EP1117793A2 - Proteine secretoire 48 - Google Patents

Proteine secretoire 48

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Publication number
EP1117793A2
EP1117793A2 EP99953037A EP99953037A EP1117793A2 EP 1117793 A2 EP1117793 A2 EP 1117793A2 EP 99953037 A EP99953037 A EP 99953037A EP 99953037 A EP99953037 A EP 99953037A EP 1117793 A2 EP1117793 A2 EP 1117793A2
Authority
EP
European Patent Office
Prior art keywords
zsig48
gene
polypeptide
cells
amino acid
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99953037A
Other languages
German (de)
English (en)
Inventor
Si Lok
Paul O. Sheppard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zymogenetics Inc
Original Assignee
Zymogenetics Inc
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Filing date
Publication date
Application filed by Zymogenetics Inc filed Critical Zymogenetics Inc
Publication of EP1117793A2 publication Critical patent/EP1117793A2/fr
Withdrawn legal-status Critical Current

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates generally to a new cytokine having diagnostic and therapeutic uses.
  • the present invention relates to a novel secreted protein designated "Secretory Protein - 48' or , Zsig48 for short, and to nucleic acid molecules encoding Zsig48.
  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue.
  • hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF) , epidermal growth factor
  • EGF granulocyte-macrophage colony stimulating factor
  • EPO erythropoietin
  • Proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.
  • cytokines molecules that promote the proliferation, maintenance, survival or differentiation of cells.
  • examples of cytokines include erythropoietin (EPO) , which stimulates the development of red blood cells; thrombopoietin (TPO) , which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF) , which stimulates development of neutrophils.
  • EPO erythropoietin
  • TPO thrombopoietin
  • G-CSF granulocyte-colony stimulating factor
  • the present invention fills this need by providing for a secretory protein, designated "Zsig48 ! which can stimulate the proliferation of peripheral blood mononuclear cells, i . e . , T-cells, B-cells and monocytes, (PBMNCs) .
  • Zsig48 a secretory protein
  • the present invention also provides Zsig48 polypeptides and Zsig48 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins .
  • the human Zsig48 polypeptide with signal sequence is comprised of a sequence which is 105 amino acid residues as shown in SEQ ID NOs : 1 and 2.
  • the signal sequence is comprised of amino acid residues 1-26 of SEQ ID NO:2.
  • a mature Zsig48 polypeptide thus is comprised of amino acid residues 27, a leucine, to and including amino acid residue 105, a histidine, of SEQ ID NO: 2 also defined by SEQ ID NO:3.
  • An alternative mature Zsig48 polypeptide is comprised of amino acid residues 29, a leucine, to and including amino acid residue 105 of SEQ ID NO: 2, also defined by SEQ ID NO:4.
  • the signal sequence is comprised of amino acid residues 1-40, the mature sequence being comprised of amino acid residue 41, a proline, to and including amino acid residue 105 of SEQ ID NO: 2.
  • This mature sequence is also defined by SEQ ID NO: 5.
  • Another mature sequence of Zsig48 extends from amino acid residue 26 to and including amino acid residue 105 of SEQ ID NO: 2, also represented by SEQ ID NO: 16.
  • the present invention further provides pharmaceutical compositions that comprise such polypeptides, and a pharmaceutically acceptable carrier.
  • the present invention also includes variant human Zsig48 polypeptides, wherein the variant polypeptide shares an identity with the amino acid sequence of SEQ ID NOs:2, 3, 4, 5 or 16 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, 4 and 5 is due to one or more conservative amino acid substitutions.
  • An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zsig48 polypeptide having an amino acid sequence described above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zsig48 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule. Examples of such polypeptides are those polypeptides comprised of one or more of SEQ ID NOs : 8, 9, 10, 11, or 12.
  • the present invention also provides for isolated polynucleotides which encode the above-described polypeptides .
  • the present invention also provides isolated nucleic acid molecules that encode a Zsig48 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 : 1 , (b) a nucleic acid molecule which encodes isolated polypeptides having an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to an amino acid sequence selected from the group consisting of the polypeptide defined by SEQ ID NOs : 2 , 3, 4 or 5 ; (c) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:l, or the complement of SEQ ID NO:l, and (d) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule which encodes an isolated polypeptides having an amino acid sequence that is at least 70%, 80%
  • 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 Zsig48 polypeptides.
  • the present invention provides pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and at least one of such an expression vector or recombinant virus. Preferably, such pharmaceutical compositions comprise a human Zsig48 gene, or a variant thereof .
  • the present invention further contemplates antibodies and antibody fragments that specifically bind with Zsig48 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 presence of Zsig48 polypeptide in a biological sample can be detected by methods that comprise the steps of (a) contacting the biological sample with an antibody, or an antibody fragment, that specifically binds with a polypeptide having the amino acid sequence of either SEQ ID NOs : 2 3, 4 or 5, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
  • the present invention also contemplates isolated nucleic acid molecules comprising a nucleotide sequence that encodes an Zsig48 secretion signal sequence and a nucleotide sequence that encodes a biologically active polypeptide, wherein the Zsig48 secretion signal sequence comprises an amino acid sequence of residues 1 to 26, 1 to 28 or 1 to 40 of SEQ ID NO : 2.
  • Illustrative biologically active polypeptides include Factor Vila, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukin, colony stimulating factor, interferon, erythropoietin, and thrombopoietin.
  • the present invention provides fusion proteins comprising a Zsig48 secretion signal sequence and a polypeptide, wherein the Zsig48 secretion signal sequence comprises an amino acid sequence of residues 1 to 26, 1 to 28 or 1 to 40 of SEQ ID NO:2.
  • the present invention also contemplates anti- idiotype antibodies, or anti-idiotype antibody fragments, that specifically bind with an anti-Zsig48 antibody or antibody fragment, wherein the anti-idiotype antibody, or anti-idiotype antibody fragment, possesses the ability to cause proliferation of T-cells, B-cells or monocytes .
  • the present invention further includes methods for detecting an alteration in a chromosome containing
  • Illustrative chromosomal aberrations at the Zsig48 gene locus include aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within flanking sequences, including upstream promoter and regulatory regions, and can be manifested as physical alterations within a coding sequence or changes in gene expression level . Such methods are effected by examining the Zsig48 gene and its gene products.
  • suitable assay methods include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing polymerase chain reaction techniques, ligation chain reaction, ribonuclease protection assays, use of single-nucleotide polymorphisms, protein truncation assays, and other genetic linkage techniques known in the art .
  • the present invention provides methods for diagnosing an alteration in the Zsig48 gene of an individual, comprising: (a) amplifying nucleic acid molecules that encode Zsig48 from RNA isolated from a biological sample of the individual, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates an alteration in the Zsig48 gene.
  • methods of detecting a chromosome abnormality in a subject comprise: (a) amplifying nucleic acid molecules that encode Zsig48 from RNA isolated from a biological sample of the subject, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates a chromosome abnormality.
  • the detecting step is performed by comparing the nucleotide sequence of the amplified nucleic acid molecules to the nucleotide sequence of SEQ ID NOs : 1.
  • the detecting step can be performed by fractionating the amplified nucleic acid molecules and control nucleic acid molecules that encode the amino acid sequence of SEQ ID NOs : 2 , and comparing the lengths of the fractionated amplified and control nucleic acid molecules.
  • Exemplary methods for amplification include polymerase chain reaction or reverse transcriptase-polymerase chain reaction.
  • the present invention further provides for a method for promoting the proliferation of leukocytes comprising bringing the leukocytes into contact with Zsig48.
  • 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.
  • 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.
  • nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate , phosphoroselenoate , phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • the term "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.
  • sequence 5 ' ATGCACGGG 3 ' is complementary to 5 ' CCCGTGCAT 3 ' .
  • 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 (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.
  • cDNA a single-stranded DNA
  • DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase.
  • 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 .
  • CREs cyclic AMP response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O'Reilly et al . , J. Biol . Chem . 267 : 19938 (1992)), AP2 (Ye et al . , J “ . Biol . Chem . 269 : 25128 (1994)), SPl, 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.
  • a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
  • Repressible promoters are also known .
  • a “core 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 Zsig48 regulatory element preferentially induces gene expression in placenta, kidney, heart or leukocytes.
  • 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) .
  • 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.
  • 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
  • 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 incorporated 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 Zsig48 from an expression vector.
  • Zsig48 can be produced by a cell that is a "natural source" of Zsig48, 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 Zsig48 polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of Zsig48 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.
  • 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
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • 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.
  • an anti-idiotype antibody binds with the variable region of an anti- Zsig48-antibody, and thus, an anti-idiotype antibody mimics an epitope of Zsig48.
  • 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 Zsig48 monoclonal antibody fragment binds with an epitope of Zsig48.
  • 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 determining 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 .
  • 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 tag any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly-histidine tract, protein A (Nilsson et al . , EMBO J. 4:1075 (1985); Nilsson et al . ,
  • 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 counterpart 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 Zsig48" or an “ Zsig48 anti-sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of a Zsig48 gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Zsig48 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 Zsig48 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 polymorphisms of Zsig48 genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NOs : 2 , 3, 4 or 5.
  • Zsig48 genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein.
  • a variant Zsig48 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 Zsigr48genes 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-Zsig48 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 polymorphism 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 counterpart 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.
  • Zsig48 as shown in SEQ ID NO : 2 has a single disulfide bond between the cysteine residue 48 and cysteine residue 81, and an amphiphilic helix, i.e. a helix which contains both a hydrophilic and a hydrophobic face, from amino acid residue 88, a proline, to amino acid residue 105, a histidine of SEQ ID NO : 2.
  • an amphiphilic helix i.e. a helix which contains both a hydrophilic and a hydrophobic face, from amino acid residue 88, a proline, to amino acid residue 105, a histidine of SEQ ID NO : 2.
  • Zsig48 especially the C-terminal helix suggests that Zsig48 may be a peptide ligand for the G- protein coupled 7-transmembrane domain (TMD) class of receptors.
  • TMD 7-transmembrane domain
  • G-protein -linked receptors act indirectly to regulate the activity of a separate plasma membrane-bound target protein, which can be an enzyme or an ion channel.
  • the interaction between the receptor and the target protein is mediated by a third protein, called a trimeric GTP-binding regulatory protein, i.e. a G protein.
  • Zsig48 binds to its G-protein coupled 7-TMD receptor.
  • the receptor then converts this extracellular event into one or intracellular signals that alter the behavior of the target cell.
  • general utility claims for ligands of this receptor class would be applicable for Zsig48. These would include: alteration of cellular metabolism and secretion, ion transport, cell proliferation, differentiation.
  • Zsig48 is highly expressed in the heart, placenta, and kidney. That Zsig48 is highly expressed in the placenta would suggests that the polypeptide may function in fetal development directly or indirectly by providing support functions to the placenta such as blood vessel development or the transport of metabolites. Transcript appearance in the heart further suggests that Zsig48 may be important as a autocrine or paracrine regulator of myocardial function. It has been further determined by RT-PCR that Zsig48 is expressed in leukocytes .
  • Example 3 below shows that Zsig48 can be used to promote proliferation of leukocytes in an individual.
  • Polynucleotides generally a cDNA sequence, of the present invention encode the described polypeptides herein.
  • a cDNA sequence which encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows.
  • Alanine (Ala) is encoded by GCA, GCC, GCG or
  • Cysteine is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT;
  • Glutamic acid is encoded by GAA or GAG
  • Phenylalanine is encoded by TTC or TTT
  • Glycine is encoded by GGA, GGC, GGG or GGT
  • Histidine is encoded by CAC or CAT;
  • Isoleucine (lie) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA, or AAG; Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT; Methionine (Met) is encoded by ATG;
  • Asparagine is encoded by AAC or AAT
  • Proline is encoded by CCA, CCC, CCG or CCT;
  • Glutamine is encoded by CAA or CAG;
  • Arginine is encoded by AGA, AGG, CGA, CGC,
  • Serine is encoded by AGC, AGT, TCA, TCC, TCG or TCT;
  • Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
  • Valine (Val) is encoded by GTA, GTC, GTG or GTT;
  • Trp Tryptophan
  • Tyrosine (Tyr) is encoded by TAC or TAT.
  • mRNA messenger RNA
  • T thymine nucleotide
  • U uracil nucleotide
  • Nucleic acid molecules encoding a human Zsig48 gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon SEQ ID NO: 1
  • a nucleic acid molecule that encodes a human Zsig48 gene can be isolated from a human cDNA library.
  • the first step would be to prepare the cDNA library by isolating RNA from placenta, kidney, leukocytes or heart tissue 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 rd Edi tion, 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 placental, leukocyte, kidney or heart 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) .
  • 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 65:1408 (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) .
  • commercially available kits can be used to synthesize double-stranded cDNA molecules.
  • such kits are available from Life Technologies, Inc. (Gaithersburg, MD) , CLONTECH Laboratories, Inc. (Palo Alto, CA) , Promega Corporation (Madison, WI) and STRATAGENE (La Jolla, CA) .
  • Various cloning vectors are appropriate for the construction of a cDNA library.
  • 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 ⁇ gtll," 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 Corp.) or other commercially available vectors.
  • a plasmid vector such as a pBLUESCRIPT vector (STRATAGENE; La Jolla, CA) , a LAMDAGEM-4 (Promega Corp.) 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 (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327) .
  • 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 Zsig48 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 Zsig48 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 Applica tions , White (ed.), pages 211- 215 (Humana Press, Inc. 1993) .
  • PCR Protocols Current Methods and Applica tions, White (ed.), pages 317-337 (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:l, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11) .
  • Anti-Zsig48 antibodies produced as described below, can also be used to isolate DNA sequences that encode human Zsig48 genes from cDNA libraries.
  • the antibodies can be used to screen ⁇ gtll expression libraries, or the antibodies can be used for immunoscreening following hybrid selection and translation
  • a Zsig48 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 Applica tions 2:266 (1993), Dillon et al .
  • 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. For the production of longer genes (>300 base pairs), however, special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • the sequence of a Zsig48 cDNA or Zsig48 genomic fragment can be determined using standard methods. Moreover, the identification of genomic fragments containing a Zsig48 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 Zsig48 proteins by "gene activation," following the methods disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous Zsig48 gene in a cell is altered by introducing into the Zsig48 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 Zsig48 5' non-coding sequence that permits homologous recombination of the construct with the endogenous Zsig48 locus, wherein the sequences within the construct become operably linked with the endogenous Zsig48 coding sequence.
  • an endogenous Zsig48 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression. Production of Zsig48 Gene Variants
  • the present invention provides a variety of nucleic acid molecules, including DNA and RNA molecules, that encode the Zsig48 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 counterparts from other species (orthologs) .
  • species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species.
  • Zsig48 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Orthologs of human Zsig48 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 Zsig48 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 Zsig48 -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 Zsig48 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 Zsig48 polypeptide. Similar techniques can also be applied to the isolation of genomic clones, and to the isolation of nucleic molecules that encode murine Zsig48.
  • SEQ ID NO:l represents a single allele of human Zsig48, and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ID NO:l, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs : 2 , 3, 4 and 5.
  • cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the Zsig48 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 Zsig48 can hybridize to nucleic acid molecules having the nucleotide sequence of SEQ ID NO:l, 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 Zsig48 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 (or its complement) at 42 °C overnight in a solution comprising 50% formamide, 5xSSC (IxSSC: 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 IxSSC: 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.
  • a higher temperature such as about 65°C
  • 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.
  • 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.
  • nucleic acid molecules encoding a variant Zsig48 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.
  • 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.
  • SSPE for SSC in the wash solution.
  • Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65°C.
  • SDS sodium dodecyl sulfate
  • nucleic acid molecules encoding a variant Zsig48 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 Zsig48 polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO: 2, SEQ ID NO: 5, 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 N02 : 2 , 3, 4 and 5, or their orthologs .
  • the present invention also contemplates Zsig48 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.
  • Such Zsig48 variants include nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 (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, 4 or 5.
  • Zsig48 variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 (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, 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, 4 or 5.
  • the present invention also contemplates human Zsig48 variant nucleic acid molecules identified by at least one of hybridization analysis and sequence identity determination, with reference to SEQ ID NOs : 2 , 3, 4 or 5.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull . Ma th . Bio . 48 : 603 (1986), and Henikoff and Henikoff, Proc . Natl . Acad. Sci . USA 85:10915 (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 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) .
  • 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 Zsig48 variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc . Na t ' l Acad . Sci . USA 85:2444 (1988), and by Pearson, Meth . Enzymol . 183 : 63 (1990) . Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence ( e . g.
  • 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 NOs:2, 3 , 4 or 5. That is, variants can be obtained that contain one or more amino acid substitutions of SEQ ID NOs:2, 3, 4 or 5 , in which an alkyl amino acid is substituted for an alkyl amino acid in an Zsig48 amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in an Zsig48 amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur- containing amino acid in an Zsig48 amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid in an Zsig48 amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a Zsig48 amino acid sequence, a basic amino acid is substituted for a basic amino acid in a Zsig48 amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarbox
  • 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.
  • 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 85:10915 (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.
  • 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) .
  • human Zsig48 are characterized by having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the corresponding human (i.e., SEQ ID NOs: 2, 3, 4 or 5) amino acid sequences, wherein the variation in amino acid sequence is due to one or more conservative amino acid substitutions.
  • Conservative amino acid changes in an Zsig48 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
  • variants to promote proliferation of T-cells, B-cells or monocytes using a standard method, such as the assay described herein.
  • a variant Zsig48 polypeptide can be identified by the ability to specifically bind anti- Zsig48 antibodies.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N- methylglycine, allo-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 .
  • an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs .
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is typically carried out in a cell -free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al . , J. Am . Chem . Soc . 113 : 2722 (1991), Ellman et al . , Methods Enzymol . 202 : 301 (1991), Chung et al . , Science
  • a second method translation is carried out in Xenopus oocytes by microinj ection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al . , J. Biol . Chem . 271 : 19991 (1996)) .
  • 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) .
  • non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 33 : 7470 (1994) .
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro 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 Zsig48 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 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering, " in Proteins : Analysis and Design, Angeletti (ed.), pages 259- 311 (Academic Press, Inc. 1998)] .
  • Zsig48 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, Zsig48 labeled with biotin or FITC can be used for expression cloning of Zsig48 ligand.
  • variants of the disclosed Zsig48 nucleotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer, Na ture 370 : 389 (1994) , Stemmer, Proc . Na t ' l Acad. Sci . USA 51:10747 (1994), and international publication No. WO 97/20078. Briefly, variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind with anti-Zsig48 antibodies can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the present invention also includes "functional fragments" of Zsig48 polypeptides and nucleic acid molecules encoding such functional fragments.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a Zsig48 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ID N0:1 can be digested with Bal31 nuclease to obtain a series of nested deletions.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of a Zsig48 gene can be synthesized using the polymerase chain reaction.
  • the present invention also contemplates functional fragments of a Zsig48 gene that has amino acid changes, compared with the amino acid sequence of SEQ ID NO: 2.
  • a variant Zsig48 gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequences of SEQ ID NOs: 2 , 3 , 4 or 5 as discussed above.
  • An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant Zsig48 gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, as discussed above.
  • the present invention also provides polypeptide fragments or peptides comprising an [-bearing portion of a Zsig48 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 38, a serine to and including amino acid residue 57, a threonine, of SEQ ID NO : 2 also defined by SEQ ID NO: 8; a polypeptide comprised of amino acid residue 38, a serine, to and including amino acid residue 79, a threonine, of SEQ ID NO: 2, also defined by SEQ ID NO : 9 ; a polypeptide comprised of amino acid residue 38, a serine, to and including amino acid residue 102, a serine of SEQ ID NO: 2 also defined by SEQ ID NO: 10; a polypeptide comprised of amino acid residue 60, a threonine, to and including amino acid residue 79, a threonine, of SEQ ID NO: 2 also defined by SEQ ID NO: 11; and a polypeptide comprised of amino acid residue comprised of amino acid residue
  • 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 215:660 (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 N0:2.
  • Such epitope-bearing peptides and polypeptides can be produced by fragmenting a Zsig48 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) ) .
  • the gene encodes a polypeptide that is characterized by its ability to bind specifically to an anti-Zsig48 antibody.
  • the present invention includes a computer-readable medium encoded with a data st7ructure that provides at least one of the following sequences: SEQ ID N0:1, 2, 3, 4, 5, 8, 9, 10, 11, and 12.
  • a computer-readable medium can be encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l, SEQ ID NO: 2.
  • Suitable forms of computer-readable media include magnetic media and optically-readable media.
  • Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT) , a disk cache, and a ZIP disk.
  • Optically readable media are exemplified by compact discs (e . g. , CD-read only memory (ROM) , CD-re-writable (RW) , and CD-recordable) , and digital versatile/video discs (DVD) ( e . g. , DVD-ROM, DVD- RAM, and DVD+RW) .
  • compact discs e . g. , CD-read only memory (ROM) , CD-re-writable (RW) , and CD-recordable
  • DVD digital versatile/video discs
  • Fusion proteins of Zsig48 can be used to express Zsig48 in a recombinant host, and to isolate expressed Zsig48. As described below, particular Zsig48 fusion proteins also have uses in diagnosis and therapy.
  • One type of fusion protein comprises a peptide that guides a Zsig48 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 Zsig48, 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 Zsig48 -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 pheromone ⁇ - factor (encoded by the MF ⁇ l gene) , invertase (encoded by the SUC2 gene) , or acid phosphatase (encoded by the PH05 gene) . See, for example, Romanos et al .
  • Zsig48 can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide. Glutathione S- transferase fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • a Zsig48 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 . 325:215 (1996), Morganti et al . , Biotechnol . Appl .
  • Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO) .
  • the present invention also contemplates that the use of the secretory signal sequence contained in the Zsig48 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 26 or 1-28 or 1-40 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.
  • exemplary polypeptides include pharmaceutically active molecules such as Factor Vila, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukins ( e . g.
  • interleukin-1 IL-1
  • IL-2 interleukin-2
  • IL-3 interleukin-4
  • IL-5 IL-6
  • IL-7 interleukin-8
  • IL-9 IL-10
  • IL-11 IL-12
  • IL-13 IL-13
  • IL-14 IL-14
  • IL-15 interleukin-15
  • colony stimulating factors e . g. , granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)
  • interferons e . g.
  • the Zsig48 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 Zsig48 secretory signal sequence can be constructed using standard techniques .
  • fusion protein comprises an Zsig48 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
  • 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: 17) .
  • 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 Zsig48 fusion protein that comprises a Zsig48 moiety and a human Fc fragment, wherein the C-terminus of the Zsig48 moiety is attached to the N-terminus of the Fc fragment via a peptide linker, such as a peptide consisting of the amino acid sequence of SEQ ID NO: 20.
  • the Zsig48 moiety can be a Zsig48 molecule or a fragment thereof.
  • a Zsig48 fusion protein comprises an IgG sequence, a Zsig48 moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently joined to the aminoterminal of the Zsig48 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 X domain.
  • Fusion proteins comprising a Zsig48 moiety and an Fc moiety can be used, for example, as an in vi tro assay tool.
  • the presence of a Zsig48 ligand in a biological sample can be detected using a Zsig48- immunoglobulin fusion protein, in which the Zsig48 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 Zsig48 to its ligand.
  • antibody-Zsig48 fusion proteins comprising antibody variable domains, are useful as therapeutic proteins, in which the antibody moiety binds with a target antigen, such as a tumor associated antigen.
  • 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 ' l Acad . Sci . USA 53:7826 (1996), Hank et al . , Clin . Cancer Res . 2:1951 (1996), and Hu et al . , Cancer Res . 56 : 4998 (1996).
  • Antibodies Hybridomas 6 129 (1995), and Xiang et al . , J. Biotechnol . 53 : 3 (1997), describe fusion proteins that include an F(ab') 2 fragment and a tumor necrosis factor alpha moiety. Additional cytokine-antibody fusion proteins include IL-8, IL-12, or interferon- ⁇ as the cytokine moiety (Holzer et al . , Cytokine 8 : 214 (1996); Gillies et al . , J. Immunol . 160 : 6195 (1998); Xiang et al . , Hum . Antibodies Hybridomas 7:2 (1996)). Also see, Gillies, U.S. Patent No. 5,650,150.
  • 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 Zsig48 compositions, in which an
  • Zsig48 polypeptide is linked with a polymer.
  • the polymer is water soluble so that the Zsig48 conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • An example of 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, or mono- (C1-C10) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al . , U.S. Patent No. 5,252,714) .
  • the polymer may be branched or unbranched.
  • a mixture of polymers can be used to produce Zsig48 conjugates.
  • Zsig48 conjugates used for therapy should preferably comprise pharmaceutically acceptable water- soluble polymer moieties.
  • Suitable water-soluble polymers include polyethylene glycol (PEG) , monomethoxy-PEG, mono- (CI-CIO) alkoxy-PEG, aryloxy-PEG, poly- (N-vinyl pyrrolidone) PEG, tresyl monomethoxy PEG, PEG propionaldehyde, Jis-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols ( e. g.
  • Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000.
  • a Zsig48 conjugate can also comprise a mixture of such water-soluble polymers.
  • PEGylation by acylation typically requires reacting an active ester derivative of PEG with a Zsig48 polypeptide.
  • An example of an activated PEG ester is PEG esterified to W-hydroxysuccinimide .
  • acylation includes the following types of linkages between Zsig48 and a water soluble polymer: amide, carbamate, urethane, and the like.
  • Methods for preparing PEGylated Zsig48 by acylation will typically comprise the steps of (a) reacting a Zsig48 polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to Zsig48, 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:Zsig48, the greater the percentage of polyPEGylated Zsig48 product.
  • the product of PEGylation by acylation is typically a polyPEGylated Zsig48 product, wherein the lysine ⁇ -amino groups are PEGylated via an acyl linking group.
  • An example of a connecting linkage is an amide.
  • the resulting Zsig48 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 Zsig48 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 Zsig48 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 W-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 Zsig48 monopolymer conjugates.
  • Reductive alkylation to produce a substantially homogenous population of monopolymer Zsig 8 conjugate molecule can comprise the steps of: (a) reacting a Zsig48 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 Zsig48, 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, dimethyl mine borane, trimethylamine borane, and pyridine borane .
  • the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the -ZNT- terminus of Zsig48.
  • Such reaction conditions generally provide for pKa differences between the lysine amino groups and the ⁇ -amino group at the i ⁇ -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:
  • Zsig48 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 molecular weight of the water-soluble polymer is the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein.
  • the typical molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25 kDa.
  • the molar ratio of water-soluble polymer to Zsig48 will generally be in the range of 1:1 to 100:1. Typically, the molar ratio of water-soluble polymer to Zsig48 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 Zsig48 expression vector may comprise a Zsig48 gene and a secretory sequence derived from a Zsig48 gene or another secreted gene.
  • Zsig48 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 . 12 : 555 (1986)]
  • GH1 ATCC CCL82
  • HeLa S3 cells ATCC CCL2.2
  • rat hepatoma cells H-4-II-E
  • COS-1 ATCC CRL 1650
  • murine embryonic 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, yosin, 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. Molec . 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.
  • a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter, can be used to control Zsig48 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 . 15:4485 (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 .
  • drugs 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.
  • Zsig48 polypeptides can also be produced by cultured mammalian cells using a viral delivery system.
  • viruses for this purpose include adenovirus, herpesvirus, 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 (1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)) .
  • 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
  • Adenovirus vector- infected human 293 cells 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 . 15:145 (1994)).
  • Zsig48 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 Zsig48 genes into insect cells.
  • Suitable expression vectors are based upon the Autographa calif ornica multiple nuclear polyhedrosis virus (AcMNPV) , and contain well-known promoters such as Drosophila hea t shock protein (hsp) 70 promoter, Autographa calif ornica nuclear polyhedrosis virus immediate -early gene promoter ( ie-1) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter.
  • AcMNPV Autographa calif ornica multiple nuclear polyhedrosis virus
  • hsp Drosophila hea t shock protein
  • ie-1 Autographa calif ornica nuclear polyhedrosis virus
  • 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 Zsig48 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill -Perkins and Possee, J. Gen . Virol .
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Zsig48 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al . , Proc . Nat ' l Acad. Sci . 82:7952 (1985)) .
  • a transfer vector containing a Zsig48 gene is transformed into E.
  • 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
  • transfer vector constructs a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native Zsig48 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen Corporation; Carlsbad, CA) , or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native Zsig48 secretory signal sequence.
  • the recombinant virus or bacmid is used to transfect host cells.
  • suitable insect host cells include cell lines derived from IPLB-S-f-21, a Spodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL 1711) , Sf21AE, and S 21 (Invitrogen Corporation; 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) .
  • Sf9 ATCC CRL 1711
  • Sf21AE Spodoptera frugiperda pupal ovarian cell line
  • S 21 Invitrogen Corporation
  • Drosophila Schneider-2 cells Drosophila Schneider-2 cells
  • HIGH FIVEO cell line Invitrogen
  • 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 Express FiveOTM (Life Technologies) for the T. ni cells.
  • 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 YCpl9.
  • Methods for transforming S . cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al . , U.S. Patent No. 4,931,373, Brake, U.S. Patent No. 4,870,008, Welch et al . , U.S. Patent No. 5,037,743, and Murray et al .
  • 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).
  • a preferred vector system for use in 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.
  • Transformation systems for other yeasts including Hansenula polymorpha , Schizosaccharomyces pombe, Kluyveromyces lactis , Kluyveromyces fragilis , Ustilago maydis, Pichia pastoris , Pichia methanolica, Pichia guillermondii and Candida mal tosa 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. methanoli ca 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 (AUG1 or AUG2) .
  • P. methanolica alcohol utilization gene AUG2
  • 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
  • a preferred selectable marker for use in Pichia methanolica is a P.
  • 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.
  • 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.
  • methanol utilization genes (AUG1 and AUG2) are deleted.
  • PEP4 and PRB1 are preferred.
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P . methanolica cells.
  • P. 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 Agrrojbacteriu-7. tumefaciens , microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch et al . , Science 227 : 1229 (1985), Klein et al . , Biotechnology 10:268 (1992), and Miki et al . , "Procedures for Introducing Foreign DNA into Plants, " in Methods in Plant Molecular Biology and Biotechnology, Glick et al . (eds.), pages 67-88 (CRC Press, 1993) .
  • Zsig48 genes can be expressed in prokaryotic host cells.
  • Suitable promoters that can be used to express Zsig48 polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4 , T3 , Sp6 and T7 polymerases, the P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5 , tac, lpp- 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, 4 th Ed. (Benjamin Cummins 1987) , and by Ausubel et al . (1995J .
  • Preferred prokaryotic hosts include E. coli and Bacillus subtilus .
  • Suitable strains of E. coli include BL2KDE3), BL21 (DE3 ) pLysS, BL21 (DE3 ) pLysE, DH1, DH4I, DH5 , DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1 , Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991) ) .
  • Suitable strains of Bacillus subtilus include BR151, YB886, MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)).
  • the polypeptide When expressing a Zsig48 polypeptide in bacteria such as E . coli , 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. In the former case, 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 denaturant such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione
  • 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 preferred 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 Zsig48 purified from natural sources (e . g. , placenta or leukocytes) , and recombinant Zsig48 polypeptides and Zsig48 polypeptides purified from recombinant host cells.
  • natural sources e . g. , placenta or leukocytes
  • 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 preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or 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, Affini ty Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988) , and Doonan, Protein Purification Protocols (The Humana Press 1996) .
  • Zsig48 isolation and purification can be devised by those of skill in the art.
  • anti-Zsig48 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 Zsig48, 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 adsorption (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 adsorption
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e . g. , maltose-binding protein, an immunoglobulin domain
  • Zsig48 polypeptides or fragments thereof may also be prepared through chemical synthesis, as described below.
  • Zsig48 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, 4 or 5.
  • the polypeptides comprise
  • Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
  • Zsig48 polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
  • the polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Merrifield, J " . Am . Chem . Soc . 85 : 2149 (1963) .
  • Solid phase synthesis is usually carried out from the carboxyl-terminus by coupling the alpha-amino protected (side-chain protected) amino acid to a suitable solid support.
  • An ester linkage is formed when the attachment is made to a chloromethyl , chlortrityl or hydroxymethyl resin, and the resulting polypeptide will have a free carboxyl group at the C-terminus.
  • an amide resin such as benzhydrylamine or p-methylbenzhydrylamine resin (for tBoc chemistry) and Rink amide or PAL resin (for Fmoc chemistry) are used, an amide bond is formed and the resulting polypeptide will have a carboxamide group at the C-terminus.
  • the "native chemical ligation" approach to producing polypeptides is one variation of total chemical synthesis strategy (see, for example, Dawson et al . , Science 266 : 776 (1994), Ralpheng et al . , Proc . Nat 'l Acad. Sci . USA 54:7845 (1997), and Dawson, Methods Enzymol . 287 : 34 (1997) ) .
  • an N-terminal cysteine-containing peptide is chemically ligated to a peptide having a C-terminal thioester group to form a normal peptide bond at the ligation site.
  • the "expressed protein ligation" method is a semi-synthesis variation of the ligation approach (see, for example, Muir et al , Proc . Na t ' l Acad . Sci . USA 95 : 6705 (1998); Severinov and Muir, J. Biol . Chem . 273:16205 (1998)).
  • synthetic peptides and protein cleavage fragments are linked to form the desired protein product.
  • This method is particularly useful for the site- specific incorporation of unnatural amino acids (e . g. , amino acids comprising biophysical or biochemical probes) into proteins .
  • a gene or gene fragment is cloned into the PCYB2- IMPACT vector (New England Biolabs, Inc.; Beverly, MA) using the Ndel and Smal restriction sites.
  • the gene or gene fragment is expressed in frame fused with a chitin binding domain sequence, and a Pro-Gly is appended to the native C terminus of the protein of interest.
  • the presence of a C- terminal glycine reduces the chance of side reactions, because the glycine residue accelerates native chemical ligation.
  • Affinity chromatography with a chitin resin is used to purify the expressed fusion protein, and the chemical ligation step is initiated by incubating the resin-bound protein with thiophenol and synthetic peptide in buffer. This mixture produces the in si tu generation of a highly reactive phenyl "thioester derivative of the protein that rapidly ligates with the synthetic peptide to produce the desired semi-synthetic protein.
  • a general class of Zsig48 analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below. Moreover, recombinant antibodies comprising anti-idiotype variable domains can be used as analogs (see, for example, Monfardini et al . , Proc . Assoc . Am. Physicians 108 : 420 (1996)). Since the variable domains of anti-idiotype Zsig48 antibodies mimic Zsig48, these domains can provide either Zsig48 agonist or antagonist activity.
  • a third approach to identifying Zsig48 analogs is provided by the use of combinatorial libraries.
  • Methods for constructing and screening phage display and other combinatorial libraries are provided, for example, by Kay et al . , Phage Display of Peptides and Proteins (Academic Press 1996), Verdine, U.S. Patent No. 5,783,384, Kay, et. al . , U.S. Patent No. 5,747,334, and Kauffman et al . , U.S. Patent No. 5,723,323.
  • the activity of Zsig48 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 Corp. (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.
  • the microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells.
  • Zsig48, its analogs, and anti-iodiotype Zsig48 antibodies can be used to identify and to isolate Zsig48 ligands.
  • proteins and peptides of the present invention can be immobilized on a column and used to bind ligand proteins from tissue and serum preparations that are run over the column (Hermanson et al . (eds.), Immobilized Affini ty Ligand Techniques , pages 195-202 (Academic Press 1992)) .
  • Radiolabeled or affinity labeled Zsig48 polypeptides can also be used to identify or to localize Zsig48 ligands in a biological sample (see, for example, Deutscher (ed.), Methods in Enzymol . , vol.
  • a solid phase system can be used to identify a Zsig48 ligand, or an agonist or antagonist of a Zsig48 ligand.
  • a Zsig48 polypeptide or Zsig48 fusion protein can be immobilized onto the surface of a receptor chip of a commercially available biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden) . The use of this instrument is disclosed, for example, by
  • a Zsig48 polypeptide or fusion protein is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within a flow cell.
  • a test sample is then passed through the cell. If a receptor is present in the sample, it will bind to the immobilized polypeptide or fusion protein, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. This system can also be used to examine antibody-antigen interactions, and the interactions of other complement/anti-complement pairs.
  • Antibodies to Zsig48 can be obtained, for example, using the product of a Zsig48 expression vector or Zsig48 isolated from a natural source as an antigen. Particularly useful anti-Zsig48 antibodies "bind specifically" to Zsig48. Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to Zsig48 with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to Zsig48.
  • antibodies specifically bind if they bind to a Zsig48 polypeptide, peptide or epitope with a binding affinity (K a ) of 10 6 M “1 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 Zsig48, but not known related polypeptides using a standard Western blot analysis.
  • Anti- Zsig48 antibodies can be produced using antigenic Zsig48 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 , for example, SEQ ID NOs: 8-12.
  • 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 Zsig48.
  • 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.
  • the immunogenicity of a Zsig48 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 Zsig48 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- Zsig48 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 : 310 (1990) .
  • monoclonal anti-Zsig48 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
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an Zsig48 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-Zsig48 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.
  • 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.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • 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, Cri t . 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 . Methods for producing scFvs are described, for example, by Whitlow et al . ,
  • a scFV can be obtained by exposing lymphocytes to Zsig48 polypeptide in vi tro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zsig48 protein or peptide) .
  • Genes encoding polypeptides having potential Zsig48 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,
  • Random peptide display libraries can be screened using the Zsig48 sequences disclosed herein to identify proteins which bind to Zsig48.
  • 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 :
  • an anti-Zsig48 antibody may be derived from 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 counterparts.
  • 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 . Na t ' l Acad . Sci . USA 86 : 3833 (1989).
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zsig48 antibodies or antibody fragments, using standard techniques. See, for example, Green et al . , "Production of Polyclonal Antisera, " in Methods In Molecular Biology: Immuno chemical 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-Zsig48 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.
  • Nucleic acid molecules can be used to detect the expression of a Zsig48 gene in a biological sample.
  • Probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO : 1 or a fragment thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:l, or a fragment thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • RNA isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target
  • Zsig48 RNA species After separating unbound probe from hybridized molecules, the amount of hybrids is detected.
  • Illustrative biological samples include blood, urine, saliva, tissue biopsy, and autopsy material.
  • RNA detection includes northern analysis and dot/slot blot hybridization (see, for example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al . (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S .
  • Zsig48 RNA can be detected with a nonradioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes (Humana Press, Inc. 1993) ) .
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates.
  • Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
  • Zsig48 oligonucleotide probes are also useful for in vivo diagnosis.
  • 18 F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al . , Nature Medicine 4:467 (1998)).
  • PCR polymerase chain reaction
  • Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols : Current Methods and Applica tions (Humana Press, Inc. 1993), Cotter (ed.), Mol ecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker
  • PCR reverse transcriptase-PCR
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with Zsig48 primers (see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)) .
  • Zsig48 primers see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997).
  • Zsig48 primers see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)
  • RNA is isolated from biological sample using, for example, the gunadinium- thiocyanate cell lysis procedure described above.
  • a solid-phase technique can be used to isolate mRNA from a cell lysate.
  • a reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or Zsig48 anti-sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • Zsig48 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length.
  • PCR amplification products can be detected using a variety of approaches.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled Zsig48 probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • CPT cycling probe technology
  • Alternative methods for detection of Zsig48 sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA) , cooperative amplification of templates by cross- hybridization (CATCH) , and the ligase chain reaction (LCR) (see, for example, Marshall et al . , U.S. Patent No. 5,686,272 (1997), Dyer et al . , J. Virol . Methods 60 : 161 (1996), Ehricht et al . , Eur . J. Biochem . 243 : 358 (1997), and Chadwick et al . , J. Virol . Me thods 70 : 59 (1998)).
  • Other standard methods are known to those of skill in the art .
  • Zsig48 probes and primers can also be used to detect and to localize Zsig48 gene expression in tissue samples.
  • Methods for such in si tu hybridization are well- known to those of skill in the art (see, for example, Choo (ed.), In Si tu Hybridization Protocols (Humana Press, Inc. 1994), Wu et al . (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Si tu Hybridization (RISH) , " in Methods in Gene Biotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al . (eds.), "Localization of DNA or Abundance of mRNA by Fluorescence In Si tu
  • Nucleic acid molecules comprising Zsig48 nucleotide sequences can also be used to determine whether a subject's chromosomes contain a mutation in the Zsig48 gene which has been mapped to chromosome 7q36.3. Detectable chromosomal aberrations at the Zsig48 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Of particular interest are genetic alterations that inactivate the Zsig48 gene.
  • Aberrations associated with the Zsig48 locus at chromosome 7q36.3 can be detected using nucleic acid molecules of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, amplification- refractory mutation system analysis (ARMS) , single-strand conformation polymorphism (SSCP) detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis (FAMA) , and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular
  • RNA is isolated from a biological sample, and used to synthesize cDNA.
  • PCR is then used to amplify the Zsig48 target sequence and to introduce an RNA polymerase promoter, a translation initiation sequence, and an in-frame ATG triplet .
  • PCR products are transcribed using an RNA polymerase, and the transcripts are translated in vi tro with a T7 -coupled reticulocyte lysate system.
  • the translation products are then fractionated by SDS-PAGE to determine the lengths of the translation products.
  • the protein truncation test is described, for example, by Dracopoli et al . (eds.), Current Protocols in Human Genetics, pages 9.11.1 - 9.11.18 (John Wiley & Sons 1998).
  • Zsig48 protein is isolated from a subject, the molecular weight of the isolated Zsig48 protein is determined, and then compared with the molecular weight a normal Zsig48 protein, such as a protein having the amino acid sequence of SEQ ID NOs : 2 , 3 , 4 or 5.
  • a substantially lower molecular weight for the isolated Zsig48 protein is indicative that the protein is truncated.
  • substantially lower molecular weight refers to at least about 10 percent lower, and preferably, at least about 25 percent lower.
  • the Zsig48 protein may be isolated by various procedures known in the art including immunoprecipitation, solid phase radioimmunoassay, enzyme-linked immunosorbent assay, or Western blotting.
  • the molecular weight of the isolated Zsig48 protein can be determined using standard techniques, such as SDS-polyacrylamide gel electrophoresis .
  • kits for performing a diagnostic assay for Zsig48 gene expression or to detect mutations in the Zsig48 gene comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO : 1 , or a fragment thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:l, or a fragment thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • Kits may comprise nucleic acid primers for performing PCR.
  • kits contains all the necessary elements to perform a nucleic acid diagnostic assay described above.
  • a kit will comprise at least one container comprising a Zsig48 probe or primer.
  • the kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zsig48 sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like.
  • a kit may also comprise a means for conveying to the user that the Zsig48 probes and primers are used to detect Zsig48 gene expression.
  • written instructions may state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes Zsig48, or a nucleic acid molecule having a nucleotide sequence that is complementary to an Zsig48 -encoding nucleotide sequence.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert .
  • anti-Zsig48 antibodies are used in liquid phase.
  • the presence of Zsig48 in a biological sample can be tested by mixing the biological sample with a trace amount of labeled Zsig48 and an anti-Zsig48 antibody under conditions that promote binding between Zsig48 and its antibody.
  • Complexes of Zsig48 and anti-Zsig48 in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein which binds with the antibody, such as an Fc antibody or Staphylococcus protein A.
  • the concentration of Zsig48 in the biological sample will be inversely proportional to the amount of labeled Zsig48 bound to the antibody and directly related to the amount of free Zsig48.
  • Illustrative biological samples include blood, urine, saliva, tissue biopsy, and autopsy material.
  • vi tro assays can be performed in which anti-Zsig48 antibody is bound to a solid-phase carrier.
  • antibody can be attached to a polymer, such as aminodextran, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube.
  • a polymer such as aminodextran
  • anti- Zsig48 antibodies can be used to detect Zsig48 in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of Zsig48 and to determine the distribution of Zsig48 in the examined tissue.
  • General immunochemistry techniques are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application, " in Mammalian Development : A Practical Approach, Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular Biology, Vol . 10 : Immunochemical Protocols (The Humana Press, Inc. 1992) ) .
  • Immunochemical detection can be performed by contacting a biological sample with an anti-Zsig48 antibody, and then contacting the biological sample with a detectably labeled molecule which binds to the antibody.
  • the detectably labeled molecule can comprise an antibody moiety that binds to anti-Zsig48 antibody.
  • the anti-Zsig48 antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin) . Numerous variations of this basic technique are well-known to those of skill in the art.
  • an anti-Zsig48 antibody can be conjugated with a detectable label to form an anti-Zsig48 immunoconjugate .
  • Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
  • the detectable label can be a radioisotope that is detected by autoradiography.
  • Isotopes that are particularly useful for the purpose of the present invention are 3 H, 125 I, 131 I, 35 S and 14 C.
  • Anti-Zsig48 immunoconjugates can also be labeled with a fluorescent compound.
  • the presence of a fiuorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence.
  • Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthal- dehyde and fluorescamine .
  • anti-Zsig48 immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoco jugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • Bioluminescent compound can be used to label anti-Zsig48 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-Zsig48 immunoconjugates can be detectably labeled by linking an anti-Zsig48 antibody component to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means .
  • enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galac- tosidase, glucose oxidase, peroxidase and alkaline phosphatase .
  • the convenience and versatility of immunochemical detection can be enhanced by using anti- Zsig48 antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al . (eds.), "Avidin-Biotin Technology,” Methods In Enzymology, Vol . 184 (Academic Press 1990), and Bayer et al . , "Immunochemical Applications of Avidin-Biotin Technology," in Methods In Molecular Biology, Vol . 10 , Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992) .
  • kits for performing an immunological diagnostic assay for Zsig48 gene expression comprise at least one container comprising an anti-Zsig48 antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zsig48 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 Zsig48 antibodies or antibody fragments are used to detect Zsig48 protein.
  • written instructions may state that the enclosed antibody or antibody fragment can be used to detect Zsig48.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • Molecules of the present invention can be used to promote proliferation of peripheral blood leukocytes. This can be useful in treating cancer patients whose leukocytes have been depleted by chemotherapy, radiation or illness.
  • Zsig48 can be administered to patients receiving bone marrow transplants to promote proliferation of leukocytes produced by the transplanted marrow. Also Zsig48 would be useful in treating immunosuppressed individuals as in the elderly or human immmundeficiency virus (HIV) infected individuals.
  • Zsig48 can also be used as a vaccine adjuvant to be administered with vaccines.
  • a dosage of Zsig48 administered for inducing the proliferation of T-cells, B-cells and monocytes will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history.
  • a dosage of Zsig48 which is in the range of from about 1 pg/kg to 10 mg/kg per day (amount of agent/body weight of patient) , preferably the dose will range from 4 - 100 ⁇ g/kg per day administered intravenously although a lower or higher dosage also may be administered as circumstances dictate.
  • Administration of a molecule having Zsig48 activity to a subject can be intravenous, intraarterial , intraperitoneal , intramuscular, subcutaneous, intrapleural , intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
  • the administration may be by continuous infusion or by single or multiple boluses.
  • Zsig48 can be administered as a controlled release formulation.
  • Cleland and Jones, Pharm. Res . 13:1464 (1996) describe a method for producing interferon- ⁇ encapsulated in polylactic-coglycolic microspheres.
  • Additional routes of administration include oral, dermal, mucosal -membrane, pulmonary, and transcutaneous .
  • Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems [see, for example, DiBase and Morrel, "Oral Delivery of
  • AERX diabetes management system which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs.
  • Studies have shown that proteins as large as 48,000 kDa have been delivered across skin at therapeutic concentrations with the aid of low- frequency ultrasound, which illustrates the feasibility of transcutaneous administration [Mitragotri et al . , Science 269 : 850 (1995)].
  • Transdermal delivery using electroporation provides another means to administer Zsig48 (Potts et al . , Pharm . Biotechnol . 10 : 213 (1997)) .
  • a pharmaceutical composition comprising a protein, polypeptide, or peptide having Zsig48 activity can be formulated according to known methods to prepare pharmaceutically useful compositions, wherein the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline, water, TWEEN ® 80 with mannitol and sodium acetate are examples of a pharmaceutically acceptable carriers and additives.
  • Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington ' s Pharmaceutical Sciences , 19th Edition (Mack Publishing Company 1995) .
  • molecules having Zsig48 activity and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount.
  • a combination of a protein, polypeptide, or peptide having Zsig48 activity and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. In the present context, an agent is physiologically significant if its presence results in the proliferation T-cells, B-cells or monocytes .
  • a pharmaceutical composition comprising molecules having Zsig48 activity can be furnished in liquid form, in an aerosol, or in solid form.
  • Proteins having Zsig48 activity such as human or murine Zsig48, can be administered as a conjugate with a pharmaceutically acceptable water-soluble polymer moiety, as described above.
  • a Zsig48 -polyethylene glycol conjugate is useful to increase the circulating half-life of the Zsig48, and to reduce the immunogenicity of the polypeptide.
  • Liquid forms, including liposome- encapsulated formulations are illustrated by injectable solutions and oral suspensions.
  • Exemplary solid forms include capsules, tablets, and controlled-release forms, such as a miniosmotic pump or an implant .
  • dosage forms can be devised by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington ' s Pharmaceutical Sciences , 19 th Edition (Mack Publishing Company 1995) , and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996) .
  • Zsig48 pharmaceutical compositions may be supplied as a kit comprising a container that comprises Zsig48, a Zsig48 agonist, or a Zsig48 antagonist (e . g. , an anti- Zsig48 antibody or antibody fragment) .
  • Zsig48 can be provided in the form of a solution for injection for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • a kit can include a dry- powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic polypeptide.
  • Such a kit may further comprise written information on indications and usage of the pharmaceutical composition. Moreover, such information may include a statement that the Zsig48 composition is contraindicated in patients with known hypersensitivity to Zsig48.
  • Immunomodulator Zsig48 genes can be introduced into a subject to enhance immunological responses by causing localized expression of Zsig48.
  • immunomodulator gene therapy has been examined in model systems using vectors that express IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, interferon- ⁇ , tumor necrosis factor- ⁇ , or granulocyte-macrophage colony- stimulating factor [see, for example, Cao et al . , J. Gastroenterol . Hepatol . 11 : 1053 (1996), Tahara et al . ,
  • the present invention includes the use of Zsig48 nucleotide sequences to augment the immune system to promote proliferation of leukocytes especially T-cells, B-cells or monocytes .
  • a therapeutic expression vector can be provided that inhibits Zsig48 gene expression, such as an anti-sense molecule, a ribozyme, or an external guide sequence molecule.
  • a Zsig48 gene to a subject, including the use of recombinant host cells that express Zsig48, delivery of naked nucleic acid encoding Zsig48,use of a cationic lipid carrier with a nucleic acid molecule that encodes Zsig48, and the use of viruses that express Zsig48, such as recombinant retroviruses, recombinant adeno-associated viruses, recombinant adenoviruses, and recombinant Herpes simplex viruses [HSV] (see, for example, Mulligan, Science 260 : 926 (1993), Rosenberg et al . , Science 242:1575 (1988), LaSalle et al .
  • HSV Herpes simplex viruses
  • an expression vector is constructed in which a nucleotide sequence encoding a Zsig48 gene is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription.
  • a core promoter and optionally a regulatory element
  • a Zsig48 gene can be delivered using recombinant viral vectors, including for example, adenoviral vectors [e.g., Kass-Eisler et al . , Proc. Nat'l Acad. Sci. USA 50:11498 (1993), Kolls et al . , Proc. Nat'l Acad. Sci. USA 91:215 (1994), Li et al . , Hum. Gene Ther. 4:403 (1993), Vincent et al . , Nat. Genet. 5:130 (1993), and Zabner et al . , Cell 75:207 (1993)], adenovirus- associated viral vectors [Flotte et al . , Proc.
  • adenoviral vectors e.g., Kass-Eisler et al . , Proc. Nat'l Acad. Sci. USA 50:11498 (1993), Kolls et al . , Proc. Nat'l
  • alphaviruses such as Semliki Forest Virus and Sindbis Virus [Hertz and Huang, J " . Vir. 66:857 (1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al . , Science 243:1188 (1989)]
  • herpes viral vectors e.g., U.S. Patent Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors (Koering et al . , Hum. Gene The rap . 5:457 (1994)], pox virus vectors [Ozaki et al .
  • adenovirus a double-stranded DNA virus
  • the adenovirus system offers several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii) the ability to be grown to high-titer, (iii) the ability to infect a broad range of mammalian cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters.
  • adenoviruses can be administered by intravenous injection, because the viruses are stable in the bloodstream.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell.
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. Although an adenoviral delivery system with an El gene deletion cannot replicate in the host cells, the host's tissue will express and process an encoded heterologous protein. Host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence . Secreted proteins will enter the circulation from tissue that expresses the heterologous gene ( e . g. , the highly vascularized liver) .
  • adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El-deleted, and in addition, contain deletions of E2A or E4 (Lusky et al . , J. Virol . 72 : 2022 (1998); Raper et al . , Human Gene Therapy 5:671 (1998)).
  • the deletion of E2b has also been reported to reduce immune responses (Amalfitano et al . , J. Virol . 72:926 (1998) ) .
  • By deleting the entire adenovirus genome very large inserts of heterologous DNA can be accommodated.
  • High titer stocks of recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using standard methods.
  • recombinant HSV can be prepared in Vero cells, as described by Brandt et al . , J. Gen . Virol . 72:2043 (1991), Herold et al . , J. Gen . Virol . 75:1211 (1994), Visalli and Brandt, Virology 185 : 419 (1991), Grau et al . , Inves t .
  • an expression vector comprising a
  • Zsig48 gene can be introduced into a subject's cells by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner et al . , Proc . Na t ' l Acad . Sci . USA 84:7413 (1987); Mackey et al . , Proc . Nat 'l Acad. Sci . USA 85:8027 (1988)].
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Liposomes can be used to direct transfection to particular cell types, which is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e . g. , hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • Electroporation is another alternative mode of administration. For example, Aihara and Miyazaki, Nature Biotechnology 16: 867 (1998), have demonstrated the use of in vivo electroporation for gene transfer into muscle.
  • a therapeutic gene may encode a Zsig48 anti-sense RNA that inhibits the expression of Zsig48.
  • Suitable sequences for anti-sense molecules can be derived from the nucleotide sequences of Zsig48 disclosed herein.
  • an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
  • Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example, Draper and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337) .
  • ribozymes include nucleotide sequences that bind with Zsig48 mRNA.
  • expression vectors can be constructed in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P- mediated cleavage of mRNA molecules that encode a Zsig48 gene.
  • an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see, for example, Altman et al . , U.S. Patent No. 5,168,053, Yuan et al . , Science 263 : 1269 (1994), Pace et al . , international publication No. WO 96/18733, George et al .
  • the external guide sequence comprises a ten to fifteen nucleotide sequence complementary to Zsig48 mRNA, and a 3 ' -NCCA nucleotide sequence, wherein N is preferably a purine.
  • the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 '-side of the base-paired region.
  • the dosage of a composition comprising a therapeutic vector having a Zsig48 nucleotide acid sequence, such as a recombinant virus will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history.
  • Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into a cavity that contains a tumor.
  • Horton et al . Proc . Na t ' l Acad . Sci . USA 56:1553 (1999)
  • intramuscular injection of plasmid DNA encoding interferon- ⁇ produces potent antitumor effects on primary and metastatic tumors in a murine model.
  • a composition comprising viral vectors, non- viral vectors, or a combination of viral and non-viral vectors of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby vectors or viruses are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition such as phosphate-buffered saline is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient subject.
  • suitable carriers are well-known to those in the art (see, for example, Remington ' s Pharmaceutical Sciences, 19th Ed . (Mack Publishing Co. 1995) , and Gilman ' s the Pharmacological Basis of Therapeutics , 7th Ed. (MacMillan Publishing Co. 1985)).
  • a therapeutic gene expression vector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount.
  • a combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject. In the present context, an agent is physiologically significant if its presence causes proliferation T-cells, B-cells or monocytes .
  • the therapy is preferably somatic cell gene therapy. That is, the preferred treatment of a human with a therapeutic gene expression vector or a recombinant virus does not entail introducing into cells a nucleic acid molecule that can form part of a human germ line and be passed onto successive generations (i.e., human germ line gene therapy) .
  • Transgenic mice can be engineered to over- express the human Zsig48 gene in all tissues or under the control of a tissue-specific or tissue-preferred regulatory element. These over-producers of Zsig48 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 Zsig48. Transgenic mice that over-express Zsig48 also provide model bioreactors for production of Zsig48 in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses a Zsig48 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 (Sigma) . Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (described, for example, by Menino and O'Claray, Biol . Reprod. 77 : 159 (1986), and Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated with 5% CO 2 , 5% 02 , and 90% N at 37°C. The eggs are then stored in a 37°C/5% CO 2 incubator until microin J ection.
  • plasmid DNA containing a Zsig48 encoding sequence is linearized, gel- purified, and resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinj ection .
  • the Zsig48 encoding sequences can encode the amino acid sequence of SEQ ID NOs : 2, 3, 4 or 5.
  • Plasmid DNA is microinj ected 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 microinj ected eggs are transferred into an organ tissue- culture dish with pre-gassed W640 medium for storage overnight in a 37°C/5% CO 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 transferred 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.
  • a baby serrefine Robot, Rockville, MD
  • 12-17 healthy two-cell embryos from the previous day's injection are transferred into the recipient.
  • the swollen ampulla is located and holding the oviduct between the ampulla and the bursa, a nick in the oviduct is made with a 28 g needle close to the bursa, making sure not to tear the ampulla or the bursa.
  • the pipette is transferred 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 Zsig48 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 born and weaned, the sexes are separated, and their tails snipped for genotyping. To check for expression of a transgene in a live animal, a partial hepatectomy is performed.
  • a surgical prep is made of the upper abdomen directly below the zyphoid process. Using sterile technique, a small 1.5- 2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized. Using 4-0 silk, 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 transferred 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 Zsig48 mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
  • transgenic mice that over-express Zsig48
  • Zsig48 gene expression can be inhibited using anti- sense genes, ribozyme genes, or external guide sequence genes.
  • inhibitory sequences are targeted to murine Zsig48 mRNA.
  • Methods for producing transgenic mice that have abnormally low expression of a particular gene are known to those in the art [see, for example, Wu et al . , “Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies," in Methods in Gene Biotechnology, pages 205-224 (CRC Press 1997)].
  • An alternative approach to producing transgenic mice that have little or no Zsig48 gene expression is to generate mice having at least one normal Zsig48 allele replaced by a nonfunctional Zsig48 gene.
  • One method of designing a nonfunctional Zsig48 gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes murine Zsig48.
  • 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
  • RNA extracted from each one of the cell lines was reversed transcribed in the following manner.
  • the first strand cDNA reaction contained 10 ⁇ l of twice poly d (T) -selected poly (A) + mRNA from K562, Daudi, HL-60, MOLT-4 or Raj i cells (Clontech, Palo Alto, CA) at a concentration of 1.0 mg/ml, and 2 ⁇ l of 20 pmole/ ⁇ l first strand primer SEQ ID NO: 7 (GTC TGG GTT CGC TAC TCG AGG CGG CCG CTA TTT TTT TTT TTT TTT TTT TTT TTT TTT) containing an Xho I restriction site. The mixture was heated at 70°C for 3.0 minutes and cooled by chilling on ice.
  • First strand cDNA synthesis was initiated by the addition of 8 ⁇ l of first strand buffer (5x SUPERSCRIPTTM buffer; Life Technologies, Gaithersburg, MD) , 4.0 ⁇ l of 100 mM dithiothreitol, and 3.0 ⁇ 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.
  • first strand buffer 5x SUPERSCRIPTTM buffer; Life Technologies, Gaithersburg, MD
  • 4.0 ⁇ l of 100 mM dithiothreitol 3.0 ⁇ l of a deoxynucleotide triphosphate (dNTP) solution containing 10 mM each of dTTP, dATP, dGTP and 5 -methyl - dCTP (Pharmacia LKB Bio
  • the reaction mixture was incubated at 37° C for 2 minutes, followed by the addition of 10 ⁇ l of 200 U/ ⁇ l RNase H " reverse transcriptase (SUPERSCRIPT II®'" Life Technologies) .
  • the efficiency of the first strand synthesis was analyzed in a parallel reaction by the addition of 10 ⁇ Ci of 32 P- ⁇ dCTP to a 5 ⁇ l aliquot from one of the reaction mixtures to label the reaction for analysis.
  • the reactions were incubated at 37°C for 10 minutes, 45°C for 50 minutes, then incubated at 50°C for 10 minutes.
  • Unincorporated 32 p- ⁇ dCTP in the labeled reaction was removed by chromatography on a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) .
  • the unincorporated nucleotides and primers in the unlabeled first strand reactions were removed by chromatography on 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) .
  • the length of labeled first strand cDNA was determined by agarose gel electrophoresis .
  • the second strand reaction contained 135 ⁇ l of the unlabeled first strand cDNA, 40 ⁇ l of 5x polymerase I buffer (125 mM Tris: HCI, pH 7.5, 500 mM KC1 , 25 mM MgCl 2 , 50mM (NH ) 2 S0 4))' 2.5 ⁇ l of 100 mM dithiothreitol, 5.0 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 ⁇ l of 5 mM ⁇ -NAD, 2.5 ⁇ l of 10 U/ ⁇ l E. coli DNA ligase (New England Biolabs; Beverly, MA), 7 ⁇ l of 10 U/ ⁇ l E.
  • 5x polymerase I buffer 125 mM Tris: HCI, pH 7.5, 500 mM KC1 , 25 mM MgCl 2 , 50mM (NH ) 2 S0 4)
  • Unincorporated 32 P- ⁇ dCTP in the labeled reaction was removed by chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) before analysis by agarose gel electrophoresis.
  • the reaction was terminated by the addition of 20.0 ⁇ l 0.5 M EDTA and extraction with phenol/chloroform and chloroform followed by ethanol precipitation in the presence of 3.0 M Na acetate and 2 ⁇ l of PELLET PAINT ® carrier (Novagen, Madison, WI) .
  • the cDNAs were ethanol precipitated a second time to remove possible trace levels of EDTA.
  • the yield of cDNA was estimated to be approximately 2 ⁇ g from starting mRNA template of 10 ⁇ g.
  • Eco Rl adapters were ligated onto the 5 ' ends of the cDNA described above to enable cloning into an expression vector.
  • the cDNA was digested with Xho I, resulting in a cDNA having a 5' Eco Rl 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, Indianapolis, IN) . Digestion was carried out at 37°C for one hour. The reaction was terminated by incubation at 70°C for 20 minutes and chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) .
  • the cDNA was ethanol precipitated, washed with 70% ethanol, air dried and resuspended in 13.5 ⁇ l water,
  • the region of the gel containing cDNAs 0.5 to 2 kb in length was excised and was placed in an empty adjacent lane of the gel at a position identical in distance to the lane origin.
  • the electrodes were reversed, and the cDNA was electrophoresed until the 0.5 to 2 kb length cDNA was concentrated near the lane origin.
  • the areas of the gel containing the concentrated cDNAs were 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,
  • the 0.5-2 kb fractions were pooled and cloned into pBLUESCRIPT (Gibco/BRL) to yield the K562L library.
  • the pooling of the cDNAs from the five cell lines was done to increase message complexity, particularly for cDNAs encoding cytokines and cytokine receptors.
  • the object of the present example was to test the effect of Zsig48 on peripheral blood leukocytes in a mixed leukocyte reaction.
  • a mixed leukocyte reaction is induced by culturing mononuclear leukocytes (which include T cells, B cells and monocytes from one individual with mononuclear leukocytes derived from another individual . These cells are generally isolated from peripheral blood. If there are differences in the alleles of the major histocompatibility complex (MHC) genes between the two individuals the mononuclear cells will proliferate over a period of from 4-7 days. This proliferative response is measured by incorporation of 3 H-thymidine into DNA during cell replication. This is called the allogeneic MLR.
  • MHC major histocompatibility complex
  • the blood was diluted in tissue culture medium and layered on top of a tube half full with Ficoll .
  • Ficoll has a density greater than that of lymphocytes but less than that of red blood cells and granulocytes. After centrifugation the red blood cells and granulocytes passed down through the Ficoll to form a pellet at the bottom of the tube while the mononuclear cells, i.e., T-cells, B- cells and monocytes remained at the interface of the medium and Ficoll.
  • the layer containing the PBMNCs was removed and resuspended in culture medium at a concentration of about 5 x 10 8 cells per mL of culture medium.
  • the cells from one of the individuals were then irradiated with 3300 rads gamma radiation. These irradiated cells were termed the stimulators. None was done to the cells of the other individual. These non- irradiated cells were termed the responder cells.
  • a suspension of both cells in medium was made containing 1 x 10 s cells per mL of responder cells and 0.15 X 10 6 cells per mL of stimulator cells. 100 ⁇ L aliquots of the mixed cells were placed in a series of wells of a multi-well plate. Into triplicate wells were placed aliquots of solutions of Zsig48 at the following concentrations: 0 ng/mL, 300 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, 10 ng/mL, 2 ng/mL, 0.1 ng/mL and 0.02 ng/mL .
  • a suspension of responder cells was also irradiated with 3300 rads of gamma radiation.
  • a suspension of cells was then made containing 0.15 X 10 6 cells per mL of irradiated responder and 1 x 10 6 of non- irradiated responder cells.
  • This mixture of irradiated and non- irradiated responder cells was called mock mixed leukocytes. 100 ⁇ L aliquots of the mock mixed leukocytes were placed in a series of wells of a multi-well plate.
  • each culture got l ⁇ Ci 3H-thymidine (Amersham- Pharmacia Biotech) . Plates were incubated another 20-24h. The cells were harvested onto 96-well filter mats and the mats were dried. About 30 ⁇ L of scintillation fluid was added to each spot containing the dried cells on the mat and the radiation was detected for one minute by a scintillation counter (Packard TOPCOUNT NXT®) as counts of radiation per minute (CPM) . This indicated the amount 3 H- thymidine which the cells took up which indicated the amount of proliferation that the leukocytes underwent.
  • a scintillation counter Packard TOPCOUNT NXT®
  • Zsig48 stimulates the proliferation of leukocytes in both a mixed leukocyte reaction and in an unmixed leukocyte reaction.
  • Zsig48 can be used to promote leukocyte proliferation both in the presence of antigen and in those cases where the cells have not been stimulated by antigen.
  • Two expression vectors were prepared to express zSig48 polypeptides in insect cells: pSig48CEE, designed to express a zSig48 polypeptide with a C-terminal GLU-GLU tag and pSig48NEE, designed to express a zSig48 polypeptide with an N-terminal GLU-GLU tag.
  • the band was excised, diluted to 0.5% agarose with 2 mM MgC12, melted at 65 °C and ligated into an BamHI/Xba I digested baculovirus expression vector, pZBV32L (a modification of the pFastBac expression vector, the polyhedron promoter has been removed and replaced with the late activating Basic Protein Promoter and the coding sequence for the Glu-Glu tag as well as a stop signal has been inserted at the 3 ' end of the multiple cloning region) . Forty- four point 6 nanograms of the restriction digested zsig48 insert and 215.9 ng of the corresponding vector were ligated overnight. The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH 7.5 and 1 mM EDTA) and 4 fmol of the diluted ligation mix was transformed into DH5a Library Efficiency competent cells
  • the transformed DNA and cells were diluted in 450 ml of SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml IM NaCl, 1.5 mM KC1, 10 mM MgCl2, 10 mM MgS04 and 20 mM glucose) and plated onto LB plates containing 100 mg/ml ampicillin.
  • SOC media 2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml IM NaCl, 1.5 mM KC1, 10 mM MgCl2, 10 mM MgS04 and 20 mM glucose
  • Clones were analyzed by restriction digests and 1 ml of the positive clone was transformed into 20 ml DHlOBac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg, MD) according to manufacturer's instruction, by heat shock for 45 seconds in a 42°C waterbath.
  • the transformed DNA was diluted in 980 ml SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml IM NaCl, 1.5 mM KC1, 10 mM MgC12, 10 mM MgS04 and 20 mM glucose) and plated onto Luria Agar plates containing 50 mg/ml kanamycin, 7 mg/ml gentamicin, 10 mg/ml tetracycline, IPTG and Bluo Gal. The cells were incubated for 48 hours at 37°C. A color selection was used to identify those cells having virus that had incorporated into the plasmid
  • Bacmid (referred to as a "bacmid") . Those colonies, which were white in color, were picked for analysis. Bacmid DNA was isolated from positive colonies using the QiaVac Miniprep ⁇ system (Qiagen) according the manufacturer's directions. Clones were screened for the correct insert by amplifying DNA using primers to the Basic Protein Promoter and to the SV40 terminus via PCR. Those having the correct insert were used to transfect Spodoptera frugiperda (Sf9) cells.
  • pSig48NEE A 263 bp zSig48 fragment with BamHI and Xbal restriction sites on the 5' and 3' ends, respectively, was generated by PCR amplification from zsig48/pZP9 (described above) . The fragment was visualized by gel electrophoresis and ligated into the expression vector, pZBV31L, as described above.
  • One microliter of pSig48NEE was used to independently transform 20 ml DHlOBac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg, MD) according to manufacturer's instruction, by heat shock at 42 °C for 45 seconds. The transformants were then diluted in 980 ml SOC media and plated on to Luria Agar plates as described above. Bacmid DNA was isolated from positive colonies and screened for the correct insert using the PCR method as described above. Those having the correct insert were used to transfect Spodoptera frugiperda (Sf9) cells.
  • Sf9 cells were seeded at 5 x 106 cells per 35 mm plate and allowed to attach for 1 hour at 27°C.
  • Five microliters of bacmid DNA was diluted with 100 ml Sf-900
  • Sf9 cells were grown in 50 ml Sf-900 II SFM in a
  • 125 ml shake flask to an approximate density of 0.41-0.52 x 105 cells/ml. They were then infected with 100 ml of the virus stock from above and incubated at 27°C for 2-3 days after which time the virus was harvested.
  • the virus titers for AcSig48CEE and AcSig48NEE were 1.08x108 pfu/ml and 1.84x108 pfu/ml, respectively.

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Abstract

L'invention concerne la protéine sécrétoire 48 (Zsig48), des polypeptides, des polynucléotides codant pour ces polypeptides, des anticorps qui se fixent spécifiquement sur Zsig48 et des anticorps anti-idiotype. Ces polypeptides Zsig48 entraînent une prolifération des leucocytes et peuvent par conséquent être utilisés pour augmenter le taux de leucocytes lors des traitements anticancéreux et chez les patients en état d'immunosuppression.
EP99953037A 1998-10-01 1999-10-01 Proteine secretoire 48 Withdrawn EP1117793A2 (fr)

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US16474098A 1998-10-01 1998-10-01
US164740 1998-10-01
PCT/US1999/022970 WO2000018796A2 (fr) 1998-10-01 1999-10-01 Proteine secretoire 48

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WO2000018796A3 (fr) 2000-09-28
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