[go: up one dir, main page]

WO2000031260A1 - Glycoproteine zpep10 a specificite testiculaire - Google Patents

Glycoproteine zpep10 a specificite testiculaire Download PDF

Info

Publication number
WO2000031260A1
WO2000031260A1 PCT/US1999/027223 US9927223W WO0031260A1 WO 2000031260 A1 WO2000031260 A1 WO 2000031260A1 US 9927223 W US9927223 W US 9927223W WO 0031260 A1 WO0031260 A1 WO 0031260A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
polypeptide
seq
sequence
zpeplo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/027223
Other languages
English (en)
Inventor
Paul O. Sheppard
Christopher S. Piddington
Jeff L. Ellsworth
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zymogenetics Inc
Original Assignee
Zymogenetics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zymogenetics Inc filed Critical Zymogenetics Inc
Priority to AU17303/00A priority Critical patent/AU1730300A/en
Priority to CA002369706A priority patent/CA2369706A1/fr
Publication of WO2000031260A1 publication Critical patent/WO2000031260A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the testis is the center for spermatogenesis, the process by which a germ cell proceeds through multiple stages of differentiation, and culminates in the formation of a terminally differentiated cell (spermatozoa or sperm) having a unique function.
  • spermatozoa or sperm a terminally differentiated cell
  • seminiferous tubules where spermatogonium mature into spermatozoa.
  • interstitial cells which secrete androgens, such as testosterone, required for maturation and function of the testis and development of secondary sexual characteristics.
  • Disorders of the testis are common and have profound effect. Infertility can result from disorders occurring during spermatogenesis.
  • Many developmental disorders, such as hypogonadism are associated with altered sex hormone production and levels in the testis.
  • Testicular cancer although rare, is the most common form of cancer in young men between the ages of 15 and 35.
  • Testis specific proteins have therapeutic value in the treatment of disorders associated with the testis such as dysfunctional sperm production, infertility and testicular cancer.
  • the present invention provides novel testis-specific membrane glycoproteins, soluble ligands, agonists and antagonists, related compositions and methods as well as other uses that should be apparent to those skilled in the art from the teachings herein.
  • the figure is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO: 2.
  • the profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.
  • affinity tag is used herein to denote a peptide segment that can be attached to a polypeptide to provide for purification or detection of the polypeptide or provide sites for attachment of the polypeptide to a substrate.
  • Affinity tags include a poly-histidine tract, protein A (Nilsson et al . , EMBO J. 4:1075, 1985; Nilsson et al . , Methods Enzymol. 198:3, 1991) , glutathione S transferase (Smith and Johnson, Gene 6 :31, 1988), Glu-Glu affinity tag (Grussenmeyer et al .
  • allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • araino-terminal and “carboxyl- terminal” are used herein to denote positions within polypeptides and proteins. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a protein is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete protein .
  • 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' .
  • contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'- ATGGCTTAGCTT-3' are 5' -TAGCTTgagtct-3' and 3'- gtcgacTACCGA-5' .
  • degenerate nucleotide sequence 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 denotes 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 may optionally include 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 n 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 Tiian, Nature 316:774-78, 1985) .
  • an “isolated” polypeptide or protein is a polypeptide or protein that is found n 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.
  • DNA segments denotes 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.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • 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 vitro, 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”.
  • RNA or 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 .
  • a “protein” is a macromolecule comprising one or more polypeptide chains .
  • a protein may also comprise non- peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • a bioactive molecule i.e., a ligand
  • Membrane-bound receptors are characterized by a multi- domain structure comprising an extracellular ligand- binding domain and an intracellular effector domain that is typically involved in signal transduction . Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
  • Most nuclear receptors also exhibit a multi-domain structure, including an amino-terminal, transactivating domain, a DNA binding domain and a ligand binding domain.
  • 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) .
  • monomeric e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor
  • 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 denotes 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.
  • secretory peptide a polypeptide 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 peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • soluble receptor is used herein to refer to a receptor 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 receptors 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 have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Receptor polypeptides are said 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 invention provides an isolated polypeptide comprising an extracellular domain, wherein the extracellular domain comprises amino acid residues 22 to 111 of the amino acid sequence of SEQ ID NO : 2.
  • polypeptide further comprises a transmembrane domain that resides in a carboxyl-terminal position relative to the extracellular domain, wherein the transmembrane domain comprises amino acid residues 112 to 133 of the amino acid sequence of SEQ ID NO: 2.
  • polypeptide further comprises a cytoplasmic domain that resides in a carboxyl-terminal position relative to the transmembrane domain, wherein the cytoplasmic domain comprises amino acid residues 134 to 142 of the amino acid sequence of SEQ ID NO: 2.
  • polypeptide further comprises a secretory signal that resides in an amino-terminal position relative to the extracellular domain, wherein the secretory signal sequence comprises amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO: 2.
  • the invention also provides an isolated polypeptide as described herein comprising amino acid residue 1 to amino acid residue 142 of SEQ ID NO: 2. Also provided is an isolated polypeptide as described herein, covalently linked amino terminally or carboxy terminally to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores . Within another aspect the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical to a amino acid residue 21 to amino acid residue 142 of SEQ ID NO:2, wherein the polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • any difference between the amino acid sequence of the isolated polypeptide and the corresponding amino acid sequence of SEQ ID NO : 2 is due to a conservative amino acid substitution.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of amino acid residue 1 to amino acid residue 20 of SEQ ID NO : 2.
  • an isolated polypeptide selected from the group consisting of: a) amino acid residues 21-111 of SEQ ID NO:2; b) amino acid residues 112-133 of SEQ ID NO: 2; c) amino acid residues 134-142 of SEQ ID NO : 2 ; d) amino acid residues 1-20 of SEQ ID NO:2; e) amino acid residues 21-133 of SEQ ID NO:2; f) amino acid residues 112-142 of SEQ ID NO : 2 ; g) amino acid residues 1-111 of SEQ ID NO:2; and h) amino acid residues 1-133 of SEQ ID NO : 2.
  • the invention provides a fusion protein consisting of a first portion and a second portion joined by a peptide bond, the first portion comprising a polypeptide as described herein, and the second portion comprising another polypeptide.
  • the invention also provides a polypeptide as described herein in combination with a pharmaceutically acceptable vehicle.
  • the invention provides an antibody that specifically binds to an epitope of a polypeptide of as described herein.
  • 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. Also provides is an anti-idiotype antibody that specifically binds to an antibody as described herein. Also provided is a binding protein that specifically binds to an epitope of a polypeptide as described herein.
  • the invention provides a method of producing an antibody to a polypeptide comprising: inoculating an animal with a polypeptide as described herein; wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal .
  • an isolated polynucleotide encoding a polypeptide comprising an extracellular domain, wherein the extracellular domain comprises amino acid residues 22 to 111 of the amino acid sequence of SEQ ID NO : 2.
  • the polypeptide further comprises a transmembrane domain that resides in a carboxyl -terminal position relative to the extracellular domain, wherein the transmembrane domain comprises amino acid residues 112 to 133 of the amino acid sequence of SEQ ID NO : 2.
  • polypeptide further comprises a cytoplasmic domain that resides in a carboxyl-terminal position relative to the transmembrane domain, wherein the cytoplasmic domain comprises amino acid residues 134 to 142 of the amino acid sequence of SEQ ID NO : 2.
  • polypeptide further comprises a secretory signal that resides in an amino-terminal position relative to the extracellular domain, wherein the secretory signal sequence comprises amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO : 2.
  • the invention also provides an isolated polynucleotide as described herein encoding a polypeptide comprising amino acid residue 1 to amino acid residue 142 of SEQ ID NO:2.
  • polypeptide is covalently linked amino terminally or carboxy terminally to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores .
  • the invention provides an isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to a amino acid residue 21 to amino acid residue 142 of SEQ ID NO : 2 , wherein the polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • any difference between the amino acid sequence of the isolated polypeptide and the corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative amino acid substitution.
  • the invention also provides an isolated polynucleotide selected from the group consisting of: a) a sequence of nucleotides from nucleotide 139 to nucleotide 411 of SEQ ID NO:l; b) a sequence of nucleotides from nucleotide
  • nucleotide 477 of SEQ ID NO:l c) a sequence of nucleotides from nucleotide 139 to nucleotide 504 of SEQ ID NO:l; d) a sequence of nucleotides from nucleotide 79 to nucleotide 504 of SEQ ID NO:l; e) a sequence of nucleotides from nucleotide 1 to nucleotide 1094 of SEQ ID N0:1; f) a polynucleotide that remains hybridized following stringent wash conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:l, or the complement of SEQ ID NO : 1 ; and g) nucleotide sequences complementary to a) , b) , c) , d) , e) , or f .
  • an isolated polynucleotide encoding a fusion protein consisting of a first portion and a second portion joined by a peptide bond, the first portion comprises a polypeptide as described herein; and the second portion comprising another polypeptide.
  • the invention also provides an isolated polynucleotide comprising the sequence of nucleotide 1 to nucleotide 426 of SEQ ID NO: 3.
  • an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described herein; and a transcription terminator.
  • DNA segment encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag.
  • DNA segment further encodes a secretory signal sequence operably linked to the polypeptide.
  • secretory signal sequence comprises residues 1 to 20 of SEQ ID NO:2.
  • the invention also provides a cultured cell into which has been introduced an expression vector as described herein; wherein the cell expresses the polypeptide encoded by the DNA segment.
  • the invention also provides a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector as described herein; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.
  • the present invention is based in part upon the discovery of a novel DNA sequence (SEQ ID NO:l) and the corresponding deduced polypeptide sequence (SEQ ID NO:2) which encode a testis-specific polypeptide designated zpeplO.
  • the novel zpeplO polypeptide-encoding polynucleotides of the present invention were initially identified by querying an EST database for polypeptides containing repetitive patterns and post-translational processing sites yielding potentially active peptides.
  • the polypeptide corresponding to an EST meeting those search criteria was further analyzed and found to be a membrane glycoprotein .
  • the EST sequence was from a testis cell library.
  • SEQ ID NO:l Analysis of the DNA encoding a zpeplO polypeptide (SEQ ID NO:l) revealed an open reading frame encoding 142 amino acids (SEQ ID NO:2) comprising a putative signal sequence (residues 1 to 20 of SEQ ID NO : 2 , nucleotides 79 to 138 of SEQ ID NO:l) and 122 amino acids of predicted mature sequence (residues 21 to 142 of SEQ ID NO:2, nucleotides 139 to 504 of SEQ ID NO : 1 ) containing an extracellular domain (residues 21 to 111 of SEQ ID NO:2, nucleotides 139 to 411 of SEQ ID NO:l) containing six cysteine residues, amino acid residues 35, 45, 84, 87, 94 and 100 of SEQ ID NO:2, a tri-basic amino acid cleavage site, amino acid residues 97-99 of SEQ ID NO : 2 ; potential N
  • ZpeplO polypeptides may be processed into an active form through cleavage after lys (amino acid residue 98 of SEQ ID NO:2) or arg (amino acid residue 99 of SEQ ID NO:2) of the tribasic site arg-lys- arg (amino acid residues 97-99 of SEQ ID NO:2).
  • Prohormone convertase PC4 exhibits highly specific testis expression (WIPO publication, WO98/50560) and may serve to cleave the zpeplO polypeptide.
  • the present invention therefore provides post- translationally modified polypeptides or polypeptide fragments having the amino acid sequence from amino acid residue 21 to amino acid residue 98 of SEQ ID NO: 2 and the amino acid sequence from amino acid residue 21 to amino acid residue 99 of SEQ ID NO: 2.
  • post translational modifications include proteolytic cleavage, glycosylation and disulfide bonding.
  • Analysis of the tissue distribution of the mRNA corresponding to this novel DNA by Northern blot and Dot blot analysis suggest that zpeplO is a testis-specific protein having a transcript of about 1.5 kb .
  • the present invention further provides polynucleotide molecules, including DNA and RNA molecules, encoding zpeplO proteins.
  • the polynucleotides of the present invention include the sense strand; the anti-sense strand; and the DNA as double-stranded, having both the sense and anti-sense strand annealed together by their respective hydrogen bonds.
  • Representative DNA sequences encoding zpeplO proteins are set forth in SEQ ID NO:l. DNA sequences encoding other zpeplO proteins can be readily generated by those of ordinary skill in the art based on the genetic code. Counterpart RNA sequences can be generated by substitution of U for T.
  • SEQ ID NO: 3 is a degenerate DNA sequence that encompasses all DNAs that encode the zpeplO polypeptide of SEQ ID NO: 2.
  • the degenerate sequence of SEQ ID NO: 3 also provides all RNA sequences encoding SEQ ID NO: 2 by substituting U for T.
  • zpeplO polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 426 of SEQ ID NO: 3 and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one- letter codes used within SEQ ID NO : 3 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter.
  • “Complement” indicates the code for the complementary nucleotide (s ) .
  • the code Y denotes either C or T
  • its complement R denotes A or G
  • A being complementary to T
  • G being complementary to C.
  • degenerate codons used in SEQ ID NO: 3, encompassing all possible codons for a given amino acid, are set forth in Taule 2.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR) , and the degenerate codon for arginine
  • MGN can, in some circumstances, encode serine (AGY) .
  • some polynucleotides encompassed by the degenerate seguence 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.
  • Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) .
  • the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, 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 .
  • 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
  • T m of the hybrid about 1°C for each 1% formamide in the buffer solution.
  • stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6X SSC and 0-50% formamide.
  • a higher degree of stringency can be achieved at temperatures of from 40-70°C with a hybridization buffer having up to 4X SSC and from 0-50% formamide.
  • Highly stringent conditions typically encompass temperatures of 42-70°C with a hybridization buffer having up to IX SSC and 0-50% formamide.
  • Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
  • the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes.
  • 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 which 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 (publicly available shareware) and Primer Premier (PREMIER Biosoft International, Palo Alto, CA) 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. Typically, hybridization of longer polynucleotide sequences, >50 bp, is done at temperatures of about 20-
  • 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 . 7-deazz- 2' -deoxyguanosine can be substituted for guanosine to reduce dependence on T m .
  • Hybridization buffers generally contain blocking agents such as Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, tRNA, 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).
  • SSC IX SSC: 0.15 M NaCl, 15 mM sodium citrate
  • SSPE IX SSPE: 1.8 M NaCl, 10 mM NaH 2 P0 4 , 1 mM EDTA, pH 7.7
  • hybridization buffers typically contain from between 10 ⁇ ruM-1 M Na + .
  • Premixed hybridization solutions are also available from commercial sources such as Clontech Laboratories (Palo Alto, CA) and Promega Corporation (Madison, WI) 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 m of a hybrid.
  • 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 zpeplO 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 lymph node, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • Total RNA can be prepared using guanidine HC1 extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry l_8:52-94, 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 zpeplO polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • the polynucleotides of the present invention can also be synthesized using automated equipment.
  • the current method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • synthetic genes double-stranded
  • Gene synthesis methods are well known in the art. See, for example, Glick and
  • the zpeplO polynucleotide sequences disclosed herein can be used to isolate polynucleotides encoding other zpeplO proteins.
  • Such other proteins include alternatively spliced cDNAs (including cDNAs encoding secreted zpeplO proteins) and counterpart polynucleotides from other species (orthologs) .
  • These orthologous polynucleotides can be used, inter alia , to prepare the respective orthologous proteins.
  • Other species of interest include, but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species.
  • zpeplO polynucleotides and proteins from other mammalian species, including human and other primates, porcine, ovine, bovine, canine, feline, and equine polynucleotides and proteins.
  • Orthologs of mouse zpeplO 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 zpeplO as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a zpeplO- 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 sequences.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR
  • 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 zpeplO polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Electronic databases can also be screened for EST sequences of zpeplO orthologs. Degenerate polynucleotide primer sequences useful for identifying zpeplO orthologs would include: zpeplO residues 15-20 of SEQ ID NO:2
  • ATGAAYAARGRNACNGA (SEQ ID NO: 6) zpeplO residues 64-69 of SEQ ID NO:2
  • GRNACNGARAARACNCA (SEQ ID NO : 7 ) zpeplO residues 86-91 of SEQ ID NO:2
  • ACNTGYAARGGNACNGA (SEQ ID NO: 8) .
  • allelic variants can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. 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.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zpeplO polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the present invention also provides isolated zpeplO polypeptides that are substantially homologous to the polypeptide of SEQ ID NO : 2 and its species orthologs.
  • substantially homologous is used herein to denote polypeptides having 60%, preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO: 2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2 or its orthologs. 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.
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zpeplO .
  • the FASTA algorithm is described by Pearson and Lipman, Proc . Nat. Acad. Sci. USA 85 . : 2444, 1988, and by Pearson, Meth. Enzymol . 183 :63 , 1990.
  • the ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3) .
  • Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zpeplO polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • Aromatic phenylalanme tryptophan tyros e
  • the proteins of the present invention can also comprise non-naturally occurring ammo acid residues.
  • Non-naturally occurring ammo acids include, without limitation, trans-3-methylprolme, 2 , 4-methanoprolme, c ⁇ s-4-hydroxyprolme, trans-4-hydroxyprolme, N-methyl- glyc e, alJo-threonme, methylthreonme, hydroxyethyl- cyste e, hydroxyethylhomocysteme, nitroglutamme, omo- glutamme, pipecolic acid, thiazolidme carboxylic acid, dehydroprol e, 3- and 4-methylprolme, 3, 3-d ⁇ methyl- prol e, tert-leuc e, norvalme, 2-azaphenyl-alanme, 3- azaphe ⁇ ylalanme, 4-azaphenylalanme, and 4-fluorophenyl- alan e.
  • an m vi tro system can be employed wherein nonsense mutations are suppressed using chemically ammoacylated 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 . col i 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 .
  • 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) .
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 3_3:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zpeplO amino acid residues .
  • Sites of ligand-receptor or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al . , Science 255 : 306- 12, 1992; Smith et al., J. Mol . Bioi. 224_: 899-904 , 1992; Wlodaver et al . , FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related proteins.
  • Amino acid residues that might be considered essential in the zpeplO polypeptide are cysteine residues at amino acid residues 17, 20, 35, 45, 84, 87, 94 and 100 of SEQ ID NO: 2; the potential arg-lys-arg tri-basic amino acid cleavage site at amino acid residues 97-99 of SEQ ID NO:2; the potential N-linked glycosylation sites at amino acid residues 83 and 86 of SEQ ID NO : 2 and the potential O- glycosylation sites at amino acid residues 28, 36, 48, 52, 60, 65, 68, 78, 79, 80, 85, 86, 90, 93 and 104 of SEQ ID NO: 2.
  • a hydrophobicity profile of SEQ ID NO: 2 is shown in the attached figure. Those skilled in the art will recognize that this hydrophobicity will be taken into account when designing alterations in the amino acid sequence of a zpeplO polypeptide, so as not to disrupt the overall profile.
  • variants of the disclosed zpeplO 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., ligand binding receptors
  • 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, for example, residues 21 to 111, 21 to 142 or 1 to 142 of SEQ ID NO:2 or allelic variants thereof and retain the properties of wild-type protein.
  • polypeptides may include additional amino acids, such as affinity tags and the like.
  • polypeptides may also include additional polypeptide segments as generally disclosed herein.
  • the invention also provides soluble polypeptides. It is preferred that these soluble polypeptides be extracellular polypeptides and be in a form substantially free of transmembrane and intracellular polypeptide segments.
  • the DNA encoding the soluble polypeptide is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide or the native zpeplO secretory signal sequence
  • an N- or C- terminal extension such as an affinity tag or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the soluble polypeptide.
  • the present invention also provides zpeplO fusion proteins.
  • fusion proteins of the present invention encompass
  • polypeptide selected from the following: a) a polypeptide comprising a sequence of amino acid residues from amino acid residue 21 to amino acid residue 111 of SEQ ID NO: 2; and b) a polypeptide comprising a sequence of amino acid residues from amino acid residue 1 to amino acid residue 20 of SEQ ID NO: 2; and
  • the other polypeptide may be a signal peptide to facilitate secretion of the fusion protein, a transmembrane and/or cytoplasmic domain, or another soluble polypeptide or the like.
  • the extracellular portion of a zpeplO polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
  • Immunoglobulin-zpeplO polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zpeplO analogs.
  • Auxiliary domains can be fused to zpeplO polypeptides to target them to specific cells, tissues, or macromolecules .
  • a soluble zpeplO polypeptide or protein could be targeted to a predetermined cell type by fusing a zpeplO polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • polypeptides and proteins can be targeted for therapeutic or diagnostic purposes .
  • a zpeplO polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al . , Connective Tissue Research 3_4:l-9, 1996.
  • the soluble zpeplO polypeptide is useful in studying the distribution of zpeplO receptors on tissues or specific cell lineages, and to provide insight into receptor/ligand biology.
  • cells expressing the ligand are identified by fluorescence immunocytometry or immunohistochemistry .
  • the effects of zpeplO on steroidogenesis or Leydig or Sertoli cell expression can be examined by probing tissue slices with soluble zpeplO fusions, see for example, Daehlin et al . , Scand. J. Urol . Nephrol . 19:7-12, 1985; Gavino et al.,
  • the 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, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al .
  • a DNA sequence encoding a zpeplO polypeptide of the present invention 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 signal sequence, leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the zpeplO 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 zpeplO DNA sequence in the correct reading frame and positioned to direct newly synthesized polypeptide into secretory pathways to host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory 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 1-20 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.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor.
  • a normally non-secreted protein such as a receptor.
  • Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 1_4:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 2:603, 1981: Graham and Van der Eb, Virology 52 :456, 1973), electroporation (Neumann et al . , EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., eds .
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
  • Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus . See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein 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
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci. (Banqalorej l_l:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV) .
  • AcNPV Autographa calif ornica nuclear polyhedrosis virus
  • DNA encoding the zpeplO polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods.
  • the first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the zpeplO flanked by AcNPV sequences.
  • Suitable insect cells e.g. SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a zpeplO polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow et al . (J. Virol. 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD) . This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zpeplO polypeptide into a baculovirus genome maintained in E .
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zpeplO.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins and Possee, J. Gen. Virol. 7_l:971-6, 1990; Bonning et al . , J. Gen.
  • transfer vectors can be constructed which replace the native zpeplO secretory signal sequences with secretory signal sequences derived from insect proteins .
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT) , honey bee Melittin (Invitrogen, Carlsbad, CA) , or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native secretory signal sequence.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zpeplO polypeptide, for example, a Glu-Glu epitope tag
  • a transfer vector containing zpeplO 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 zpeplO 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 Trichopl usia 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
  • the recombinant virus-infected cells typically produce the recombinant zpeplO polypeptide at 12-72 hours post- infection and secrete it with varying efficiency into the medium.
  • the culture is usually harvested 48 hours post- infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the zpeplO polypeptide is filtered through micropore filters, usually 0.45 ⁇ m pore size. Procedures used are generally described in available laboratory manuals (King and Possee, ibid. ; O'Reilly et al . , ibid . ; Richardson, C. D., ibid. ) . Subsequent purification of the zpeplO polypeptide from the supernatant can be achieved using methods described herein.
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanol ica .
  • Methods for transforming S . cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S.
  • 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 Sa ccharomyces 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. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No.
  • Kl uyveromyces lactis , Kl uyveromyces fragilis , Ustilago maydis , Pichia pastoris , Pichia methanol ica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol . 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
  • 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. methanol ica 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. methanol ica 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.
  • host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes ( AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes ( PEP4 and PRB1 ) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. 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 (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia , Bacill us 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 zpeplO polypeptide in bacteria such as E. col i , 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.
  • P. methanolica cells are cultured m a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors .
  • a preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenme and 0.006% L-leucme).
  • ZpeplO polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • ZpeplO polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine ammo acid residue.
  • Expressed recombinant zpeplO 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 de ⁇ vatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like.
  • DEAE Fast-Flow Sepharose Pharmacia, Piscataway, NJ
  • PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resms, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl-Sepharose FF Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like
  • polyacrylic resms such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity
  • the zpeplO polypeptides of the present invention can be isolated by exploitation of their structural features.
  • a fusion of the polypeptide of interest and an affinity tag e.g., polyhistidine, Glu-Glu, FLAG, 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.
  • a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
  • Proteins/polypeptides which bind zpeplO can also be used for purification of zpeplO.
  • the zpeplO- binding protein/polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids containing zpeplO polypeptide are passed through the column one or more times to allow zpeplO polypeptide to bind to the ligand-binding or receptor polypeptide.
  • the bound zpeplO polypeptide is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to disrupt ligand-receptor binding.
  • soluble zpeplO transfected expression host cells may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine micro- encapsulation, permselective membrane encapsulation and diffusion chambers have been described as a means to entrap transfected mammalian cells or primary mammalian cells.
  • 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. Most importantly, the capsules or microenvironments mask and shield the foreign, embedded cells from the recipient animal's immune response. Such microenvironments can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Algmate threads provide a simple and quick means for generating embedded cells. The materials needed to generate the algmate threads are readily available and relatively inexpensive.
  • the algmate threads are relatively strong and durable, both in vi tro and, based on data obtained using the threads, in vivo .
  • the algmate threads are easily manipulable and the methodology is scalable for preparation of numerous threads.
  • 3% algmate 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 mtrape ⁇ toneally injected into a recipient m 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 Becker et al . , Meth. Cell Bioi. 43:161-89, 1994; and Douglas and 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 (iii) the host immune response to the administered virus, precluding readministration of the adenoviral vector.
  • adenovirus By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can 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 (the human 293 cell line is exemplary) .
  • the host cell the human 293 cell line is exemplary
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells.
  • the host's tissue e.g., liver
  • the host's tissue will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein.
  • 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 vitro .
  • the cells 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. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al . , Cytotechnol . l_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.
  • 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 . Bioi. 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.
  • complement/anti-complement pair denotes non- identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 9 M _1 .
  • ZpeplO polypeptide and other ligand homologs can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al . , Science 253 : 545-8 , 1991; Cunningham et al . , Science 245:821-5, 1991) .
  • the invention also provides anti-zpeplO antibodies.
  • Antibodies to zpeplO can be obtained, for example, using as an antigen the product of a zpeplO expression vector, or zpeplO isolated from a natural source.
  • Particularly useful anti-zpeplO antibodies "bind specifically" with zpeplO.
  • Antibodies are considered to be specifically binding if the antibodies bind to a zpeplO polypeptide, peptide or epitope with a binding affinity (K a ) of 10 M or greater, preferably 10 M or greater, o _ 1 more preferably 10 M or greater, and most preferably 10 9 M-1 or greater.
  • Suitable antibodies include antibodies that bind with zpeplO, in particular the extracellular domain of zpeplO (amino acid residues 21-111 of SEQ ID NO:2) .
  • Anti-zpeplO antibodies can be produced using antigenic zpeplO epitope-bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within SEQ ID NO: 2.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with zpeplO.
  • amino acid sequence of the epitope- bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided) .
  • the hydrophobicity plot provided in the Figure provides such information. Using the plot antigenic regions can be selected, such as those found in the fragments, amino acid residue 39-44, 65-70, 38-43, 62-67 and 96-101 of SEQ ID
  • amino acid sequences containing proline residues may be also be desirable for antibody production.
  • Polyclonal antibodies to recombinant zpeplO protein or to zpeplO isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al., "Production of Polyclonal Antisera, " in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al . , "Expression of foreign proteins in E . coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al . (eds.), page 15 (Oxford University Press 1995) .
  • the immunogenicity of a zpeplO polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zpeplO 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
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, hamsters, guinea pigs, goats or sheep
  • an anti-zpeplO antibody of the present invention may also be derived from a subhuman primate antibody.
  • Antibodies can also be raised in transgenic animals such as transgenic sheep, cows, goats or pigs, and may be expressed in yeast and fungi in modified forms as will as in mammalian and insect cells.
  • monoclonal anti-zpeplO antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al . , Na ture 256: 49b (1975), Coligan et al . (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991), Picksley et al . , "Production of monoclonal antibodies against proteins expressed in E . col i , " in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al . (eds.), page 93 (Oxford University Press 1995) ) .
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising a zpeplO gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures .
  • an anti-zpeplO antibody of the present invention may be derived from a human monoclonal antibody.
  • Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al . , Nat . Genet . 7:13, 1994, Lonberg et al . , Nature 368:856, 1994, and Taylor et al . , Int. lmmun. 6:579, 1994.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size- exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al . , "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). For particular uses, it may be desirable to prepare fragments of anti-zpeplO antibodies. 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.
  • 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.
  • Fv fragments comprise an association of V H and V L chains. This association can be noncovalent, as described by Inbar et al . , Proc . Nat ' 1 Acad. Sci. USA 69:2659, 1972.
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as gluteraldehyde (see, for example, Sandhu, Crit . Rev. Biotech. 1_2:437, 1992).
  • the Fv fragments may comprise V H and V chains which are connected by a peptide linker.
  • scFv single- chain antigen binding proteins
  • scFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E . coli .
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing scFvs are described, for example, by Whitlow et al . , Methods: A Companion to Methods in Enzymology 2:97, 1991, also see, Bird et al . , Science 242 :423, 1988, Ladner et al., U.S. Patent No. 4,946,778, Pack et al . , Bio/Technology 1L:1271, 1993, and Sandhu, supra .
  • a scFV can be obtained by exposing lymphocytes to zpeplO polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zpeplO protein or peptide) .
  • Genes encoding polypeptides having potential zpeplO polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , U.S. Patent No. 5,223,409, Ladner et al . , U.S. Patent No. 4,946,778, Ladner et al . , U.S. Patent No. 5,403,484, Ladner et al . , U.S. Patent No. 5,571,698, and Kay et al .
  • Random peptide display libraries can be screened using the zpeplO sequences disclosed herein to identify proteins which bind to zpeplO.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody- producing cells (see, for example, Larrick et al . , Methods: A Companion to Methods in Enzymology 2:106, 1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal
  • an anti-zpeplO antibody may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
  • General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al . , Proc. Natl. Acad. Sci. USA 8j5:3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986, Carter et al . , Proc. Natl .
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-zpeplO antibodies or antibody fragments, using standard techniques. See, for example, Green et al . , "Production of Polyclonal
  • Antisera " in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed. ) , pages 1-12 (Humana Press 1992) . Also, see Coligan, ibid. at pages 2.4.1-2.4.7.
  • monoclonal anti-idiotype antibodies can be prepared using anti-zpeplO antibodies or antibody fragments as immunogens with the techniques, described above.
  • humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques. Methods for producing anti-idiotype antibodies are described, for example, by Irie, U.S. Patent No. 5,208,146, Greene, et. al . , U.S. Patent No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol. 77:1875, 1996.
  • Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance) .
  • zpeplO polypeptides or anti-zpeplO antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like) , as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance) .
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adria ycin.
  • cytotoxic drugs such as adria ycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • Soluble zpeplO polypeptides or antibodies to zpeplO can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance) .
  • zpeplO polypeptides or anti-zpeplO antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.
  • Suitable detectable molecules can be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules can be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like) , as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance) .
  • Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues) .
  • a fusion protein including only the targeting domain can be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
  • the anti- complementary molecule can be conjugated to a detectable or cytotoxic molecule.
  • Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti- complementary-detectable/cytotoxic molecule conjugates.
  • the bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.
  • the zpeplO gene is almost exclusively expressed in the testis. Low levels of transcript are also seen in a number of other tissues, with the kidney accounting for most of the ancillary expression.
  • the tissue specificity observed for zpeplO suggests a general role in development and regulatory control of testicular differentiation and gonadal steroidogenesis and spermatogenesis.
  • ZpeplO polypeptides, agonists and antagonists have enormous potential in both in vi tro and in vivo applications.
  • testicular hormone production can be divided into early and late steps, with the latter dependent on the activation of functionally-determined Leydig cell precursors by LH .
  • the factors that control the early steps in this process remain unknown (Huhtaniemi, Reprod. Fertil . Dev. 7: 1025-35, 1995) suggesting that testis specific polypeptides such as zpeplO might be responsible for activation of a non- steroidogenic, non-LH responsive precursor cell.
  • testicular products have been shown to control LH and FSH production and in turn, these products regulate, testicular function, this suggests a regulatory role for zpeplO in hormone production by the hypothalamic, pituitary, gonadal axis. It is well known that steroidogenesis and spermatogenesis take place within two different cellular compartments of the testes, with Leydig and Sertoli cells responsible for the former and latter, respectively (Saez, Endocrin. Rev. 15: 574-626, 1994) . The activity of each of these cell types appears to be regulated by the secretory products of the other.
  • Sertoli cell derived tumor necrosis factor-a, fibroblast growth factor, interleukin-1 transforming growth factor- ⁇ , epidermal growth factor/transforming growth factor- ⁇ , activin, inhibin, insulin-like growth factor-1, platelet derived growth factor, endothelin, and ariginine-vasopressin have all been shown to regulate Leydig cell function (Saez, Endocrin. Rev. 15: 574-626, 1994) .
  • zpeplO might control or modulate the activities of one or more of these genes.
  • the membrane glycoprotein zpeplO may also function as a binding site for one or more growth factor peptides or hormones in much the same way that heparin binds with platelet-derived growth factor (PDGF), fibroblast growth factors (such as aFGF and bFGF) and vascular endothelial growth factor (VFGF) and sequesters them on the cell surface.
  • PDGF platelet-derived growth factor
  • fibroblast growth factors such as aFGF and bFGF
  • VFGF vascular endothelial growth factor
  • testicular function In men, aging is associated with a progressive decline in testicular function. These changes are manifest clinically by decreased virility, vigor, and libido that point towards a relative testicular deficiency
  • ZpeplO polypeptides, agonists or antagonists may be evaluated therapeutically . Soluble zpeplO polypeptides, zpeplO agonists and/or zpeplO antagonists may also have therapeutic value in treatment of testicular cancer, infertility, or in the recovery of function following testicular surgery.
  • zpeplO polypeptides and zpeplO agonists to stimulate proliferation or differentiation of testicular cells 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, 10801 University Boulevard, Manassas, VA. 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 2 ; 347-354, 1990, incorporation of radiolabelled nucleotides (Cook et al . , Anal. Biochem. 179 : 1-7, 1989), incorporation of 5- bromo-2 ' -deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al . , J. Immunol. Methods 82 : 169-79, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al .
  • Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5_ ; 281-4, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv . Anim. Cell Bioi. Technol . Bioprocesses, 161-71, 1989) .
  • ZpeplO polypeptides, agonists and antagonists will also prove useful in the study of spermatogenesis and infertility.
  • zpeplO agonists may find application in the treatment of male infertility.
  • ZpeplO antagonists may be useful as male contraceptive agents.
  • ZpeplO antagonists are useful as research reagents for characterizing sites of ligand-receptor interaction.
  • In vivo assays well known in the art, are available for evaluating the effect of zpeplO ligands and agonists on testes. For example, compounds can be injected intraperitoneally for a specific time duration. After the treatment period, animals are sacrificed and testes removed and weighed.
  • Testicles are homogenized and sperm head counts are made (Meistrich et al . , Exp . Cell Res . 92:72-8, 1976).
  • Other activities for example, chemotaxic activity that may be associated with proteins of the present invention can be analyzed.
  • late stage factors in spermatogenesis are involved in egg- sperm interactions and sperm motility.
  • Activities such as enhancing viability of cryopreserved sperm, stimulating the acrosome reaction, enhancing sperm motility and enhancing egg-sperm interactions may be associated with the ligands and agonists of the present invention.
  • Assays evaluating such activities are known (Rosenberger , J. Androl .
  • zpeplO its agonists and/or antagonists may have applications in enhancing fertilization during assisted reproduction in humans and in animals.
  • assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer and gamete intrafallopian transfer. Such methods are useful for assisting men and women who may have physiological or metabolic disorders that prevent natural conception.
  • Such methods are also used in animal breeding programs, such as for livestock, zoological animals, endangered species or racehorses and could be used as methods for the creation of transgenic animals.
  • agonists or antagonists of the present invention are evaluated with respect to their ability to enhance viability of cryopreserved sperm, sperm motility, the ability of sperm to penetrate cervical mucus, particularly in association with methods of assisted reproduction, according to procedures known in the art (see for example, Juang et al . , Anim. Reprod. Sci. Pl :2 l -9 ' 1989; Juang et al . , Anim. Reprod. Sci. 22:47-53, 1990; Colon et al., Fertil. Steril. 46:1133-39, 1986; Lessing et al . , Fertil.
  • zpeplO polypeptide performance in this regard can be compared to relaxins and the like.
  • zpeplO polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more proteins to identify synergistic effects.
  • soluble zpeplO, agonists and/or antagonists can be added to "capacitation media", a cocktail of compounds known to activate sperm, such as caffeine, dibutyl cyclic adenosine monophosphate (dbcAMP) or theophylline .
  • Such mixtures have resulted in improved reproductive function of the sperm, in particular, sperm motility and zonae penetration (Park et al . , Am. J. Obstet. Gynecol. 158 : 974-9, 1988; Vandevoort et al . , Mol . Repro . Develop. 21:299-304, 1993; Vandevoort and Overstreet, J. Androl . l_6:327-33, 1995).
  • the capacitation mixture can then be combined with sperm, an egg or an egg-sperm mixture prior to fertilization of the egg .
  • zpeplO polypeptides or polypeptide fragments may function as germ-cell-specific antigens for use as components in "immunocontraceptive" or "anti-fertility” vaccines to induce formation of antibodies and/or cell mediated immunity to selectively inhibit a process, or processes, critical to successful reproduction in humans and animals.
  • sperm and testis antigens in the development of an immunocontraceptive have been described (O'Hern et al . , Bioi Reprod. 52:311-39, 1995; Diekman and Herr, Am. J. Reprod. Immunol. 37:111-17, 1997; Zhu and Naz, Proc. Natl. Acad. Sci.
  • the zpeplO protein or fragments can be conjugated to a carrier protein or peptide, such as tetanus or diphtheria toxoid.
  • a carrier protein or peptide such as tetanus or diphtheria toxoid.
  • An adjuvant, as described above, can be included and the protein or fragment can be noncovalently associated with other molecules to enhance intrinsic immunoreactivity .
  • Methods for administration and methods for determining the number of administrations are known in the art. Such a method might include a number of primary injections over several weeks followed by booster injections as needed to maintain a suitable antibody titer.
  • zpeplO therapeutic antibodies small molecule antagonists or agonists of zpeplO polypeptides, or zpeplO polypeptide fragments or soluble zpeplO receptors
  • 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 zpeplO polypeptide, agonist or antagonist.
  • Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use.
  • pharmaceutically acceptable carrier or vehicle 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.
  • One skilled in the art may formulate the compounds of the present invention in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington: The Science and Practice of
  • a pharmaceutically effective amount of a zpeplO 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 polypeptide of the present invention is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • Doses of zpeplO polypeptide will generally be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • the proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years .
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high- resolution, contiguous maps of mammalian chromosomes (Cox et al., Science l ⁇ O'- 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. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) .
  • 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. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
  • 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.
  • the present invention provides reagents for use in diagnostic applications.
  • the zpeplO gene, a probe comprising zpeplO DNA or RNA, or a subsequence thereof can be used to determine if the zpeplO gene is present on a particular chromosome or if a mutation has occurred.
  • Detectable chromosomal aberrations at the zpeplO gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.
  • these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
  • Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO: 1, the complement of SEQ ID NO : 1 , or an RNA equivalent thereof.
  • Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR technigues, ligation chain reaction (Barany, PCR Methods and Applications 1:5- 16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid.; Ausubel et . al . , ibid.
  • Ribonuclease protection assays comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion.
  • PCR assays a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient.
  • Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991).
  • SSCP single strand conformational polymorphism
  • Polynucleotides encoding zpeplO polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zpeplO activity. If a mammal has a mutated or absent zpeplO gene, the zpeplO gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zpeplO 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.
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al . , Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . , J. Clin. Invest.
  • HSV1 herpes simplex virus 1
  • a zpeplO 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 . Cell 32:153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol. 62:1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty 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 2i :74 3 -7 ' 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988).
  • the use of 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.
  • directing transfection to particular cells represents one area of benefit.
  • 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
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • DNA vectors 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, e.g., Wu et al . , J. Bioi. Chem. 267 :963-7, 1992; Wu et al . , J. Bioi. Chem. 2 ⁇ 2:14621-4, 1988.
  • Antisense methodology can be used to inhibit zpeplO gene translation, such as to inhibit cell proliferation in vivo .
  • Polynucleotides that are complementary to a segment of a zpeplO-encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ID NO:l
  • Such antisense polynucleotides are used to inhibit expression of zpeplO polypeptide-encoding genes in cell culture or in a subj ect .
  • mice engineered to express the zpeplO gene, and mice that exhibit a complete absence of zpeplO gene function, referred to as "knockout mice"
  • mice may also be generated (Lowell et al . , Nature 366:740-42, 1993). These mice may be employed to study the zpeplO gene and the protein encoded thereby in an in vivo system.
  • the zpeplO polypeptide-encoding polynucleotides of the present invention were initially identified by querying an EST database for polypeptides containing repetitive patterns and post-translational processing sites yielding potentially active peptides.
  • the polypeptide encoded by an EST meeting those search criteria was further analyzed and found to be a membrane glycoprotein .
  • the EST sequence was from a testis cell library. Several clones considered likely to contain the entire coding region were used for sequencing and resulted in incompletely spliced messages. A minimal nucleotide sequence having all potential introns spliced out was generated from these sequences.
  • Oligonucleotide primers ZC16,186, (SEQ ID NO:9) and ZC16,187, (SEQ ID NO:10) were designed from the sequence of the EST.
  • One nanogram of template DNA was combined with 20 pmoles of each primer in a PCR mixture. The reaction mixture was incubated at
  • the probe was purified using a NUCTRAP push column
  • MTN I, MTN II and MTN III; Clontech Human Multiple Tissue Northern Blots (MTN I, MTN II and MTN III; Clontech) were probed to determine the tissue distribution of human zpeplO expression.
  • An approximately 530 bp probe, entirely 3'UTR was derived by restriction digest of the clone described above with Not I and Eco Rl .
  • the restriction digested fragment was visualized by agarose gel electrophoresis and purified using Qiaex II (Qiagen, Chatsworth, CA) according to manufacturer's instructions.
  • the probe was radioactively labeled using the MULTIPRIME DNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions.
  • the probe was purified using a NUCTRAP push column (Stratagene) .
  • EXPRESSHYB (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight o o at 65 C using and the blots were then washed at 50 C in IX SSC, 0.1% SDS. A 1.5 kb transcript corresponding to zpeplO was seen in testis and a non-discrete smear was seen in kidney.
  • RNA Master Dot Blot (Clontech) that contained RNAs from various tissues that were normalized to 8 housekeeping genes was also probed and hybridized as described above. The highest level of expression was seen in testis with significantly reduced expression in kidney.

Landscapes

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

Abstract

La présente invention concerne des polypeptides zpep10 et des polynucléotides codant ces derniers. Le polypeptide zpep10 est une glycoprotéine membranaire à spécificité testiculaire. Les polypeptides zpep10 pourraient être utiles pour moduler la spermatogenèse et l'interaction oeuf-sperme et seraient utiles pour étudier ou moduler ces fonctions dans des systèmes in vitro ou in vivo. La présente invention concerne également des anticorps dirigés contre les polypeptides zpep10.
PCT/US1999/027223 1998-11-20 1999-11-16 Glycoproteine zpep10 a specificite testiculaire Ceased WO2000031260A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU17303/00A AU1730300A (en) 1998-11-20 1999-11-16 Testis specific glycoprotein zpep10
CA002369706A CA2369706A1 (fr) 1998-11-20 1999-11-16 Glycoproteine zpep10 a specificite testiculaire

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US19712398A 1998-11-20 1998-11-20
US09/197,123 1998-11-20

Publications (1)

Publication Number Publication Date
WO2000031260A1 true WO2000031260A1 (fr) 2000-06-02

Family

ID=22728139

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/027223 Ceased WO2000031260A1 (fr) 1998-11-20 1999-11-16 Glycoproteine zpep10 a specificite testiculaire

Country Status (3)

Country Link
AU (1) AU1730300A (fr)
CA (1) CA2369706A1 (fr)
WO (1) WO2000031260A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993006859A1 (fr) * 1991-10-04 1993-04-15 The Trustees Of Princeton University Proteine de surface de spermatozoide

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993006859A1 (fr) * 1991-10-04 1993-04-15 The Trustees Of Princeton University Proteine de surface de spermatozoide

Also Published As

Publication number Publication date
CA2369706A1 (fr) 2000-06-02
AU1730300A (en) 2000-06-13

Similar Documents

Publication Publication Date Title
US6498235B2 (en) Testis specific glycoprotein zpep10
US7084253B2 (en) Protease-activated receptor PAR4 (ZCHEMR2)
US6235708B1 (en) Testis-specific cystatin-like protein cystatin T
US6355788B1 (en) Follistatin-related protein zfsta2
US6245529B1 (en) Testis-specific cystatin-like protein cystatin T
WO2000031260A1 (fr) Glycoproteine zpep10 a specificite testiculaire
WO2000031264A2 (fr) Proteine cystatine t de type cystatine specifique des testicules
US20010044134A1 (en) Novel secreted polypeptide zsig87
US6372889B1 (en) Soluble protein ZTMPO-1
US6420525B1 (en) Human transcription factor ZGCL-1
WO1998055612A1 (fr) Precurseurs de neurokinine b
MXPA01005033A (en) Testis specific glycoprotein zpep10
WO2000023591A2 (fr) Proteine secretee zsig49
US7122342B1 (en) Protease-activated receptor PAR4 (ZCHEMR2)
EP1123393A1 (fr) La proteine zfsta2 apparentee a la follistatine
EP1144450A1 (fr) Proteine-12 a mammalienne helice alpha
WO2000020583A1 (fr) Homologue de ribonucleoproteine zrnp1, ayant egalement une homologie vis-a-vis du recepteur de l'hormone de liberation des gonadotrophines (gnrh)
US20020173624A1 (en) Secreted proteins encoded by human chromosome 13
US20040058354A1 (en) Mammalian alpha-helical protein-53
WO1999016870A1 (fr) Proteine zsig-11 secretee
MXPA01003766A (en) Follistatin-related protein zfsta2
MXPA01004974A (en) Testis-specific cystatin-like protein cystatin t
WO2002079248A2 (fr) Proteine 53 alpha helicoidale secretee par des mammiferes
CA2337741A1 (fr) Polypeptide pancreatique et ovarien, zsig58
WO2001042292A2 (fr) Nouveau polypeptide secrete zsig87

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref country code: AU

Ref document number: 2000 17303

Kind code of ref document: A

Format of ref document f/p: F

AK Designated states

Kind code of ref document: A1

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

AL Designated countries for regional patents

Kind code of ref document: A1

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

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

Ref document number: PA/a/2001/005033

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 2369706

Country of ref document: CA

Ref country code: CA

Ref document number: 2369706

Kind code of ref document: A

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase