WO1999016870A1 - Proteine zsig-11 secretee - Google Patents

Proteine zsig-11 secretee Download PDF

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Publication number
WO1999016870A1
WO1999016870A1 PCT/US1998/020449 US9820449W WO9916870A1 WO 1999016870 A1 WO1999016870 A1 WO 1999016870A1 US 9820449 W US9820449 W US 9820449W WO 9916870 A1 WO9916870 A1 WO 9916870A1
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Prior art keywords
amino acid
polypeptide
seq
sequence
zsig
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PCT/US1998/020449
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English (en)
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Paul O. Sheppard
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Zymogenetics, Inc.
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Priority to AU95922/98A priority Critical patent/AU9592298A/en
Publication of WO1999016870A1 publication Critical patent/WO1999016870A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • Proteins secreted from cells can act as intercellular signaling molecules which control the ontogeny and maintenance of tissue form and function. These secreted proteins control, among other things, proliferation, differentiation, migration, and expression of cells of multicellular organisms and act in concert to form cells, tissues and organs, and to repair and regenerate damaged tissue. Examples of secreted proteins include hormones and polypeptide growth factors which include steroid hormones (e.g.
  • Hormones and growth factors influence cellular metabolism by binding to receptors.
  • Receptors may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems.
  • Other classes of receptors are soluble molecules, such as the transcription factors.
  • the present invention provides novel secreted proteins, agonists, antagonists and receptors of such proteins, as well as related compositions and methods. It is to these secreted proteins and the polynucleotides encoding them that the present invention is directed.
  • an isolated polynucleotide molecule that encodes a polypeptide wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 2 ; b) a polynucleotide molecule having the sequence of SEQ ID NO : 6 ; and c) a polynucleotide molecule that hybridizes under stringent conditions to a polynucleotide molecule having the nucleotide sequence of SEQ ID NO : 1 , or the complement of SEQ ID NO : 1.
  • any difference between the amino acid sequence encoded by the polynucleotide molecule and the corresponding amino acid sequence of SEQ ID NO : 2 is due to a conservative amino acid substitution.
  • the polypeptide further comprises an affinity tag or binding domain.
  • the polynucleotide molecule comprises nucleotides 138-1001 of SEQ ID NO : 1.
  • the invention provides a polynucleotide molecule encoding a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, the first portion comprising a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2; and the second portion comprising another polypeptide .
  • a polynucleotide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-25 of SEQ ID NO : 2 , wherein the secretory signal sequence is operably linked to;an additional polypeptide.
  • the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule that encodes a polypeptide, wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 2 ; b) a polynucleotide molecule having the sequence of SEQ ID NO : 6 ; and c) a polynucleotide molecule that hybridizes under stringent conditions to a polynucleotide molecule having the nucleotide sequence of SEQ ID NO: 1, or the complement of
  • polypeptide further comprises a secretory signal sequence operably linked to the DNA segment.
  • secretory signal sequence comprises amino acid residues 1-25 of SEQ ID NO : 2.
  • polynucleotide encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag.
  • the invention also provides a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule that encodes a polypeptide, wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 2 ; b) a polynucleotide molecule having the sequence of -SEQ ID NO : 6 ; and c) a polynucleotide molecule that hybridizes under stringent conditions to a polynucleotide molecule having the nucleotide sequence of
  • a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule that encodes a polypeptide, wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO : 2 from, amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2; b) a polynucleotide molecule having the sequence of SEQ ID NO : 6 ; and c) a polynucleotide molecule that hybridizes under stringent conditions to a polynucleotide
  • the invention further provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 2.
  • the polypeptide comprises a sequence of amino acid residues that is at least 90% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 2.
  • the polypeptide comprises a sequence of amino acid residues that is at least 95% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polypeptide further comprises an affinity tag or binding domain .
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3
  • the invention also provided an antibody or antibody fragment that specifically binds to a polypeptide as described above.
  • the antibody is selected from the group consisting of : a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody.
  • the antibody fragment is selected from the group consisting of F(ab') , F(ab) , Fab' , Fab, Fv, scFv, and minimal recognition unit.
  • an anti-idiotype antibody that specifically binds to the antibody as described above.
  • a pharmaceutical composition comprising a polypeptide, the polypeptide comprising a sequence of amino acid residues that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 26 (Gly) to amino acid residue 313 (Glu) , and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 2 ; in combination with a pharmaceutically acceptable vehicle.
  • a method of detecting the presence of ZSIG-11 RNA is a biological sample, comprising the steps of: a) contacting a ZSIG- 11 nucleic acid probe under stringent hybridizing conditions with either i) test RNA molecules isolated from the biological sample, or ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of SEQ ID NO : 1 , or its complement, and b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of ZSIG-11 RNA is the biological sample.
  • a method of detecting the presence of ZSIG-11 in a biological sample comprising 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 SEQ ID NO : 2 , 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 antibody or the antibody fragment further comprises a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold.
  • 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.
  • 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
  • Allelic variant 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 (i.e., no change in the encoded polypeptide) , or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • Amino-terminal and carboxyl-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.
  • Complement/anti-complement pair Denotes non- identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) 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. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 ⁇ 9 M.
  • Complements of polynucleotide molecules Denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5 ' CCCGTGCAT 3 ' .
  • Degenerate nucleotide sequence As applied herein denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide 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) .
  • Expression vector A DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Isolated: when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems .
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316 -.774-78, 1985) .
  • Isolated polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term "isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator .
  • Ortholo 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.
  • Paralogs 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.
  • Polynucleotide Denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp") , nucleotides ("nt”), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded.
  • double-stranded molecules When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term “base pairs” . It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • Polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides" .
  • Probes and/or primers can be RNA or DNA.
  • DNA can be either cDNA or genomic DNA.
  • Polynucleotide probes and primers are single or double- stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements.
  • Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
  • PCR primers are at least 5 nucleotides in length, preferably 15 or more nt , more preferably 20-30 nt . Short polynucleotides can be used when a small region of the gene is targeted for analysis.
  • a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art .
  • Promoter Denotes a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • 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.
  • Receptor A cell-associated protein, or a polypeptide subunit of such protein, that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell . Binding of ligand to receptor results in a change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain (s) of the receptor and other molecule (s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell.
  • ligand a bioactive molecule
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
  • 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) .
  • Secretory signal sequence A DNA sequence that encodes a polypeptide (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. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • Soluble receptor or ligand A receptor or a ligand polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains.
  • Soluble ligands are most commonly receptor- binding polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors or ligands can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate. Many cell-surface receptors and ligands have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs . Receptor and ligand polypeptides are the to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
  • 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 protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention is based in part upon the discovery of a novel DNA sequence (SEQ ID NO:l) and corresponding polypeptide sequence (SEQ ID NO: 2).
  • This novel secreted protein has been designated ZSIG-11.
  • Novel ZSIG-11 ligand-encoding polynucleotides and polypeptides of the present invention were initially identified by querying an expressed sequence tag (EST) database for secretory signal sequences characterized by an upstream methionine start site, a hydrophobic region of approximately 13 amino acids and a cleavage site, in an effort to select for secreted proteins.
  • Polypeptides corresponding to ESTs meeting those search criteria were compared to known sequences to identify secreted proteins having homology to known ligands.
  • SEQ ID NO:l The protein encoded by this DNA fragment is predicted to be a secreted protein; however, no homology to any known protein was identified.
  • Analysis of the deduced amino acid sequence revealed a 25 amino acid residue signal sequence (residues 1-25 of SEQ ID NO:2), a beta sheet domain containing three cysteines from residues 26 to 148 or 161 of SEQ ID NO : 2 , two additional cysteines are found at amino acid residues 165 and 182; and a domain containing 11 potential phosphorylation sites from residues 151 or 164 to 313 of SEQ ID NO: 2) .
  • the resulting peptides would be from amino acid residue 26 to residue 148, from amino acid residue 151 to residue 161 and from amino acid residue 163 to residue 313 of SEQ ID NO : 2.
  • the domains could be partitioned into a beta sheet domain containing all five cysteine residues, comprising amino acid residues 26 to 214 of SEQ ID NO : 2 and a region condensed region containing phosphorylation sites, comprising amino acid residues 215 to 313 of SEQ ID NO : 2.
  • a moderate level of transcription was detected in skeletal muscle, pancreas, small intestine, peripheral blood lymphocytes, brain, placenta, liver, kidney, thymus, ovary, colon, spinal cord, trachea and adrenal gland.
  • Three transcripts of 5 kb, 2 kb and 1.5 kb corresponding to ZSIG-11 were detected in the human osteogenic cell lines HOS, MG-63, Sa052 and U205.
  • ZSIG-11 maps 252.51 cR_3000 from the top of the human chromosome 20 linkage group on the WICGR radiation hybrid map. Proximal and distal framework markers were D20S908 and D20S99, respectively. This positions ZSIG-11 in the 20ql3.12 region on the integrated LDB chromosome 20 map .
  • a degenerate polynucleotide sequence that encompasses all polynucleotides that encode the ZSIG-11 polypeptide of SEQ ID NO: 2 (amino acid residue 1-313) is disclosed in SEQ ID NO : 6.
  • ZSIG-11 polypeptide- encoding polynucleotides ranging from nucleotide 1 or 76 to nucleotide 939 of SEQ ID NO : 6 are contemplated by the present invention.
  • Also contemplated by the present invention are fragments as described above with respect to SEQ ID N0:1, which are formed from analogous regions of SEQ
  • nucleotides 63 to 1001 of SEQ ID NO: 1 correspond to nucleotides 1 to 939 of SEQ ID NO : 6 and nucleotides 138 to 1001 of SEQ ID NO : 1 correspond to nucleotides 76 to 939 of SEQ ID NO : 6
  • nucleotides 138 to 503 of SEQ ID NO : 1 correspond to nucleotides 76 to 431 of SEQ ID NO: 6
  • nucleotides 513 to 545 of SEQ ID NO : 1 correspond to nucleotides 450 to 483 of SEQ ID NO: 6
  • nucleotides 552 to 1001 of SEQ ID NO : 1 correspond to nucleotides 490 to 939 of SEQ ID NO: 6.
  • the nucleotide base codes in SEQ ID NO : 6 are summarized in Table 1 below. TABLE 1
  • degenerate codons used in SEQ ID NO: 6, encompassing all possible codons for a given amino acid, are set forth in Table 2 below.
  • degenerate codon representative of all possible codons encoding each amino acid.
  • WSN can, in some circumstances, encode arginine
  • MGN can, in some circumstances, encode serine
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO : 2. Such variant sequences can be tested for functionality as disclosed herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO : 1 , other polynucleotide probes, primers, fragments and sequences recited herein or sequences complementary thereto.
  • Polynucleotide hybridization is well known in the art and widely used for many applications, see for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; Ausubel et al . , eds .
  • Polynucleotide hybridization exploits the ability of single stranded complementary sequences to form a double helix hybrid.
  • Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.
  • Hybridization will occur between sequences which contain some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch.
  • the T m of the mismatched hybrid decreases by 1°C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases.
  • Stringent hybridization conditions encompass temperatures of about 5- 25°C below the thermal melting point (T m ) of the hybrid and a hybridization buffer having up to 1 M Na + .
  • the above conditions are meant to serve as a guide and it is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid.
  • the T m for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence.
  • Those conditions that influence the T m include, the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution. Numerous equations for calculating T m are known in the art, see for example (Sambrook et al . , ibid. ; Ausubel et al . , ibid.
  • Sequence analysis software such as Oligo 4.0 and Primer Premier, as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T m based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and suggest suitable probe sequences.
  • hybridization of longer polynucleotide sequences >50 bp, is done at temperatures of about 20-25°C below the calculated T m .
  • hybridization is typically carried out at the T m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
  • the length of the polynucleotide sequence influences the rate and stability of hybrid formation. Smaller probe sequences, ⁇ 50 bp, come to equilibrium with complementary sequences rapidly, but may form less stable hybrids. Incubation times of anywhere from minutes to hours can be used to achieve hybrid formation. Longer probe sequences come to equilibrium more slowly, but form more stable complexes even at lower temperatures. Incubations are allowed to proceed overnight or longer. Generally, incubations are carried out for a period equal to three times the calculated Cot time. Cot time, the time it takes for the polynucleotide sequences to reassociate, can be calculated for a particular sequence by methods known in the art .
  • the base pair composition of polynucleotide sequence will effect the thermal stability of the hybrid complex, thereby influencing the choice of hybridization temperature and the ionic strength of the hybridization buffer.
  • A-T pairs are less stable than G-C pairs in aqueous solutions containing NaCl . Therefor, the higher the G-C content, the more stable the hybrid. Even distribution of G and C residues within the sequence also contribute positively to hybrid stability.
  • Base pair composition can be manipulated to alter the T m of a given sequence, for example, 5-methyldeoxycytidine can be substituted for deoxycytidine and 5-bromodeoxuridine can be substituted for thymidine to increase the T m .
  • Hybridization buffers generally contain blocking agents such as Denhardt ' s solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, milk powders (BLOTTO) , heparin or SDS, and a Na + source, such as SSC (IX SSC: 0.15 M NaCl , 15 mM sodium citrate) or SSPE (IX SSPE : 1.8 M NaCl , 10 mM NaH 2 P0 4 , 1 mM EDTA, pH 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid is increased. Typically, hybridization buffers contain from between 10 mM-1 M Na + . Premixed hybridization solutions are also available from commercial sources such as Clontech
  • RNA probes for use according to manufacturer's instruction. Addition of destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the T ra of a hybrid.
  • destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations
  • formamide is used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures.
  • Formamide also acts to reduce non-specific background when using RNA probes.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for isolating DNA and RNA are well known in the art. It is generally preferred to isolate RNA from testis tissue, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry lj3:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder
  • cDNA Complementary DNA
  • poly (A) + RNA using known methods.
  • Polynucleotides encoding ZSIG-11 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • SEQ ID NO : 1 represents a single allele of the human ZSIG-11 gene, and that allelic variation and alternative splicing, "splice variants", are expected to exist.
  • Allelic variants of the DNA 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 NO: 2.
  • Splice variants of the DNA sequence shown in SEQ ID NO : 1 include DNA sequences that result from mature RNA molecules created by known eukaryotic RNA splicing processes wherein intron sequence is removed and exon sequence is joined. Such DNA sequences encoding proteins which retain properties of the ZSIG-11 protein of SEQ ID NO : 2 are within the scope of the present invention. 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.
  • the present invention further provides counterpart ligands and polynucleotides from other species (“species orthologs”) .
  • species orthologs These species would include, but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species.
  • ZSIG-11 ligand polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate ligands.
  • Species orthologs of human ZSIG-11 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 the ligand. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A ZSIG-11- encoding cDNA can then 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 sequence. A cDNA can also be cloned using the polymerase chain reaction (PCR) (Mullis, U.S. Patent No.
  • PCR polymerase chain reaction
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to ZSIG-11. Similar techniques can also be applied to the isolation of genomic clones.
  • the present invention also provides isolated polypeptides that are substantially homologous to the ligand polypeptide of SEQ ID NO : 2 and its species orthologs or paralogs.
  • isolated is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated protein or polypeptide is substantially free of other proteins or polypeptides, particularly other proteins or polypeptides of animal origin. It is preferred to provide the proteins or polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • substantially homologous is used herein to denote proteins or polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO : 2 or its species orthologs or paralogs. Such proteins or polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 2 or its species orthologs or paralogs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA £9:10915-9, 1992.
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl -terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag) , such as a poly-histidine tract, protein A (Nilsson et al . , EMBO J . 4 .
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl - glycine, allo-threonine, methylthreonine, hydroxyethyl- cysteine, hydroxyethylhomocysteine, nitroglutamine, homo- glutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3 , 3-dimethyl- proline, tert-leucine, norvaline, 2 -azaphenyl-alanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenyl- alan
  • 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 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 .
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) .
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine
  • the non- naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. £:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification.
  • variants of the disclosed ZSIG-11 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication 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 above can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides e.g., receptor binding
  • 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.
  • polypeptides that are substantially homologous to residues 26-313 of SEQ ID NO: 2 or allelic variants thereof and retain the receptor-binding properties of the wild-type protein.
  • polypeptides may include additional amino acids from the signal sequence, affinity tags and the like.
  • polypeptides may also include additional polypeptide segments as generally disclosed above .
  • the present invention provides fusions comprising the secretory peptide of ZSIG-11 (residues 1 through 25 of SEQ ID NO: 2) .
  • This secretory peptide can be used to direct the secretion of other proteins of interest by joining a polynucleotide sequence encoding it to the 5' end of a sequence encoding a protein of interest .
  • polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Ausubel et al . , eds . , Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
  • a DNA sequence encoding a ZSIG-11 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the ZSIG-11 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized e novo .
  • the secretory signal sequence is joined to the ZSIG-11 DNA sequence in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA 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).
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-25 of SEQ ID NO : 2 is be 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. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are preferred hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell £4:725, 1978; Corsaro and Pearson, Somatic Cell Genetics £:603, 1981; Graham and Van der Eb, Virology £2:456, 1973), electroporation (Neumann et al . , EMBO J . . 1:841-45, 1982), DEAE-dextran mediated transfection (Ausubel et al . , ibid) , liposome-mediated transfection (Hawley-Nelson et al .
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol. £6:59-72, 1977) and Chinese hamster ovary (e.g., CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus .
  • promoters include those from metallothionem genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “ transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants . " A preferred selectable marker is a gene encoding 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 may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa cal if ornica nuclear polyhedrosis virus (AcNPV) .
  • AcNPV Autographa cal if ornica nuclear polyhedrosis virus
  • King and Possee The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et al . , Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols.
  • ZSIG-11 baculovirus utilizes a transposon-based system described by Luckow (Luckow et al., J. Virol. 67_: 4566-79 , 1993). This system, which utilizes transfer vectors, is sold in the Bac-to-BacTM kit
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed ZSIG-11 polypeptide, for example, a Glu- Glu epitope tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci . ⁇ _2_:7952-4, 1985) .
  • a transfer vector containing ZSIG-11 is transformed into E . coli , and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses ZSIG-11 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994.
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435).
  • Commercially available serum-free media are used to grow and 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 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
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Sac char o yces cerevisiae, Pichia pastoris, and Pichia methanolica .
  • 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.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S.
  • Transformation systems for other yeasts including
  • Hansenula polymorpha Schizosaccharomyces pombe, Kl uyveromyces lactis , Kluyver omyces fragilis , Ustilago maydis , Pichia pastoris , Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art.
  • Patent No. 4,486,533 The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • 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) .
  • 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), which allows ade2 host cells to grow in the absence of adenine .
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P . methanolica cells. It is preferred to transform P. methanolica cells 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 ( ⁇ ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) .
  • the polypeptide When expressing a ZSIG-11 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.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • Expressed recombinant ZSIG-11 polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • 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 anion exchange media include derivatized dextran ⁇ , agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly 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.
  • Phenyl-Sepharose FF Pharmacia
  • Toyopearl butyl 650 Toso Haas, Montgomeryville, PA
  • Octyl-Sepharose Pharmacia
  • polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • 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.
  • Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol . , Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529- 39) .
  • an affinity tag e.g., polyhistidine, maltose-binding protein, an immunoglobulin domain
  • Protein refolding (and optionally reoxidation) procedures may be advantageously used. It is preferred to purify the protein to >80% purity, more preferably to >90% purity, even more preferably >95%, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin. ZSIG-11 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • ZSIG-11 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.
  • the presence of five cysteine residues suggests that covalent disulfide bonds between ZSIG-11 monomers may be formed.
  • the cysteine residues may also be indicative of up to two internal disulfide bonds in the ZIG-11 polypeptide structure.
  • a combination of ZSIG-11 monomer-ZSIG-11 monomer disulfide bonds and ZSIG-11 polypeptide internal disulfide bonds may also be formed.
  • the invention also provides ZSIG-11 fusion or chimeric proteins with human Ig or as His-tagged proteins.
  • a N- terminal or C-terminal extension such as a poly-histidine tag, substance P, FlagTM peptide (Hopp et al . , Biotechnology £: 1204-10, 1988; available from Eastman Kodak Co., New Haven, CT) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the ZSIG-11 polypeptide.
  • ZSIG-11 polypeptides can be expressed as a fusion or chimeric proteins with immunoglobulin heavy chain constant regions, typically an F c fragment, which contains two constant region domains and a hinge region, but lacks the variable region.
  • immunoglobulin heavy chain constant regions typically an F c fragment, which contains two constant region domains and a hinge region, but lacks the variable region.
  • Such chimera are typically secreted as multimeric molecules, wherein the Fc portions are disulfide bonded to each other and two ligand polypeptides are arrayed in close proximity to each other. Fusions of this type can be used to affinity purify the cognate receptor from solution, as an in vi tro assay tool, and to block signals in vi tro by specifically titrating out or blocking endogenous ligand.
  • a ZSIG-11- Ig fusion protein (chimera) is added to a sample containing the soluble receptor under conditions that facilitate receptor-ligand binding (typically near- physiological temperature, pH, and ionic strength) .
  • the chimera-receptor complex is then separated from the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads) .
  • the receptor is then eluted using conventional chemical techniques, such as with a salt or pH gradient.
  • the chimera itself can be bound to a solid support, with binding and elution carried out as above. Collected fractions can be re-fractionated until the desired level of purity is reached.
  • the chimeras are bound to a support via the Fc region and used in an ELISA format.
  • soluble receptor- Ig fusion proteins may be made using receptors for which a ligand has not been identified. ZSIG-11 is then mixed with a receptor fusion protein and binding is assayed as described above.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/anti- complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complement/anti- complement pair
  • a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ)
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145 :229-40, 1991 and Cunningham and Wells, J. Mol . Biol. 234 : 554-63 , 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell .
  • a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, 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.
  • ZSIG-11 is expressed in a wide variety of tissues, including endocrine tissues, suggesting it may be a growth, metabolism and/or reproduction related factor.
  • Assays measuring cell proliferation or differentiation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al . , Investigational New Drugs £:347-54, 1990), incorporation of radiolabelled nucleotides (Cook et al . , Analytical Biochem. 179 : 1-7 , 1989), incorporation of 5-bromo-2 ' -deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al . , J. Immunol .
  • ZSIG-11 is highly expressed in testis, with lesser expression in prostate, ovaries, placenta, thyroid, adrenal gland and brain, suggesting a role in regulation of the reproductive process.
  • the high level of expression in testis may indicate an association with conditions related to testicular tissues.
  • Proliferation and differentiation in vi tro in response to administered ZSIG- 11 polypeptides, agonists, antagonists, antibodies and binding proteins disclosed herein can be measured using cultured testicular cells or in vivo by administering molecules of the present invention to the appropriate animal model.
  • Cultured testicular cells include dolphin DBl.Tes cells (CRL-6258); mouse GC-1 spg cells (CRL-2053); TM3 cells (CRL-1714) ; TM4 cells (CRL-1715) ; and pig ST cells (CRL-1746) , available from American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD .
  • ZSIG-11 may also show ability to suppress basal and LPS stimulated TNF ⁇ production from monocytes.
  • Administration of purified ZSIG-11 to mice resulted in significant increases in circulating levels of BUN, and decreases in phosphorus and albumin and point to changes in the renal system and/or cardiac system, as described in greater detail in the examples below. This could be a primary effect targeting kidney or heart. Histopathological analysis of tissues of treated mice indicated no significant effects on the tissues examined, including kidney, heart and liver.
  • ZSIG-11 transfected expression host cells may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers have been described as a means to entrap transfected mammalian cells or primary mammalian cells.
  • These types of non-immunogenic "encapsulations" or microenvironments permit the transfer of nutrients into the microenvironment , and also permit the diffusion of proteins and other macromolecules secreted or released by the captured cells across the environmental barrier to the recipient animal.
  • the capsules or microenvironments mask and shield the foreign, embedded cells from the recipient animal's immune response.
  • Alginate threads provide a simple and quick means for generating embedded cells.
  • the materials needed to generate the alginate threads are readily available and relatively inexpensive.
  • the alginate threads are relatively strong and durable, both in vi tro and, based on data obtained using the threads, in vivo .
  • the alginate threads are easily manipulable and the methodology is scalable for preparation of numerous threads. In an exemplary procedure, 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension
  • the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle attached) .
  • a large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution.
  • 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 T.C. Becker et al . , Meth. Cell Biol. £:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4 . : 44- 53, 1997) .
  • adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. Some disadvantages (especially for gene therapy) associated with adenovirus gene delivery include: (i) very low efficiency integration into the host genome; (ii) existence in primarily episomal form; and
  • adenovirus By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts may be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (i.e., the human 293 cell line) .
  • the host cell i.e., the human 293 cell line
  • the virus cannot replicate in the host cells.
  • the host's tissue i.e., liver
  • the host's tissue i.e., liver
  • Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • the adenovirus system can also be used for protein production in vi tro . By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see A. Gamier et al . , Cytotechnol . £5:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non-secreted proteins may also be effectively obtained.
  • ZSIG-11 polypeptides can also be used to prepare antibodies that specifically bind to ZSIG-11 epitopes, peptides or polypeptides.
  • polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Technigues and Applications, CRC Press, Inc., Boca Raton, FL, 1982) .
  • polyclonal antibodies can be generated from a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
  • the immunogenicity of a ZSIG-11 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZSIG-11 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
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof, such as F(ab')2 .
  • F(ab)2, Fab', Fab, Fv and scFv proteolytic fragments Such 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. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.
  • the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
  • 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.
  • Non-human antibodies may be humanized by grafting only non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a humanlike surface by replacement of exposed residues, wherein the result is a "veneered” antibody) .
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics.
  • Humanized monoclonal antibodies directed against ZSIG-11 polypeptides could be used as a protein therapeutic, in particular for use as an immunotherapy.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to ZSIG-11 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZSIG-11 protein or peptide) .
  • ZSIG-11 polypeptides can also be used to prepare antibodies that specifically bind to ZSIG-11 epitopes, peptides or polypeptides.
  • Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules.
  • Antibodies herein specifically bind if they bind to a ZSIG-11 polypeptide, peptide or epitope with a binding affinity (K a ) of 10 M or greater, preferably 10 M ⁇ 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, G., Ann. NY Acad. Sci. 51: 660-72, 1949).
  • Antibodies specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect ZSIG-11 but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid. ) . Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family such as other known secreted proteins, mutant ZSIG-11 polypeptides, and related non-human secreted proteins. Moreover, antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds ZSIG-11 polypeptides.
  • antibodies raised to ZSIG-11 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to ZSIG-11 will flow through the matrix under the proper buffer conditions.
  • Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Coligan, et al . (eds.), National Institutes of Health, John Wiley and Sons, Inc.,
  • ZSIG-11 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.
  • These 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. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al .
  • Random peptide display libraries can be screened using the ZSIG-11 sequences disclosed herein to identify proteins which bind to ZSIG-11.
  • These "binding proteins" which interact with ZSIG-11 polypeptides may be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing ZSIG- 11 activity.
  • the binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease.
  • assays known to those skilled in the art can be utilized to detect antibodies and binding proteins which specifically bind to ZSIG-11 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include : concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, ELISA, dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wildtype versus mutant ZSIG-11 protein or peptide. Antibodies to ZSIG-11 may be used for tagging or labeling cells.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates. Uses of such tagged antibodies include immunologically tagging with a fluorescent tag for use in FAC analysis for example, and immunohistochemical tagging of cells that express human ZSIG-11 which can be used as diagnostic assays. Antibodies may also be used for isolating ZSIG-11 by affinity purification; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZSIG-11 in vitro and in vivo . Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • vi tro assay methods for detecting the presence of ZSIG-11 in a biological sample, comprising 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 SEQ ID NO : 2 , 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.
  • anti-ZSIG-11 antibodies are used in liquid phase.
  • the presence of ZSIG-11 in a biological sample can be tested by mixing the biological sample with a trace amount of labeled ZSIG-11 and an anti-ZSIG-11 antibody under conditions that promote binding between ZSIG-11 and its antibody.
  • Complexes of ZSIG-11 and anti-ZSIG-11 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 ZSIG-11 in the biological sample will be inversely proportional to the amount of labeled ZSIG-11 bound to the antibody and directly related to the amount of free labeled ZSIG-11.
  • vi tro assays can be performed in which anti-ZSIG-11 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-ZSIG-11 antibodies can be used to detect ZSIG-11 in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of ZSIG-11 and to determine the distribution of ZSIG-11 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 ibid. ; Ausubel ibid. ; 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-ZSIG-11 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-ZSIG-11 antibody.
  • the anti-ZSIG-11 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-ZSIG-11 antibody can be conjugated with a detectable label to form an anti-ZSIG-11 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-ZSIG-11 immunoconjugates can also be labeled with a fluorescent compound.
  • the presence of a fluorescently-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-phth- aldehyde and fluorescamine .
  • anti-ZSIG-11 immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoconjugate 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-ZSIG-11 immunoconjugates of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-ZSIG-11 immunoconjugates can be detectably labeled by linking an anti-ZSIG-11 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 .
  • anti- ZSIG-11 antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al .
  • Antibodies Production, Engineering, and Clinical
  • the present invention also contemplates the use of immunoconjugates for in vivo detection of ZSIG-11 polypeptide.
  • the method of diagnostic imaging with radiolabeled monoclonal antibodies is well- known.
  • radioisotopes that can be bound to antibodies and are appropriate for diagnostic imaging include K-emitters and positron-emitters such as 99m Tc, 94m Tc, 67 Ga, 64 Cu, lxl In, 123 I, 124 I, 125 I, 131 I, 51 Cr, 89 Zr, 18 F and 68 Ga.
  • K-emitters and positron-emitters such as 99m Tc, 94m Tc, 67 Ga, 64 Cu, lxl In, 123 I, 124 I, 125 I, 131 I, 51 Cr, 89 Zr, 18 F and 68 Ga.
  • Other suitable radioisotopes are known to those of skill in the art.
  • Anti-ZSIG-11 antibodies also can be labeled with paramagnetic ions for purposes of in vivo diagnosis. Elements that are particularly useful for magnetic resonance imaging include Gd, Mn, Dy and Fe ions.
  • ZSIG-11 ligand polypeptides may be used to identify and characterize receptors which bind ZSIG-11 polypeptides. Proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Technigues, Hermanson et al . , eds., Academic Press, San Diego, CA, 1992, 195-202) .
  • Proteins and peptides can also be radiolabeled or photoaffinity labeled as described above and specific cell-surface proteins can be identified.
  • the soluble ligand is useful in studying the distribution of receptors on tissues or specific cell lineages, and to provide insight into receptor/ligand biology.
  • the invention also provides isolated and purified ZSIG-11 polynucleotide probes.
  • polynucleotide probes can be RNA or DNA.
  • DNA can be either cDNA or genomic DNA.
  • Polynucleotide probes are single or double-stranded DNA or RNA, generally synthetic oligonucleotides , but may be generated from cloned cDNA or genomic sequences and will generally comprise at least 16 nucleotides, more often from 17 nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion, domain or even the entire ZSIG-11 gene or cDNA.
  • the synthetic oligonucleotides of the present invention have at least 80% identity to a representative ZSIG-11 DNA sequence (SEQ ID NO:l) or its complement .
  • Preferred regions from which to construct probes include the 5' and/or 3' coding sequences, receptor binding regions, signal sequences and the like. Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example, Ausubel et al . , eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1991.
  • the molecules can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
  • a detectable signal such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
  • Such probes can also be used in hybridizations to detect the presence or quantify the amount of ZSIG-11 gene or mRNA transcript in a sample.
  • ZSIG-11 polynucleotide probes could be used to hybridize to DNA or RNA targets for diagnostic purposes, using such techniques such as fluorescent in si tu hybridization (FISH) or immunohistochemistry .
  • Polynucleotide probes could be used to identify genes encoding ZSIG-11-like proteins.
  • Such probes can also be used to screen libraries for related ZSIG-11 sequences. Such screening would be carried out under conditions of low stringency which would allow identification of sequences which are substantially homologous, but not requiring complete homology to the probe sequence.
  • Libraries may be made of genomic DNA or cDNA.
  • Polynucleotide probes are also useful for Southern, Northern, or slot blots, colony and plaque hybridization and in si tu hybridization. Mixtures of different ZSIG-11 polynucleotide probes can be prepared which would increase sensitivity or the detection of low copy number targets, in screening systems.
  • Nucleic acid molecules can be used to detect the expression of a ZSIG-11 gene in a biological sample.
  • a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target ZSIG-11 RNA species. After separating unbound probe from hybridized molecules, the amount of hybrids is detected.
  • a method of detecting the presence of ZSIG-11 RNA in a biological sample comprising the steps of : a) contacting a ZSIG-11 nucleic acid probe under stringent hybridizing conditions with either i) test RNA molecules isolated from the biological sample, or ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of SEQ ID NO:l, or its complement, and b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of ZSIG-11 RNA is the biological sample.
  • RNA detection includes northern analysis and dot/slot blot hybridization (see, for example, Ausubel ibid, at pages 4-
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S .
  • ZSIG-11 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.
  • ZSIG-11 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).
  • positron emission tomography Teavitian et al . , Nature Medicine 4:467, 1998.
  • Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods.
  • PCR primers can be designed to amplify a sequence encoding a particular ZIG-11 region, such as the ZIG-11 beta sheet domain (encoded by about nucleotide 138 to nucleotide 145 of SEQ ID N0:1) .
  • PCR reverse transcriptase-PCR
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with ZSIG-11 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) .
  • ZSIG-11 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) .
  • PCR is then performed and the products are analyzed using standard techniques.
  • RNA is isolated from a biological sample using, for example, the guanidinium- 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 ZSIG-11 anti-sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • ZSIG-11 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 ZSIG-11 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
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross- hybridization
  • LCR ligase chain reaction
  • ZSIG-11 probes and primers can also be used to detect and to localize ZSIG-11 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,
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high- resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows the designing of PCR primers suitable for use with chromosomal radiation hybrid mapping panels.
  • Commercially available radiation hybrid mapping panels which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) , are available. These panels enable rapid, PCR based, chromosomal localizations and ordering of genes, sequence-tagged sites (STSs) , and other nonpolymorphic- and polymorphic markers within a region of interest.
  • the precise knowledge of a gene's position can be useful in a number of ways including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms such as YAC-, BAC- or cDNA clones, 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region, and 3) for cross-referencing model organisms such as mouse which may be beneficial in helping to determine what function a particular gene might have.
  • ZSIG-11 gene maps 252.51 cR_3000 from the top of the human chromosome 20 linkage group on the WICGR radiation hybrid map. Proximal and distal framework markers were D20S908 and D20S99, respectively. This positions ZSIG-11 in the 20ql3.12 region on the integrated LDB chromosome 20 map.
  • ZSIG-11 oligonucleotide probes can be used to determine if the ZSIG-11 gene is present on chromosome 20 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the ZSIG-11 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides 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, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid.; Ausubel et . al . , ibid.; A.J. Marian, Chest 108:255-65, 1995) .
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • ZSIG-11 polypeptides may be used within diagnostic systems. Antibodies or other agents that specifically bind to ZSIG-11 may be used to detect the presence of circulating ZSIG-11 polypeptides and/or receptors. More specifically, the present invention contemplates methods for detecting ZSIG-11 polypeptides comprising: exposing a solution possibly containing ZSIG-11 polypeptide to an antibody attached to a solid support, wherein the antibody binds to a first epitope of a ZSIG-11 polypeptide; washing the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a ZSIG- 11 polypeptide-antibody complex, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
  • Serum or biopsy ZSIG-11 polypeptide concentration may be indicative of dysfunction related to altered levels of ZSIG-11 polypeptides.
  • Other acceptable detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay .
  • ELISA enzyme-linked immunosorbent assay
  • Immunohistochemically labeled antibodies can be used to detect ZSIG-11 ligand in tissue samples.
  • ZSIG-11 levels can also be monitored by such methods as RT-PCR, where ZSIG-11 mRNA can be detected and quantified. Such methods could be used as diagnostic tools to monitor and quantify receptor or ligand polypeptide levels. The information derived from such detection methods would provide insight into the significance of ZSIG-11 polypeptides in various diseases, and as such would serve as diagnostic methods for those diseases in which altered levels of ZSIG-11 are significant .
  • the present invention also provides methods for studying known or identifying new prohormone convertases, or endoproteases, enzymes which process prohormones and protein precursors .
  • Precursor proteins are cleaved or processed into active form through the action of prohormone convertases (endoproteases) .
  • the most prevalent cleavage or processing site is a dibasic amino acid prohormone convertase site.
  • There are only a few dibasic amino acid combinations including lys-lys, arg- arg, arg-lys and lys-arg.
  • Non-dibasic cleavage and processing sites have also been observed, for example, Asn-Arg is a non-dibasic site found in gastrin.
  • ZSIG-11 polypeptides may be processed by prohormone convertases into an active from.
  • Known prohormone convertases include, but are not limited to, prohormone convertase 3
  • PC3 prohormone convertase 2
  • PC4 prohormone convertase 4
  • PACE4 PACE4
  • Prohormone convertases sometimes exhibit tissue specificity.
  • ZSIG-11 polypeptides which are expressed at high levels in testis, for example, are likely to be processed by prohormone convertases exhibiting testis specificity.
  • ZSIG-11 polypeptides or fragments may be incubated with known or suspected prohormone convertases (enzyme) to produce a 122 amino acid residue fragment from amino acid residue 26 to amino acid residue 148, a 10 amino acid fragment from amino acid residue 151 to amino acid residue 161, and a 150 amino acid fragment from amino acid residue 163 to amino acid residue 313 of SEQ ID NO : 2.
  • the enzyme and substrate are incubated together or co-expressed in a test cell for a time sufficient to achieve cleavage/processing of the ZSIG-11 polypeptide or fragment or fusion thereof.
  • Detection and/or quantification of cleavage products follows, using procedures that are known in the art. For example, enzyme kinetics techniques, measuring the rate of cleavage, can be used to study or identify prohormone convertases capable of cleaving ZSIG-11 polypeptides, fragments or fusion proteins of the present invention.
  • the present invention also provides post-translationally modified polypeptides or polypeptide fragments having the amino acid sequence from amino acid residue 26 to amino acid residue 148 of SEQ ID NO : 2 ; the amino acid sequence from amino acid residue 151 to amino acid residue 161 of SEQ ID NO: 2 and the amino acid sequence from amino acid residue 163 to amino acid residue 313 of SEQ ID NO : 2.
  • post translational modifications include proteolytic cleavage, glycosylation, disulfide bonding and hydroxylation.
  • the present invention also provides antisense polynucleotide compositions that are complementary to a segment of the polynucleotides set forth in SEQ ID NO: 1.
  • Such synthetic antisense oligonucleotides are designed to bind to mRNA encoding ZSIG-11 polypeptides and to inhibit translation of such mRNA. Such antisense oligonucleotides are used to inhibit expression of ZSIG- 11 polypeptide- encoding genes in cell culture or in a subject.
  • mice 11 gene function, referred to as “knockout mice”.
  • mice may also be generated (Lowell et al . , Nature ££6:740-2, 1993). These mice may be employed to study the ZSIG- 11 gene and the protein encoded thereby in an in vivo system.
  • the invention also provides antagonists, which either bind to ZSIG-11 polypeptides or, alternatively, to a receptor to which ZSIG-11 polypeptides bind, thereby inhibiting or eliminating the function of ZSIG-11.
  • ZSIG- 11 antagonists would include antibodies; oligonucleotides which bind either to the ZSIG-11 polypeptide or to its receptor; natural or synthetic analogs of ZSIG- 11 polypeptides which retain the ability to bind the receptor but do not result in either ligand or receptor signaling.
  • Such analogs could be peptides or peptide-like compounds.
  • One such peptide would comprise amino acid residues 26 to 214 of SEQ ID NO : 2 which contains the five cysteine residues.
  • ZSIG-11 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a ZSIG- 11 ligand or receptor would be beneficial.
  • the ZSIG-11 polynucleotides and/or polypeptides disclosed herein can be useful as therapeutics, wherein ZSIG-11 agonists (including ZSIG-11 polypeptides (such as a polypeptide comprising an amino acid sequence from amino acid residue 24 to 214 of SEQ ID N0:2), substrates, cofactors, and the like) and antagonists could modulate one or more biological processes in cells, tissues and/or biological fluids.
  • ZSIG-11 is expressed in a wide variety of tissues, especially those of the endocrine system.
  • the ZSIG-11 polypeptides, agonists and antagonists could be employed in therapeutic protocols for treatment of growth, metabolism and/or reproduction related disorders.
  • ZSIG-11 polypeptides or ZSIG-11 antagonists may be useful as a fertility inducing therapeutic.
  • Such polypeptides and antagonists could be used in methods of assisted reproduction, such as artificial insemination or in vi tro fertilization in humans and animals, to enhance the likelihood of successful fertilization.
  • ZSIG- 11 polypeptides or antagonists would find application in the treatment of infertility.
  • ZSIG-11 The high level of expression of ZSIG-11 in the testis suggests a role in spermatogenesis and/or sperm maturation, perhaps as a proliferation or differentiation factor. ZSIG-11 is also expressed in ovaries. Regulation of reproductive function in males and females is controlled in part by feedback inhibition of the hypothalamus and anterior pituitary by blood-bone hormones. Testis proteins, such as activins and inhibins, have been shown to regulate secretion of active molecules including follicle stimulating hormone (FSH) for the pituitary (Ying, Endodcr . Rev. : 267-93, 1988; Plant et al . , Hum. Reprod. £: 41-44 , 1993) .
  • FSH follicle stimulating hormone
  • Inhibins also expressed in the ovaries, have also been shown to regulate ovarian functions (Woodruff et al . , Endocr . ££2:2332-42, 1993; Russell et al . , J. Reprod. Fertil . 100 : 115-22 , 1994).
  • Relaxin has also been shown to be a systemic and local acting hormone regulating follicular and uterine growth (Bagnell et al . , J. Reprod. Fertil. 4£:127-38, 1993), as such, the polypeptides of the present invention may also have effects on female gametes and reproductive tract. These functions may also be associated with the ZSIG-11 proteins and may be used to regulate testicular or ovarian functions.
  • the invention also provides nucleic acid-based therapeutic treatment. If a mammal has a mutated or lacks a ZSIG-11 gene, the ZSIG-11 gene can be introduced into the cells of the mammal.
  • a gene encoding a ZSIG-11 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV) , papillomavirus , Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV) , and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell .
  • defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al . , Molec . Cell . Neurosci . £:320-30, 1991), an attenuated adenovirus vector, such as the vector described by Stratford- Perricaudet et al . (J. Clin. Invest. £0:626-30, 1992), and a defective adeno-associated virus vector (Samulski et al . , J. Virol. £1:3096-101, 1987; Samulski et al . , ££ Virol. £:3822-28, 1989) .
  • HSV1 herpes virus 1
  • the gene can be introduced in a retroviral vector, e . g. , as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . ,
  • the vector can be introduced 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. Natl. Acad. Sci. USA £4:7413-17, 1987; and Mackey et al .
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be 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 , and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • Targeted peptides e.g., hormones or neurotransmitters , and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter (see, for example, Wu et al . , J. Biol. Chem.
  • ZSIG-11 polypeptides are also contemplated for pharmaceutical use.
  • Pharmaceutically effective amounts of ZSIG-11 polypeptides, agonists or ZSIG-11 antagonists of the present invention can be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal, topical, transdermal administration or the like, according to conventional methods.
  • Formulations may further include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets, for example.
  • Slow or extended-release delivery systems including any of a number of biopolymers (biological -based systems) , systems employing liposomes, and polymeric delivery systems, can also be utilized with the compositions described herein to provide a continuous or long-term source of the ZSIG- 11 polypeptide or antagonist.
  • Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use.
  • pharmaceutically acceptable carrier refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
  • a "pharmaceutically effective amount" of a ZSIG- 11 polypeptide, agonist or antagonist is an amount sufficient to induce a desired biological result.
  • the result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • an effective amount of a ZSIG-11 polypeptide is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • Effective amounts of the ZSIG- 11 polypeptides can vary widely depending on the disease or symptom to be treated.
  • the amount of the polypeptide to be administered and its concentration in the formulations depends upon the vehicle selected, route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and the stability of the compound in the formulation.
  • the clinician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients.
  • Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health of the patient, and other factors evident to those skilled in the art.
  • a dose will be in the range of 0.1-100 mg/kg of subject.
  • the dosages of the present compounds used to practice the invention include dosages effective to result in the desired effects. Estimation of appropriate dosages effective for the individual patient is well within the skill of the ordinary prescribing physician or other appropriate health care practitioner. As a guide, the clinician can use conventionally available advice from a source such as the Physician's Desk Reference, 48 th Edition, Medical Economics Data Production Co., Montvale, New Jersey 07645-1742 (1994) .
  • compositions are presented for administration in unit dosage forms.
  • unit dosage form refers to physically discrete units suitable as unitary dosed for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce a desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle.
  • unit dosage forms include vials, ampules, tablets, caplets, pills, powders, granules, eyedrops, oral or ocular solutions or suspensions, ocular ointments, and oil-in-water emulsions.
  • Means of preparation, formulation and administration are known to those of skill, see generally Remington's Pharmaceutical Science 15 th ed., Mack Publishing Co., Easton, PA (1990) .
  • Novel ZSIG-11 ligand-encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST database for initially identified by querying an expressed sequence tag (EST) database for secretory signal sequences characterized by an upstream methionine start site, a hydrophobic region of approximately 13 amino acids and a cleavage site, in an effort to select for secreted proteins.
  • EST expressed sequence tag
  • the template was sequenced on an Applied BiosystemsTM model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Ct . ) using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.) according to the manufacturer's instructions. Oligonucleotides ZC976 (SEQ ID NO: 7) and ZC447 (SEQ ID NO : 8 ) promoters on the vector were used as sequencing primers.
  • Oligonucleotides ZC11300 (SEQ ID NO:), ZC11796 (SEQ ID NO : 9 ) , ZC11797 (SEQ ID NO:10), ZC11873 (SEQ ID NO:ll), ZC11874 (SEQ ID NO:12), ZC13966 (SEQ ID NO:13) and ZC13967 (SEQ ID NO:14) were used to complete the sequence from the clone. Sequencing reactions were carried out in a Hybaid OmniGene Temperature Cycling System (National Labnet Co., Woodbridge, NY) . SequencherTM 3.0 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI) was used for data analysis. The resulting 1,341 bp sequence is disclosed in SEQ ID NO : 1.
  • MTN I Human Multiple Tissue Northern Blots
  • a 30 bp DNA probe (ZC11,300; SEQ. ID. NO .3 ) derived from the published sequence for LIN461779.R, which is located just 3 ' of the signal sequence cleavage site at the 5' end of the mature ZSIG- 11 polypeptide as represented in SEQ. ID.
  • T4 polynucleotide kinase and forward reaction buffer (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's specifications.
  • the probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA) .
  • ExpressHybTM (Clontech) solution was used for prehybridization and as a hybridizing solution for the o
  • Northern blots Hybridization took place overnight at 42 C using 2 x 10 cpm/ml of labeled probe, and the blots were then washed at 70°C in IX SSC, 0.1% SDS.
  • a 1.8 kb transcript was detected in numerous tissues. The highest level was in testis, with high levels in prostate, thyroid and heart, and lesser levels in skeletal muscle, pancreas, small intestine, peripheral blood lymphocytes, brain, placenta, liver, kidney, liver, thymus, ovary, colon, spinal cord, trachea and adrenal gland.
  • RNA was blotted overnight onto a nytran filter (Schleicher & Schuell, Keene, NH) , and the filter was UN crosslinked (1,200 ⁇ Joules) in a STRATALI ⁇ KER® UV crosslinker (Stratagene Cloning Systems) . Hybridization took place overnight at 55°C using 2.2 x 10 cpm/ml of labeled probe. The blots were then washed at 50°C in 0. IX SSC, 0.1% SDS and exposed to film for two days. Three transcript signals (5kb, 2kb and 1.5kb) were detected corresponding to ZSIG-11 were detected in all four bone tissues.
  • ZSIG- 11 was mapped to chromosome 20 using the commercially available GeneBridge 4 Radiation Hybrid Panel
  • a publicly available WWW server http : //www-genome . wi .mit . edu/cgi-bin/contig/ rhmapper.pl
  • WICGR Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome
  • KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH 2 0 for a total volume of 20 ⁇ l .
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 54°C and 1.5 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 3% NuSieve GTG agarose gel (FMC Bioproducts, Rockland, ME) .
  • ZSIG-11 maps 252.51 cR_3000 from the top of the human chromosome 20 linkage group on the WICGR radiation hybrid map.
  • Proximal and distal framework markers were D20S908 and D20S99, respectively. This positions ZSIG-11 in the 20ql3.12 region on the integrated LDB chromosome 20 map (The Genetic Location Database, University of Southhampton, WWW server :http : //cedar .genetics . soton. ac .uk/public html/) .
  • ZSIG-11 polypeptide ZSIG-llCF/pZP9 and ZSIG-llNF/pZP9 , wherein the constructs are designed to express a ZSIG-11 polypeptide with a C- or N-terminal FLAG tag (SEQ ID NO: 15) .
  • a 947 bp PCR generated ZSIG-11 DNA fragment was created using ZC13450 (SEQ ID NO: 28) and ZC13445 (SEQ ID NO: 29) as PCR primers and the template described in Example 1 above.
  • the PCR reaction was incubated at 94°C for 5 minutes, and then run for 10 cycles of 30 seconds at 94°C and 2 minutes at 75°C, followed by 15 cycles at 94°C o for 30 seconds and 60 C for 2 minutes.
  • the resultant PCR product was then run on a 0.9% GTG/TBE agarose gel with lx TBE buffer.
  • the DNA was digested with the restriction enzymes Xho I and Bam HI, followed by extraction and precipitated.
  • the excised DNA was subcloned into plasmid CF/pZP9 which had been cut with Xho I and Bam HI .
  • the ZSIG-ll/CFpZP9 expression vector uses the native ZSIG-11 signal peptide, and the FLAG epitope (SEQ ID NO: 15) is attached at the C-terminus as a purification aid.
  • Plasmid CF/pZP9 (deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, multiple restriction sites for insertion of coding sequences, a sequence encoding the flag peptide (SEQ ID NO: 15) , a stop codon and a human growth hormone terminator.
  • the plasmid also has an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.
  • a 873 bp PCR generated ZSIG- 11 DNA fragment was created in accordance with the procedure set forth above using ZC13440 (SEQ ID NO:16) and ZC13439 (SEQ ID NO:17) as PCR primers .
  • the purified PCR fragment was digested with the restriction enzymes BAM HI and Xho I, followed by extraction and precipitation.
  • the excised ZSIG- 11 DNA was subcloned into plasmid NF/pZP9 which had been cut with Bam HI and Xho I .
  • the ZSIG-ll/NFpZP9 expression vector incorporates the TPA leader and attaches the FLAG epitope (SEQ ID NO: 15) to the N-terminal of the ZSIG-11 polypeptide-encoding polynucleotide sequence.
  • Plasmid NF/pZP9 (deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, a TPA leader peptide followed by the sequence encoding the FLAG peptide (SEQ ID NO:15), multiple restriction sites for insertion of coding sequences, and a human growth hormone terminator.
  • the plasmid also contains an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.
  • ZSIG-llCF/pZP9 screens the primers were, ZC13450 (SEQ ID NO: 28) and ZC13445 (SEQ ID NO: 29) and for ZSIG-llNF/pZP9 screens the primers were ZC13440 (SEQ ID NO: 16) and ZC13439 (SEQ ID NO: 17) .
  • the insert sequence of positive clones, 873 bp fragment for ZSIG-11NF and a 947 bp fragment for ZSIG- ll/CF were verified by sequence analysis.
  • a large scale plasmid preparation was done using a QIAGEN * Maxi prep kit
  • T-162 flask containing confluent cells expressing ZSIG-ll/NF, obtained from the expression procedure described above, was expanded into six T-162 flasks. One of the six resulting flasks was used to freeze down four cryovials, and the other five flasks were used to generate a Nunc cell factory.
  • the cells from the five T-165 flasks were used to seed a Nunc cell factory (10 layers, commercially available from VWR) . Briefly, the cells from the T-162 flasks described above containing cells expressing ZSIG- 11-NF were detached using trypsin, pooled, and added to 1.5 liters ESTEP1 media (668.7g/50L DMEM (Gibco) , 5.5 g/50L pyruvic acid, sodium salt 96% (Mallinckrodt) , 185.0 g/50L NaHC0 3 (Mallinkrodt) , 5.0 mg/ml and 25 ml/50L insulin (JRH Biosciences) , 10.0 mg/ml and 25 ml/50L transferrin (JRH Biosciences), 2.5L/50L fetal bovine serum (characterized) (Hyclone) , 1 ⁇ M MTX, with pH adjusted to 7.05 +/- 0.05 ) prewarmed to 37°C.
  • a visual contamination test (phenol red color change) was performed on the Nunc cell factories. Since no contamination was observed, supernatant from the confluent factories was poured into a small harvest container, sampled and discarded. The adherent cells were then washed once with 400 ml PBS. To detach the cells from the factories, 100 mis of trypsin 78
  • BHK 570 cells (ATCC No. CRL-10314) were plated in 10 cm tissue culture dishes and allowed to grow to approximately 50 to 80% confluency overnight at 37°C, 5%
  • DMEM/FBS media DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, MD) , 5% fetal bovine serum (Hyclone, Logan, UT) , 1 ⁇ M L-glutamine (JRH Biosciences, Lenexa, KS) , 1 ⁇ M sodium pyruvate (Gibco BRL) ) .
  • the cells were then transfected with the plasmid ZSIG-llNF/pZP9 (N- terminal FLAG tag) or ZSIG-llCF/pZP9 (C-terminal FLAG tag) , using LipofectamineTM (Gibco BRL) , in serum free (SF) media formulation (DMEM, 10 ⁇ g/ml transferrin, 5 ⁇ g/ml insulin, 10 ⁇ g/ml fetuin, 2 ng/ml selenium, 1% L-glutamine and 1% sodium pyruvate) .
  • DMEM serum free
  • the cells were incubated at 37°C for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to the plate. The plate was incubated at 37°C overnight and the DNA: LipofectamineTM mixture was replaced with fresh FBS/DMEM media the next day. On day 2 post-transfection, the cells were split into the selection media (DMEM/FBS media from above with the addition of 1 ⁇ M methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50. The plates were refed at day 5 post-transfection with fresh selection media.
  • DMEM/FBS media from above with the addition of 1 ⁇ M methotrexate (Sigma Chemical Co., St. Louis, Mo.)
  • the ZSIG-11NF cells were collected following two, 200 ml washes of ESTEP1 media. To each of ten ESTEP1 media- containing bottles (1.5 liters each, at 37°C) was added 40 mis of collected cells. One 1.5 liter bottle was then used to fill one Nunc factory. Each cell factory was placed in a 37°C/5.0% C0 2 incubator.
  • a visual contamination test (phenol red color change) was performed on the Nunc cell factories. Since no contamination was observed, supernatant from the confluent factories were poured into a small harvest container, sampled and discarded. Cells were then washed once with 400 ml PBS. To the factories was added 1.5 liters of ESTEP2 media (668.7g/50L DMEM).
  • the contents of the small harvest containers for each factory were pooled and immediately filtered. A second harvest was then performed, substantially as described above at 50 hours and the cell factories were discarded thereafter.
  • An aseptically assembled filter train apparatus was used for aseptic filtration of the harvest supernatant (conditioned media) . Assembly was a follows: tubing was wire-tied to an Opti-Cap filter (Millipore Corp., Bedford, MA) and a Gelman Supercap 50 filter (Gelman Sciences, Ann Arbor, MI) . The Supercap 50 filter was also attached to a sterile capped container located in a hood; tubing located upstream of the Millipore Opti-cap filter was inserted into a peristaltic pump; and the free end of the tubing was placed in the large harvest container. The peristaltic pump was run between 200 and 300 rpm, until all of the conditioned media passed through the 0.22 ⁇ m final filter into a sterile collection container. The filtrate was placed in a 4°C cold room pending purification.
  • Conditioned media containing ZSIG-11/NF was collected for concentration at various time points (at the 5 T-162 flask stage; 1 factory, fetal bovine serum media; 10 factories, fetal bovine serum media; 10 factories, serum free media and a second 10 factory, serum free media time point) . Since the expected mass of the protein was in excess of 8 kDA, Millipore 5 kDa cut off concentrators were used. The starting volume for each sample was 15 ml, which was concentrated to a final volume of 1.5 ml. The concentrators were spun at 4°C in Beckman tabletop centrifuge at 2000 x g (3000 rpm) for 40 minutes.
  • the concentrate was transferred to a 1.5 ml non-stick microfuge tube, and the volume was adjusted to 1 ml using flow through media to achieve a lOx concentration.
  • the lOx concentrate was split into two Costar Spin-X tubes, and the tubes were spun at 8000 x g for two minutes in a Eppendorf 5415 microfuge (VWR, Seattle, WA) .
  • ZSIG-11 polypeptides in insect cells pSGCFll, designed to express a C-terminally FLAG-tagged ZSIG-11 polypeptide and pSGNFll designed to express a N-terminally FLAG-tagged polypeptide .
  • the restriction digest fragment was run on a 1% SeaPlaque/1% NuSieve agarose gel.
  • the 977 bp band was excised, diluted to 0.5% agarose with 2 mM MgCl 2 , melted at 65°C and ligated into a Eco Rl/Xba I digested baculovirus expression vector, pZBV4L (a modification of the pFastBac expression vector, the polyhedron promoter has been removed and replaced with the late activating Basic Protein Promoter) .
  • the ligated DNA was diluted in 450 ⁇ l of SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KC1, 10 mM MgCl 2 , 10 mM MgS0 4 and 20 mM glucose) and plated onto LB plates containing 100 ⁇ g/ml ampicillin. Clones were analyzed by restriction digests and 1 ⁇ l of the positive clone was transformed into 20 ⁇ l DHlOBac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg,
  • the ligated DNA was diluted in 980 ml SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl , 10 mM MgCl 2 , 10 mM MgS0 4 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.
  • SOC media 2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl , 10 mM MgCl 2 , 10 mM MgS0 4 and 20 mM glucose
  • a color selection was used to identify those cells having virus that had incorporated into the plasmid (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.
  • the restriction digest fragment was run on a 1% SeaPlaque/1% NuSieve agarose gel.
  • the 1028 bp band was excised, diluted to 0.5% agarose with 2 mM MgCl 2 , melted at 65°C and ligated into a Nco I/Xba I digested baculovirus expression vector, pZBV3L (a modification of the pFastBac expression vector, the polyhedron promoter has been removed and replaced with the late activating Basic
  • Sf9 cells were seeded at 5 x 10 s cells per 35 mm plate and allowed to attach for 1 hour at 27°C.
  • Five microliters of bacmid DNA was diluted with 100 ⁇ l Sf-900
  • Sf9 cells were grown in 50 ml Sf-900 II SFM in a 50 ml shake flask to an approximate density of approximately 0.04 x 10 6 cells/ml. They were then transfected with 100 ⁇ l of the virus stock from above and incubated at 27°C for 3 days after which time the virus was harvested, with titers of about 2 x 10 7 pfu/ml . To scale up, Sf9 cells were grown to a density of 1.3 x 10 ⁇ SF9 cells/ml in five liters of SF 900 II SFM, approximately 91 hours. The cells were then transfected with the harvested virus (MOI 0.2) and incubated as above for 47.4 hours post infection.
  • the sample was centrifuged at 10,000 rpm for 30 min at 4°C in a Beckman JLA-10.5 rotor (Beckman Instruments, Palo Alto, CA) in a Beckman Avanti J25I centrifuge (Beckman Instruments) to remove cell debris.
  • a 50.0 ml sample of anti-Flag Sepharose Eastman Kodak, Rochester,
  • the anti-Flag Sepharose gel was washed with 2.0 column volumes of 0.1M glycine, pH 2.5, and the glycine wash was collected separately.
  • the pH of the glycine-eluted fraction was adjusted to 7.0 by the addition of a small volume of 10X PBS and stored at 4°C for future analysis if needed.
  • the peptide elution was concentrated to 5.0 ml using a 5,000 molecular weight cutoff membrane concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions.
  • the concentrated peptide elution was separated from free peptide by chromatography on a 1.5 x 50 cm Sephadex G-50 (Pharmacia, Piscataway, NJ) column equilibrated in PBS at a flow rate of 1.0 ml/min using a BioCad Sprint HPLC (PerSeptive BioSystems, Framingham, MA) . Two-ml fractions were collected and the absorbance at 280 nM was monitored. The first peak of material absorbing at 280 nM and eluting near the void volume of the column was collected. This material represented purified ZSIG-11-NF.
  • the purified ZSIG-11-NF was composed of two major Coomassie blue-stained bands of apparent molecular weights 50,000 and 45,000. Both bands showed cross-reactivity with the anti-Flag antibody on Western blots. The relative abundance of these two bands was about 3 to 1 in favor of the 50 kDa protein. Under non-reducing conditions, the SDS-PAGE and Western blotting analysis showed that the material was highly aggregated and migrated as a smear from about 70,000 Da to the top of the gel.
  • the protein concentration of the purified proteins was performed by BCA analysis (Pierce, Rockford, IL) and the material was aliquoted, and stored at -80°C according to our standard procedures. The concentration of ZSIG-11-NF was 0.24 mg/ml .
  • ZSIG- 11 in Pichia methanolica utilizes the expression system described in co-assigned WIPO publication WO 97/17450.
  • An expression plasmid containing all or part of a polynucleotide encoding ZSIG- 11 was constructed via homologous recombination.
  • the expression vector was built from pCZR190, which contains the AUG1 promoter, followed by the alpha factor prepro ( ⁇ Fpp) leader sequence, followed by an amino-terminal FLAG tag (NF) , a blunt-ended Sma I restriction site for insertion of the gene sequence of interest, a translational stop codon, followed by the AUG1 terminator, the ADE2 selectable marker, and finally the AUG1 3' untranslated region.
  • pCZR190 contains the AUG1 promoter, followed by the alpha factor prepro ( ⁇ Fpp) leader sequence, followed by an amino-terminal FLAG tag (NF) , a blunt-ended Sma I restriction site for insertion
  • the N-terminal linker comprises 70 base pairs of the ⁇ Fpp coding sequence joined to a nucleotide sequence encoding a FLAG tag (SEQ ID NO: 15) followed by 70 base pairs of nucleotide sequence encoding a portion of the amino-terminus from the mature ZSIG- 11 sequence.
  • the C-terminal linker comprises about 70 base pairs of carboxy terminus coding sequence of the ZSIG-11 joined with 70 base pairs of AUG1 terminator sequence .
  • the N-terminal linker was synthesized by a PCR reaction.
  • the PCR reaction was incubated at 94°C for 1.5 minutes, followed by 10 cycles of 30 seconds at 94°C, 1 minute at 50°C and 1 minute at 72°C, concluded with a 10 minute extension at 72°.
  • the resulting 140 bp double stranded, NF-tagged linker is disclosed in SEQ ID NO: 22.
  • the C-terminal untagged ZSIG-11 linker was made via a PCR reaction as described using oligonucleotides linkers ZC14218 (SEQ ID NO:23) and ZC14734 (SEQ ID NO:24) , and primers ZC14273 (SEQ ID NO: 25) and ZC14355 (SEQ ID NO:26).
  • the resulting 140 bp double stranded, C-terminal untagged linker is disclosed in SEQ ID NO: 27.
  • the NF-ZSIG-11 plasmid was made by homologously recombining 100 ng of Sma I digested pCZR190 acceptor vector, the 1 ⁇ g of Eco Rl-Xho I ZSIG-11 cDNA donor fragment, 1 ⁇ g of N-terminal FLAG-tagged ZSIG- 11 linker
  • Ura + yeast transformants from a single plate were resuspended in 2.5 ml H 2 0 and spun briefly to pellet the yeast cells.
  • the cell pellet was resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA) .
  • lysis buffer 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA
  • Five hundred microliters of the lysis mixture was added to an Eppendorf tube containing 300 ⁇ l acid washed glass beads and 200 ⁇ l phenol-chloroform, vortexed for 1 minute intervals two or three times, followed by a 5 minute spin in a Eppendorf centrifuge at maximum speed.
  • DH10B Gibco BRL
  • the cells were electropulsed at 2.0 kV and 400 ohms. Following electroporation, 1 ml SOC (2% BactoTM Tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgS0 4 , 20 mM glucose) was added and the cells were allowed to recover for 1 hour at 37°C prior to plating 250 ⁇ l aliquots on four LB AMP plates (LB broth (Lennox), 1.8% BactoTM Agar (Difco) , 100 mg/L Ampicillin) .
  • SOC 2% BactoTM Tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgS0 4 , 20 mM glucose
  • Transformants were then picked and screened via Western blot for high- level NF-tagged ZSIG-11 expression.
  • the resulting NF- tagged-ZSIG-11 strains were designated PMAD16 : :pGMN18-16 and 21 and subjected to large scale fermentation.
  • Example 10 In vivo Administration of ZSIG-11
  • mice received the final protein injection were bled under ether anesthesia for complete hematology counts and clinical chemistry.
  • the mice were sacrificed by cervical dislocation. Selected organ weights were taken and tissues were harvested for necropsy.

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Abstract

La présente invention concerne des nouveaux polypeptides de ligand, des polynucléotides codant ces polypeptides, des compositions associées, des anticorps et des procédés.
PCT/US1998/020449 1997-09-29 1998-09-29 Proteine zsig-11 secretee WO1999016870A1 (fr)

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WO2001057077A1 (fr) * 2000-02-02 2001-08-09 Smithkline Beecham Biologicals S.A. Proteines specifiquement exprimees ou hautement surexprimees dans des tumeurs et acides nucleiques les codant
WO2003002737A1 (fr) * 2001-06-27 2003-01-09 Riken Nouvelle proteine inhibitrice de la topoisomerase humaine 2$g(a) et utilisation associee

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WO1998039446A2 (fr) * 1997-03-07 1998-09-11 Human Genome Sciences, Inc. 70 proteines humaines secretees

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Publication number Priority date Publication date Assignee Title
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