Suitable polypeptides include those in which the amino acid sequence occurs at the C-terminus of the polypeptide.
  • the present invention also provides isolated nucleic acid molecules that encode a Zsig ⁇ O polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, (b) a nucleic acid molecule which encodes isolated polypeptides having an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of the polypeptide defined by SEQ ID NO:2; the polypeptide sequence of amino acid residues 16, an isoleucine, to and including amino acid residue 271, an isoleucine, of SEQ ID NO:2 also defined by SEQ ID NO:3; and the polypeptide sequence of amino acid residues 19, an isoleucine, to and including amino acid residue 271 of SEQ ID NO: 2, also defined by SEQ ID NO:4 or a variant thereof (c) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the
  • the present invention also provides vectors and expression vectors comprising such nucleic acid molecules, recombinant host cells comprising such vectors and expression vectors, and recombinant viruses comprising such expression vectors. These expression vectors and recombinant host cells can be used to prepare Zsig ⁇ O polypeptides.
  • the present invention provides pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and at least one of such an expression vector or recombinant virus.
  • such pharmaceutical compositions comprise a human Zsig ⁇ O gene, or a variant thereof.
  • the present invention further contemplates antibodies and antibody fragments that specifically bind with Zsig ⁇ O polypeptides.
  • Such antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
  • antibody fragments include F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, and minimal recognition units.
  • the present invention also contemplates anti-idiotype antibodies, or anti- idiotype antibody fragments, that specifically bind with an anti-Zsig ⁇ O antibody or antibody fragment.
  • nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • nucleic acid molecule also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • nucleic acid molecule refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence. For example, the sequence 5'
  • ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • the term "contig” denotes a nucleic acid molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule. Contiguous sequences are said to "overlap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid molecule that encodes a polypeptide.
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • structural gene refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • an "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • a nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
  • nucleic acid molecule construct is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • Linear DNA denotes non-circular DNA molecules having free 5' and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
  • Codon DNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA refers to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • cDNA also refers to a clone of a cDNA molecule synthesized from an RNA template.
  • a “promoter” is a nucleotide sequence that directs the transcription of a structural gene.
  • a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al, Mol Endocrinol. 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol.
  • DSEs differentiation-specific elements
  • CREs cyclic AMP response elements
  • SREs serum response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O'Reilly et al, J. Biol Chem. 2(57:19938 (1992)), AP2 (Ye et al, J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson et al, eds., Molecular Biology of the Gene, 4th ed. (The
  • a promoter contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
  • a “regulatory element” is a nucleotide sequence that modulates the activity of a core promoter.
  • a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific,” “tissue-specific,” or “organelle-specific” manner.
  • the Zsig ⁇ O regulatory element preferentially induces gene expression in pituitary, melanocyte, fetal heart or pregnant uterus.
  • An “enhancer” is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
  • Heterologous DNA refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell.
  • DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA).
  • endogenous DNA DNA derived from the host cell species
  • non-host DNA i.e., exogenous DNA
  • a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.
  • a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter.
  • a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.
  • a "polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides.”
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
  • an "integrated genetic element” is a segment of DNA that has been inco ⁇ orated into a chromosome of a host cell after that element is introduced into the cell through human manipulation.
  • integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its progeny.
  • a "cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • an “expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked to” the promoter.
  • a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
  • a “recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
  • a recombinant host is a cell that produces Zsig ⁇ O from an expression vector.
  • Zsig ⁇ O can be produced by a cell that is a "natural source" of Zsig ⁇ O, and that lacks an expression vector.
  • “Integrative transformants” are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
  • a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • a fusion protein can comprise at least part of an Zsig ⁇ O polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of Zsig ⁇ O using affinity chromatography.
  • Receptor denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • Membrane-bound receptors are characterized by a multi -domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • secretory signal sequence denotes a DNA sequence that encodes a peptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • secretory peptide a DNA sequence that encodes a peptide that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • isolated polypeptide is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • SDS sodium dodecyl sulfate
  • amino-terminal or N-terminal and “carboxyl-terminal or C- terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
  • immunomodulator includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of less than 10 9 M "1 .
  • An "anti-idiotype antibody” is an antibody that binds with the variable region domain of an immunoglobulin. In the present context, an anti-idiotype antibody binds with the variable region of an anti- Zsig ⁇ O-antibody, and thus, an anti-idiotype antibody mimics an epitope of Zsig ⁇ O.
  • an “antibody fragment” is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, a Zsig ⁇ O monoclonal antibody fragment binds with an epitope of Zsig ⁇ O.
  • antibody fragment also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • scFv proteins peptide linker
  • a “chimeric antibody” is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
  • Humanized antibodies are recombinant proteins in which murine complementarity determimng regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • a "therapeutic agent” is a molecule or atom which is conjugated to an antibody moiety to produce a conjugate which is useful for therapy.
  • therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
  • a "detectable label” is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al, EMBOJ. 4:1075 (1985); Nilsson et al, Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al, Proc.
  • naked antibody is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
  • antibody component includes both an entire antibody and an antibody fragment.
  • an “immunoconjugate” is a conjugate of an antibody component with a therapeutic agent or a detectable label.
  • antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a therapeutic agent.
  • therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein") and toxins (“antibody-toxin fusion protein”).
  • tumor associated antigen is a protein normally not expressed, or expressed at lower levels, by a normal counte ⁇ art cell.
  • tumor associated antigens include alpha- fetoprotein, carcinoembryonic antigen, and Her-2/neu. Many other illustrations of tumor associated antigens are known to those of skill in the art. See, for example, Urban et al, Ann. Rev. Immunol. 10:617 (1992).
  • an "infectious agent” denotes both microbes and parasites.
  • a “microbe” includes viruses, bacteria, rickettsia, mycoplasma, protozoa, fungi and like microorganisms.
  • a “parasite” denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible to immune-mediated clearance or lytic or phagocytic destruction, such as malarial parasites, spirochetes, and the like.
  • infectious agent antigen is an antigen associated with an infectious agent.
  • a “target polypeptide” or a “target peptide” is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen.
  • T cells recognize peptide epitopes presented by a major histocompatibility complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.
  • antigenic peptide is a peptide which will bind a major histocompatibility complex molecule to form an MHC-peptide complex which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell.
  • antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell which binds or expresses the antigen.
  • the antigenic peptide can be bound in the context of a class I or class II major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.
  • RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA.
  • a nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA.
  • the RNA transcript is termed an "anti- sense RNA” and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene.”
  • Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
  • an "anti-sense oligonucleotide specific for Zsig ⁇ O" or an “Zsig ⁇ O anti- sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the Zsig ⁇ O gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Zsig ⁇ O gene.
  • a "ribozyme” is a nucleic acid molecule that contains a catalytic center.
  • the term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
  • a nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene.”
  • an "external guide sequence” is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P.
  • a nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene.”
  • the term “variant human Zsig ⁇ O gene” refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO: 2. Such variants include naturally-occurring polymo ⁇ hisms of ' Zsig ⁇ O genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO: 2.
  • Zsig ⁇ O genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein.
  • a variant Zsig ⁇ O gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, or its complement, under stringent conditions.
  • variant Zsig ⁇ Ogenes can be identified by sequence comparison. Two amino acid sequences have "100% amino acid sequence identity” if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence. Similarly, two nucleotide sequences have "100% nucleotide sequence identity” if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin).
  • a variant gene or polypeptide encoded by a variant gene is functionally characterized by its ability to bind specifically to an anti-Zsig60 antibody.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymo ⁇ hism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counte ⁇ art of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • Nucleic acid molecules encoding a human Zsig ⁇ O gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon SEQ ID NO:l . These techniques are standard and well established.
  • a nucleic acid molecule that encodes a human Zsig ⁇ O gene can be isolated from a human cDNA library.
  • the first step would be to prepare the cDNA library by isolating RNA from pituitary gland using methods well known to those of skill in the art.
  • RNA isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants.
  • total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells, extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and separating RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3 r " Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)”]; Wu et al, Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)”]).
  • total RNA can be isolated from pituitary tissue by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation (see, for example, Chirgwin et al, Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages 33-41).
  • poly(A) + RNA In order to construct a cDNA library, poly(A) + RNA must be isolated from a total RNA preparation. Poly(A) + RNA can be isolated from total RNA using the standard technique of oligo(dT)-cellulose chromatography (see, for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 59:1408 (1972); Ausubel (1995) at pages 4-11 to 4- 12).
  • Double-stranded cDNA molecules are synthesized from poly(A) + RNA using techniques well-known to those in the art. (see, for example, Wu (1997) at pages 41-46). Moreover, commercially available kits can be used to synthesize double- stranded cDNA molecules. For example, such kits are available from Life
  • a cDNA library can be prepared in a vector derived from bacteriophage, such as a ⁇ gtlO vector. See, for example, Huynh et al, "Constructing and Screening cDNA Libraries in ⁇ gtlO and ⁇ gtl 1," in DNA Cloning: A Practical Approach Vol I, Glover (ed.), page 49 (IRL Press, 1985); Wu (1997) at pages 47-52.
  • double-stranded cDNA molecules can be inserted into a plasmid vector, such as a pBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Co ⁇ .) or other commercially available vectors.
  • a plasmid vector such as a pBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Co ⁇ .) or other commercially available vectors.
  • Suitable cloning vectors also can be obtained from the American Type Culture Collection (Manassas, VA).
  • the cDNA library is inserted into a prokaryotic host, using standard techniques.
  • a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained, for example, from Life Technologies, Inc. (Gaithersburg, MD).
  • a human genomic library can be prepared by means well-known in the art
  • Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl, digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient.
  • Genomic DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases.
  • Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307- 327).
  • Nucleic acid molecules that encode a human Zsig ⁇ O gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the human Zsig ⁇ O gene, as described herein.
  • PCR polymerase chain reaction
  • General methods for screening libraries with PCR are provided by, for example, Yu et al, "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).
  • human genomic libraries can be obtained from commercial sources such as Research Genetics (Huntsville, AL) and the American Type Culture Collection (Manassas, VA).
  • a library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon SEQ ID NO: 1, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).
  • Anti-Zsig60 antibodies produced as described below, can also be used to isolate DNA sequences that encode human Zsig ⁇ O genes from cDNA libraries.
  • the antibodies can be used to screen ⁇ gtl 1 expression libraries, or the antibodies can be used for immunoscreening following hybrid selection and translation (see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al, "Screening ⁇ expression libraries with antibody and protein probes," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University Press 1995)).
  • a Zsig ⁇ O gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).
  • Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al, Plant Molec. Biol. 21:1131 (1993), Bambot et al, PCR Methods and Applications 2:266 (1993), Dillon et al, "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al, PCR Methods Appl 4:299 (1995)).
  • the nucleic acid molecules of the present invention can also be synthesized with "DNA synthesizers" using protocols such as the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • synthetic genes double-stranded are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
  • One method for building a synthetic gene requires the initial production of a set of overlapping, complementary oligonucleotides, each of which is between 20 to 60 nucleotides long.
  • the sequences of the strands are planned so that, after annealing, the two end segments of the gene are aligned to give blunt ends.
  • Each internal section of the gene has complementary 3' and 5' terminal extensions that are designed to base pair precisely with an adjacent section.
  • synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease sites of a cloning vector and other sequences should also be added that contain signals for the proper initiation and termination of transcription and translation.
  • An alternative way to prepare a full-size gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). After the 3' and 5' extensions (6 to 10 nucleotides) are annealed, large gaps still remain, but the base- paired regions are both long enough and stable enough to hold the structure together. The duplex is completed and the gaps filled by enzymatic DNA synthesis with E. coli DNA polymerase I. This enzyme uses the 3'-hydroxyl groups as replication initiation points and the single-stranded regions as templates. After the enzymatic synthesis is completed, the nicks are sealed with T4 DNA ligase.
  • the complete gene sequence is usually assembled from double-stranded fragments that are each put together by joining four to six overlapping oligonucleotides (20 to 60 base pairs each). If there is a sufficient amount of the double-stranded fragments after each synthesis and annealing step, they are simply joined to one another. Otherwise, each fragment is cloned into a vector to amplify the amount of DNA available. In both cases, the double- stranded constructs are sequentially linked to one another to form the entire gene sequence. Each double-stranded fragment and the complete sequence should be characterized by DNA sequence analysis to verify that the chemically synthesized gene has the correct nucleotide sequence.
  • the sequence of a Zsig ⁇ O cDNA or Zsig ⁇ O genomic fragment can be determined using standard methods. Moreover, the identification of genomic fragments containing a Zsig ⁇ O promoter or regulatory element can be achieved using well- established techniques, such as deletion analysis [see, generally, Ausubel (1995)].
  • Cloning of 5' flanking sequences also facilitates production of Zsig ⁇ O proteins by "gene activation," following the methods disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous Zsig ⁇ O gene in a cell is altered by introducing into the Zsig ⁇ O locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a Zsig ⁇ O 5' non-coding sequence that permits homologous recombination of the construct with the endogenous Zsig ⁇ O locus, wherein the sequences within the construct become operably linked with the endogenous Zsig ⁇ O coding sequence.
  • an endogenous Zsig ⁇ O promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • the present invention provides a variety of nucleic acid molecules, including DNA and RNA molecules that encode the Zsig ⁇ O polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.
  • preferential codon usage or "preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid.
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.
  • the present invention further provides variant polypeptides and nucleic acid molecules that represent counte ⁇ arts from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are Zsig ⁇ O polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zsig ⁇ O can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zsig ⁇ O as disclosed herein. Suitable sources of mRNA can be identified by probing northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line.
  • a Zsig ⁇ O-encoding cDNA can be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • a cDNA can also be cloned using the polymerase chain reaction with primers designed from the representative human Zsig ⁇ O sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to Zsig ⁇ O polypeptide. Similar techniques can also be applied to the isolation of genomic clones, and to the isolation of nucleic molecules that encode murine Zsig ⁇ O.
  • SEQ ID NO: 1 represents a single allele of human Zsig ⁇ O, and that allelic variation and alternative splicing are expected to occur.
  • cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the Zsig ⁇ O polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • isolated nucleic acid molecules that encode human Zsig ⁇ O can hybridize to nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary thereto, under "stringent conditions.”
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • a nucleic acid molecule encoding a variant Zsig ⁇ O polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) at 42°C overnight in a solution comprising 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • 5xSSC lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution lOOx
  • hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide.
  • premixed hybridization solutions are available (e.g., EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed according to the manufacturer's instructions.
  • the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions.
  • Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C. That is, nucleic acid molecules encoding a variant Zsig ⁇ O polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1 % SDS at 55°C, or 2xSSC with 0.1 % SDS at 65°C.
  • SDS sodium dodecyl sulfate
  • Typical highly stringent washing conditions include washing in a solution of 0.1 x - 0.2x SSC with 0.1 % sodium dodecyl sulfate (SDS) at 50 - 65°C.
  • SDS sodium dodecyl sulfate
  • nucleic acid molecules encoding a variant Zsig ⁇ O polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 0.1 % SDS at 50°C, or 0.2xSSC with 0.1 % SDS at 65°C.
  • the present invention also provides isolated Zsig ⁇ O polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO:2, SEQ ID NO:3, or their orthologs.
  • substantially similar sequence identity is used herein to denote polypeptides having at least 70%), at least 80%, at least 90%, at least 95%) or greater than 95% sequence identity to the sequences shown in SEQ ID NO:2, SEQ ID NO:5, or their orthologs.
  • the present invention also contemplates Zsig ⁇ O variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ID NO: 2, and a hybridization assay, as described above.
  • Zsig ⁇ O variants include nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%) or greater than 95% sequence identity to the amino acid sequence of SEQ ID NOs:2, 3 or 4.
  • Zsig ⁇ O variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1 x - 0.2x SSC with 0.1% SDS at 50 - 65°C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • the present invention also contemplates human Zsig ⁇ O variant nucleic acid molecules identified by at least one of hybridization analysis and sequence identity determination, with reference to SEQ ID NOs: 2, 3 or 4.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 59:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one- letter codes). The percent identity is then calculated as: ([Total number of identical matches]/ [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100). Table 1
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative Zsig ⁇ O variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman- Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAMJ. Appl.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as described above.
  • the present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared with the amino acid sequence of SEQ ID NO: 2.
  • variants can be obtained that contain one or more amino acid substitutions of SEQ ID NO: 2, in which an alkyl amino acid is substituted for an alkyl amino acid in an Zsig ⁇ O amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in an Zsig ⁇ O amino acid sequence, a sulfur- containing amino acid is substituted for a sulfur-containing amino acid in an Zsig ⁇ O amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy- containing amino acid in an Zsig ⁇ O amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a Zsig ⁇ O amino acid sequence, a basic amino acid is substituted for a basic amino acid in a Zsig ⁇ O amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in an Zsig ⁇ O amino acid sequence.
  • a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • variant Zsig ⁇ O polypeptides that have an amino acid sequence that differs from either SEQ ID NO:2 can be obtained by substituting a threonine residue for Ser 39 , by substituting a valine residue for He 82 , by substituting an aspartate residue for Glu 104 , or by substituting a valine residue for He 187 . Additional variants can be obtained by producing polypeptides having two or more of these amino acid substitutions.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Conservative amino acid changes in an Zsig ⁇ O gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO: 1.
  • Such "conservative amino acid” variants can be obtained, for example, by oligonucleotide- directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).
  • the ability of such variants to promote anti-viral or anti-proliferative activity can be determined using a standard method, such as the assay described herein.
  • a variant Zsig ⁇ O polypeptide can be identified by the ability to specifically bind anti- Zsig ⁇ O antibodies.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tr ⁇ rcs-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ tVo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3 -azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3 -azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3 -azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine.
  • the non-naturally occurring amino acid is inco ⁇ orated into the protein in place of its natural counte ⁇ art. See, Koide et al, Biochem. 33:7470 (1994).
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions [Wynn and Richards, Protein Sci. 2:395 (1993)].
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zsig ⁇ O amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al, Proc. Nat'l Acad. Sci. USA 55:4498 (1991), Coombs and Corey, "Site- Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 [Academic Press, Inc. 1998)].
  • Zsig ⁇ O ligand binding domains can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al, Science 255:306 (1992), Smith et al, J. Mol. Biol 224:899 (1992), and Wlodaver et al, FEBS Lett. 309:59 (1992). Moreover, Zsig ⁇ O labeled with biotin or FITC can be used for expression cloning of Zsig ⁇ O ligand.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zsig ⁇ O polypeptide described herein.
  • Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen.
  • Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al, Proc. Nat'l Acad. Sci.
  • Examples of such epitopes are a polypeptide comprised of amino acid residue 39, a serine to and including amino acid residue 56, a threonine, of SEQ ID NO: 2 also defined by SEQ ID NO: 10; a polypeptide comprised of amino acid residue 39, a serine, to and including amino acid residue 108, a serine, of SEQ ID NO: 2, also defined by SEQ ID NO: 11; and a polypeptide comprised of amino acid residue 39, a serine, to and including amino acid residue 76, a serine, of SEQ ID NO:2 also defined by SEQ ID NO:12.
  • polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind.
  • Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein [see, for example, Sutcliffe et al, Science 219:660 (1983)]. Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.
  • Antigenic epitope-bearing peptides and polypeptides preferably contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID NO: 2.
  • Such epitope-bearing peptides and polypeptides can be produced by fragmenting a Zsig ⁇ O polypeptide, or by chemical peptide synthesis, as described herein.
  • epitopes can be selected by phage display of random peptide libraries [see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al, Curr. Opin. Biotechnol. 7:616 (1996)]. Standard methods for identifying epitopes and producing antibodies from small peptides that comprise an epitope are described, for example, by Mole, "Epitope Mapping," in Methods in
  • Fusion proteins of Zsig ⁇ O can be used to express Zsig ⁇ O in a recombinant host, and to isolate expressed Zsig ⁇ O. As described below, particular Zsig ⁇ O fusion proteins also have uses in diagnosis and therapy.
  • fusion protein comprises a peptide that guides a Zsig ⁇ O polypeptide from a recombinant host cell.
  • a secretory signal sequence also known as a signal peptide, a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence may be derived from Zsig ⁇ O, a suitable signal sequence may also be derived from another secreted protein or synthesized de novo.
  • the secretory signal sequence is operably linked to an Zsig ⁇ O-encoding sequence such that the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide sequence of interest (see, e.g., Welch et al, U.S. Patent No. 5,037,743; Holland et al, U.S. Patent No. 5,143,830).
  • yeast signal sequence is preferred for expression in yeast cells.
  • suitable yeast signal sequences are those derived from yeast mating phermone ⁇ -factor (encoded by the MF l gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene).
  • Zsig ⁇ O can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide.
  • Glutathione S-transferease fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • a Zsig ⁇ O fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resin column, while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose.
  • Established techniques for expressing a heterologous polypeptide as a fusion protein in a bacterial cell are described, for example, by Williams et al, "Expression of Foreign Proteins in E. coli Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies," in DNA Cloning 2: A Practical Approach, 2 nd Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995).
  • the PINPOINT Xa protein purification system provides a method for isolating a fusion protein comprising a polypeptide that becomes biotinylated during expression with a resin that comprises avidin.
  • Peptide tags that are useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaryotic cells include polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies). See, for example, Luo et al, Arch. Biochem. Biophys. 329:215 (1996), Morganti et al, Biotechnol. Appl Biochem. 23:67 (1996), and Zheng et al, Gene 186:55 (1997). Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma- Aldrich Co ⁇ oration (St. Louis, MO).
  • the present invention also contemplates that the use of the secretory signal sequence contained in the Zsig ⁇ O polypeptides of the present invention to direct other polypeptides into the secretory pathway.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1 to 21 of SEQ ID NO:2 (or amino acid residues 1 to 15 of SEQ ID NO: 2) is operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor.
  • a normally non-secreted protein such as a receptor
  • Such fusions may be used in a transgenic animal or in a cultured recombinant host to direct peptides through the secretory pathway.
  • the Zsig ⁇ O secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Fusion proteins comprising a Zsig ⁇ O secretory signal sequence can be constructed using standard techniques.
  • fusion protein comprises a Zsig ⁇ O polypeptide and an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two or three constant region domains and a hinge region but lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • F c fragment an immunoglobulin heavy chain constant region
  • Chang et al, U.S. Patent No. 5,723,125 describe a fusion protein comprising a human interferon and a human immunoglobulin Fc fragment.
  • the C- terminal of the interferon is linked to the N-terminal of the Fc fragment by a peptide linker moiety.
  • An example of a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert.
  • An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO: 13 ).
  • a preferred Fc moiety is a human ⁇ 4 chain, which is stable in solution and has little or no complement activating activity.
  • the present invention contemplates a Zsig ⁇ O fusion protein that comprises a Zsig ⁇ O moiety and a human Fc fragment, wherein the C-terminus of the Zsig ⁇ O moiety is attached to the N-terminus of the Fc fragment
  • the Zsig ⁇ O moiety can be a Zsig ⁇ O molecule or a fragment thereof.
  • a Zsig ⁇ O fusion protein comprises an IgG sequence, a Zsig ⁇ O moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently joined to the aminoterminal of the Zsig ⁇ O moiety, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH 2 domain, and a CH 3 domain. Accordingly, the IgG sequence lacks a CH, domain.
  • Fusion proteins comprising a Zsig ⁇ O moiety and an Fc moiety can be used, for example, as an in vitro assay tool.
  • the presence of a Zsig ⁇ O ligand in a biological sample can be detected using a Zsig ⁇ O-immunoglobulin fusion protein, in which the Zsig ⁇ O moiety is used to target the cognate ligand, and a macromolecule, such as Protein A or anti-Fc antibody, is used to detect the bound fusion protein-receptor complex.
  • fusion proteins can be used to identify agonists and antagonists that interfere with the binding of Zsig ⁇ O to its ligand.
  • antibody-Zsig ⁇ O fusion proteins comprising antibody variable domains
  • a target antigen such as a tumor associated antigen.
  • Methods of making antibody- cytokine fusion proteins are known to those of skill in the art.
  • antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al, Ann. Oncol 6:945 (1995), Nicolet et al, Cancer Gene Ther. 2:161 (1995), Becker et al, Proc. Nat 'I Acad. Sci. USA 93:7826 (1996), Hank et al, Clin. Cancer Res. 2: 1951 (1996), and Hu et al, Cancer Res.
  • cytokine-antibody fusion proteins include IL-8, IL-12, or interferon- ⁇ as the cytokine moiety (Holzer et al, Cytokine 5:214 (1996); Gillies et al, J. Immunol.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. General methods for enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) at pages 16-19 to 16-25.
  • the present invention also contemplates chemically modified Zsig ⁇ O compositions, in which an Zsig ⁇ O polypeptide is linked with a polymer.
  • the polymer is water-soluble so that the Zsig ⁇ O conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled.
  • An example of a reactive aldehyde is polyethylene glycol propionaldehyde (see, for example, Harris, et al, U.S. Patent No. 5,252,714).
  • the polymer may be branched or unbranched. Moreover, a mixture of polymers can be used to produce Zsig ⁇ O conjugates. Zsig ⁇ O conjugates used for therapy should preferably comprise pharmaceutically acceptable water-soluble polymer moieties.
  • Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(Cl- C10)alkoxy-PEG, aryloxy-PEG, poly-(N- vinyl py ⁇ olidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bw-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate- based polymers.
  • Suitable PEG may have a molecular weight from about 600 to about
  • PEGylation by acylation typically requires reacting an active ester derivative of PEG with a Zsig ⁇ O polypeptide.
  • An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide.
  • acylation includes the following types of linkages between Zsig ⁇ O and a water soluble polymer: amide, carbamate, urethane, and the like.
  • Methods for preparing PEGylated Zsig ⁇ O by acylation will typically comprise the steps of (a) reacting a Zsig ⁇ O polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to Zsig ⁇ O, and (b) obtaining the reaction product(s).
  • PEG such as a reactive ester of an aldehyde derivative of PEG
  • the optimal reaction conditions for acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG:Zsig60, the greater the percentage of polyPEGylated Zsig ⁇ O product.
  • the product of PEGylation by acylation is typically a polyPEGylated Zsig ⁇ O product, wherein the lysine ⁇ -amino groups are PEGylated via an acyl linking group.
  • An example of a connecting linkage is an amide.
  • the resulting Zsig ⁇ O will be at least 95% mono-, di-, or tri-pegylated, although some species with higher degrees of PEGylation may be formed depending upon the reaction conditions.
  • PEGylated species can be separated from unconjugated Zsig ⁇ O polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, and the like.
  • PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with Zsig ⁇ O in the presence of a reducing agent.
  • PEG groups are preferably attached to the polypeptide via a -CH 2 -NH group.
  • Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization.
  • the reaction is performed at a pH that allows one to take advantage of the pKa differences between the ⁇ -amino groups of the lysine residues and the ⁇ -amino group of the N- terminal residue of the protein.
  • a water-soluble polymer that contains a reactive group such as an aldehyde
  • the conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups.
  • the present invention provides a substantially homogenous preparation of Zsig ⁇ O monopolymer conjugates.
  • Reductive alkylation to produce a substantially homogenous population of monopolymer Zsig ⁇ O conjugate molecule can comprise the steps of: (a) reacting a Zsig ⁇ O polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the ⁇ -amino group at the amino terminus of the Zsig ⁇ O, and (b) obtaining the reaction product(s).
  • the reducing agent used for reductive alkylation should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation.
  • Preferred reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, and pyridine borane.
  • the reductive alkylation reaction conditions are those that permit the selective attachment of the water soluble polymer moiety to the N-terminus of Zsig ⁇ O.
  • Such reaction conditions generally provide for pKa differences between the lysine amino groups and the ⁇ -amino group at the N-terminus.
  • the pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal ⁇ -group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer: Zsig ⁇ O need not be as large because more reactive groups are available. Typically, the pH will fall within the range of 3 - 9, or 3 - 6.
  • the molar ratio of water-soluble polymer to Zsig ⁇ O will generally be in the range of 1 : 1 to 100: 1. Typically, the molar ratio of water-soluble polymer to Zsig ⁇ O will be 1 : 1 to 20: 1 for polyPEGylation, and 1 :1 to 5: 1 for monoPEGylation.
  • polypeptides of the present invention can be produced in recombinant host cells following conventional techniques.
  • a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
  • Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
  • expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell.
  • a Zsig ⁇ O expression vector may comprise a Zsig ⁇ O gene and a secretory sequence derived from a Zsig ⁇ O gene or another secreted gene.
  • Zsig ⁇ O proteins of the present invention may be expressed in mammalian cells.
  • suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293- HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin et al, Som. Cell. Molec. Genet.
  • GH1 rat pituitary cells
  • ATCC CCL82 HeLa S3 cells
  • ATCC CCL2.2 HeLa S3 cells
  • H-4-II-E rat hepatoma cells
  • COS-1 SV40-transformed monkey kidney cells
  • NIH- 3T3 ATCC CRL 1658
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • viral sources such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Mo lee. Appl Genet. 1:273 (1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 early promoter (Benoist et al, Nature 290:304 (1981)), the Rous sarcoma virus promoter (Gorman et al, Proc. Nat'l Acad. Sci.
  • a prokaryotic promoter such as the bacteriophage T3
  • RNA polymerase promoter can be used to control Zsig ⁇ O gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al, Mol. Cell. Biol. 10:4529 (1990), and Kaufman et al, Nucl. Acids Res. 79:4485 (1991)).
  • An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like.
  • the transfected cells are selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).
  • one suitable selectable marker is a gene that provides resistance to the antibiotic neomycin.
  • selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Zsig ⁇ O polypeptides can also be produced by cultured mammalian cells using a viral delivery system.
  • Exemplary viruses for this pu ⁇ ose include adenovirus, he ⁇ esvirus, vaccinia virus and adeno-associated virus (AAV).
  • Adenovirus a double- stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et al. , Meth. Cell Biol. 43 : 161
  • Advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters.
  • Adenovirus vector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein [see Gamier et al, Cytotechnol 75:145 (1994)].
  • Zsig ⁇ O genes may also be expressed in other higher eukaryotic cells, such as avian, fungal, insect, yeast, or plant cells.
  • the baculovirus system provides an efficient means to introduce cloned Zsig ⁇ O genes into insect cells.
  • Suitable expression vectors are based upon the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein
  • hsp 70 promoter Autographa californica nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter.
  • ie-1 Autographa californica nuclear polyhedrosis virus immediate-early gene promoter
  • baculovirus plO promoter Autographa californica nuclear polyhedrosis virus immediate-early gene promoter
  • Drosophila metallothionein promoter Drosophila metallothionein promoter.
  • a second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, et al, J. Virol 67:4566 (1993)). This system, which utilizes transfer vectors, is sold in the
  • BAC-to-BAC kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Zsig ⁇ O polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 77:971 (1990), Bonning, et al, J. Gen. Virol. 75:1551 (1994), and Chazenbalk, and
  • transfer vectors can include an in- frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Zsig ⁇ O polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al, Proc. Nat'l Acad. Sci. 82:7952 (1985)).
  • a transfer vector containing a Zsig ⁇ O gene is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques.
  • the illustrative pFASTBAC vector can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et al, J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995).
  • a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native Zsig ⁇ O secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen Co ⁇ oration; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native Zsig ⁇ O secretory signal sequence.
  • Suitable insect host cells include cell lines derived from IPLB-S -21, a Spodoptera frugiperda pupal ovarian cell line, such as S 9 (ATCC CRL 1711), Sf21 AE, and Sf21 (Invitrogen Co ⁇ oration; San Diego, CA), as well as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).
  • Commercially available serum-free media can be used to grow and to maintain the cells. Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or
  • the cells are typically grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • yeast cells can also be used to express the genes described herein.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
  • GAL1 galactose
  • PGK phosphoglycerate kinase
  • ADH alcohol dehydrogenase
  • AOX1 alcohol oxidase
  • HIS4 histidinol dehydrogenase
  • These vectors include Yip-based vectors, such as YIp5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCp 19.
  • Yip-based vectors such as YIp5
  • YRp vectors such as YRpl7
  • YEp vectors such as YEpl3
  • YCp vectors such as YCp 19.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Additional suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al, U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454.
  • Transformation systems for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol 132:3459 (1986), and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al, Yeast 14:11-23 (1998), and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUGl o ⁇ AUGl).
  • P. methanolica alcohol utilization gene such as a P. methanolica alcohol utilization gene (AUGl o ⁇ AUGl).
  • Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes.
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • methanolica ADE2 gene which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine.
  • a UG1 and A UG2 methanol utilization genes
  • PEP4 and PRB1 methanol utilization genes
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells.
  • methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells. Methods for introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like.
  • Zsig ⁇ O genes can be expressed in prokaryotic host cells.
  • Suitable promoters that can be used to express Zsig ⁇ O polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp ⁇ and T7 polymerases, the P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
  • subtilis subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene.
  • Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et al, Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins
  • Prefe ⁇ ed prokaryotic hosts include E. coli and Bacillus subtilus. Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600,
  • Suitable strains of Bacillus subtilus include BR151, YB886, Mil 19, Ml 120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)).
  • a Zsig ⁇ O polypeptide in bacteria such as E.
  • the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • polypeptides of the present invention it is prefe ⁇ ed to purify the polypeptides of the present invention to at least about 80% purity, more preferably to at least about 90% purity, even more preferably to at least about 95% purity, or even greater than 95% purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • the polypeptides of the present invention may also be purified to a pharmaceutically pure state, which is greater than 99.9% pure.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Fractionation and/or conventional purification methods can be used to obtain preparations of Zsig ⁇ O purified from natural sources (e.g., pituitary gland), and recombinant Zsig ⁇ O polypeptides and Zsig ⁇ O polypeptides purified from recombinant host cells.
  • ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are prefe ⁇ ed.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, MontgomeryviUe, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl-Sepharose FF Pharmacia
  • Toyopearl butyl 650 Toso Haas, MontgomeryviUe, PA
  • Octyl-Sepharose Pharmacia
  • polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996).
  • Zsig ⁇ O isolation and purification can be devised by those of skill in the art.
  • anti -Zsig ⁇ O antibodies obtained as described below, can be used to isolate large quantities of protein by immunoaffinity purification.
  • the use of monoclonal antibody columns to purify interferons from recombinant cells and from natural sources has been described, for example, by Staehelin et al, J. Biol Chem. 256:9750 (1981), and by Adolf et al, J. Biol. Chem. 265:9290 (1990).
  • methods for binding receptors, such as Zsig ⁇ O, to ligand polypeptides bound to support media are well known in the art.
  • the polypeptides of the present invention can also be isolated by exploitation of particular properties.
  • immobilized metal ion adso ⁇ tion (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • IMAC immobilized metal ion adso ⁇ tion
  • Zsig ⁇ O polypeptides or fragments thereof may also be prepared through chemical synthesis, as described below.
  • Zsig ⁇ O polypeptides may be monomers or multimers; glycosylated or non-glycosylated; PEGylated or non-PEGylated; and may or may not include an initial methionine amino acid residue.
  • Peptides and polypeptides of the present invention comprise at least six, preferably at least nine, and more preferably at least 15 contiguous amino acid residues of SEQ ID NOs:2, 3 or 4. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of these amino acid sequences. Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
  • the activity of Zsig ⁇ O can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent cellular responses.
  • An exemplary device is the CYTOSENSOR Microphysiometer manufactured by Molecular Devices Co ⁇ . (Sunnyvale, CA).
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method (see, for example, McConnell et al, Science 257:1906 (1992), Pitchford et al, Meth. Enzymol. 228:84 (1997), Arimilli et al, J. Immunol. Meth.
  • microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells.
  • Antibodies to Zsig ⁇ O can be obtained, for example, using the product of a Zsig ⁇ O expression vector or Zsig ⁇ O isolated from a natural source as an antigen.
  • Particularly useful anti- Zsig ⁇ O antibodies "bind specifically" to Zsig ⁇ O.
  • Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to Zsig ⁇ O with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to Zsig ⁇ O.
  • antibodies specifically bind if they bind to a Zsig ⁇ O polypeptide, peptide or epitope with a binding affinity (K a ) of 10 6 M “ ' or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, and most preferably 10 9 M “1 or greater.
  • K a binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis [Scatchard, Ann. NY Acad. Sci. 51:660 (1949)].
  • antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zsig ⁇ O, but not known related polypeptides using a standard Western blot analysis.
  • Anti- Zsig ⁇ O antibodies can be produced using antigenic Zsig ⁇ O epitope- bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within SEQ ID NO: 2.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with Zsig ⁇ O.
  • amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.
  • polypeptides containing such antigenic sites include the polypeptides of SEQ ID NOs: 2, 3, 4, 6, 7, 10, 11 and 12.
  • Polyclonal antibodies to recombinant Zsig ⁇ O protein or to Zsig ⁇ O isolated from natural sources can be prepared using methods described by Green et al, "Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al, "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995).
  • the immunogenicity of a Zsig ⁇ O polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zsig ⁇ O or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • polypeptide portion is "hapten-like,” such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • an anti- Zsig ⁇ O antibody of the present invention may also be derived from a subhuman primate antibody.
  • General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No. WO 91/11465, and in Losman et al, Int. J. Cancer 46:3 0 (1990).
  • monoclonal anti-Zsig ⁇ O antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al, Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) ["Coligan”], Picksley et al, "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 [Oxford University Press 1995)].
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an Zsig ⁇ O gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B- lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • an anti-Zsig ⁇ O antibody of the present invention may be derived from a human monoclonal antibody.
  • Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al, Nature Genet.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography [see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al, "Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)].
  • antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.
  • the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
  • These methods are described, for example, by Goldenberg, U.S. patent No. 4,331 ,647, Nisonoff et al, Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al, in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
  • Fv fragments comprise an association of V H and V L chains. This association can be noncovalent, as described by Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659 (1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde [see, for example, Sandhu, Crit. Rev. Biotech. 12:437 (1992)].
  • the Fv fragments may comprise V H and V L chains which are connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • a scFV can be obtained by exposing lymphocytes to Zsig ⁇ O polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zsig ⁇ O protein or peptide).
  • Genes encoding polypeptides having potential Zsig ⁇ O polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al, U.S. Patent No. 5,223,409, Ladner et al., U.S. Patent No. 4,946,778, Ladner et al, U.S. Patent No. 5,403,484, Ladner et al, U.S. Patent No. 5,571,698, and Kay et al, Phage Display of Peptides and Proteins (Academic Press, Inc.
  • Random peptide display libraries can be screened using the Zsig ⁇ O sequences disclosed herein to identify proteins that bind to Zsig ⁇ O.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al, Methods: A Companion to Methods in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al.
  • An anti-Zsig60 antibody may be a "humanized” monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counte ⁇ arts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
  • General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al, Nature 321:522 (1986), Carter et al, Proc.
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zsig ⁇ O antibodies or antibody fragments, using standard techniques. See, for example, Green et al, "Production of Polyclonal Antisera,” in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7.
  • monoclonal anti- idiotype antibodies can be prepared using anti-Zsig ⁇ O antibodies or antibody fragments as immunogens with the techniques, described above.
  • humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques.
  • a bioluminescent compound can be used to label anti-Zsig60 immunoconjugates of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-Zsig ⁇ O immunoconjugates can be detectably labeled by linking an anti-Zsig60 antibody component to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • the binding of marker moieties to anti- Zsig ⁇ O antibodies can be accomplished using standard techniques known to the art.
  • Monoclonal Antibodies Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages 180-208, (Cambridge University Press, 1995), Perry, “The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology," in Monoclonal Antibodies: Principles and Applications, Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (Academic Press, Inc.
  • biotin- or FITC-labeled Zsig ⁇ O can be used to identify cells that bind Zsig ⁇ O. Such can binding can be detected, for example, using flow cytometry.
  • kits comprise at least one container comprising an anti-Zsig ⁇ O antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zsig ⁇ O antibody or antibody fragments.
  • indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like.
  • a kit may also comprise a means for conveying to the user that Zsig ⁇ O antibodies or antibody fragments are used to detect Zsig ⁇ O protein.
  • written instructions may state that the enclosed antibody or antibody fragment can be used to detect Zsig ⁇ O.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • Transgenic mice can be engineered to over-express the human Zsig ⁇ O gene in all tissues or under the control of a tissue-specific or tissue-prefe ⁇ ed regulatory element. These over-producers of Zsig ⁇ O can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serve as models for human disease caused by excess Zsig ⁇ O. Transgenic mice that over-express Zsig ⁇ O also provide model bioreactors for production of Zsig ⁇ O in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses a Zsig ⁇ O gene can begin with adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl , 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl, 2-4 months, (Taconic Farms)).
  • the donors are acclimated for one week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, MO) I. P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) I. P. to induce superovulation.
  • Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating.
  • Fertilized eggs are collected under a surgical scope.
  • the oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase
  • plasmid DNA containing a Zsig ⁇ O encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCI (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinjection.
  • the Zsig ⁇ O encoding sequences can encode amino acid residues 16, an isoleucine, to 271 an isoleucine, of SEQ ID NO:2.
  • the mature sequence is also designated by SEQ ID NO: 3.
  • the mature sequence of Zsig ⁇ O extends from amino acid residue 19, an isoleucine, to and including amino acid residue 271.
  • This mature sequence is also represented by SEQ ID NO: 4.
  • Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO ⁇ -equilibrated mineral oil.
  • the DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei.
  • Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transfe ⁇ ed into an organ tissue-culture dish with pre-gassed W640 medium for storage overnight in a 37°C/5% CO 2 incubator.
  • two-cell embryos are transferred into pseudopregnant recipients.
  • the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
  • Recipients are anesthetized and shaved on the dorsal left side and transfe ⁇ ed to a surgical microscope.
  • a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
  • the reproductive organs are exteriorized onto a small surgical drape.
  • the fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
  • the pipette is transfe ⁇ ed into the nick in the oviduct, and the embryos are blown in, allowing the first air bubble to escape the pipette.
  • the fat pad is gently pushed into the peritoneum, and the reproductive organs allowed to slide in.
  • the peritoneal wall is closed with one suture and the skin closed with a wound clip.
  • the mice recuperate on a 37°C slide warmer for a minimum of four hours.
  • the recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning.
  • the weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
  • Genomic DNA is prepared from the tail snips using, for example, a QIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to amplify a Zsig ⁇ O gene or a selectable marker gene that was introduced in the same plasmid. After animals are confirmed to be transgenic, they are back-crossed into an inbred strain by placing a transgenic female with a wild-type male, or a transgenic male with one or two wild-type female(s). As pups are bom and weaned, the sexes are separated, and their tails snipped for genotyping.
  • a partial hepatectomy is performed.
  • a surgical prep is made of the upper abdomen directly below the zyphoid process.
  • a small 1.5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized.
  • a tie is made around the lower lobe securing it outside the body cavity.
  • An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed proximal to the first tie.
  • a distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish.
  • the excised liver section is transfe ⁇ ed to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice.
  • the surgical site is closed with suture and wound clips, and the animal's cage placed on a 37°C heating pad for 24 hours post operatively.
  • the animal is checked daily post operatively and the wound clips removed 7-10 days after surgery.
  • the expression level of Zsig ⁇ O mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
  • transgenic mice that over-express Zsig ⁇ O
  • Such transgenic mice provide useful models for diseases associated with a lack of Zsig ⁇ O.
  • Zsig ⁇ O gene expression can be inhibited using anti-sense genes, ribozyme genes, or external guide sequence genes.
  • inhibitory sequences are targeted to murine
  • An alternative approach to producing transgenic mice that have little or no Zsig ⁇ O gene expression is to generate mice having at least one normal Zsig ⁇ O allele replaced by a nonfunctional Zsig ⁇ O gene.
  • One method of designing a nonfunctional Zsig ⁇ O gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes murine Zsig ⁇ O.
  • Standard methods for producing these so- called “knockout mice” are known to those skilled in the art [see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice," in Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al, "New Strategies for Gene Knockout,” in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)].
  • Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of Zsig ⁇ O can be used as standards or as "unknowns" for testing pu ⁇ oses.
  • Zsig ⁇ O polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein Zsig ⁇ O is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of Zsig ⁇ O polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.
  • Zsig ⁇ O polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed Zsig ⁇ O polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo.
  • native and tagged protein i.e., receptor binding, signal transduction, proliferation, and differentiation
  • Zsig ⁇ O polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, in particular the locations of the disulfide bonds, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • analytical skills such as mass spectrometry, circular dichroism to determine conformation, in particular the locations of the disulfide bonds, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • a kit containing the Zsig ⁇ O can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of Zsig ⁇ O would be unique unto itself.
  • the antibodies which bind specifically to Zsig ⁇ O can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify Zsig ⁇ O, cloning and sequencing the polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies.
  • the Zsig ⁇ O gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum.
  • Such educational kits containing the Zsig ⁇ O gene, polypeptide, or antibody are considered within the scope of the present invention.
  • Antibodies to Zsig ⁇ O can be tagged to identify or isolate cells of the pituitary gland.
  • the antibodies can be labeled molecules such as fluorescent dyes, alkaline phosphatase or horseradish peroxidase.
  • fluorescent dyes are fluorescein using fluorescein isothiocyanate (FITC), rhodamine using tetramethylrhodamine (TRITC), Texas Red and phycoerythrin.
  • antibodies that bind to Zsig ⁇ O are coupled to a fluorescent dye and then the cells are mixed with the labeled antibodies under conditions wherein the antibodies bind to the membrane-bound Zsig ⁇ O polypeptide.
  • the labeled cells are then separated from the unlabeled cells in an electronic fluorescence-activated cell sorter (FACS).
  • FACS electronic fluorescence-activated cell sorter
  • Antibodies that are radiolabeled with radioisotopes or labeled with a fluorescent dye can be injected into an individual to detect the size of the pituitary gland to determine the size and mo ⁇ hology of the gland to determine if pathology might be present.
  • Antibodies are generally radiolabeled with radioactive iodine, generally
  • 125 I The decay of 125 I yields low-energy gamma and X-ray radiation and therefore is easy to detect.
  • Antibodies radiolabeled with 125 I are generally done so by a simple oxidation reaction using Na I25 I and a strong oxidant such as l,3,4,6-3a,6 ⁇ -diphenyl- glycoluril.
  • the iodide- 125 is oxidized to form iodine- 125 (I 2 ), which attacks tyrosyl and histidyl side chains by means of a halogenation reaction.
  • the radiolabeled antibody can then be administered to a patient.
  • the dosage of labeled anti-Zsig ⁇ O for radiographic imaging will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of Zsig ⁇ O that is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.
  • the radiolabeled antibodies bind to the Zsig ⁇ O expressed on the membrane of the cells of the pituitary gland.
  • a single photon emission computed tomography (SPECT) scan of the pituitary gland can then be made.
  • the resultant image would indicate the size and shape of the pituitary gland.
  • the normal adult pituitary gland has a height of about 3 to 7 mm.
  • An enlarged pituitary gland could indicate an adenoma, which could result in acromegaly or giantism.
  • Example 1 Production a Pituitary Gland cDNA Library RNA extracted from cells of pituitary gland was purchased from Clontech, Palo Alto, CA and reversed transcribed in the following manner.
  • the first strand cDNA reaction contained 10 ⁇ l of human pituitary twice poly d(T)-selected poly
  • First strand cDNA synthesis was initiated by the addition of 8 ⁇ l of first strand buffer (5x SUPERSCRIPTTM buffer; Life Technologies, Gaithersburg, MD), 4 ⁇ l of 100 mM dithiothreitol, and 2 ⁇ l of a deoxynucleotide triphosphate (dNTP) solution containing 10 mM each of dTTP, dATP, dGTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, NJ) to the RNA-primer mixture.
  • the reaction mixture was incubated at 37° C for 2 minutes, followed by the addition of 10 ⁇ l of 200 U/ ⁇ l RNase
  • the second strand reaction contained 100 ⁇ l of the unlabeled first strand cDNA, 30 ⁇ l of 5x polymerase I buffer [125 mM Tris: HCI, pH 7.5, 500 mM KC1, 25 mM MgCl2, 50mM (NH 4 ) 2SO4)], 2.0 ⁇ l of 100 mM dithiothreitol, 3.0 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 ⁇ l of 5 mM ⁇ -NAD, 2.0 ⁇ l of 10 U/ ⁇ l E. coli DNA ligase (New England Biolabs; Beverly, MA), 5 ⁇ l of 10 U/ ⁇ l E.
  • 5x polymerase I buffer [125 mM Tris: HCI, pH 7.5, 500 mM KC1, 25 mM MgCl2, 50mM (NH 4 ) 2SO4)] 2.0 ⁇ l of 100 mM dithiothreitol, 3.0
  • Eco RI adapters were ligated onto the 5' ends of the cDNA described above to enable cloning into an expression vector.
  • a 12.5 ⁇ l aliquot of cDNA (-2.0 ⁇ g) and 3 ⁇ l of 69 pmole/ ⁇ l of Eco RI adapter (Pharmacia LKB Biotechnology Inc.) were mixed with 2.5 ⁇ l lOx ligase buffer (660 mM Tris-HCI pH 7.5, 100 mM MgCl2), 2.5 ⁇ l of 10 mM ATP, 3.5 ⁇ l 0.1 M DTT and 1 ⁇ l of 15 U/ ⁇ l T4 DNA ligase (Promega Co ⁇ ., Madison, WI).
  • the reaction was incubated 1 hour at 5°C, 2 hours at 7.5°C, 2 hours at 10°C, 2 hours at 12.5°C and 16 hours at 10°C.
  • the reaction was terminated by the addition of 65 ⁇ l H 2 O and 10 ⁇ l 10X H buffer (Boehringer Mannheim) and incubation at 70°C for 20 minutes.
  • the cDNA was digested wit Xho I, resulting in a cDNA having a 5' Eco RI cohesive end and a 3' Xho I cohesive end.
  • the Xho I restriction site at the 3' end of the cDNA had been previously introduced.
  • Restriction enzyme digestion was carried out in a reaction mixture by the addition of 1.0 ⁇ l of 40 U/ ⁇ l Xho I (Boehringer Mannheim). Digestion was carried out at 37°C for 45 minutes. The reaction was terminated by incubation at 70°C for 20 minutes and chromatography through a 400 pore size gel filtration column (Clontech Laboratories).
  • the cDNA was ethanol precipitated, washed with 70% ethanol, air dried and resuspended in 13.5 ⁇ l water, 2 ⁇ l of 10X kinase buffer (660 mM Tris-HCI, pH 7.5, 100 mM MgCl 2 ), 0.5 ⁇ l 0.1 M DTT, 2 ⁇ l 10 mM ATP, 2 ⁇ l T4 polynucleotide kinase (10 U/ ⁇ l, Life Technologies). Following incubation at 37° C for 30 minutes, the cDNA was ethanol precipitated in the presence of 2.5 M Ammonium Acetate, and electrophoresed on a 0.8% low melt agarose gel.
  • 10X kinase buffer 660 mM Tris-HCI, pH 7.5, 100 mM MgCl 2
  • 0.5 ⁇ l 0.1 M DTT 2 ⁇ l 10 mM ATP
  • 2 ⁇ l T4 polynucleotide kinase 10 U/ ⁇ l
  • the contaminating adapters and cDNA below 0.6 Kb in length were excised from the gel.
  • the electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin.
  • the area of the gel containing the concentrated cDNA was excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined.
  • An aliquot of water approximately three times the volume of the gel slice (300 ⁇ l) and 35 ⁇ l lOx ⁇ -agarose I buffer (New England Biolabs) was added to the tube, and the agarose was melted by heating to 65°C for 15 minutes. Following equilibration of the sample to 45°C,
  • cDNA was cloned into the Eco RI and.A7zo I sites of pBLUESCRIPT SK+ vector (Gibco/BRL) and electroporated into DH10B cells. Bacterial colonies containing ESTs of known genes were identified and eliminated from sequence analysis by reiterative cycles of probe hybridization to hi-density colony filter arrays (Genome Systems). cDNAs of known genes were pooled in groups of 50 - 100 inserts and were labeled with 32 P using a
  • MEG AP RIME labeling kit (Amersham). Colonies that did not hybridize to the probe mixture were selected for sequencing. Sequencing was done using an ABI 377 sequencer using either the T3 or the reverse primer.
  • Example 2 Assembly of the Zsig ⁇ O Polynucleotide Expressed Sequence Tags (ESTs) of SEQ ID NOs: 5, 8 and 9 were discovered by sequencing of cDNA clones isolated from a pituitary cDNA library produced according to the procedure of Example 1. SEQ ID NOs: 5, 8 and 9 were aligned together by computer to produce the polynucleotide of SEQ ID NO: 1.
  • ESTs Sequence Tags

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne la protéine-60 membranaire, les polynucléotides présentant cette protéine, et les anticorps et anticorps anti-idiotypiques de cette protéine. Des anticorps marqués de cette protéine peuvent être utilisés pour représenter les glandes hypophysaires par imagerie, et pour séparer les cellules des glandes hypophysaires des autres cellules au moyen d'un trieur de cellules marqué par fluorescence.
PCT/US2000/026664 1999-09-28 2000-09-28 Proteine-60 membranaire WO2001023567A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU77281/00A AU7728100A (en) 1999-09-28 2000-09-28 Membrane-bound protein-60

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15636799P 1999-09-28 1999-09-28
US60/156,367 1999-09-28

Publications (1)

Publication Number Publication Date
WO2001023567A1 true WO2001023567A1 (fr) 2001-04-05

Family

ID=22559276

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/026664 WO2001023567A1 (fr) 1999-09-28 2000-09-28 Proteine-60 membranaire

Country Status (2)

Country Link
AU (1) AU7728100A (fr)
WO (1) WO2001023567A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0618291A2 (fr) * 1993-02-26 1994-10-05 Takeda Chemical Industries, Ltd. Protéine réceptrice-PACAP, méthode pour la préparation de cette protéine et l'utilisation de celle-ci
WO1999028467A1 (fr) * 1997-12-03 1999-06-10 Zymogenetics, Inc. Proteine thyroidienne humaine zsig45 et adn codant celle-ci
WO1999046374A2 (fr) * 1998-03-10 1999-09-16 Metagen Gesellschaft Für Genomforschung Mbh Sequences d'acide nucleique humaines issues de tissus tumoraux prostatiques
WO2000009552A1 (fr) * 1998-08-14 2000-02-24 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO2000056891A2 (fr) * 1999-03-22 2000-09-28 Incyte Pharmaceuticals, Inc. Proteines transmembranaires humaines

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0618291A2 (fr) * 1993-02-26 1994-10-05 Takeda Chemical Industries, Ltd. Protéine réceptrice-PACAP, méthode pour la préparation de cette protéine et l'utilisation de celle-ci
WO1999028467A1 (fr) * 1997-12-03 1999-06-10 Zymogenetics, Inc. Proteine thyroidienne humaine zsig45 et adn codant celle-ci
WO1999046374A2 (fr) * 1998-03-10 1999-09-16 Metagen Gesellschaft Für Genomforschung Mbh Sequences d'acide nucleique humaines issues de tissus tumoraux prostatiques
WO2000009552A1 (fr) * 1998-08-14 2000-02-24 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO2000056891A2 (fr) * 1999-03-22 2000-09-28 Incyte Pharmaceuticals, Inc. Proteines transmembranaires humaines

Also Published As

Publication number Publication date
AU7728100A (en) 2001-04-30

Similar Documents

Publication Publication Date Title
US20050020497A1 (en) Mammalian glycoprotein hormone-1
US20020161203A1 (en) Rattlesnake venom gland proteins
US20030017980A1 (en) Mammalian Wnt polypeptide-5
US20020004228A1 (en) Zvwf1: a member of the von willebrand factor type A domain superfamily
US20030157686A1 (en) Rattlesnake venom gland proteins
WO2001023567A1 (fr) Proteine-60 membranaire
US20020150991A1 (en) Insulin homolog polypeptide Zins5
CA2360577A1 (fr) Zlrr3: proteine humaine repetee riche en leucine
US20020146418A1 (en) Human V2 vomeronasal receptor
US20020091239A1 (en) Human chemokine
US20030032778A1 (en) New member of the human syntaxin/epimorphin family
US20020142396A1 (en) Mammalian cystatin-8 and its use to inhibit cancer procoagulant protein
CA2358873A1 (fr) Polypeptide humain avec multiples domaines du type facteur de croissance epidermique (egf), denomme zntr2
US20040180398A1 (en) Mammalian secretory protein Zsig43
US20030143678A1 (en) Zlrr3: a human leucine-rich repeat protein
US20020150974A1 (en) Placental protein having multiple EGF-like domains
WO2001032707A1 (fr) Semaphorine humaine
WO2002012479A2 (fr) Zcys9: un membre de la superfamille des cystatines
AU6506999A (en) Secretory protein - 48
WO2001094388A2 (fr) Zcys6: membre de la superfamille des cystatines
CA2393527A1 (fr) Trousse pedagogique et methode utilisant un gene et une proteine stimule par le facteur de necrose tumorale
WO2002020612A2 (fr) Recepteur-3 vomerien humain
WO2002032956A2 (fr) Proteine de liaison au nucleotide cyclique humain
WO2001042466A2 (fr) Zvwf1: un membre de la superfamille caracterisee par le domaine de type a du facteur willebrand
WO2002020760A2 (fr) Recepteur vomeronasal human

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载