WO2000028030A1 - Proteine secretoire humaine 61 - Google Patents

Proteine secretoire humaine 61 Download PDF

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Publication number
WO2000028030A1
WO2000028030A1 PCT/US1999/026585 US9926585W WO0028030A1 WO 2000028030 A1 WO2000028030 A1 WO 2000028030A1 US 9926585 W US9926585 W US 9926585W WO 0028030 A1 WO0028030 A1 WO 0028030A1
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Prior art keywords
polypeptide
zsigδl
cells
polypeptides
cell
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PCT/US1999/026585
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English (en)
Inventor
Paul O. Sheppard
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Zymogenetics, Inc.
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Priority to CA002350621A priority Critical patent/CA2350621A1/fr
Priority to IL14291999A priority patent/IL142919A0/xx
Priority to EP99963883A priority patent/EP1129192A1/fr
Priority to JP2000581197A priority patent/JP2002529088A/ja
Priority to AU20232/00A priority patent/AU2023200A/en
Publication of WO2000028030A1 publication Critical patent/WO2000028030A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity

Definitions

  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue .
  • hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF) , epidermal growth factor (EGF) , granulocyte-macrophage colony stimulating factor (GM-CSF) , erythropoietin (EPO) and calcitonin.
  • Proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.
  • cytokines molecules that promote the proliferation, maintenance, survival or differentiation of cells.
  • examples of cytokines include erythropoietin (EPO) , which stimulates the development of red blood cells; thrombopoietin (TPO) , which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF) , which stimulates development of neutrophils .
  • EPO erythropoietin
  • TPO thrombopoietin
  • G-CSF granulocyte-colony stimulating factor
  • the present invention addresses this need by providing novel polypeptides and related compositions and methods.
  • the present invention provides an isolated polynucleotide encoding a mammalian secretory protein termed mammalian secretory protein-61 (hereinafter referred to as Zsig61) .
  • the human Zsig ⁇ l polypeptide with signal sequence is comprised of a sequence of amino acids 81 amino acids long with the initial Met as shown in SEQ ID NO: 1 and SEQ ID NO : 2.
  • the signal sequence is comprised of amino acid residues 1-19, the mature sequence being comprised of amino acid residue 20, a valine through and including amino acid residue 81, a valine of SEQ ID NO:2.
  • the mature sequence is further defined by SEQ ID NO:4.
  • the signal sequences extends from amino acid residue 1-24, the mature sequence then being comprised of amino acid residue 25, a glycine, through and including amino acid residue 81, a valine, of SEQ ID NO : 2.
  • This mature sequence is further defined by SEQ ID NO : 5.
  • a mature sequence is defined by amino acid residue 48, a cysteine to and including amino acid residue 78, a cysteine, of SEQ ID NO: 2, also defined by SEQ ID NO: 6.
  • polypeptide further comprises an affinity tag.
  • polynucleotide is DNA.
  • an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding Zsig61 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.
  • a cultured eukaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a protein polypeptide encoded by the DNA segment .
  • a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond.
  • the first portion of the chimeric polypeptide consists essentially of (a) a Zsig ⁇ l polypeptide as shown in SEQ ID NOs : 2,4,5 and 6 (b) allelic variants of SEQ ID NOs : 2 , 4 , 5 and 6; and (c) protein polypeptides that are at least 90% identical to (a) or (b) .
  • the second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag. Within one embodiment the affinity tag is an immunoglobulin F c polypeptide.
  • the invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
  • an antibody that specifically binds to a Zsig ⁇ l polypeptide as disclosed above, and also an anti- idiotypic antibody which neutralizes the antibody to a Zsig ⁇ l polypeptide.
  • An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zsig ⁇ l polypeptide having an amino acid sequence described above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zsig ⁇ l polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope- bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention.
  • polypeptides examples include the polypeptide extending from amino acid residue 25, a glycine, to and including amino acid residue 62 an arginine of SEQ ID NO:2, also defined by SEQ ID NO: 8; the polypeptide extending from amino acid residue 51, a glutamine, to and including amino acid residue 75 a serine of SEQ ID NO:2, also defined by SEQ ID NO : 9 ; and the polypeptide extending from amino acid residue 25, a glycine, to and including amino acid residue 75 a serine of SEQ ID NO: 2, also defined by SEQ ID NO: 10. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • affinity tag any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly-histidine tract, protein A, Nilsson et al . , EMBO J. 4:1075 (1985); Nilsson et al . ,
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and carboxyl- terminal are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl- terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • 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 .
  • polynucleotide molecule is a polynucleotide molecule 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' .
  • sequence 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.
  • 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 is used to denote 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 include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 315:774-78 (1985).
  • an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e . g. , transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, b- globin, and myoglobin are paralogs of each other.
  • polynucleotide is a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt”), or kilobases ("kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded.
  • double-stranded molecules When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • a “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides” .
  • the term “promoter” is used herein for its art- recognized meaning to denote 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 .
  • receptors can be membrane bound, cytosolic or nuclear; monomeric ⁇ e . g. , thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric ⁇ e. g. , PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor) .
  • secretory signal sequence 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.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene .
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the Zsig ⁇ l polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules .
  • Polynucleotides generally a cDNA sequence, of the present invention encode the described polypeptides herein.
  • a cDNA sequence which encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides.
  • the amino acid residues are encoded by their respective codons as follows .
  • Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;
  • Cysteine (Cys) is encoded by TGC or TGT;
  • Aspartic acid is encoded by GAC or GAT;
  • Glutamic acid (Glu) is encoded by GAA or GAG;
  • Phenylalanine is encoded by TTC or TTT;
  • Glycine is encoded by GGA, GGC, GGG or GGT;
  • Histidine is encoded by CAC or CAT;
  • Isoleucine (lie) is encoded by ATA, ATC or ATT;
  • Lysine is encoded by AAA, or AAG;
  • Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;
  • Methionine (Met) is encoded by ATG;
  • Asparagine is encoded by AAC or AAT;
  • Proline is encoded by CCA, CCC, CCG or CCT;
  • Glutamine is encoded by CAA or CAG;
  • Arginine is encoded by AGA, AGG, CGA, CGC,
  • Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;
  • Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
  • Valine (Val) is encoded by GTA, GTC, GTG or GTT;
  • Trp Tryptophan
  • Tyrosine TAC or TAT.
  • mRNA messenger RNA
  • T thymine nucleotide
  • U uracil nucleotide
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid.
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. 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, or a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for preparing DNA and RNA are well known in the art.
  • RNA is isolated from a tissue or cell that produces large amounts of Zsig ⁇ l RNA. Such tissues and cells are identified by Northern blotting, Thomas, Proc .
  • RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient, Chirgwin et al . , Biochemistry 18:52-94
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc . Na tl . Acad . Sci . USA
  • cDNA Complementary DNA
  • poly (A) + RNA using known methods.
  • genomic DNA can be isolated.
  • Polynucleotides encoding Zsig ⁇ l polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding Zsig ⁇ l can be obtained by conventional cloning procedures.
  • Complementary DNA (cDNA) clones are preferred, although for some applications ⁇ e . g. , expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
  • Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Expression libraries can be probed with antibodies to Zsig ⁇ l, receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesizers.
  • the 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 are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. See Glick and Pasternak, Molecular
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs) .
  • species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species.
  • Zsig ⁇ l polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Orthologs of human Zsig61 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 Zsig61 as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A Zsig ⁇ l-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 (Mullis, U.S. Patent No.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to Zsig ⁇ l polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ ID N0:1 represents a single allele of human Zsig ⁇ l and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the 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 Zsig61 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 Zsig ⁇ l polypeptides that are substantially homologous to the polypeptides of SEQ ID NO : 2 and their orthologs.
  • substantially homologous is used herein to denote polypeptides having 50%, preferably 60%, more 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 NOs : 2 , 3,4 or 5 or their orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . ,
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • Variant Zsig ⁇ l polypeptides or substantially homologous Zsig ⁇ l polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl -terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from 20 to 30 amino acid residues that comprise a sequence that is at least 90%, preferably at least 95%, and more preferably 99% or more identical to the corresponding region of SEQ ID NO: 4.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the Zsig ⁇ l polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites . Table 2
  • Aromatic phenylalanine tryptophan tyrosine
  • the present invention further provides a variety of other polypeptide fusions [and related multimeric proteins comprising one or more polypeptide fusions] .
  • a Zsig61 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-Zsig61 polypeptide fusions can be expressed in genetically engineered cells [to produce a variety of multimeric Zsig ⁇ l analogs] .
  • Auxiliary domains can be fused to Zsig ⁇ l polypeptides to target them to specific cells, tissues, or macromolecules ⁇ e . g.
  • a Zsig ⁇ l polypeptide or protein could be targeted to a predetermined cell type by fusing a Zsig61 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes.
  • a Zsig ⁇ l 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
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis-4- hydroxyproline, trans-4 -hydroxyprolme, N-methylglycine , allo- threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3 , 3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine .
  • an in vi tro system can be employed
  • 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 . 33:7470-7476 (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-natural amino acids, and unnatural amino acids may be substituted for Zsig ⁇ l amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science
  • Variants of the disclosed Zsig61 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, (1994), Stemmer, Proc . Natl .
  • variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment . These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • any Zsig ⁇ l polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above .
  • the Zsig ⁇ l 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
  • a DNA sequence encoding a Zsig ⁇ l polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers .
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of Zsig ⁇ l, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo .
  • the secretory signal sequence is operably linked to the Zsig ⁇ l DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5 ' to the 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.
  • 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.
  • 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. 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 14:725 (1978); Corsaro and
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen . Virol . 36 : 59 (1977) and Chinese hamster ovary
  • CHO-K1 e.g. CHO-K1; ATCC No. CCL 61
  • Additional 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.
  • 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 can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • hygromycin resistance 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.
  • Other higher 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.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) .
  • DNA encoding the Zsig61 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 Zsig ⁇ l 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 Zsig ⁇ l polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • a transfer vector comprising a Zsig ⁇ l 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, V.A, et al . , J Virol 67:4566 (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 Zsig ⁇ l polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid. "
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case Zsig ⁇ l.
  • 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, M.S. and Possee, R.D., J " Gen Virol 71:971 (1990);
  • transfer vector constructs a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native Zsig ⁇ l 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 Zsig61 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 Zsig ⁇ l polypeptide, for example, a Glu-Glu epitope tag, Grussenmeyer, T. et al . , Proc Natl Acad
  • a transfer vector containing Zsig ⁇ l is transformed into E. coli , and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
  • Recombinant virus that expresses Zsig ⁇ l 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 army worm, Spodoptera frugiperda . See, in general, Glick and Pasternak,
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOD of 0.1 to 10, more typically near 3.
  • MOD multiplicity of infection
  • the recombinant virus-infected cells typically produce the recombinant Zsig61 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 Zsig ⁇ l polypeptide is filtered through micropore filters, usually 0.45 ⁇ m pore size. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid. ; O'Reilly, D.R. et al . , ibid. ; Richardson,
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris , and Pichia methanolica .
  • 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.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) .
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al . (U.S. Patent No.
  • 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;
  • Patent No. 4,977,092) and alcohol dehydrogenase genes See also U.S. Patents Nos . 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis , Ustilago maydis , Pichia pastoris , Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen . Microbiol . 132:3459 (1986) and Cregg, U.S. Patent
  • Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene ⁇ AUGl or AUG2) .
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a preferred selectable marker for use in Pichia methanolica is a P .
  • methanolica ADE2 gene which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine .
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art, see, e.g.,
  • the polypeptide When expressing a Zsig61 polypeptide in bacteria such as E. coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the 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 in 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% adenine and 0.006% L-leucine) .
  • polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, 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 polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant Zsig61 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 chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl-Sepharose FF Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like
  • polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • the polypeptides of the present invention can be isolated by exploitation of their properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3 : 1 (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography. Methods in
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e . g. , maltose-binding protein, an immunoglobulin domain
  • polypeptide fusions, or hybrid Zsig61 proteins are constructed using regions or domains of the inventive Zsig ⁇ l, Sambrook et al . , ibid. , Altschul et al . , ibid. , Picard, Cur.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain (s) conferring a biological function may be swapped between Zsig ⁇ l of the present invention with the functionally equivalent domain (s) from another family member. Such domains include, but are not limited to, the secretory signal sequence, conserved, and significant domains or regions in this family. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • Zsig ⁇ l polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • Zsig ⁇ l polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • Polypeptides especially polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
  • the polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Merrifield, J. Am . Chem. Soc . 85:2149
  • the activity of molecules of the present invention can be measured using a variety of assays.
  • Zsig61 can be measured in vi tro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model.
  • Zsig ⁇ l transfected (or co-transfected) expression host cells may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers have been described as a means to entrap transfected mammalian cells or primary mammalian cells.
  • 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) .
  • viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV) .
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C. Becker et al . , Meth . Cell Biol . 43:161 (1994); and J.T. Douglas and D.T.
  • 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 in ection.
  • 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 vi tro .
  • the cells can produce proteins for extended periods of time.
  • 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 . ,
  • agonists including the natural ligand/ substrate/ cofactor/ etc.
  • antagonists have enormous potential in both in vi tro and in vivo applications.
  • Zsig ⁇ l and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Antagonists include the natural ligand/ substrate/ cofactor/ etc.
  • Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction. Also as a treatment for prostate cancer.
  • Inhibitors of Zsig ⁇ l activity include anti-Zsig ⁇ l antibodies and soluble Zsig61 receptors, as well as other peptidic and non- peptidic agents (including ribozymes) .
  • Zsig ⁇ l can also be used to identify inhibitors (antagonists) of its activity.
  • Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of Zsig ⁇ l.
  • samples can be tested for inhibition of Zsig61 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of Zsig ⁇ l- dependent cellular responses.
  • Zsig61-responsive cell lines can be transfected with a reporter gene construct that is responsive to a Zsig ⁇ l-stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a Zsig61-DNA response element operably linked to a gene encoding a protein which can be assayed, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE) , hormone response elements (HRE) insulin response element (IRE), Nasrin et al . , Proc . Na tl . Acad . Sci . USA
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of Zsig ⁇ l on the target cells as evidenced by a decrease in Zsig ⁇ l stimulation of reporter gene expression. Assays of this type will detect compounds that directly block Zsig ⁇ l binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor- ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of Zsig61 binding to receptor using Zsig61 tagged with a detectable label (e.g.,
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors .
  • a Zsig ⁇ l polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two constant region domains and lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • Methods for preparing such fusions are disclosed in U.S. Patents Nos . 5,155,027 and 5,567,584.
  • Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify the ligand.
  • the chimeras are bound to a support via the F c region and used in an ELISA format .
  • a Zsig61 ligand-binding polypeptide can also be used for purification of ligand.
  • the 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.
  • 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 are known in the art, and 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 ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
  • the ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl) , or pH to disrupt ligand-receptor binding.
  • 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 (BIAcore, 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 (BIAcore, 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.
  • 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.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art . Such systems include Scatchard analysis for determination of binding affinity, Scatchard, Ann. NY Acad. Sci . 51 : 660 (1949) and calorimetric assays, Cunningham et al . , Science 253:545 (1991); Cunningham et al . , Science 245:821 (1991).
  • Zsig61 polypeptides can also be used to prepare antibodies that specifically bind to Zsig61 epitopes, peptides or polypeptides.
  • the Zsig61 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • Suitable antigens would be the Zsig ⁇ l polypeptides encoded by SEQ ID NOs: 2-24.
  • Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a Zsig61 polypeptide or a fragment thereof.
  • the immunogenicity of a Zsig61 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zsig ⁇ l 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. If the polypeptide portion is "hapten-like" , such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody) .
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to Zsig ⁇ l protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zsig61 protein or peptide) .
  • Genes encoding polypeptides having potential Zsig ⁇ l 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 . , US
  • Patent NO. 5,571,698 and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA) , Invitrogen Inc. (San Diego, CA) , New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) .
  • Random peptide display libraries can be screened using the Zsig61 sequences disclosed herein to identify proteins which bind to Zsig ⁇ l. These "binding proteins" which interact with Zsig ⁇ l polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity.
  • the binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease.
  • These binding proteins can also act as Zsig ⁇ l "antagonists" to block Zsig ⁇ l binding and signal transduction in vi tro and in vivo . These anti-Zsig ⁇ l binding proteins would be useful for inhibiting the activity of Zsig ⁇ l .
  • Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules.
  • antibodies herein specifically bind if they bind to a Zsig ⁇ l polypeptide, peptide or epitope
  • K a binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis.
  • antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides.
  • Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zsig61 but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid. ) .
  • Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family (e.g. IL-
  • Zsig ⁇ l polypeptides and non-human Zsig ⁇ l.
  • antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides. For example, antibodies raised to Zsig ⁇ l are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to Zsig ⁇ l will flow through the matrix under the proper buffer conditions. Such screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to closely related polypeptides, Antibodies : A Laboratory Manual , Harlow and Lane (eds.) (Cold
  • Antibodies to Zsig ⁇ l may be used for tagging cells that express Zsig61; for isolating Zsig ⁇ l by affinity purifica ion; for diagnostic assays for determining circulating levels of Zsig ⁇ l polypeptides; for detecting or quantitating soluble Zsig ⁇ l as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block Zsig61 in vi tro and in vivo .
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to Zsig ⁇ l or fragments thereof may be used in vi tro to detect denatured Zsig ⁇ l or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • 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) .
  • Zsig ⁇ l polypeptides or anti-Zsig ⁇ l 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 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-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 may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest .
  • the domain only fusion protein includes a complementary molecule
  • 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.
  • Zsig61-cytokine fusion proteins or antibody-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers) , if the Zsig ⁇ l polypeptide or anti-Zsig61 antibody targets the hyperproliferative blood or bone marrow cell. See, generally, Hornick et al . , Blood
  • fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine.
  • Suitable Zsig ⁇ l polypeptides or anti-Zsig ⁇ l antibodies target an undesirable cell or tissue ⁇ i . e . , a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells .
  • Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) , for instance.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • 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.
  • Proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column, Immobilized Affini ty Ligand Techniques, Hermanson et al . , eds., pp.195-202 (Academic Press, San Diego, CA,
  • Proteins and peptides can also be radiolabeled, Methods in Enzymol . , vol. 182, "Guide to Protein
  • Polynucleotides encoding Zsig61 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit Zsig61 activity. If a mammal has a mutated or absent Zsig ⁇ l gene, the Zsig61 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a Zsig ⁇ l 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 (HSVl) vector, Kaplitt et al . , Molec . Cell . Neurosci . 2 : 320 (1991); an attenuated adenovirus vector, such as the vector described by Stratford- Perricaudet et al . , J. Clin . Invest . 50:626 (1992); and a defective adeno-associated virus vector, Samulski et al . , J.
  • HSVl herpes simplex virus 1
  • a Zsig ⁇ l gene can be introduced in a retroviral vector, e.g., as described in
  • 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 . ,
  • 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. More particularly, directing transfection to particular cells represents one area of benefit. For instance, 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
  • 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. Biol . Chem . 267:963 (1992); Wu et al . , J. Biol . Chem . 263:14621-4, 1988.
  • Antisense methodology can be used to inhibit Zsig61 gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a Zsig ⁇ l-encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ID NO:l
  • Such antisense polynucleotides are used to inhibit expression of Zsig ⁇ l polypeptide-encoding genes in cell culture or in a subject .
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the Zsig ⁇ l gene a probe comprising Zsig ⁇ l DNA or RNA or a subsequence thereof can be used to determine if the Zsig61 gene is present on chromosome 17pl3.3 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the Zsig ⁇ l gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ;
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ;
  • mice may be employed to study the Zsig ⁇ l gene and the protein encoded thereby in an in vivo system.
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al . , Science
  • 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 for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
  • Zsig ⁇ l has been mapped to 17pl3.3.
  • Sequence tagged sites can also be used independently for chromosomal localization.
  • An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome.
  • An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences .
  • STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS) , GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences .
  • dbSTS Database of Sequence Tagged Sites
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or disease states) ; as molecular weight markers on Southern gels; as chromosome markers (when labeled) to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to " subtract -out " known sequences in the process of discovering other novel polynucleotides; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays [such as, for example, that described in Gyuris et al . Cell 75:791-803 (1993)] to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • the proteins provided by the present invention can similarly be used to raise antibodies or to elicit another immune response: as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues using labeled antibodies; and to isolate correlative receptors or ligands.
  • a reagent including the labeled reagent
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • a protein of the present invention may exhibit cytokine-cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine-cell proliferation either inducing or inhibiting
  • cell differentiation either inducing or inhibiting
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including without limitation, 32D, DA2 , DA1G, T10, B9, B9/11, BaF3 , MC9/G, M+ (preB M+) , 2E8, RB5, DAI, 123, T1165, HT2 , CTLL2 , TF- 1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by assays for T-cell or thymocyte proliferation, assays for cytokine production or proliferation of spleen cells, lymph node cells or thymocytes, assays for proliferation and differentiation of hematopoietic and lymphopoietic cells, and assays for T-cell clone responses to antigens which will identify, among others, proteins that affect antigen-presenting cells (APC) /T-cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production.
  • APC antigen-presenting cells
  • immunological assays include assays for T-cell dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) ; mixed lymphocyte reaction (MLR) assays (which will identify proteins that generate predominantly Thl and CTL responses) ; dendritic cell-dependent assays (which will identify, among others, proteins expressed a by dendritic cells that activate na ⁇ ve T-cells) ; assays for lymphocyte survival/apoptosis (which will identify proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) ; assays for B cell function and assays for protein that influence early steps of T-cell commitment and development.
  • MLR mixed lymphocyte reaction
  • dendritic cell-dependent assays which will identify, among others, proteins expressed a by dendritic cells that activate na ⁇ ve T-cells
  • lymphocyte survival/apoptosis
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity including, without limitation, the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders [including severe combined immunodeficiency (SCID) ] , e.g., in regulating (up or down) growth and proliferation of T or B lymphocytes., as well as effecting the cytolytic activity of natural killer (NK) cells and other cell populations.
  • SCID severe combined immunodeficiency
  • NK natural killer
  • These immune deficiencies may be genetic or by caused by viral as well as bacterial or fungal infections or may result from autoimmune disorders.
  • the protein of the present invention by may possibly be used to treat such diseases or to boost the immune system.
  • the protein of the present invention may be useful in promoting hematopoiesis, including causing proliferation of red blood cells, megakaryocytes, and myeloid cells such as monocytes/macrophages.
  • Assays for relating to stem cell growth or differentiation include: Freshney, M.G., in Culture of
  • the protein of the present invention may be used to repair or regenerate any number of different tissues including bone, ligaments, tendons, neurons and skin. Assays for tissue regeneration include those described in International Patent Publication No. WO95/16035 (bone, cartilage, tendon); WO95/05846 (neuron) ; and WO91/07491 (skin, endothelium) .
  • a protein of the present invention may also exhibit activin or inhibin related activities.
  • Inhibin is a glycoprotein that circulates in plasma and inhibits gonadotropin-releasing hormone (GnRH) -stimulated follicle stimulating hormone (FSH) secretion by the pituitary gland.
  • GnRH gonadotropin-releasing hormone
  • FSH follicle stimulating hormone
  • the protein of the present invention may be useful as a contraceptive or as a based upon the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesi ⁇ in male mammals.
  • Assays for activin/inhibin activity are described in the following: Vale et al . ,
  • the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
  • pharmaceutical formulations will include a Zsig ⁇ l protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed.
  • Therapeutic doses will generally be in the range of 0.1 to 100 ⁇ g/kg of patient weight per day, preferably 0.5-20 mg/kg per day, with the exact dose 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 .
  • Northern blots confirm the predicted message size and demonstrate abundant levels of Zsig61 mRNA in human liver, kidney and pancreas. Since the original clone was derived from a library representing the endocrine pancreas (islets of Langerhans) , the signal in whole pancreas is likely due at least in part to expression in the islet cells. On RNA "dot blots" the presence of zsig ⁇ l mRNA in liver, kidney and pancreas is confirmed. Numerous other tissues, including pituitary, thyroid, adrenal, prostate, stomach, small intestine and colon show a relatively weaker degree of hybridization with the zsig ⁇ l-specific probe. In addition, zsig ⁇ l mRNA was found in fetal liver and fetal kidney RNA samples .
  • Zsig ⁇ l mRNA is found in a large number of glandular organs, most of which share a common function of regulating energy homeostasis (i.e. the absorption, utilization, and excretion of nutrients from the body) .
  • zsig61 is likely to be secreted from these tissues in response to events or conditions which alter metabolic parameters such as blood glucose levels or the concentrations of other carbohydrates or lipids. Conditions such as pH, temperature or oxygen tension may also affect secretion of zsig61 from these tissues.
  • Zsig61 may then act through a receptor mediated mechanism or by modulating the activity of some other blood component to alleviate the condition.
  • the presence of zsig ⁇ l mRNA in fetal liver and kidney samples suggests a possible role for this protein in growth and/or differentiation of tissues.
  • Modulation of zsig ⁇ l levels in proximity to the target tissue should be useful in the treatment of conditions associated with abnormal metabolic activity, including abnormal proliferation or degenerative conditions. This may be achieved by administration of polypeptide, fragments . , antibodies ., binding proteins, DNA based therapy, etc.
  • RNA extracted from pancreatic islet cells was reversed transcribed in the following manner.
  • the first strand cDNA reaction contained 10 ml of human pancreatic islet cell poly d(T) -selected poly (A) + mRNA (Clontech, Palo Alto, CA) at a concentration of 1.0 mg/ml , and 2 ml of 20 pmole/ml first strand primer SEQ ID NO : 7 (GTC TGG GTT CGC TAC TCG AGG CGG CCG CTA TTT TTT TTT TTT TTT TTT TTT)SE containing an Xho I restriction site.
  • the mixture was heated at 70°C for 2.5 minutes and cooled by chilling on ice.
  • First strand cDNA synthesis was initiated by the addition of 8 ml of first strand buffer (5x SUPERSCRIPTS buffer; Life Technologies, Gaithersburg, MD) , 4 ml of 100 mM dithiothreitol, and 3 ml of a deoxynucleotide triphosphate (dNTP) solution containing 10 mM each of dTTP, dATP, dGTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, NJ) to the RNA-primer mixture.
  • the reaction mixture was incubated at 40° C for 2 minutes, followed by the addition of 10 ml of 200 U/ml RNase H- reverse transcriptase (SUPERSCRIPT Ila; Life Technologies) .
  • the efficiency of the first strand synthesis was analyzed in a parallel reaction by the addition of 10 mCi of 32P-adCTP to a 5 ml aliquot from one of the reaction mixtures to label the reaction for analysis.
  • the reactions were incubated at 40°C for 5 minutes, 45°C for 50 minutes, then incubated at 50°C for 10 minutes. Unincorporated 32P-adCTP in the labeled reaction was removed by chromatography on a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) .
  • the unincorporated nucleotides and primers in the unlabeled first strand reactions were removed by chromatography on 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) .
  • the length of labeled first strand cDNA was determined by agarose gel electrophoresis .
  • the second strand reaction contained 102 ml of the unlabeled first strand cDNA, 30 ml of 5x polymerase I buffer (125 mM Tris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl2, 50mM (NH4) 2S04) ) , 2.0 ml of 100 mM dithiothreitol, 3.0 ml of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 ml of 5 mM b-NAD, 2.0 ml of 10 U/ml E. coli DNA ligase (New England Biolabs; Beverly, MA) , 5 ml of 10 U/ml E.
  • 5x polymerase I buffer 125 mM Tris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl2, 50mM (NH4) 2S04)
  • Unincorporated 32P-adCTP in the labeled reaction was removed by chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) before analysis by agarose gel electrophoresis .
  • the reaction was terminated by the addition of 10.0 ml 0.5 M EDTA and extraction with phenol/chloroform and chloroform followed by ethanol precipitation in the presence of 3.0 M Na acetate and 2 ml of Pellet Paint carrier (Novagen, Madison, WI) .
  • the yield of cDNA was estimated to be approximately 2 mg from starting mRNA template of 10 mg.
  • Eco RI adapters were ligated onto the 5 ' ends of the cDNA described above to enable cloning into an expression vector.
  • a 12.5 ml aliquot of cDNA ( ⁇ 2.0 mg) and 3 ml of 69 pmole/ml of Eco RI adapter (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) were mixed with 2.5 ml lOx ligase buffer (660 mM Tris-HCl pH 7.5, 100 mM MgCl2), 2.5 ml of 10 mM ATP, 3.5 ml 0.1 M DTT and 1 ml of 15 U/ml T4 DNA ligase (Promega Corp., Madison, WI) .
  • the reaction was incubated 1 hour at 5°C, 2 hours at 7.5°C, 2 hours at 10°C, 2 hours at 12.5°C and 16 hours at 10° C.
  • the reaction was terminated by the addition of 65 ml H 2 0 and 10 ml 10X H buffer (Boehringer Mannheim, Indianapolis, IN) and incubation at 70°C for 20 minutes.
  • the cDNA was digested with Xho I, resulting in a cDNA having a 5' Eco RI cohesive end and a 3' Xho I cohesive end.
  • the Xho I restriction site at the 3' end of the cDNA had been previously introduced.
  • Restriction enzyme digestion was carried out in a reaction mixture by the addition of 1.0 ml of 40 U/ml Xho I (Boehringer Mannheim, Indianapolis, IN) . Digestion was carried out at 37°C for 45 minutes. The reaction was terminated by incubation at 70°C for 20 minutes and chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) .
  • the cDNA was ethanol precipitated, washed with 70 ⁇ % ethanol, air dried and resuspended in 10.0 ml water, 2 ml of 10X kinase buffer (660 mM Tris-HCl, pH 7.5, 100 mM MgC12) , 0.5 ml 0.1 M DTT, 2 ' ml 10 mM ATP, 2 ml T4 polynucleotide kinase (10 U/ml, Life Technologies, Gaithersburg, MD) . Following incubation at 37° C for 30 minutes, the cDNA was ethanol precipitated in the presence of 2.5 M Ammonium Acetate, and electrophoresed on a 0.8 ⁇ % low melt agarose gel.
  • 10X kinase buffer 660 mM Tris-HCl, pH 7.5, 100 mM MgC12
  • 0.5 ml 0.1 M DTT 2 ' ml 10 mM ATP
  • the contaminating adapters and cDNA below 0.6 Kb in length were excised from the gel.
  • the electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin.
  • the area of the gel containing the concentrated cDNA was excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined.
  • An aliquot of water approximately three times the volume of the gel slice (300 ml) and 35 ml lOx b-agarose I buffer (New England Biolabs) was added to the tube, and the agarose was melted by heating to 65°C for 15 minutes.
  • cDNA was cloned into the Eco RI and Xho I sites of pBLUESCRIPT SK+ vector (Gibco/BRL, Gaithersburg, MD) and electroporated into DH10B cells. Bacterial colonies containing ESTs of known genes were identified and eliminated from sequence analysis by reiterative cycles of probe hybridization to hi-density colony filter arrays (Genome Systems, St. Louis, MI) . cDNAs of known genes were pooled in groups of 50 - 10Q inserts and were labeled with 32P-adCTP using a MEGAPRIME labeling kit (Amersham, Arlington Heights, IL) .
  • Colonies which did not hybridize to the probe mixture were selected for sequencing. Sequencing was done using an ABI 377 sequencer using either the T3 or the reverse primer. The resulting data were analyzed which resulted in the identification of the novel gene Zsig ⁇ l .

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Abstract

Cette invention se rapporte à des molécules de polynucléotides et de polypeptides pour la protéine sécrétoire mammifère 61 (Zsig 61). Ces polypeptides, et les polynucléotides qui les codent, ont une action de régulation hormonale et placentaire et peuvent servir à réguler le développement du placenta. Cette invention concerne également des anticorps contre les polypeptides de Zsig61; ainsi que des anticorps anti-idiotypiques d'anticorps se fixant à Zsig61.
PCT/US1999/026585 1998-11-12 1999-11-09 Proteine secretoire humaine 61 WO2000028030A1 (fr)

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CA002350621A CA2350621A1 (fr) 1998-11-12 1999-11-09 Proteine secretoire humaine 61
IL14291999A IL142919A0 (en) 1998-11-12 1999-11-09 Human secretory protein-61
EP99963883A EP1129192A1 (fr) 1998-11-12 1999-11-09 Proteine secretoire humaine 61
JP2000581197A JP2002529088A (ja) 1998-11-12 1999-11-09 ヒト分泌タンパク質−６１
AU20232/00A AU2023200A (en) 1998-11-12 1999-11-09 Human secretory protein-61

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMBL Database, Heidelberg, FRG Emest2 accession number AA658245 18 November 1997 NCI-CGAP: "nu21a05.s1 NCI_CGAP_Pr2 Homo sapiens cDNA clone IMAGE:1208624" *
NIELSEN, H. ET AL.: "Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites", PROTEIN ENGINEERING, vol. 10, no. 1, January 1997 (1997-01-01), pages 1 - 6, XP002072638 *

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