EP1180147A1 - Secreted alpha-helical protein-31 - Google Patents

Secreted alpha-helical protein-31

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Publication number
EP1180147A1
EP1180147A1 EP00939397A EP00939397A EP1180147A1 EP 1180147 A1 EP1180147 A1 EP 1180147A1 EP 00939397 A EP00939397 A EP 00939397A EP 00939397 A EP00939397 A EP 00939397A EP 1180147 A1 EP1180147 A1 EP 1180147A1
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EP
European Patent Office
Prior art keywords
zalpha31
polypeptide
seq
amino acid
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00939397A
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German (de)
French (fr)
Inventor
Darrell C. Conklin
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Zymogenetics Inc
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Zymogenetics Inc
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Publication of EP1180147A1 publication Critical patent/EP1180147A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • Hormones and polypeptide growth factors control proliferation, maintenance, survival and differentiation of cells of multicellular organisms. 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. Hormones and growth factors influence cellular metabolism by binding to proteins. 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
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly- histidine tract, protein A. Nilsson et al, EMBO J. 4:1075 (1985); Nilsson et al, Methods Enzymol. 198:3 (1991), glutathione S transferase, Smith and Johnson, Gene 67:31 (1988), Glu-Glu affinity tag, Grussenmeyer et al., Proc. Natl.
  • 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.
  • 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.
  • Angiogenic denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in concert with one or more additional compounds. Angiogenic activity is measurable as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell migration, capillary sprout formation, and endothelial cell proliferation.
  • 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° M ⁇ *.
  • polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGAGCTT-3' are 5'- AGCTTgagt-3' and 3'-tcgacTACC-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 316:774-78 (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. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • 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. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • a "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 vitro, or prepared from a combination of natural and synthetic molecules.
  • bp base pairs
  • nt nt
  • kb kilobases
  • 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”.
  • 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.
  • the term "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.
  • 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).
  • monomeric e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor
  • multimeric e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor.
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • 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.
  • Molecular weights and lengths of polymers determined by imprecise analytical methods will be understood to be approximate values. When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ⁇ 10%.
  • the present invention provides novel cytokine polypeptides/proteins.
  • alpha helical protein-31 The novel cytokine, termed "alpha helical protein-31" hereinafter referred to as
  • Zalpha31 was discovered and identified to be a cytokine by the presence of polypeptide and polynucleotide features characteristic of four-helix-bundle cytokines (e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth hormone).
  • four-helix-bundle cytokines e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth hormone.
  • sequence of the zalpha31 polypeptide was obtained from a single clone believed to contain its corresponding polynucleotide sequence. Libraries that might be searched for such sequences include heart, brain, thyroid, liver, spinal cord, adrenal gland, testis, macrophages, lymphoid cells, activated immune cells, and the like.
  • nucleotide sequence of a representative zalpha31 -encoding DNA is described in SEQ ID NO:l, and its deduced 142 amino acid sequence is described in
  • the zalpha31 polypeptide represents a full-length polypeptide segment (residue 1 (Met) to residue 142 (Arg) of SEQ ID NO:2).
  • the domains and structural features of zalpha31 are further described below.
  • SEQ ID NO:2 Analysis of the zalpha31 polypeptide encoded by the DNA sequence of SEQ ID NO:l revealed an open reading frame encoding 142 amino acids (SEQ ID NO:2) comprising a predicted signal peptide of 19 amino acid residues (residue 1 (Met) to residue 19 (Asp) of SEQ ID NO:2), and a mature polypeptide of 122 amino acids (residue 20 (Asp) to residue 142 (Arg) of SEQ ID NO:2).
  • cytokines are predicted to have a four-alpha helix structure, with helices A, C and D being most important in ligand-receptor interactions, and are more highly conserved among members of the family.
  • Helices A-D in zalpha31 define a biologically active receptor-binding domain that comprises amino acids 37 (He) to 132 (Leu) of SEQ ID NO:2.
  • the mature Zalpha31 polypeptide has an unglycosylated molecular weight of approximately 14,009 Daltons (D).
  • SEQ ID NO:2 indicates the presence of four amphipafhic, alpha-helical regions, namely helices A, B, C and D:
  • Helix A can correspond to amino acids 26 (Ala) to 40
  • Helix B can correspond to amino acids 59 (Leu) to 73
  • Helices A-D in zalpha31 can define an extended biologically active receptor-binding domain that comprises amino acids 26 (Ala) to 132
  • Each helix contains an external region having amino acid residues which are generally hydrophilic, and an internally located region which generally contains hydrophobic amino acid residues.
  • the amino acid residues which are positioned on the exterior of the helices are considered crucial for receptor binding and should not be changed to another amino acid residue except to one that is almost identical in charge.
  • the amino acid residues which are positioned on the interior of the helix may be changed to any hydrophobic amino acid residue.
  • amino acid residues 37, 40, 41, 44, 47, 48 and 51 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 38, 39,
  • SEQ ID NO:2 are positioned on the external portion of helix A.
  • amino acid residues 65, 68, 69, 72, 75, 76, and 79 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 66, 67, 70, 71, 73, 74, 77, and 78 of SEQ ID NO:2 are positioned on the external portion of helix B.
  • amino acid residues 87, 90, 91, 94, 97, 98, and 101 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 88, 89, 92. 92, 95, 96, 99 and 100 of SEQ ID NO:2 are positioned on the external portion of helix C.
  • amino acid residues 118, 121, 122, 125, 128, 129, and 132 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 119, 120, 123, IX, 126, 127, 130, and 131 of SEQ ID NO:2 are positioned on the external portion of helix D.
  • Helices 1 through 4 are spaced apart from N-terminus to C-terminus in a configuration represented by the following:
  • H# denotes the specific helix disclosed above (e.g., HI is Helix A, H2 is Helix B etc.), and
  • ⁇ # ⁇ denotes the approximate number of amino acid residues between the motifs, up to plus or minus 2 residues.
  • cytokines Four-helical bundle cytokines are also grouped by the length of their component helices.
  • "Long-helix” form cytokines generally consist of between 24-30 residue helices, and include IL-6, ciliary neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone (hGH).
  • "Short-helix” form cytokines generally consist of between 18-21 residue helices and include IL-2, IL-4 and GM-CSF. Zalpha31 is believed to be a new member of the short-helix form cytokine group.
  • CNTF and IL-6 Studies using CNTF and IL-6 demonstrated that a CNTF helix can be exchanged for the equivalent helix in IL-6, conferring CTNF-binding properties to the chimera.
  • functional domains of four-helical cytokines are determined on the basis of structural homology, irrespective of sequence identity, and can maintain functional integrity in a chimera (Kallen et al., J. Biol. Chem. 274:11859- 11867, 1999). Therefore, the helical domains of zalpha31 will be useful for preparing chimeric fusion molecules, particularly with other short-helix form cytokines to determine and modulate receptor binding specificity.
  • fusion proteins engineered with helix A and/or helix D and fusion proteins that combine helical and loop domains from other short-form cytokines such as IL-2, IL-4, IL-15 and GM-CSF.
  • IL-2, IL-4, IL-15 and GM-CSF The amino acid residues comprising helices A, B, C, and D, and loops A/B, B/C and C/D for zalpha31.
  • IL-2, IL-4, IL- 15 and GM-CSF are shown in Table 1.
  • POLYNUCLEOTIDES The present invention also provides polynucleotide molecules, including
  • SEQ ID NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha31 polypeptide of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:3 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T.
  • zalpha31 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 426 of SEQ ID NO:3 and their RNA equivalents are contemplated by the present invention.
  • Table 2 sets forth the one-letter codes used within SEQ ID NO: 3 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. "Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 3.
  • any X NNN On of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine WSN
  • the degenerate codon for arginine AGR
  • the degenerate codon for arginine MGN
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 3).
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. 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 (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Numerous equations for calculating T, depart are known in the art, and are specific for DNA.
  • RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al, (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur. Crit. Rev. Biochem. Mol Biol. 26:227 (1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake.
  • Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 ⁇ g/ml denatured commercially-available carrier DNA.
  • stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2X SSC.
  • a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65°C. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
  • the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes.
  • Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
  • the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of Zalpha31 RNA. Such tissues and cells are identified by Northern blotting, Thomas, Proc. Natl Acad. Sci. USA 77:5201 (1980) and are discussed below. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in : CsCl gradient, Chirgwin et al., Biochemistry 18:52-94 (1979). Poly
  • RNA + RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972).
  • Complementary DNA cDNA is prepared from poly(A) + RNA using known methods.
  • genomic DNA can be isolated. Polynucleotides encoding Zalpha31 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding Zalpha31 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 Zalpha31 , receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesis machines. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a DNA or a DNA fragment, then each complementary strand is made separately, for example via the phosphoramidite method known in the art.
  • the production of short polynucleotides 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies are usually employed for producing longer polynucleotides (longer than about 300 bp). For example, synthetic DNAs (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
  • One method for building a synthetic DNA involves producing a set of overlapping, complementary oligonucleotides. Each internal section of the DNA has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section. After the DNA is assembled, the process is completed by ligating the nicks along the backbones of the two strands.
  • synthetic DNAs can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector.
  • Alternative ways to prepare a full-length DNA are also known in the art. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are Zalpha31 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Orthologs of human Zalpha31 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 Zalpha31 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 Zalpha31 -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. 4,683,202), using primers designed from the representative human Zalpha31 sequence disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to Zalpha31 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ ID NO:l represents a single allele of human Zalpha31 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 Zalpha31 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 Zalpha31 polypeptides that are substantially similar to the polypeptides of SEQ ID NO:2 and their orthologs.
  • the term "substantially similar” is used herein to denote polypeptides having 70%, preferably 80%, more preferably at least 85%, sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci.
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zalpha31.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat 'I Acad. Sci. USA 85:2444 (1988), and by Pearson. Meth. Enzymol 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAMJ. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • ktup l
  • gap opening penalty 10
  • gap extension penalty l
  • substitution matrix BLOSUM62
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • the BLOSUM62 table (Table 4) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat 'I Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed below), the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • the present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO:3.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat 'I Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
  • the language "conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0,1,2, or 3.
  • Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1,2 or 3), while more preferred conservative substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • the present invention includes those polypeptides which are at least 90%, preferably 95% and most preferably 99% identical to SEQ ID NO: 3 and which are able to stimulate antibody production in a mammal, and said antibodies are able to bind the native sequence of SEQ ID NO:3.
  • Variant Zalpha31 polypeptides or substantially homologous Zalpha31 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 5) 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 about 30 to about 175 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:2.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the Zalpha31 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • Aromatic phenylalanine tryptophan tyrosine
  • the present invention further provides a variety of other polypeptide fusions and relat e d multimeric proteins comprising one or more polypeptide fusions.
  • a Zalpha31 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-Zalpha31 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric Zalpha31 analogs.
  • Auxiliary domains can be fused to Zalpha31 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a Zalpha31 polypeptide or protein could be targeted to a predetermined cell type by fusing a Zalpha31 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • polypeptides and proteins can be targeted for therapeutic or diagnostic purposes.
  • a Zalpha31 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
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tr ⁇ « -3-methylproline, 2,4-methanoproline, c -y-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ /Zo-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 vitro system can be employed wherein nonsense mutation
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, oi 4-i ' luorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • desired non-naturally occurring amino acid(s) e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, oi 4-i ' luorophenylalanine.
  • non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al, Biochem. 35:7470-7476 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site- directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci. 2:395-403 (1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zalpha31 amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al, Proc. Natl. Acad. Sci. USA 55:4498-502 (1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al, Proc. Natl. Acad. Sci. USA 55:4498-502 (1991).
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, J. Biol. Chem. 277:4699-708. 1996.
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al, Science 255:306-312 (1992); Smith et al, J. Mol. Biol. 224:899-904 (1992); Wlodaver et al, FEBS Lett. 309:59-64 (1992).
  • Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.. Current Opin. Struct. Biol. 6:3-10, 1996).
  • available software e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA
  • secondary structure propensities e.g., binary patterns, complementary packing and buried polar interactions
  • Amino acid sequence changes are made in zalpha31 polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • the zalpha31 polypeptide comprises one or more helices
  • changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners.
  • the effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al.,
  • a Hopp/Woods hydrophilicity profile of the zalpha31 protein sequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981 ; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering J_: 153-169, 1998).
  • the profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
  • hydrophilic regions include: (1) amino acid residues 14 (Asp) to 19 (Asp) of SEQ ID NO: 2, (2) amino acid residues 26 (Ala) to 31 (Glu) of SEQ ID NO: 2, (3) amino acid 27 (Glu) to 32 (Pro) of SEQ ID NO: 2, (4) amino acid residues 136 (Tyr) to 141 (Lys) of SEQ ID NO: 2, and (5) amino acid residues 137 (Lys) to 142 (Arg) of SEQ ID NO: 2.
  • hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a zalpha31 polypeptide, so as not to disrupt the overall structural and biological profile.
  • hydrophobic residues selected from the group consisting of Val, Leu and He or the group consisting of Met,
  • residues tolerant of substitution could include such residues as shown in SEQ ID NO:2.
  • Cysteine residues could be relatively intolerant of substitution.
  • the identities of essential amino acids can also be inferred from analysis of sequence similarity between other cytokine family members with zalpha31. Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions.
  • An alternative approach to identifying a variant zalpha31 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zalpha31 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 , as discussed above.
  • the present invention also includes functional fragments of zalpha31 polypeptides and nucleic acid molecules encoding such functional fragments.
  • a "functional" zalpha31 or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti- zalpha31 antibody or zalpha31 receptor (either soluble or immobilized).
  • zalpha31 is characterized by a four-helical-bundle structure comprising helix A (amino acid residues 37-51), helix B (amino acid residues 65-79), helix C (amino acid residues 87- 101) and helix D (amino acid residues 118-132), as shown in SEQ ID NO: 2.
  • the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the helices described above; and (b) functional fragments comprising one or more of these helices.
  • the other polypeptide portion of the fusion protein may be contributed by another four-helical-bundle cytokine, such as IL-15, IL-2, IL-4 and GM-CSF, or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zalpha31 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ID NO:l or fragments thereof can be digested with Bal3 ⁇ nuclease to obtain a series of nested deletions.
  • DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zalpha31 activity, or for the ability to bind anti-zalpha31 antibodies or zalpha31 receptor.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zalpha31 fragment.
  • particular fragments of a zalpha31 polynucleotide can be synthesized using the polymerase chain reaction.
  • variants of the disclosed Zalpha31 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, (1994), Stemmer, Proc. Natl Acad. Sci. USA 97:10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro 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.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below.
  • 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.
  • proteins of the present invention can be joined to other bioactive molecules, particularly other cytokines, to provide multi-functional molecules.
  • other cytokines particularly other cytokines
  • one or more helices from zalpha31 can be joined to other cytokines to enhance their biological properties or efficiency of production.
  • the present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the helices of zalpha31 is fused to another polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmacodynamics.
  • Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
  • the Zalpha31 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.
  • a DNA sequence encoding a Zalpha31 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 Zalpha31 , 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 Zalpha31 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. Patent No. 5,037,743; Holland et al, U.S. Patent No. 5,143,830).
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence that encodes a signal peptide from amino acids 1 (Met) to 19 (Asp) of SEQ ID NO:2 is operably linked to another DNA segment encoding a polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions can be used in vivo or in vitro 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 Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al, EMBO J.
  • 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 -./., J. Gen. Virol. 36:59 (1977) and Chinese hamster ovary (e.g. CHO- Kl; ATCC No. CCL 61) cell lines.
  • Suitable cell hues are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants".
  • stable 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
  • Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MH C, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. /Bangalore) 11 :47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide. London, Chapman & Hall; O'Reilly, D.R. et al.,
  • Baculovirus Expression Vectors A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995.
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system is sold in the Bac-to- BacTM kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zalpha31 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 zalpha31.
  • 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.
  • the baculovirus basic protein promoter also known as Pcor, p6.9 or MP promoter
  • Pcor baculovirus basic protein promoter
  • MP promoter baculovirus basic protein promoter
  • transfer vectors can be constructed which replace the native zalpha31 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 zalpha31 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 zalpha31 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985).
  • a transfer vector containing zalpha31 is transformed into 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 zalpha31 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994.
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 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
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • 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, C. D., ibid.).
  • Subsequent purification of the zalpha31 polypeptide from the supernatant can be achieved using methods described herein.
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae. Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are unclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al, U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al, U.S. Patent No. 5,037,743; and Murray et al, U.S. Patent No. 4,845,075.
  • 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 POT! vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al, U.S.
  • Patent No. 4,615,974; and Bitter U.S. Patent No. 4,977,092 and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or A UG2).
  • 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.
  • host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (A UG1 and A UG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP 4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. ⁇ acthanolica 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., Sambrook et al, ibid.).
  • the polypeptide When expressing a Zalpha31 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
  • 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).
  • Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a Zalpha31 polypeptide of the invention.
  • the epitope of the this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • a region of a protein to which an antibody can bind is defined as an "antigenic epitope”. See for instance, Gey sen, H.M. et al, Proc. Natl. Acad Sci. USA 57:3998-4002 (1984).
  • peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See Sutcliffe, J.G. et al Science 219:660-666 (1983).
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues, usually are effective.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that react with the protein.
  • the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred.
  • antigenic epitopes to be used according to the present invention, however, specifically designed antigenic epitopes include the peptides predicted, for example, from a Jameson- Wolf plot, comprising: (1) amino acid residues 11 (Thr) to 20 (Asp) of SEQ ID NO:2; (2) amino acid residues 60 (Ser) to 64 (Lys) of SEQ ID NO:2; (3) amino acid residues 88 (Ser) to 96 (Gin) of SEQ ID NO:2; (4) amino acid residues 127 (Ala) to 135 (Lys) of SEQ ID NO:2; and (5) amino acid residues 127 (Ala) to 139 (Leu) of SEQ ID NO:2.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zalphal polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al, supra. See also U.S. Patent No. 4,708,781 (1987) further describes how to identify a peptide bearing an immunogenic epitope of a desired protein.
  • 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 pretc.
  • ed 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 Zalpha31 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.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used.
  • These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support.
  • polypeptides of the present invention can be isolated by exploitation of their structural and biological 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. 5:1 (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 Enzymol, Vol. 7 ,2, "Guide to Protein Purification", M. Deutscher, (ed.),page 529-539 (Acad. Press, San Diego, 1990).
  • an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
  • polypeptide fusions, or hybrid Zalpha31 proteins are constructed using regions or domains of the inventive
  • 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.
  • a domain(s) conferring a biological function may be swapped between Zalpha31 of the present invention with the functionally equivalent domain(s) from another family member, e.g., IL-2, IL-4, GM-CSF, or other four-helix bundle cytokine family member.
  • Such domains include, but are not limited to, the secretory signal sequence, helices A through D, 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.
  • polypeptides that have substantially similar sequence identity to residues 1-142 or 20-142 of SEQ ID NO: 2, or functional fragments and fusions thereof, wherein such polypeptides or fragments or fusions retain the properties of the wild-type protein such as the ability to stimulate proliferation, differentiation, induce specialized cell function or bind a cell or zalpha31 receptor or anti-zalpha31 antibodies.
  • Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zalpha31 polypeptide and those polypeptides to which they are fused.
  • a DNA segment that encodes a domain of interest e.g., a zalpha31 helix A through D, or motif described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide and inserted into an appropriate expression vector, as described herein.
  • DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof.
  • a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by Helix A, followed by Helix B, followed by Helix C, followed by Helix D, or as interchanged with equivalent regions from another protein.
  • fusion proteins can be expressed, isolated, and assayed for activity as described herein.
  • Zalpha31 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • Zalpha31 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 of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970.
  • the peptide-resin is with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups.
  • a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure for example, signal transduction, Ig binding or cAMP modulation. Such assays are well known in the art. For a general reference, see Nihei, Y., et al.. supra.; and Rindisbacher, L., et al., supra..
  • the activity of molecules of the present invention can be measured using a variety of assays. Of particular interest are changes in steroidogenesis, spermatogenesis, in the testis, LH and FSH production and GnRH in the hypothalamus.
  • Proteins of the present invention are useful for example in increasing sperm production, treating thyroid, adrenal, lymphoid, inflammatory, pancreatic, blood or bone disorders, can be measured in vitro using cultured cells or in vivo by administering molecules of the present invention to the appropriate animal model.
  • host cells expressing a secreted form of zalpha31 polypeptide can be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells.
  • Non-immunogenic "encapsulations” permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal. Most importantly, the capsules mask and shield the foreign, embedded cells from the recipient animal's immune response. Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Alginate threads provide a simple and quick means for generating embedded cells.
  • 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5 x 10* to about 5 x 10 7 cells/ml) is mixed with the 3% alginate solution. One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl2 solution over a time period of ⁇ 15 min, forming a "thread".
  • the extruded thread is then transferred into a solution of 50 mM CaCl2, and then into a solution of 25 mM CaC_2-
  • the thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine.
  • the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle).
  • a large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution.
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, 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 review, see T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997).
  • adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene 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).
  • 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. However, the host's tissue (e.g., liver) 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.
  • tissue e.g., liver
  • adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El deleted, and in addition contain deletions o _ I--.2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998).
  • deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998).
  • by deleting the entire adenovirus genome very large inserts of heterologous DNA can be accommodated.
  • the adenovirus system can also be used for protein production in vitro.
  • the cells By culturing ade ⁇ .. virus-infected non-293 cells under conditions where the cells are not rapidly dividing, 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 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure proliferation of and/or binding to cells.
  • Suitable cell lines to be engineered to be zalpha31 -dependent include the IL-3 -dependent BaF3 cell line (Palacios and Steinmetz. Cell 41 : 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993).
  • growth factor-dependent cell lines such as FDC-P1 (Hapel et al., Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993) are suitable for this purpose.
  • Growth factor-dependent cell lines can be established according to published methods (e.g. Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexter et al., in Baum et al. Eds.. Experimental Hematology Today, 8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).
  • Proteins of the present invention are useful for stimulating proliferation, activation, differentiation and/or induction or inhibition of specialized cell function of cells of the involved homeostasis of the hematopoiesis and immune function.
  • zalpha31 polypeptides are useful for stimulating proliferation, activation, differentiation, induction or inhibition of specialized cell functions of cells of the hematopoietic lineages, including, but not limited to, T cells, B cells, NK cells, dendritic cells, monocytes, and macrophages, as well as epithelial cells.
  • Proliferation and/or differentiation of hematopoietic cells can be measured in vitro using cultured cells or in vivo by administering molecules of the present invention to the appropriate animal model.
  • assays measuring cell proliferation or differentiation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990, incorporated herein by reference), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incorporated herein by reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol.
  • the activity of zalpha31 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S.
  • the microphysiometer can be used for assaying adherent or non- adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha31 polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zalpha31 -responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zalpha31 polypeptide.
  • ZALPHA31 -responsive eukaryotic cells comprise cells into which a receptor for zalpha31 has been transfected creating a cell that is responsive to zalpha31 ; or cells naturally responsive to zalpha31 such as cells derived from small intestine, PBLs, or bone marrow tissue. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zalpha31 polypeptide, relative to a control not exposed to zalpha31, are a direct measurement of zalpha31 -modulated cellular responses. Moreover, such zalpha31 -modulated responses can be assayed under a variety of stimuli.
  • a method of identifying agonists of zalpha31 polypeptide comprising providing cells responsive to a zalpha31 polypeptide. culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate.
  • culturing a third portion of the cells in the presence of zalpha31 polypeptide and the absence of a test compound can be used as a positive control for the zalpha31 -responsive cells, and as a control to compare the agonist activity of a test compound with that of the zalpha31 polypeptide.
  • a method of identifying antagonists of zalpha31 polypeptide comprising providing cells responsive to a zalpha31 polypeptide, culturing a first portion of the cells in the presence of zalpha31 and the absence of a test compound, culturing a second portion of the cells in the presence of zalpha31 and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • Antagonists and agonists, for zalpha31 polypeptide can be rapidly identified using this method.
  • zalpha31 can be used to identify cells, tissues, or cell lines which respond to a zalpha31 -stimulated pathway.
  • the microphysiometer, described above can be used to rapidly identify ligand-responsive cells, such as cells responsive to zalpha31 of the present invention.
  • Cells can be cultured in the presence or absence of zalpha31 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zalpha31 are responsive to zalpha31.
  • Such cell lines can be used to identify antagonists and agonists of zalpha31 polypeptide as described above.
  • Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction. Also as a treatment for prostate cancer.
  • Inhibitors of Zalpha31 activity include anti-Zalpha31 antibodies and soluble Zalpha31 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
  • Zalpha31 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 Zalpha31.
  • samples can be tested for inhibition of Zalpha31 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of Zalpha31- dependent cellular responses.
  • Zalpha31 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a Zalpha31 -stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a Zalpha31-DNA response element operably linked to a gene encoding a protein which can be assayed, such as luciferase.
  • .UNA 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. Natl. Acad. Sci. USA 87:5273 (1990) and serum response elements (SRE) (Shaw et al.
  • Cyclic AMP response elements are reviewed in Roestler et al, J. Biol. Chem. 263 (19):9063 (1988) and Habener, Molec. Endocrinol 4 (8):1087 (1990). Hormone response elements are reviewed in Beato, Cell 56:335 (1989).
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of Zalpha31 on the target cells as evidenced by a decrease in Zalpha31 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block Zalpha31 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
  • compounds or other samples can be tested for direct blocking of Zalpha31 binding to receptor using Zalpha31 tagged with a detectable label (e.g., I25 I, biotin, horseradish peroxidase, FITC, and the like).
  • a detectable label e.g., I25 I, biotin, horseradish peroxidase, FITC, and the like.
  • the ability of a test sample to inhibit the binding of labeled Zalpha31 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • a Zalpha31 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 Zalpha31 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.
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods
  • 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.
  • 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 255:545 (1991); Cunningham et al. Science 245:821 (1991).
  • Zalpha31 polypeptides can also be used to prepare antibodies that bind to zalpha31 epitopes, peptides or polypeptides.
  • the zalpha31 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zalpha31 polypeptide (e.g., SEQ ID NO:2).
  • Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein.
  • Suitable antigens include the mature zalpha31 polypeptide encoded by SEQ ID NO:2 from amino acid number 1 (Met) to 142 (Arg) of SEQ ID NO:2, or a contiguous 9 to 122 amino acid fragment thereof.
  • Other suitable antigens include alpha helical domains, extracellular domains, motifs, regions, epitopes, etc., as disclosed herein.
  • hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot.
  • Zalpha31 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: those predicted 6 amino acid antigenic epitopes determined from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored.
  • suitable hydrophilic regions include: (1) amino acid residues 14 (Asp) to 19 (Asp) of SEQ ID NO: 2, (2) amino acid residues 26 (Ala) to 31 (Glu) of SEQ ID NO: 2, (3) amino acid 27 (Glu) to 32 (Pro) of SEQ ID NO: 2, (4) amino acid residues 136 (Tyr) to 141 (Lys) of SEQ ID NO: 2, and (5) amino acid residues 137 (Lys) to 142 (Arg) of SEQ ID NO: 2.
  • Suitable hydrophilic peptides also include those antigenic epitopes predicted from a Jameson- Wolf plot, comprising: (1) amino acid residues 11 (Thr) to 20 (Asp) of SEQ ID NO:2; (2) amino acid residues 60 (Ser) to 64 (Lys) of SEQ ID NO:2; (3) amino acid residues 88 (Ser) to 96 (Gin) of SEQ ID NO:2; (4) amino acid residues 127 (Ala) to 135 (Lys) of SEQ ID NO:2; and (5) amino acid residues 127 (Ala) to 139 (Leu) of SEQ ID NO:2.
  • Antibodies from an immune response generated by inoculation of an animal with these antigens 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 Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.
  • 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 zalpha31 polypeptide or a fragment thereof.
  • the immunogenicity of a zalpha31 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 zalpha31 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. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • 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. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination. Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules.
  • a threshold level of binding is determined if anti- zalpha31 antibodies herein bind to a zalpha31 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zalpha31) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 M "
  • binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis
  • anti-zalpha31 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zalpha31 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
  • known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family (e.g., other four-helix bundle cytokines), Screening can also be done using non-human zalpha31 , and zalpha31 mutant polypeptides.
  • antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zalpha31 polypeptides.
  • antibodies raised to zalpha31 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zalpha31 will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995).
  • assays known to those skilled in the art can be utilized to detect antibodies which bind to zalpha31 proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zalpha31 protein or polypeptide.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zalpha31 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zalpha31 protein or peptide).
  • Genes encoding polypeptides having potential zalpha31 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. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO.
  • Random peptide display libraries can be screened using the zalpha31 sequences disclosed herein to identify proteins which bind to zalpha31. These "binding polypeptides" which interact with zalpha31 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 polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor.
  • the binding polypeptides can also be used for diagnostic assays for determining circulating levels of zalpha31 polypeptides; for detecting or quantitating soluble zalpha31 polypeptides as marker of underlying pathology or disease.
  • These binding polypeptides can also act as zalpha31 "antagonists" to block zalpha31 binding and signal transduction in vitro and in vivo. These anti-zalpha31 binding polypeptides would be useful for inhibiting zalpha31 activity or protein-binding.
  • Antibodies to zalpha31 may be used for tagging cells that express zalpha31; for isolating zalpha31 by affinity purification; for diagnostic assays for determining circulating levels of zalpha31 polypeptides; for detecting or quantitating soluble zalpha31 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 zalpha31 activity in vitro 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 zairha31 or fragments thereof may be used in vitro to detect denatured zalpha31 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).
  • Zalpha31 polypeptides or anti-Zalpha31 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 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.
  • Zalpha31 -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 Zalpha31 polypeptide or anti- Zalpha31 antibody targets the hyperproliferative blood or bone marrow cell. See, generally, Homick et al, Blood 89:4437 (1997). They described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine.
  • Suitable Zalpha31 polypeptides or anti-Zalpha31 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
  • the Zalpha31 polypeptide or anti- Zalpha31 antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy.
  • iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.
  • 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.
  • Molecules of the present invention can be used to identify and isolate receptors involved in spermatogenesis, steroidogenesis, testicular differentiation and regulatory control of the hypothalamic-pituitary-gonadal axis, thyroid, heart and adrenal function.
  • proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column, Immobilized Affinity Ligand Techniques, Hermanson et al, eds., pp.195-202 (Academic
  • Proteins and peptides can also be radiolabeled, Methods in Enzymol, vol. 182, "Guide to Protein Purification", M. Lieber, ed., pp 721-737 (Acad. Press, San Diego, 1990) or photoaffinity labeled, Brunner et al, Ann. Rev. Biochem. 62:483-514 (1993) and Fedan et al, Biochem. Pharmacol 55:1167 (1984) and specific cell-surface proteins can be identified.
  • the molecules of the present invention will be useful for testing disorders of the reproductive system, thyroid, adrenal, heart and immunological systems.
  • Zalpha31 represents a novel polypeptide with a putative signal peptide leader sequence and alpha helical structure. Therefore this gene may encode a secreted polypeptide with secondary structure indicating it is a member of the four helix bundle cytokine family. Alternatively, this polypeptide may have other activities associated with other biological functions including: enzymatic activity, association with the cell membrane, or function as a carrier protein.
  • Therapeutic utility includes treatment of diseases which require immune regulation including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythomatosis and diabetes.
  • Zalpha31 may be important in the regulation of inflammation and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis.
  • Zalpha31 may be a potential therapeutic in suppressing the immune system which would be important for reducing graft rejection.
  • zalpha31 may activate the immune system which would be important in boosting immunity to infectious diseases or in improving vaccines.
  • assays that measure Done formation and/or resorption are important assays to assess zalpha31 activity.
  • One example is an assay system that permits rapid identification of substances having selective calcitonin receptor activity on cells expressing the calcitonin receptor.
  • the calcitonin receptor is a member of the G-protein receptor family and transduces signal via activation of adenylate cyclase, leading to elevation of cellular cAMP levels (Lin et al., Science 254:1022-24, 1991).
  • This assay system exploits the receptor's ability to elevate cAMP levels as a way to detect other molecules that are able to stimulate the calcitonin receptor and initiate signal transduction.
  • Other assays that measure bone formation or resorption include calvarial assays, QCT, and assays that measure osteoblast size and number. Such assays are known in the art and discussed below.
  • Receptor activation can be detected by: (1) measurement of adenylate cyclase activity (Salomon et al., Anal. Biochem. 58:541-48. 1974; Alvarez and Daniels, Anal. Biochem.
  • An alternative assay system involves selection of polypeptides that are able to induce expression of a cyclic AMP response element (CRE)-luciferase reporter gene, as a consequence of elevated cAMP levels, in cells expressing a calcitonin receptor, but not in cells lacking calcitonin receptor expression, as described in U.S. patent No. 5,622,839, U.S. Patent No. 5,674,689, and U.S. patent No. 5,674,981.
  • CRE cyclic AMP response element
  • zalpha31 polypeptides, agonists or antagonists that interact with the calcitonin receptor.
  • these models may be used to test effects of zalpha31 on bone other than through the calcitonin receptor.
  • the hypocalcemic rat or mouse model can be used to determine the effect on serum calcium
  • the ovariectomized rat or mouse can be used as a model system for osteoporosis. Bone changes seen in these models and in humans during the early stages of estrogen deficiency are qualitatively similar.
  • Calcitonin has been shown to be an effective agent for the prevention of bone loss in ovariectomized women and rats (Mazzuoli et al., Calcif. Tissue Int. 47:209-14, 1990; Wronski et al., Endocrinology 129:2246-50, 1991). High dose estrogen has been shown to inhibit bone resorption and to stimulate bone formation in an ovariectomized mouse model (Bain et al., J. Bone Miner. Res. 8:435-42, 1993).
  • Biologically active zalpha31 polypeptides, agonists or antagonists, of the present invention that interact with the calcitonin receptor, or exert other effects on bone are therefore contemplated to be advantageous for use in therapeutic applications for which calcitonin is useful.
  • Such applications for example, are in the treatment of osteoporosis, Paget's disease, hyperparathyroidism, osteomalacia, idiopathic hypercalcemia of infancy and other conditions.
  • Additional applications are to inhibit gastric secretion in the treatment of acute pancreatitis and gastrointestinal disorders, and uses as analgesics, in particular for bone pain.
  • In vivo assays for measuring changes in bone formation rates include performing bone histology (see, Recker, R., eds. Bone Histomorphometry: Techniques and Interpretation. Boca Raton: CRC Press, Inc., 1983) and quantitative computed tomography (QCT; Ferretti . Bone ]7:353S-364S, 1995; Orphanoludakis et al., Investig. Radiol. 14:122-130, 1979; and Durand et al., Medical Physics 19:569-573, 1992).
  • An exemplary ex vivo assay for measuring changes in bone formation is a calavarial assay (Gowen et al., J. Immunol. 136:2478-2482, 1986) or resorption calvarial assay (Linkhart, T.A., and Mohan, S., Endocrinology 125:1484-1491, 1989).
  • polypeptides of the present invention can be assayed and used for their ability to modify inflammation.
  • Methods to determine proinflammatory and antiinflammatory qualities of zalpha31 are known in the art and discussed herein.
  • suppression of cAMP production is an indication of anti-inflammatory effects (Nihei, Y., et al., Arch. Dermatol. Res.. 287:546-552, 1995).
  • Suppression of cAMP and inhibition of ICAM and HLA-Dr induced by IFN- ⁇ in keratinocytes can be used to assess inhibition of inflammation.
  • enhancement of cAMP production and induction of ICAM and HLA-Dr in this system can be an measurement of proinflammatory effects of a protein.
  • Zalpha31. likewise can exhibit similar inflammatory effects, as shown in vivo (Example 8) and may exert these effects in tissues in which it is expressed, or in other tissues.
  • zalpha31 is expressed in the colon, and can be useful in promoting wound healing in this tissue, or exhibit anti-bacterial or anti-viral effects.
  • zalpha31 or its antagonists can be useful in treatment of inflammatory bowel disease, diverticulitis, inflammation during and after intestinal surgery, and the like.
  • zalpha31, expressed in thyroid can have wound-healing or antimicrobial or antiviral actions in tissues outside of thyroid, such, as heart, brain, liver, kidney, and the like.
  • zalpha31 polypeptide and zalpha31 antibodies can be useful in diagnosing inflammatory diseases such as melanoma, inflammatory bowel disease, diverticulitis, asthma, pelvic inflammatory disease, (PID), psoriasis, arthritis, reperfusion ischemia, and other inflammatory diseases.
  • inflammatory diseases such as melanoma, inflammatory bowel disease, diverticulitis, asthma, pelvic inflammatory disease, (PID), psoriasis, arthritis, reperfusion ischemia, and other inflammatory diseases.
  • zalpha31, antagonists can be useful in treatment of myocarditis, atherosclerosis, pelvic inflammatory disease. (PID), psoriasis, arthritis, eczema, scleroderma, and other inflammatory diseases.
  • zalpha31 polypeptide, agonists or its antagonists have potential uses in inflammatory diseases such as asthma and arthritis.
  • inflammatory diseases such as asthma and arthritis.
  • antagonists would be valuable in asthma therapy or other anti- inflammatory therapies where migration of lymphocytes is damaging.
  • zalpha31 can serve other important roles in lung function, for instance, bronchodilation, tissue elasticity, recmitment of lymphocytes in lung infection and damage.
  • Assays to assess the activity of zalpha31 in lung cells are discussed in Laberge, S. et al., Am. J. Respir. Cell Mol. Biol. 17:193-202, 1997; Rumsaeng, V. et al, J.
  • the data for zalpha31 suggests a highly inducible gene that is prevalent in B-cells (tonsils) and those cells of the monocyte/macrophage/dendritic lineage, (consistent with a ubiquitous distribution in the "normal", non-induced state) and particularly following treatment with a proinflammatory stimulus such as PMA, TNF, or LPS.
  • a proinflammatory stimulus such as PMA, TNF, or LPS.
  • This conclusion is based on its presence at higher than expected frequency in the following tissues: germinal center B cell (tonsil) library, multiple sclerosis lesions; stimulated THP-1 cells (pro-monocyte line); and peripheral blood dendritic cells (stimulated).
  • the gene was also found in a T-cell line (stimulated) and peripheral blood mononuclear cells (stimulated), fetal liver CD34+ progenitor cells and cartilage from an osteoarthritis patient showing a tendency to be associated with inflammatory events.
  • ESTs found in libraries of neural origin, both tissues (normal and diseased) and differentiated cell lines (neurons).
  • the disease association shows up with: multiple sclerosis, Huntingtons, gliosis, oligoastrocytoma, brain tumor (e.g., mets hypemephroma), and spinal cord w/lymphoma, of which zalpha31 may be associated with activated immune responses associated with those diseases.
  • cytokines are produced by brain tumors, found in MS lesions, and exhibit other neuropathies (Fontana, A. et al., J. Immunol. 132:1837-1844, 1984; Suarez, GA et al., Neurology 46:559-561, 1996) Moreover, abnormalities in single cytokines can lead to neurological disease, such as by inducing immune cell infiltration into neurological tissues (Hanisch, UK et al., Synapse 24:104-114, 1996; Sugita, Y et al, J. Neuropathol. Exp. Neurol. 58:480-488, 1999); or both neurological and immune disease (Zhu, J. et al., J. Neurol. Sci. 125:132-137, 1994; Zhu, J. et al., J. Neurosci. Res. 54:373-381, 1998).
  • an antagonist of zalpha31 would be predicted to be an anti- inflammatory agent.
  • Agonists and antagonists may be useful for a broad range on neural diseases such as; MS or Huntington's Disease, while a cytokine-radionuclide (or similar) may be useful for neural tumors.
  • zalpha31 may act indirectly or in conjunction with other cytokines in exerting it's effects.
  • an interferon, IL-1 or IL-2 could exacerbate a disease in which there is an ir. ⁇ ammation component.
  • zalpha31 antagonists that dissociate or block the combined effects of various cytokines are also useful.
  • zalpha31 polypeptides, polynucleotides or antibodies can be used as diagnostics for neurologic diseases or to determine genetic susceptibility to such diseases.
  • the molecules of the present invention can be useful for proliferation of cardiac tissue cells, such as cardiac myocytes or myoblasts; skeletal myocytes or myoblasts and smooth muscle cells; chrondrocytes; endothelial cells; adipocytes and osteoblasts in vitro.
  • cardiac tissue cells such as cardiac myocytes or myoblasts; skeletal myocytes or myoblasts and smooth muscle cells; chrondrocytes; endothelial cells; adipocytes and osteoblasts in vitro.
  • molecules of the present invention are useful as components of defined cell culture media, and can be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Molecules of the present invention are particularly useful in specifically promoting the growth and/or development of myocytes in culture, and may also prove useful in the study of cardiac myocyte hyperplasia and regeneration.
  • polypeptides, nucleic acids and/or antibodies of the present invention can be used in treatment of disorders associated with myocardial infarction, congestive heart failure, hypertrophic cardiomyopathy and dilated cardiomyopathy.
  • Molecules of the present invention may also be useful for limiting infarct size following a heart attack, aiding in recovery after heart transplantation, promoting angiogenesis and wound healing following angioplasty or endarterectomy, to develop coronary collateral circulation, for revascularization in the eye, for complications related to poor circulation such ⁇ - diabetic foot ulcers, for stroke, following coronary reperfusion using pharmacologic methods, and other indications where angiogenesis is of benefit.
  • Molecules of the present invention may be useful for improving cardiac function, either by inducing cardiac myocyte neogenesis and/or hyperplasia, by inducing coronary collateral development, or by inducing remodeling of necrotic myocardial area.
  • Other therapeutic uses for the present invention include induction of skeletal muscle neogenesis and/or hyperplasia, kidney regeneration and/or for treatment of systemic and pulmonary hypertension.
  • Zalpha31 induced coronary collateral development is measured in rabbits, dogs or pigs using models of chronic coronary occlusion (Landau et al., Amer.
  • Zalpha31 efficacy for treating stroke is tested in vivo in rats, utilizing bilateral carotid artery occlusion and measuring histological changes, as well as maze performance (Gage et al., Neurobiol. Aging 9:645-655, 1988).
  • Zalpha31 efficacy in hypertension is tested in vivo utilizing spontaneously hypertensive rats (SHR) for systemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol. Suppl. 1 :S114- 116, 1995).
  • SHR spontaneously hypertensive rats
  • zalpha31 polypeptide may exhibit antiviral activity by inhibiting viral replication by specific signaling via it's receptor(s) on a host cell (e.g. T-cell).
  • Zalpha31 can exhibit immune cell proliferative activity (See. example 8), can be assayed for this activity as disclosed herein, and may stimulate the immune system to fight viral infections.
  • zalpha31 may bind CD4 or another lymphocyte receptor and exhibit antiviral effects, for example, against human immunodeficiency virus (HIV) or human T-cell lymphotropic virus (HTLV).
  • HIV human immunodeficiency virus
  • HTLV human T-cell lymphotropic virus
  • zalpha31 polypeptide may compete for a viral receptor or co-receptor to block viral infection.
  • Zalpha31 may be given parentally to prevent viral infection or to reduce ongoing viral replication and re-infection (Gayowski, T. et al., Transplantation 64:422-426, 1997).
  • zalpha31 may be used as an antiviral therapeutic, for example, for viral leukemias (HTLV), AIDS (HIV), or gastrointestinal viral infections caused by, for example, rotavirus, calicivirus (e.g., Norwalk Agent) and certain strains of pathogenic adenovirus.
  • HTLV viral leukemias
  • HIV HIV
  • gastrointestinal viral infections caused by, for example, rotavirus, calicivirus (e.g., Norwalk Agent) and certain strains of pathogenic adenovirus.
  • proinflammatory effects of zalpha31 polypeptide can be directly tested in assays using a TranswellTM (Costar), wherein endothelial cells are plated on a semi-permeable membrane and zalpha31 polypeptide is present in the lower chamber of the transwell and Cr 51 or fluorescently-labeled neutrophils (PMNs), lymphocytes, HL60 cells, K562 cells, or the like are added on to the upper chamber of the transwell.
  • PMNs fluorescently-labeled neutrophils
  • lymphocytes lymphocytes
  • HL60 cells, K562 cells, or the like are added on to the upper chamber of the transwell.
  • Migration of the PMNs and the like to the lower chamber of the transwell in the presence of zalpha31 polypeptide, but not its absence (Negative control) would demonstrate zalpha31 polypeptide as a direct chemoattractant of the PMNs.
  • IL-8 could be employed in this assay as a positive control.
  • zalpha31 as indirect stimulator of inflammatory response, a similar method can be employed. For example, an experiment can be set up as per above where in addition to the presence of zalpha31 on the lower chamber of the transwell, fibroblast or adipocytes are plated there. In this way, effects of zalpha31 polypeptide in inducing these cells to secrete factors that enhance migration of PMNs, i.e., inflammation, can be measured.
  • the bFGF can be used as a positive control for indirect assay.
  • Anti-inflammatory effects of zalpha31 polypeptide can also be measured when added on the upper chamber in the presence of PMN's using a similar transwell assay known in the art.
  • the activity of molecules of the present invention may be measured using a variety of assays that, for example, measure neogenesis or hyperplasia (i.e., proliferation) of cardiac cells based on the potential effects of extrathyroidal activity of zalpha31.
  • Additional activities likely associated with the polypeptides of the present invention include proliferation of endothelial cells, cardiomyocytes, fibroblasts, skeletal myocytes directly or indirectly through other growth factors; action as a chemotaxic factor for endothelial cells, fibroblasts and/or phagocytic cells; osteogenic factor; and factor for expanding mesenchymal stem cell and precursor populations.
  • Proliferation can be measured using cultured cardiac cells or in vivo by administering molecules of the present invention to the appropriate animal model. Generally, proliferative effects are seen as an increase in cell number, and may include inhibition of apoptosis as well as stimulation of mitogenesis.
  • Cultured cells for use in these assays include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, and human umbilical vein endothelial cells from primary cultures. Suitable established cell lines include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No.
  • assays measuring cell proliferation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al.,
  • Differentiation is a progressive and dynamic process, ⁇ eginning with pluripotent stem cells and ending with terminally differentiated cells.
  • Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made.
  • Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products, and receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al.. Ciba Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J.
  • cardiac myocyte stem cells early stage cardiac myocyte progenitor cells
  • cardiac myocyte stem cells early stage cardiac myocyte progenitor cells
  • the novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and cardiac myocyte progenitor cells, both in vivo and ex vivo. There is evidence to suggest that factors that stimulate specific cell types down a pathway towards terminal differentiation or dedifferentiation affect the entire cell population originating from a common precursor or stem cell.
  • the present invention includes stimulating or inhibiting the proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chrondrocytes and endoth-,i ⁇ al cells.
  • Molecules of the present invention may, while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of the affect on their common precursor/stem cells.
  • molecules of the present invention may have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
  • Assays measuring differentiation include, for example, measuring cell- surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991 ; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
  • In vivo assays for evaluating cardiac neogenesis or hyperplasia include treating neonatal and mature rats with the molecules of the present invention.
  • the animals' cardiac function is measured as heart rate, blood pressure, and cardiac output to determine left ventricular function.
  • Post-mortem methods for assessing cardiac decline or improvement include: increased or decreased cardiac weight, nuclei/cytoplasmic volume, and staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res. 75:1050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci. 93:8630- 8635, 1996.)
  • PCNA proliferating cell nuclear antigen
  • Proteins of the present invention are useful for stimulating proliferation, activation, differentiation and/or induction or inhibition of specialized cell function of cells of the involved homeostasis of the hematopoiesis and immune function.
  • zalpha31 polypeptides are useful for stimulating proliferation, activation, differentiation, induction or inhibition of specialized cell functions of cells of the hematopoietic lineages, including, but not limited to, T cells, B cells, NK cells, dendritic cells, monocytes. and macrophages, as well as epithelial cells.
  • Proliferation and/or differentiation of hematopoietic cells can be measured in vitro using cultured cells or in vivo b> administering molecules of the claimed invention to the appropriate animal model.
  • assays measuring cell proliferation or differentiation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990, incorporated herein by reference), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incorporated herein by reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol.
  • Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
  • zalpha31 polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage-specific expression of a tissue.
  • direct measurement of zalpha31 polypeptide, or its loss of expression in a tissue as it differentiates, can serve as a marker for differentiation of tissues.
  • direct measurement of zalpha31 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression.
  • Increases in invasiveness and motility of cells, or the gain or loss of expression of zalpha31 in a pre-cancerous or cancerous condition, in comparison to normal tissue can serve as a diagnostic for transformation, invasion and metastasis in tumor progression.
  • knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient.
  • Methods of measuring gain and loss of expression are well known in the art and described herein and can be applied to zalpha31 expression.
  • polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999).
  • zalpha31 gain or loss of expression may serve as a diagnostic for lymphoid, B-cell, endothelial, hematopoietic and other cancers.
  • tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor.
  • the cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models.
  • Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL- 1642) and B16 melanoma (ATCC No. CRL-6323), amongst others.
  • C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus. Three days following this treatment, 10 to 10 cells are implanted under the dorsal skin.
  • the cells themselves may be infected with recombinant adenovirus, such as one expressing zalpha31 , before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
  • adenovirus such as one expressing zalpha31
  • the mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a
  • the implanted cells can be transiently transfected with zalpha31.
  • Use of stable zalpha31 transfectants as well as use of induceable promoters to activate zalpha31 expression in vivo are known in the art and can be used in this system to assess zalpha31 induction of metastasis.
  • purified zalpha31 or zalpha31 conditioned media can be directly injected in to this mouse model, and hence be used in this system.
  • O'Reilly MS et al. Cell 79:315-328, 1994
  • Rusciano D et al. Murine Models of Liver Metastasis.
  • the tumor cells will proliferate rapidly and can be found circulating in the blood and populating numerous organ systems.
  • Therapeutic strategies appropriate for testing in such a model include antibody induced toxicity, ligand-toxin conjugates or cell-based therapies.
  • the latter method commonly referred to adoptive immunotherapy, involves treatment of the animal with components of the human immune system (i.e. lymphocytes, NK cells) and may include ex vivo incubation of cells with zalpha31 or other immunomodulatory agents.
  • Polynucleotides encoding Zalpha31 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit Zalpha31 activity. If a mammal has a mutated or absent Zalpha31 gene, the Zalpha31 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a Zalpha31 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA vims, such as, but not limited to, herpes simplex vims (HSV), papillomavirus, Epstein Barr vims (EBV), adenovims, adeno-associated vims (AAV), and the like.
  • Defective vimses 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 vims 1 (HSV1) vector, Kaplitt et al, Molec. Cell. Neurosci. 2:320 (1991); an attenuated adenovims vector, such as the vector described by Stratford-Perricaudet et al, J. Clin. Invest. 90:626 (1992); and a defective adeno-associated vims vector, Samulski et al, J. Virol. 61:3096 (1987); Samulski et al, J Virol 63:3822 (1989).
  • HSV1 herpes simplex vims 1
  • Kaplitt et al Molec. Cell
  • a Zalpha31 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al, U.S. Patent No. 5,399,346; Mann et al. Cell 55:153, 1983; Temin et al, U.S. Patent No. 4,650,764; Temin et al, U.S. Patent No. 4,980,289; Markowitz et al, J. Virol. 62:1120 (1988); Temin et al, U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker, Feigner et al, Proc. Natl. Acad. Sci. USA 54:7413 (1987); Mackey et al, Proc. Natl. Acad. Sci. USA 55:8027 (1988).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit.
  • directing transfection to particular cells represents one area of benefit.
  • directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al, J. Biol. Chem. 267:963 (1992); Wu et ⁇ /., J. Biol Chem. 2(55:14621-4, 1988.
  • Antisense methodology can be used to inhibit Zalpha31 gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a Zalpha31 -encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ID NO:l
  • Such antisense polynucleotides are used to inhibit expression of Zalpha31 polypeptide-encoding genes in cell culture or in a subject.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the zalpha31 gene a probe comprising zalpha31 DNA or RNA or a subsequence thereof can be used to determine if the zalpha31 gene is present on human chromosome 10 or if a mutation has occurred.
  • Zalpha31 is located at the 10q23-q24 region of chromosome 10 (See, Example 3).
  • Detectable chromosomal aberrations at the zalpha31 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • 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.
  • 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
  • GenBank National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov
  • the zalpha31 gene is located at the 10q23-q24 region of chromosome 10.
  • Several disease related genes in a cluster in this region that are associated with neuropathies, brain cancer, and other neural effects.
  • PTEN phosphatase and tensin homolog
  • Ruvalcaba syndrome (10q23), glioma-inactivated leucine rich gene (LGI1; 10q24); macrocephaly, (10q23.3); partial epilepsy (10q23.3-q24.1); infant-onset spinocerebellar ataxia (IOSCA; 10q24); urofacial syndrome (Ochoa syndrome) (10q23-q24); and autosomal dominant spastic paraplegia 9 (10q23.3-q24.1) all map to this region of chromosome 10.
  • several of these diseases are linked to large chromosomal rearrangements, such as chromosome loss or loss of heterogeneity in the 10q23-q24 region chromosome 10.
  • Loss of 1 copy of chromosome 10 is the most common genetic event in high grade glioma, wherein rearrangement and loss of at least some parts of the second copy of the chromosome, particularly in the 10q23-26 region, has been demonstrated in approximately 80% of glioblastoma tumors (Bigner, S. and Vogelstein, B. Brain Path. 1 : 12-18, 1990). Moreover, loss of heterogeneity at 10q23 occurs in about 70% of glioblastomas and 60% of advanced prostate cancers (Li, J et al. Science 275:1943-1946, 1997), as well as other cancers.
  • polynucleotide probes can be used to detect chromosome 10q23-q24 loss or translocation associated with human diseases, such as glioblastoma, macrocephaly, and T-cell leukemia, or other cancers, neurologic or immune diseases.
  • zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 10 trisomy. For example, split-hand/foot malformation, type 3 (SHFM3) appears to be a result of a trisomy at 10q24-q25 (Nunes, ME et al., Hum. Molec. Genet. 4:2165-2170, 1995). As the zalpha31 gene is also located at the 10q23-q24 region zalpha31, polynucleotide probes can be used to detect chromosome 10q23-q24 gain, or trisomy associated with such human diseases.
  • SHFM3 split-hand/foot malformation, type 3
  • polynucleotide probes can be used to detect chromosome 10q23-q24 gain, or trisomy associated with such human diseases.
  • zalha31 is located in a chromosomal region that is associated with neural and brain implications as well as several tumors.
  • ESTs for zalpha31 are found in libraries of neural origin with disease association with multiple sclerosis, Huntington's Disease, gliosis, oligoastrocytoma, brain tumors (e.g., mets hypemephroma), and spinal cord w/lymphoma.
  • proinflammatory cytokines are produced by brain tumors, found in MS lesions, and exhibit other neuropathies.
  • Molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zalpha31 genetic defect.
  • inventive anti-zalpha31 antibodies, polynucleotides, and polypeptides can be used for the detection of zalpha31 polypeptide, mRNA or anti-zalpha3 i antibodies, thus serving as markers and be directly used for detecting or genetic diseases or cancers, as described herein, using methods known in the art and described herein.
  • zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 10q23-q24 deletions and translocations associated with human diseases, other translocations involved with malignant progression of tumors or other 10q23-q24 mutations, which are expected to be involved in chromosome rearrangements in malignancy; or in other cancers, or in spontaneous abortion.
  • zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 10q23-q24 trisomy and chromosome loss associated with human diseases or spontaneous abortion.
  • zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.
  • zalpha31 polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zalpha31 chromosomal locus. As such, the zalpha31 sequences can be used as diagnostics in forensic DNA profiling.
  • diagnostic met c ds comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (
  • a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre- implantation embryo.
  • a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the zalpha31 genetic locus, and the like are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples.
  • Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l, or an RNA equivalent thereof.
  • Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
  • Mutations associated with the zalpha31 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 705:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-as
  • mice engineered to express the zalpha31 gene referred to as "transgenic mice,” and mice that exhibit a complete absence of zalpha31 gene function, referred to as “knockout mice,” may also be generated (Snouwaert et al.. Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986).
  • transgenic mice that over-express zalpha31, either ubiquitously or under a tissue- specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype.
  • over-expression of a wild-type zalpha31 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zalpha31 expression is functionally relevant and may indicate a therapeutic target for the zalpha31, its agonists or antagonists.
  • a preferred transgenic mouse to engineer is one that over- expresses the zalpha31 mature polypeptide (approximately amino acid residue 20 (Asp) to residue 142 (Arg) of SEQ ID NO:2). Moreover, such over-expression may result in a phenotype that shows similarity with human diseases.
  • knockout zalpha31 mice can be used to determine where zalpha31 is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a zalpha31 antagonist, such as those described herein, may have.
  • the human zalpha31 cDNA can be used to isolate murine zalpha31 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the zalpha31 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases. Moreover, transgenic mice expression of zalpha31 antisense polynucleotides or ribozymes directed against zalpha31, described herein, can be used analogously to transgenic mice described above.
  • 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 Zalpha31 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.
  • 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.
  • the EST362610 sequence (SEQ ID NOT) encoded a full-length protein designated zalpha31, as described herein and in SEQ ID NO:2.
  • Northerns were performed using Human Multiple Tissue Blots(MTNl, MTN2 and MTN3) and Master Dot blot (Clontech, Palo Alto, CA).
  • An cDNA probe was prepared using PCR. Oligo nucleotides ZC22,230 (SEQ ID NO:9) and ZC22,249 (SEQ ID NO:8), designed off the EST INC515639H2 (Incyte Pharmaceuticals, Palo Alto, CA), were used as primers.
  • the template was human brain Marathon cDNA (Clontech) mad.- in house using manufacturer ' s instmctions.
  • PCR thermocycler conditions were as follows: one cycle at 94°C for 1.5 min.; 35 cycles at 94°C for 10 sec, 62°C for 20 sec, 72°C for 30 se ; one cycle at 72°C for 10 min.; followed by a 4°C hold.
  • the approximately 500bp bp probe was purified using a Gel Extraction Kit (Qiagen, Chatsworth, CA) according to manufacturer's instmctions.
  • the probe was radioactively labeled using a Rediprime II DNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's specifications.
  • the probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA).
  • EXPRESSHYB (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 55°C, using 1.5 x 10-6 cpm/ml labeled probe. The blots were then washed in 2XSSC and 0.1% SDS at room temperature, then with 2XSSC and 0.1% SDS at 65 °C, followed by a wash in 0.1X SSC and 0.1% SDS at 65°C. Two transcript sizes were observed on the blots at ⁇ 1.35kb and ⁇ 2kb in all tissues with strongest expression in heart, thyroid, spinal cord, adrenal gland, brain, and testis.
  • Dot Blots were also performed using Human RNA Master BlotsTM (Clontech). The methods and conditions for the Dot Blots are the same as for the Multiple Tissue Blots disclosed above.
  • the Dot blots showed signals in all tissues with strongest in brain, liver and heart. Th ⁇ -.
  • EST electronic northern data for zalpha31 suggests that it is a highly inducible gene that is prevalent in B-cells (tonsils) and those cells of the monocyte/macrophage/dendritic lineage, (consistent with a ubiquitous distribution in the "normal", non-induced state) and particularly following treatment with a proinflammatory stimulus such as PMA, TNF. or LPS.
  • Example 3 Chromosomal Assignment and Placement of Zalpha31 Zalpha31 was mapped to chromosome 10 using the commercially available "GeneBridge 4 Radiation Hybrid (RH) Mapping Panel"(Research Genetics, Inc., Huntsville, AL).
  • the GeneBridge 4 RH panel contains DNA from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient).
  • a publicly available WWW server http://www-genome.wi.mit.edu/cgi- bin contig/rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR” radiation hybrid map) which was constmcted with the GeneBridge 4 RH panel.
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C,
  • Example 4 Generation of untagged zalpha31 Recombinant Adenovims A. Generation of expression vector constmct for Adenovims expression
  • PCR primers ZC23457 (SEQ ID NO:12) and ZC23458 (SEQ ID NO:13) were used with template pT7T3D plasmid containing the full-length human zalpha31 cDNA in a PCR reaction as follows: one cycle at 95°C for 5 minutes; followed by 15 cycles at 95°C for 0.5 min., 58°C for 0.5 min., and 72°C for 0.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak.
  • the PCR reaction product was loaded onto a 1.2 %
  • the CMV promoter driving GFP expression was replaced with the SV40 promoter and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal.
  • the native polylinker was replaced with Fsel, EcoRV, and Ascl sites.
  • This modified form of pAdTrack CMV was named pZy Track. Ligation was performed using the Fast-LinkTM DNA ligation and screening kit (Epicentre Technologies, Madison, WI). Clones containing the zalpha31 cDNA were identified by standard mini prep procedures. In order to linearize the plasmid, approximately 5 ⁇ g of the pZy Track zalpha31 plasmid was digested with Pmel.
  • Approximately 1 ⁇ g of the linearized plasmid was cotransformed with 200ng of supercoiled pAdEasy (He et al., supra.) into BJ5183 cells.
  • the co-transformation was done using a Bio-Rad Gene Pulser at 2.5kV, 200 ohms and 25mFa.
  • the entire co-transformation was plated on 4 LB plates containing 25 ⁇ g/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin and recombinant adenovims DNA identified by standard DNA miniprep procedures. Digestion of the recombinant adenovims DNA with Fsel-Ascl confirmed the presence of zalpha31.
  • the recombinant adenovims miniprep DNA was transformed into DH10B competent cells and DNA prepared using a Qiagen maxi prep kit as per kit instmctions.
  • Pad enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 ⁇ l containing 20-30U of Pad.
  • the digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol. i he DNA pellet was resuspended in 5 ⁇ l distilled water.
  • a T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada), inoculated the day before and grown to 60-70% confluence, were transfected with the Pad digested DNA.
  • the Pad-digested DNA was diluted up to a total volume of 50 ⁇ l with sterile HBS (150mM NaCl, 20mM HEPES).
  • DOTAP Boehringer Mannheim, lmg/ml
  • the media was removed from the 293A cells and washed with 5 ml serum-free MEMalpha (Gibco BRL) containing ImM Sodium Pymvate (GibcoBRL), 0.1 mM MEM non-essential amino acids (GibcoBRL) and 25mM HEPES buffer (GibcoBRL).
  • 5 ml of semm-free MEM was added to the 293A cells and held at 37°C.
  • the DNA/lipid mixture was added drop-wise to the T25 flask of 293A cells, mixed gently and incubated at 37°C for 4 hours. After 4 hours the media containing the DNA/lipid mixture was aspirated off and replaced with 5 ml complete MEM containing 5% fetal bovine semm.
  • the transfected cells were monitored for Green Fluorescent Protein (GFP) expression and formation of foci, i.e., viral plaques.
  • GFP Green Fluorescent Protein
  • the cmde lysate was amplified (Primary (1°) amplification) to obtain a working "stock" of zalpha31 rAdV lysate.
  • Ten 10cm plates of nearly confluent (80- 90%) 293 A cells were set up 20 hours previously, 200ml of cmde rAdV lysate added to each 10cm plate and monitored for 48 to 72 hours looking for CPE under the white light microscope and expression of GFP under the fluorescent microscope. When all of the 293 A cells showed CPE (Cytopathic Effect) this 1° stock lysate was collected and freeze/thaw cycles performed as described under Crude rAdV Lysate.
  • NP-40 detergent was added to a final concentration of 0.5% to the bottles of cmde lysate in order to lyse all cells. Bottles were placed on a rotating platform for 10 min. agitating as fast as possible without the bottles falling over. The debris was pelleted by centrifugation at 20,000 X G for 15 minutes. The supernatant was transferred to 250ml polycarbonate centrifuge bottles and 0.5 volumes of 20%PEG8000/2.5M NaCl solution added. The bottles were shaken overnight on ice. The bottles were - .ntrifuged at 20,000 X G for 15 minutes and supernatant discarded into a bleach solution.
  • the white precipitate in two vertical lines along the wall of the bottle on either side of the spin mark is the precipitated vims/PEG.
  • the vims solution was placed in 2 ml microcentrifuge tubes and centrifuged at 14,000 X G in the microfuge for 10 minutes to remove any additional cell debris.
  • the supernatant from the 2ml microcentrifuge tubes was transferred into a 15ml polypropylene snapcap tube and adjusted to a density of 1.34g/ml with cesium chloride (CsCl). The volume of the virus solution was estimated and 0.55 g/ml of CsCl added.
  • CsCl cesium chloride
  • the CsCl was dissolved and 1 ml of this solution weighed 1.34 g.
  • the solution was transferred polycarbonate thick- walled centrifuge tubes 3.2ml (Beckman No. 362305) and spin at 80,000 rpm (348,000 X G) for 3-4 hours at 25°C in a Beckman Optima TLX microultracentrifuge with the TLA- 100.4 rotor.
  • the vims formed a white band. Using wide-bore pipette tips, the vims band was collected.
  • the vims from the gradient has a large amount of CsCl which must be removed before it can be used on cells.
  • Pharmacia PD-10 columns prepacked with Sephadex G-25M (Pharmacia) were used to desalt the vims preparation.
  • the column was equilibrated with 20 ml of PBS.
  • the vims was loaded and allowed to mn into the column.
  • 5 ml of PBS was added to the column and fractions of 8-10 drops collected.
  • the optical densities of 1 :50 dilutions of each fraction was determined at 260nm on a spectrophotometer. A clear absorbance peak was present between fractions 7-12. These fractions were pooled and the optical density (OD) of a 1:25 dilution determined.
  • the OD of a 1:25 dilution of the zalpha31 rAdV was 0.059, giving a vims concentration of 3.0 X 10 12 virions/ml.
  • glycerol was added to the purified vims to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80°C.
  • TCID50 formulation used was as per Quantum Biotechnologies, Inc.,
  • the titer (T) is determined from a plate where vims used is diluted from 10 to 10 -14 , and read 5 days after the infection. At each dilution a ratio (R) of positive wells for CPE per the total number of wells is determined.
  • the zalpha31 adenovims had a titer of 1.3 X 10 pfu ml.
  • Constmct for generating human zalpha31 Transgenic Mice A. Constmct for expressing human zalpha31 from the MT-1 promoter
  • Approximately 10 °g Zytrack vector containing the sequence confirmed zalpha31 coding region (Example 4) was digested with Fsel and Ascl. The vector was then ethanol precipitated and the pellet was resuspended in TE. The released 429 bp zalpha31 fragment was isolated by running the digested vector on a 1.2% SeaPlaque gel and exicising the fragment. DNA was purified using the QiaQuick (Qiagen) gel extraction kit. The zalpha31 fragment was then ligated into our standard transgenic vector pTG12-8, which was previously digested with Fsel and Ascl.
  • the pTG12-8 plasmid designed for expression of a gene of interest in transgenic mice, contains an expression cassette flanked by 10 kb of MT-1 5' DNA and 7 kb of MT-1 3' DNA.
  • the expression cassette comprises the MT-1 promoter, the rat insulin II intron, a polylinker for the insertion of the desired clone, and the human growth hormone poly A sequence.
  • About one microliter of the ligation reaction was electroporated into DH10B ElectroMax ® competent cells (GIBCO BRL, Gaithersburg, MD) according to manufacturer's direction, plated onto LB plates containing 100 ⁇ g/ml ampicillin, and incubated overnight at 37°C.
  • Colonies were picked and grown in LB media containing 100 ⁇ g/ml ampicillin.
  • Miniprep DNA was prepared from the picked clones and screened for the zalpha31 insert by restriction digestion with EcoRI, and subsequent agarose gel electrophoresis. Maxipreps of the correct pTG12-8 zalpha31 constmct were performed.
  • a Sail fragment containing with 5' and 3' flanking sequences, the MT promoter, the rat insulin II intron, zalpha31 cDNA and the human growth hormone poly A sequence was prepared using standard techniques described herein, and used for microinjection into fertilized murine oocytes. Micro injection and production of transgenic mice were produced as described in Hogan, B. et al. Manipulating the Mouse Embryo, 2 nd ed., Cold Spring Harbor Laboratory Press, NY, 1994.
  • the zalpha31 DNA fragment digested with Fsel and Ascl (Example 5A) is cloned into pKFO51 , a lymphoid-specific transgenic vector, previously digested with Fsel and Ascl as described above.
  • the pKFO51 transgenic vector is derived from pl026X (Iritani, B.M., et al., EMBO J.
  • T cell-specific lck proximal promoter contains the T cell- specific lck proximal promoter, the B/T cell-specific immunoglobulin ⁇ heavy chain enhancer, a polylinker for the insertion of the desired clone, and a mutated hGH gene that encodes an inactive growth hormone protein (providing 3' introns and a polyadenylation signal).
  • About one microliter of each ligation reaction is electroporated, plated, clones picked and screened for the human zalpha31 insert by restriction digestion as described above. A correct clone of pKFO51-zalpha31 is verified by sequencing, and a maxiprep of this clone is performed.
  • a Notl fragment, containing the lck proximal promoter and immunoglobulin ⁇ enhancer (E ⁇ LCK), zalpha31 cDNA, and the mutated hGH gene is prepared to be used for microinjection into fertilized murine oocytes.

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Abstract

The present invention relates to polynucleotide and polypeptide molecules for mammalian secreted alpha helical protein-31 (zalpha31). The polypeptides, and polynucleotides encoding them, are a novel four-helix bundle cytokine and may be used to regulate the functioning of the immune system. The present invention also includes antibodies to the Zalpha31 polypeptides.

Description

SECRETED ALPHA-HELICAL PROTEIN - 31
BACKGROUND OF THE INVENTION
Hormones and polypeptide growth factors control proliferation, maintenance, survival and differentiation of cells of multicellular organisms. 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. Examples of 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. Hormones and growth factors influence cellular metabolism by binding to proteins. 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.
Of particular interest are 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. These cytokines are useful in restoring normal blood cell levels in patients suffering from anemia or receiving chemotherapy for cancer. The demonstrated in vivo activities of these cytokines illustrates the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists. The present invention addresses this need by providing novel polypeptides and related compositions and methods. These and other aspects of the invention will become evident upon reference to the following detailed description of the invention. DETAILED DESCRIPTION OF THE INVENTION
The teachings of all the references cited herein are incorporated in their entirety herein by reference.
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:
The term "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. In principal, 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, Methods Enzymol. 198:3 (1991), glutathione S transferase, Smith and Johnson, Gene 67:31 (1988), Glu-Glu affinity tag, Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985), substance P, Flag™ peptide, Hopp et al., Biotechnology 6:1204-1210 (1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107 (1991). DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).
The term "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. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. The terms "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. "Angiogenic" denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in concert with one or more additional compounds. Angiogenic activity is measurable as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell migration, capillary sprout formation, and endothelial cell proliferation.
The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody /antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement anti-complement pair is desirable, the complement anti-complement pair preferably has a binding affinity of <10° M~*.
The term "complements of a 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'.
The term "contig" denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGAGCTT-3' are 5'- AGCTTgagt-3' and 3'-tcgacTACC-5'.
The term "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).
The term "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. The term "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. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78 (1985). 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. In a preferred form, 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. When used in this context, 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.
The term "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.
The term "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. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, α- globin, β-globin, and myoglobin are paralogs of each other.
A "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 vitro, or prepared from a combination of natural and synthetic molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides
("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-strande 1 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.
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. The term "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. 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. In general, 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).
The term "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.
The term "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.
Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%. The present invention provides novel cytokine polypeptides/proteins.
The novel cytokine, termed "alpha helical protein-31" hereinafter referred to as
"Zalpha31" was discovered and identified to be a cytokine by the presence of polypeptide and polynucleotide features characteristic of four-helix-bundle cytokines (e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth hormone).
The sequence of the zalpha31 polypeptide was obtained from a single clone believed to contain its corresponding polynucleotide sequence. Libraries that might be searched for such sequences include heart, brain, thyroid, liver, spinal cord, adrenal gland, testis, macrophages, lymphoid cells, activated immune cells, and the like.
The nucleotide sequence of a representative zalpha31 -encoding DNA is described in SEQ ID NO:l, and its deduced 142 amino acid sequence is described in
SEQ ID NO:2. In its entirety, the zalpha31 polypeptide (SEQ ID NO:2) represents a full-length polypeptide segment (residue 1 (Met) to residue 142 (Arg) of SEQ ID NO:2). The domains and structural features of zalpha31 are further described below.
Analysis of the zalpha31 polypeptide encoded by the DNA sequence of SEQ ID NO:l revealed an open reading frame encoding 142 amino acids (SEQ ID NO:2) comprising a predicted signal peptide of 19 amino acid residues (residue 1 (Met) to residue 19 (Asp) of SEQ ID NO:2), and a mature polypeptide of 122 amino acids (residue 20 (Asp) to residue 142 (Arg) of SEQ ID NO:2).
In general, cytokines are predicted to have a four-alpha helix structure, with helices A, C and D being most important in ligand-receptor interactions, and are more highly conserved among members of the family. Helices A-D in zalpha31 define a biologically active receptor-binding domain that comprises amino acids 37 (He) to 132 (Leu) of SEQ ID NO:2. The mature Zalpha31 polypeptide has an unglycosylated molecular weight of approximately 14,009 Daltons (D). Further analysis of SEQ ID NO:2 indicates the presence of four amphipafhic, alpha-helical regions, namely helices A, B, C and D:
1) "helix A" (corresponding to amino acids 37 (He) to 51 (Tyr) of SEQ ID NO:2); 2) "helix B" (corresponding to amino acids 65 (Leu) to 79 (Glu) of SEQ ID NO:2);
3) "helix C" (corresponding to amino acids 87 (He) to 101 (Leu) of SEQ ID NO:2); and 4) "helix D" (corresponding to amino acids 118 (Leu) to 132 (Leu) of
SEQ ID NO:2).
Alternatively, Helix A can correspond to amino acids 26 (Ala) to 40
(Leu) of SEQ ID NO:2; and Helix B can correspond to amino acids 59 (Leu) to 73
(Thr) of SEQ ID NO:2. As such Helices A-D in zalpha31 can define an extended biologically active receptor-binding domain that comprises amino acids 26 (Ala) to 132
(Leu) of SEQ ID NO:2.
Each helix contains an external region having amino acid residues which are generally hydrophilic, and an internally located region which generally contains hydrophobic amino acid residues. The amino acid residues which are positioned on the exterior of the helices are considered crucial for receptor binding and should not be changed to another amino acid residue except to one that is almost identical in charge. The amino acid residues which are positioned on the interior of the helix may be changed to any hydrophobic amino acid residue.
In helix A, amino acid residues 37, 40, 41, 44, 47, 48 and 51 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 38, 39,
42, 43, 45, 46, 49 and 50 of SEQ ID NO:2 are positioned on the external portion of helix A.
In helix B, amino acid residues 65, 68, 69, 72, 75, 76, and 79 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 66, 67, 70, 71, 73, 74, 77, and 78 of SEQ ID NO:2 are positioned on the external portion of helix B.
In helix C, amino acid residues 87, 90, 91, 94, 97, 98, and 101 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 88, 89, 92. 92, 95, 96, 99 and 100 of SEQ ID NO:2 are positioned on the external portion of helix C. In helix D, amino acid residues 118, 121, 122, 125, 128, 129, and 132 of SEQ ID NO:2 are positioned towards the interior of helix A, while amino acid residues 119, 120, 123, IX, 126, 127, 130, and 131 of SEQ ID NO:2 are positioned on the external portion of helix D. Helices 1 through 4 are spaced apart from N-terminus to C-terminus in a configuration represented by the following:
Asp20-{ 16}-Hl-{ 13}-H2-{7}-H3-{16}-H4-{9}- Arg142, where Asp20 is the starting residue of the mature polypeptide (as shown in SEQ ID NO:2), Arg|42 is the ending residue of the mature polypeptide(as shown in SEQ
ID NO:2),
H# denotes the specific helix disclosed above (e.g., HI is Helix A, H2 is Helix B etc.), and
{#} denotes the approximate number of amino acid residues between the motifs, up to plus or minus 2 residues.
The corresponding polynucleotides encoding the zalpha31 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO. l .
Four-helical bundle cytokines are also grouped by the length of their component helices. "Long-helix" form cytokines generally consist of between 24-30 residue helices, and include IL-6, ciliary neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone (hGH). "Short-helix" form cytokines generally consist of between 18-21 residue helices and include IL-2, IL-4 and GM-CSF. Zalpha31 is believed to be a new member of the short-helix form cytokine group. Studies using CNTF and IL-6 demonstrated that a CNTF helix can be exchanged for the equivalent helix in IL-6, conferring CTNF-binding properties to the chimera. Thus, it appears that functional domains of four-helical cytokines are determined on the basis of structural homology, irrespective of sequence identity, and can maintain functional integrity in a chimera (Kallen et al., J. Biol. Chem. 274:11859- 11867, 1999). Therefore, the helical domains of zalpha31 will be useful for preparing chimeric fusion molecules, particularly with other short-helix form cytokines to determine and modulate receptor binding specificity. Of particular interest are fusion proteins engineered with helix A and/or helix D, and fusion proteins that combine helical and loop domains from other short-form cytokines such as IL-2, IL-4, IL-15 and GM-CSF. The amino acid residues comprising helices A, B, C, and D, and loops A/B, B/C and C/D for zalpha31. IL-2, IL-4, IL- 15 and GM-CSF are shown in Table 1.
Table 1
POLYNUCLEOTIDES: The present invention also provides polynucleotide molecules, including
DNA and RNA molecules, that encode the zalpha31 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. SEQ ID NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha31 polypeptide of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:3 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, zalpha31 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 426 of SEQ ID NO:3 and their RNA equivalents are contemplated by the present invention. Table 2 sets forth the one-letter codes used within SEQ ID NO: 3 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
TABLE 2
Nucleotide Resolution Complement Resolution
A A T T
C C G G
G G C C
T T A A
R A|G Y C|T
Y C|T R A|G
A|C K G|T
K G|T M A|C
S C|G S C|G w A|T w A|T
H A|C|T D A|G|T
B C|G|T V A|C|G
V A|C|G B C|G|T
D A|G|T H A|C|T
N A|C|G|T N A|C|G|T
The degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 3.
TABLE 3
One
Amino Letter Codons Degenerate
Acid Code Codon
Cys C TGC TGT TGY
Ser S AGC AGT TCA TCC TCG TCT WSN
Thr T ACA ACC ACG ACT ACN
Pro P CCA CCC CCG CCT CCN
Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN
Asn N AAC AAT AAY
Asp D GAC GAT GAY
Glu E GAA GAG GAR
Gin Q CAA CAG CAR
His H CAC CAT CAY
Arg R AGA AGG CGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
He I ATA ATC ATT ATH
Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN
Phe F TTC TTT TTY
Tyr Y TAC TAT TAY
Tip W TGG TGG
Ter TAA TAG TGA TRR
Asn|Asp B RAY
Glu|Gln Z SAR
Any X NNN On of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." In general, see, Grantham, et al, Nuc. Acids Res. 5:1893-1912 (1980); Haas, et al. Curr. Biol. (5:315-324 (1996); Wain-Hobson, et al, Gene 73:355-364 (1981); Grosjean and Fiers, Gene 75:199-209 (1982); Holm, Nuc. Acids Res. 74:3075-3087 (1986); Ikemura, J. Mol. Biol. 158:573-597 (1982). As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 3). For example, 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.
Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Numerous equations for calculating T,„ are known in the art, and are specific for DNA. RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al, (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur. Crit. Rev. Biochem. Mol Biol. 26:227 (1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake. MN) and Primer Premier 4.0 (Premier Biosoft International: Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating Tm based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25°C below the calculated Tm. For smaller probes, <50 base pairs, hybridization is typically carried out at the Tm or 5- 10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the Tm of the hybrid about 1 °C for each 1% formamide in the buffer solution. Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 μg/ml denatured commercially-available carrier DNA. Generally, such stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2X SSC. For example, a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65°C. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
Typically, the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes. Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art. As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of Zalpha31 RNA. Such tissues and cells are identified by Northern blotting, Thomas, Proc. Natl Acad. Sci. USA 77:5201 (1980) and are discussed below. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in : CsCl gradient, Chirgwin et al., Biochemistry 18:52-94 (1979). Poly
(A)+ RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly(A)+ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding Zalpha31 polypeptides are then identified and isolated by, for example, hybridization or PCR.
A full-length clone encoding Zalpha31 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 Zalpha31 , receptor fragments, or other specific binding partners. The polynucleotides of the present invention can also be synthesized using DNA synthesis machines. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a DNA or a DNA fragment, then each complementary strand is made separately, for example via the phosphoramidite method known in the art. The production of short polynucleotides (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. However, for producing longer polynucleotides (longer than about 300 bp), special strategies are usually employed. For example, synthetic DNAs (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. One method for building a synthetic DNA involves producing a set of overlapping, complementary oligonucleotides. Each internal section of the DNA has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section. After the DNA is assembled, the process is completed by ligating the nicks along the backbones of the two strands. In addition to the protein coding sequence, synthetic DNAs can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector. Alternative ways to prepare a full-length DNA are also known in the art. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990. The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are Zalpha31 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zalpha31 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zalpha31 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 Zalpha31 -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. 4,683,202), using primers designed from the representative human Zalpha31 sequence disclosed herein. Within an additional method, 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 Zalpha31 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO:l represents a single allele of human Zalpha31 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 Zalpha31 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 Zalpha31 polypeptides that are substantially similar to the polypeptides of SEQ ID NO:2 and their orthologs. The term "substantially similar" is used herein to denote polypeptides having 70%, preferably 80%, more preferably at least 85%, sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 59:10915-10919 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 4 (amino acids are indicated by the standard one- letter codes). The percent identity is then calculated as:
Total number of identical matches x lOO
[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]
> ^
3-
H 1 U_ ^ H ro CN CN
1 1 1 e- r- < ro
(x. *JD ^ CN M ro
LΠ
-n H ro ro CN CN
1
^ CN CN 00 CN CN
1
<--< CN ro H ro
1
CO ro H CN CN CN CN CN ro
1 1 1 1 1 1 ϋ CN CN ro ro CN CN CN ro ro
1 1 1 1 1 1 1 w 1-1 O ro H CN H ro CN CN
1 1 1 1 1 1 α LD CN CN rH CN H CN 1 1 1 1 u σ. ro ro ro rH CN ro H CN CN 1 1 1
Q u> ro CN H ro ro CO H
1 1 1 1 1 1 I
1-3 rH ro o rH ro ro CN ro CN o ^-i CN ro
1 1 1 1 1 1 i-i o CM ro O CN O ro CN CN H r CN ro CN ro
1 1 1 C <Φ H CN CN o H CN H CN ro CN
1 1 1 1 1 rf. Oi a Q U U Ol H -I- J « Σ fc H EH ---
1-1 o
CN Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zalpha31. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat 'I Acad. Sci. USA 85:2444 (1988), and by Pearson. Meth. Enzymol 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAMJ. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62, with other parameters set as default. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990). FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
The BLOSUM62 table (Table 4) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat 'I Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed below), the language "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO:3. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat 'I Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the language "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0,1,2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1,2 or 3), while more preferred conservative substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3). Accordingly the present invention includes those polypeptides which are at least 90%, preferably 95% and most preferably 99% identical to SEQ ID NO: 3 and which are able to stimulate antibody production in a mammal, and said antibodies are able to bind the native sequence of SEQ ID NO:3. Variant Zalpha31 polypeptides or substantially homologous Zalpha31 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 5) 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 about 30 to about 175 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:2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the Zalpha31 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
Table 5
Conservative amino acid substitutions
Basic: arginine lysine histidine
Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Table 5 cont
Aromatic: phenylalanine tryptophan tyrosine
Small: glycine alanine serine threonine methionine
The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions. For example, a Zalpha31 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-Zalpha31 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric Zalpha31 analogs. Auxiliary domains can be fused to Zalpha31 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a Zalpha31 polypeptide or protein could be targeted to a predetermined cell type by fusing a Zalpha31 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 Zalpha31 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 34:1-9 (1996).
The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trα« -3-methylproline, 2,4-methanoproline, c -y-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, α/Zo-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. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722 (1991); Εllman et al, Methods Enzymol. 202:301 (1991 ; Chung et al, Science 259:806- 809 (1993); and Chung et al, Proc. Natl. Acad. Sci. USA 90:10145-1019 (1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs, Turcatti et al, J. Biol Chem. 277:19991-19998 (1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, oi 4-i'luorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al, Biochem. 35:7470-7476 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site- directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci. 2:395-403 (1993).
A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zalpha31 amino acid residues.
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al, Proc. Natl. Acad. Sci. USA 55:4498-502 (1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, J. Biol. Chem. 277:4699-708. 1996. Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al, Science 255:306-312 (1992); Smith et al, J. Mol. Biol. 224:899-904 (1992); Wlodaver et al, FEBS Lett. 309:59-64 (1992).
Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.. Current Opin. Struct. Biol. 6:3-10, 1996). In general, when designing modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
Amino acid sequence changes are made in zalpha31 polypeptides so as to minimize disruption of higher order structure essential to biological activity. For example, when the zalpha31 polypeptide comprises one or more helices, changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners. The effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al.,
Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are well known in the art compare folding of a variant protein to a standard molecule (e.g., the native protein). For example, comparison of the cysteine pattern in a variant and standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues which are associated with disulfide bonds or are free of such associations (Bean et al., Anal. Biochem. 201 :216- 226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generally believed that if a modified molecule does not have the same disulfide bonding pattern as the standard molecule folding would be affected. Another well known and accepted method for measuring folding is circular dichrosism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule is routine (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al, Science 257:961-964, 1992).
A Hopp/Woods hydrophilicity profile of the zalpha31 protein sequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981 ; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering J_: 153-169, 1998). The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. For example, in zalpha31, hydrophilic regions include: (1) amino acid residues 14 (Asp) to 19 (Asp) of SEQ ID NO: 2, (2) amino acid residues 26 (Ala) to 31 (Glu) of SEQ ID NO: 2, (3) amino acid 27 (Glu) to 32 (Pro) of SEQ ID NO: 2, (4) amino acid residues 136 (Tyr) to 141 (Lys) of SEQ ID NO: 2, and (5) amino acid residues 137 (Lys) to 142 (Arg) of SEQ ID NO: 2.
Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a zalpha31 polypeptide, so as not to disrupt the overall structural and biological profile. Of particular interest for replacement are hydrophobic residues selected from the group consisting of Val, Leu and He or the group consisting of Met,
Gly, Ser, Ala, Tyr and Tip. For example, residues tolerant of substitution could include such residues as shown in SEQ ID NO:2. However, Cysteine residues could be relatively intolerant of substitution.
The identities of essential amino acids can also be inferred from analysis of sequence similarity between other cytokine family members with zalpha31. Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions. An alternative approach to identifying a variant zalpha31 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zalpha31 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 , as discussed above.
Other methods of identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, J. Biol. Chem. 271 :4699 (1996).
The present invention also includes functional fragments of zalpha31 polypeptides and nucleic acid molecules encoding such functional fragments. A "functional" zalpha31 or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti- zalpha31 antibody or zalpha31 receptor (either soluble or immobilized). As previously described herein, zalpha31 is characterized by a four-helical-bundle structure comprising helix A (amino acid residues 37-51), helix B (amino acid residues 65-79), helix C (amino acid residues 87- 101) and helix D (amino acid residues 118-132), as shown in SEQ ID NO: 2. Thus, the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the helices described above; and (b) functional fragments comprising one or more of these helices. The other polypeptide portion of the fusion protein may be contributed by another four-helical-bundle cytokine, such as IL-15, IL-2, IL-4 and GM-CSF, or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein. Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zalpha31 polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO:l or fragments thereof, can be digested with Bal3\ nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zalpha31 activity, or for the ability to bind anti-zalpha31 antibodies or zalpha31 receptor. One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zalpha31 fragment. Alternatively, particular fragments of a zalpha31 polynucleotide can be synthesized using the polymerase chain reaction.
Standard methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993); Content et al., "Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems. Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation j_j Boynton et al., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem.
270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel et al., Plant Molec. Biol. 30:1 (1996).
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and
Sauer, Science 241:53-57 (1988) or Bowie and Sauer, Proc. Natl. Acad. Sci. USA 5(5:2152-2156 (1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display, e.g., Lowman et al, Biochem. 50:10832-10837 (1991); Ladner et al, U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis, Derbyshire et al, Gene 46:145 (1986); Ner et al, DNA 7:127 (1988).
Variants of the disclosed Zalpha31 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, (1994), Stemmer, Proc. Natl Acad. Sci. USA 97:10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro 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. Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below. 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.
In addition, the proteins of the present invention (or polypeptide fragments thereof) can be joined to other bioactive molecules, particularly other cytokines, to provide multi-functional molecules. For example, one or more helices from zalpha31 can be joined to other cytokines to enhance their biological properties or efficiency of production.
The present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the helices of zalpha31 is fused to another polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmacodynamics. Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NOs:2, 4 or 6 or that retain the properties of the wild-type Zalpha31 protein. For any Zalpha31 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 2 and 3 above.
PROTEIN PRODUCTION
The Zalpha31 polypeptides of the present invention, including full- length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and Ausubel et al, eds., Current
Protocols in Molecular Biology (John Wiley and Sons, Inc., NY, 1987). In general, a DNA sequence encoding a Zalpha31 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.
To direct a Zalpha31 polypeptide into the secretory pathway of a host cell, 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 Zalpha31 , 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 Zalpha31 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. Patent No. 5,037,743; Holland et al, U.S. Patent No. 5,143,830).
Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence that encodes a signal peptide from amino acids 1 (Met) to 19 (Asp) of SEQ ID NO:2 is operably linked to another DNA segment encoding a polypeptide using methods known in the art and disclosed herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions can be used in vivo or in vitro 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 Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al, EMBO J. 7:841-845 (1982), DEAE- dextran mediated transfection (Ausubel et al, ibid., and liposome-mediated transfection, Hawley-Nelson et al, Focus 75:73 (1993); Ciccarone et al, Focus 75:80 (1993), and viral vectors, Miller and Rosman, BioTechniques 7:980(1989); Wang and Finer, Nature Med. 2:714 (1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al, U.S. Patent No. 4,713,339; Hagen et al, U.S. Patent No. 4,784,950; Palmiter et al, U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. 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 -./., J. Gen. Virol. 36:59 (1977) and Chinese hamster ovary (e.g. CHO- Kl; ATCC No. CCL 61) cell lines. Additional suitable cell hues are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter. Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems 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) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MH C, 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. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. /Bangalore) 11 :47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide. London, Chapman & Hall; O'Reilly, D.R. et al.,
Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system is sold in the Bac-to- Bac™ kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zalpha31 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zalpha31. However, pFastBacl™ 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 :971-6, 1990; Bonning, B.C. et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B., J. Biol. Chem. 270:1543-9, 1995. In such transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed which replace the native zalpha31 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, 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 zalpha31 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zalpha31 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the art, a transfer vector containing zalpha31 is transformed into 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 zalpha31 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.
The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or
Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 105 cells to a density of 1-2 x 106 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. 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, C. D., ibid.). Subsequent purification of the zalpha31 polypeptide from the supernatant can be achieved using methods described herein.
Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae. Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are unclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al, U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al, U.S. Patent No. 5,037,743; and Murray et al, U.S. Patent No. 4,845,075. 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 POT! vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al, U.S.
Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol. 132:3459 (1986) and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al, U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or A UG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. 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. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (A UG1 and A UG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP 4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. <acthanolica 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., Sambrook et al, ibid.). When expressing a Zalpha31 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. In the latter case, 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. A variety of 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% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a Zalpha31 polypeptide of the invention. The epitope of the this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. A region of a protein to which an antibody can bind is defined as an "antigenic epitope". See for instance, Gey sen, H.M. et al, Proc. Natl. Acad Sci. USA 57:3998-4002 (1984). As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in the art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See Sutcliffe, J.G. et al Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues, usually are effective.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that react with the protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred. All of the polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention, however, specifically designed antigenic epitopes include the peptides predicted, for example, from a Jameson- Wolf plot, comprising: (1) amino acid residues 11 (Thr) to 20 (Asp) of SEQ ID NO:2; (2) amino acid residues 60 (Ser) to 64 (Lys) of SEQ ID NO:2; (3) amino acid residues 88 (Ser) to 96 (Gin) of SEQ ID NO:2; (4) amino acid residues 127 (Ala) to 135 (Lys) of SEQ ID NO:2; and (5) amino acid residues 127 (Ala) to 139 (Leu) of SEQ ID NO:2. . The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zalphal polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al, supra. See also U.S. Patent No. 4,708,781 (1987) further describes how to identify a peptide bearing an immunogenic epitope of a desired protein.
PROTEIN ISOLATION
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 pretc. ed is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
Expressed recombinant Zalpha31 polypeptides (or chimeric Zalpha31 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. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988). The polypeptides of the present invention can be isolated by exploitation of their structural and biological properties. For example, 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. 5:1 (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 Enzymol, Vol. 7 ,2, "Guide to Protein Purification", M. Deutscher, (ed.),page 529-539 (Acad. Press, San Diego, 1990). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.
Moreover, using methods described in the art, polypeptide fusions, or hybrid Zalpha31 proteins, are constructed using regions or domains of the inventive
Zalpha31, Sambrook et al, ibid., Altschul et al, ibid., Picard, Cur. Opin. Biology, 5:511 (1994). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure. 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 Zalpha31 of the present invention with the functionally equivalent domain(s) from another family member, e.g., IL-2, IL-4, GM-CSF, or other four-helix bundle cytokine family member. Such domains include, but are not limited to, the secretory signal sequence, helices A through D, 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. Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptides that have substantially similar sequence identity to residues 1-142 or 20-142 of SEQ ID NO: 2, or functional fragments and fusions thereof, wherein such polypeptides or fragments or fusions retain the properties of the wild-type protein such as the ability to stimulate proliferation, differentiation, induce specialized cell function or bind a cell or zalpha31 receptor or anti-zalpha31 antibodies.
Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zalpha31 polypeptide and those polypeptides to which they are fused. Generally, a DNA segment that encodes a domain of interest, e.g., a zalpha31 helix A through D, or motif described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide and inserted into an appropriate expression vector, as described herein. Generally DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof. For example, a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by Helix A, followed by Helix B, followed by Helix C, followed by Helix D, or as interchanged with equivalent regions from another protein. Such fusion proteins can be expressed, isolated, and assayed for activity as described herein.
Zalpha31 polypeptides or fragments thereof may also be prepared through chemical synthesis. Zalpha31 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 of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art. The activity of molecules of the present invention can be measured using a variety of assays that measure for example, signal transduction, Ig binding or cAMP modulation. Such assays are well known in the art. For a general reference, see Nihei, Y., et al.. supra.; and Rindisbacher, L., et al., supra..
ASSAYS
The activity of molecules of the present invention can be measured using a variety of assays. Of particular interest are changes in steroidogenesis, spermatogenesis, in the testis, LH and FSH production and GnRH in the hypothalamus.
Such assays are well known in the art.
Proteins of the present invention are useful for example in increasing sperm production, treating thyroid, adrenal, lymphoid, inflammatory, pancreatic, blood or bone disorders, can be measured in vitro using cultured cells or in vivo by administering molecules of the present invention to the appropriate animal model. For instance, host cells expressing a secreted form of zalpha31 polypeptide can be embedded in an alginate environment and injected (implanted) into recipient animals. Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells. These types of non-immunogenic "encapsulations" permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal. Most importantly, the capsules mask and shield the foreign, embedded cells from the recipient animal's immune response. Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Alginate threads provide a simple and quick means for generating embedded cells.
Tho materials needed to generate the alginate threads are known in the art. In an exemplary procedure, 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5 x 10* to about 5 x 107 cells/ml) is mixed with the 3% alginate solution. One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl2 solution over a time period of ~15 min, forming a "thread". The extruded thread is then transferred into a solution of 50 mM CaCl2, and then into a solution of 25 mM CaC_2- The thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine. Finally, the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle). A large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution.
An alternative in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, 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 review, see T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997). The adenovirus system offers several advantages: (i) adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
Using adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, 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). 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. However, the host's tissue (e.g., liver) 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.
Moreover, adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses are El deleted, and in addition contain deletions o _ I--.2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998). In addition, deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover, by deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated. Generation of so called "gutless" adenoviruses where all viral genes are deleted are particularly advantageous for insertion of large inserts of heterologous DNA. For review, see Yeh, P. and Perricaudet, M., FASEB J. 11 :615-623, 1997.
The adenovirus system can also be used for protein production in vitro. By culturing adeπ.. virus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
The activity of molecules of the present invention can be measured using a variety of assays that measure proliferation of and/or binding to cells. Of particular interest are changes in zalpha31 -dependent cells. Suitable cell lines to be engineered to be zalpha31 -dependent include the IL-3 -dependent BaF3 cell line (Palacios and Steinmetz. Cell 41 : 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). However, other growth factor-dependent cell lines, such as FDC-P1 (Hapel et al., Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993) are suitable for this purpose. Growth factor-dependent cell lines can be established according to published methods (e.g. Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexter et al., in Baum et al. Eds.. Experimental Hematology Today, 8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).
Proteins of the present invention are useful for stimulating proliferation, activation, differentiation and/or induction or inhibition of specialized cell function of cells of the involved homeostasis of the hematopoiesis and immune function. In particular, zalpha31 polypeptides are useful for stimulating proliferation, activation, differentiation, induction or inhibition of specialized cell functions of cells of the hematopoietic lineages, including, but not limited to, T cells, B cells, NK cells, dendritic cells, monocytes, and macrophages, as well as epithelial cells. Proliferation and/or differentiation of hematopoietic cells can be measured in vitro using cultured cells or in vivo by administering molecules of the present invention to the appropriate animal model. Assays measuring cell proliferation or differentiation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990, incorporated herein by reference), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incorporated herein by reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporated herein by reference), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988; all incorporated herein by reference). Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt. FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes. Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
As a ligand, the activity of zalpha31 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary device is the Cytosensor™ Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non- adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha31 polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zalpha31 -responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zalpha31 polypeptide. ZALPHA31 -responsive eukaryotic cells comprise cells into which a receptor for zalpha31 has been transfected creating a cell that is responsive to zalpha31 ; or cells naturally responsive to zalpha31 such as cells derived from small intestine, PBLs, or bone marrow tissue. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zalpha31 polypeptide, relative to a control not exposed to zalpha31, are a direct measurement of zalpha31 -modulated cellular responses. Moreover, such zalpha31 -modulated responses can be assayed under a variety of stimuli. Using the microphysiometer, there is provided a method of identifying agonists of zalpha31 polypeptide, comprising providing cells responsive to a zalpha31 polypeptide. culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Moreover, culturing a third portion of the cells in the presence of zalpha31 polypeptide and the absence of a test compound can be used as a positive control for the zalpha31 -responsive cells, and as a control to compare the agonist activity of a test compound with that of the zalpha31 polypeptide. Moreover, using the microphysiometer, there is provided a method of identifying antagonists of zalpha31 polypeptide, comprising providing cells responsive to a zalpha31 polypeptide, culturing a first portion of the cells in the presence of zalpha31 and the absence of a test compound, culturing a second portion of the cells in the presence of zalpha31 and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Antagonists and agonists, for zalpha31 polypeptide, can be rapidly identified using this method.
Moreover, zalpha31 can be used to identify cells, tissues, or cell lines which respond to a zalpha31 -stimulated pathway. The microphysiometer, described above, can be used to rapidly identify ligand-responsive cells, such as cells responsive to zalpha31 of the present invention. Cells can be cultured in the presence or absence of zalpha31 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zalpha31 are responsive to zalpha31. Such cell lines, can be used to identify antagonists and agonists of zalpha31 polypeptide as described above.
Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction. Also as a treatment for prostate cancer. Inhibitors of Zalpha31 activity (Zalpha31 antagonists) include anti-Zalpha31 antibodies and soluble Zalpha31 receptors, as well as other peptidic and non-peptidic agents (including ribozymes). Zalpha31 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 Zalpha31. In addition to those assays disclosed herein, samples can be tested for inhibition of Zalpha31 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of Zalpha31- dependent cellular responses. For example, Zalpha31 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a Zalpha31 -stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a Zalpha31-DNA response element operably linked to a gene encoding a protein which can be assayed, such as luciferase. .UNA 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. Natl. Acad. Sci. USA 87:5273 (1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563 (1989). Cyclic AMP response elements are reviewed in Roestler et al, J. Biol. Chem. 263 (19):9063 (1988) and Habener, Molec. Endocrinol 4 (8):1087 (1990). Hormone response elements are reviewed in Beato, Cell 56:335 (1989). Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of Zalpha31 on the target cells as evidenced by a decrease in Zalpha31 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block Zalpha31 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 Zalpha31 binding to receptor using Zalpha31 tagged with a detectable label (e.g., I25I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled Zalpha31 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
A Zalpha31 polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two constant region domains and lacks the variable region. 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. For use in assays, the chimeras are bound to a support via the Fc region and used in an ELISA format.
A Zalpha31 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. 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. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods
145:229 (1991) and Cunningham and Wells, J. Mol. Biol. 234:554 (1993). A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. 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 255:545 (1991); Cunningham et al. Science 245:821 (1991).
Zalpha31 polypeptides can also be used to prepare antibodies that bind to zalpha31 epitopes, peptides or polypeptides. The zalpha31 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. One of skill in the art would recognize that antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zalpha31 polypeptide (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a zalpha31 polypeptide, i.e., from 30 to 10 residues up to the entire length of the amino acid sequence are included. Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the mature zalpha31 polypeptide encoded by SEQ ID NO:2 from amino acid number 1 (Met) to 142 (Arg) of SEQ ID NO:2, or a contiguous 9 to 122 amino acid fragment thereof. Other suitable antigens include alpha helical domains, extracellular domains, motifs, regions, epitopes, etc., as disclosed herein. Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot. Zalpha31 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: those predicted 6 amino acid antigenic epitopes determined from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored. For example, in zalpha31, suitable hydrophilic regions include: (1) amino acid residues 14 (Asp) to 19 (Asp) of SEQ ID NO: 2, (2) amino acid residues 26 (Ala) to 31 (Glu) of SEQ ID NO: 2, (3) amino acid 27 (Glu) to 32 (Pro) of SEQ ID NO: 2, (4) amino acid residues 136 (Tyr) to 141 (Lys) of SEQ ID NO: 2, and (5) amino acid residues 137 (Lys) to 142 (Arg) of SEQ ID NO: 2. Suitable hydrophilic peptides also include those antigenic epitopes predicted from a Jameson- Wolf plot, comprising: (1) amino acid residues 11 (Thr) to 20 (Asp) of SEQ ID NO:2; (2) amino acid residues 60 (Ser) to 64 (Lys) of SEQ ID NO:2; (3) amino acid residues 88 (Ser) to 96 (Gin) of SEQ ID NO:2; (4) amino acid residues 127 (Ala) to 135 (Lys) of SEQ ID NO:2; and (5) amino acid residues 127 (Ala) to 139 (Leu) of SEQ ID NO:2. Antibodies from an immune response generated by inoculation of an animal with these antigens 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 Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.
As would be evident to one of ordinary skill in the art, 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 zalpha31 polypeptide or a fragment thereof. The immunogenicity of a zalpha31 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 zalpha31 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.
As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. 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). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination. Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules. A threshold level of binding is determined if anti- zalpha31 antibodies herein bind to a zalpha31 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zalpha31) polypeptide. It is preferred that the antibodies exhibit a binding affinity (Ka) of 10 M"
1 or greater, preferably 107 M-1 or greater, more preferably 108 M-1 or greater, and most preferably 10 9 M -1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis
(Scatchard, G., Ann. NY Acad. Sci. 51 : 660-672, 1949). Whether anti-zalpha31 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zalpha31 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.). Examples of known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family (e.g., other four-helix bundle cytokines), Screening can also be done using non-human zalpha31 , and zalpha31 mutant polypeptides. Moreover, antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zalpha31 polypeptides. For example, antibodies raised to zalpha31 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zalpha31 will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al.. Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice. Goding, J.W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically binding anti-zalpha31 antibodies can be detected by a number of methods in the art, and disclosed below.
A variety of assays known to those skilled in the art can be utilized to detect antibodies which bind to zalpha31 proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zalpha31 protein or polypeptide. Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zalpha31 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zalpha31 protein or peptide). Genes encoding polypeptides having potential zalpha31 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. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and 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 zalpha31 sequences disclosed herein to identify proteins which bind to zalpha31. These "binding polypeptides" which interact with zalpha31 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. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of zalpha31 polypeptides; for detecting or quantitating soluble zalpha31 polypeptides as marker of underlying pathology or disease. These binding polypeptides can also act as zalpha31 "antagonists" to block zalpha31 binding and signal transduction in vitro and in vivo. These anti-zalpha31 binding polypeptides would be useful for inhibiting zalpha31 activity or protein-binding. Antibodies to zalpha31 may be used for tagging cells that express zalpha31; for isolating zalpha31 by affinity purification; for diagnostic assays for determining circulating levels of zalpha31 polypeptides; for detecting or quantitating soluble zalpha31 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 zalpha31 activity in vitro 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. Moreover, antibodies to zairha31 or fragments thereof may be used in vitro to detect denatured zalpha31 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
BIOACTIVE CONJUGATES:
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. For instance, 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). More specifically, Zalpha31 polypeptides or anti-Zalpha31 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. For indirect attachment of a detectable or cytotoxic molecule, 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. For these purposes, biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
In another embodiment, 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). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain), 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. In instances where 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. In another embodiment, Zalpha31 -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 Zalpha31 polypeptide or anti- Zalpha31 antibody targets the hyperproliferative blood or bone marrow cell. See, generally, Homick et al, Blood 89:4437 (1997). They described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable Zalpha31 polypeptides or anti-Zalpha31 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. In yet another embodiment, if the Zalpha31 polypeptide or anti- Zalpha31 antibody targets vascular cells or tissues, such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. For instance, iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.
The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action. Molecules of the present invention can be used to identify and isolate receptors involved in spermatogenesis, steroidogenesis, testicular differentiation and regulatory control of the hypothalamic-pituitary-gonadal axis, thyroid, heart and adrenal function. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column, Immobilized Affinity Ligand Techniques, Hermanson et al, eds., pp.195-202 (Academic
Press, San Diego, CA, 1992,). Proteins and peptides can also be radiolabeled, Methods in Enzymol, vol. 182, "Guide to Protein Purification", M. Deutscher, ed., pp 721-737 (Acad. Press, San Diego, 1990) or photoaffinity labeled, Brunner et al, Ann. Rev. Biochem. 62:483-514 (1993) and Fedan et al, Biochem. Pharmacol 55:1167 (1984) and specific cell-surface proteins can be identified.
The molecules of the present invention will be useful for testing disorders of the reproductive system, thyroid, adrenal, heart and immunological systems.
Zalpha31 represents a novel polypeptide with a putative signal peptide leader sequence and alpha helical structure. Therefore this gene may encode a secreted polypeptide with secondary structure indicating it is a member of the four helix bundle cytokine family. Alternatively, this polypeptide may have other activities associated with other biological functions including: enzymatic activity, association with the cell membrane, or function as a carrier protein.
Most four-helix bundle cytokines as well as other proteins produced by activated T lymphocytes play an important biological role in cell differentiation, activation, recruitment and homeostatsis of cells throughout the body. Therapeutic utility includes treatment of diseases which require immune regulation including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythomatosis and diabetes. Zalpha31 may be important in the regulation of inflammation and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis. There may be a role of zalpha31 in mediating tumorgenesis and therefore would be useful in the treatment of cancer. Zalpha31 may be a potential therapeutic in suppressing the immune system which would be important for reducing graft rejection. Alternatively zalpha31 may activate the immune system which would be important in boosting immunity to infectious diseases or in improving vaccines.
Thyroid malfunction and some of the currently associated therapies can elicit detrimental effects on bone in vivo. Thus, given the thyroidal and pituitary localization of the present invention, assays that measure Done formation and/or resorption are important assays to assess zalpha31 activity. One example is an assay system that permits rapid identification of substances having selective calcitonin receptor activity on cells expressing the calcitonin receptor. The calcitonin receptor is a member of the G-protein receptor family and transduces signal via activation of adenylate cyclase, leading to elevation of cellular cAMP levels (Lin et al., Science 254:1022-24, 1991). This assay system exploits the receptor's ability to elevate cAMP levels as a way to detect other molecules that are able to stimulate the calcitonin receptor and initiate signal transduction. Other assays that measure bone formation or resorption include calvarial assays, QCT, and assays that measure osteoblast size and number. Such assays are known in the art and discussed below. Receptor activation can be detected by: (1) measurement of adenylate cyclase activity (Salomon et al., Anal. Biochem. 58:541-48. 1974; Alvarez and Daniels, Anal. Biochem. 1_87:98-103, 1990); (2) measurement of change in intracellular cAMP levels using conventional radioimmunoassay methods (Steiner et al., J. Biol. Chem. 247:1106-13, 1972; Harper and Brooker, J. Cyc. Nucl. Res. L207-18, 1975); or (3) through use of a cAMP scintillation proximity assay (SPA) method (Amersham Corp., Arlington Heights, IL). While these methods provide sensitivity and accuracy, they involve considerable sample processing prior to assay, are time consuming, involve the use of radioisotopes, and would be cumbersome for large scale screening assays.
An alternative assay system involves selection of polypeptides that are able to induce expression of a cyclic AMP response element (CRE)-luciferase reporter gene, as a consequence of elevated cAMP levels, in cells expressing a calcitonin receptor, but not in cells lacking calcitonin receptor expression, as described in U.S. patent No. 5,622,839, U.S. Patent No. 5,674,689, and U.S. patent No. 5,674,981.
Well established animal models are available to test in vivo efficacy of zalpha31 polypeptides, agonists or antagonists, that interact with the calcitonin receptor. Moreover, these models may be used to test effects of zalpha31 on bone other than through the calcitonin receptor. For example, the hypocalcemic rat or mouse model can be used to determine the effect on serum calcium, and the ovariectomized rat or mouse can be used as a model system for osteoporosis. Bone changes seen in these models and in humans during the early stages of estrogen deficiency are qualitatively similar. Calcitonin has been shown to be an effective agent for the prevention of bone loss in ovariectomized women and rats (Mazzuoli et al., Calcif. Tissue Int. 47:209-14, 1990; Wronski et al., Endocrinology 129:2246-50, 1991). High dose estrogen has been shown to inhibit bone resorption and to stimulate bone formation in an ovariectomized mouse model (Bain et al., J. Bone Miner. Res. 8:435-42, 1993). Biologically active zalpha31 polypeptides, agonists or antagonists, of the present invention that interact with the calcitonin receptor, or exert other effects on bone, are therefore contemplated to be advantageous for use in therapeutic applications for which calcitonin is useful. Such applications, for example, are in the treatment of osteoporosis, Paget's disease, hyperparathyroidism, osteomalacia, idiopathic hypercalcemia of infancy and other conditions. Additional applications are to inhibit gastric secretion in the treatment of acute pancreatitis and gastrointestinal disorders, and uses as analgesics, in particular for bone pain.
In vivo assays for measuring changes in bone formation rates include performing bone histology (see, Recker, R., eds. Bone Histomorphometry: Techniques and Interpretation. Boca Raton: CRC Press, Inc., 1983) and quantitative computed tomography (QCT; Ferretti . Bone ]7:353S-364S, 1995; Orphanoludakis et al., Investig. Radiol. 14:122-130, 1979; and Durand et al., Medical Physics 19:569-573, 1992). An exemplary ex vivo assay for measuring changes in bone formation is a calavarial assay (Gowen et al., J. Immunol. 136:2478-2482, 1986) or resorption calvarial assay (Linkhart, T.A., and Mohan, S., Endocrinology 125:1484-1491, 1989).
In addition, polypeptides of the present invention can be assayed and used for their ability to modify inflammation. Methods to determine proinflammatory and antiinflammatory qualities of zalpha31 are known in the art and discussed herein.
For example, suppression of cAMP production is an indication of anti-inflammatory effects (Nihei, Y., et al., Arch. Dermatol. Res.. 287:546-552, 1995). Suppression of cAMP and inhibition of ICAM and HLA-Dr induced by IFN-γ in keratinocytes can be used to assess inhibition of inflammation. Alternatively, enhancement of cAMP production and induction of ICAM and HLA-Dr in this system can be an measurement of proinflammatory effects of a protein. Zalpha31. likewise can exhibit similar inflammatory effects, as shown in vivo (Example 8) and may exert these effects in tissues in which it is expressed, or in other tissues. For example, zalpha31 is expressed in the colon, and can be useful in promoting wound healing in this tissue, or exhibit anti-bacterial or anti-viral effects. Moreover, zalpha31 or its antagonists can be useful in treatment of inflammatory bowel disease, diverticulitis, inflammation during and after intestinal surgery, and the like. In addition, zalpha31, expressed in thyroid, can have wound-healing or antimicrobial or antiviral actions in tissues outside of thyroid, such, as heart, brain, liver, kidney, and the like. Moreover, direct measurement of zalpha31 polypeptide and zalpha31 antibodies can be useful in diagnosing inflammatory diseases such as melanoma, inflammatory bowel disease, diverticulitis, asthma, pelvic inflammatory disease, (PID), psoriasis, arthritis, reperfusion ischemia, and other inflammatory diseases. Moreover zalpha31, antagonists can be useful in treatment of myocarditis, atherosclerosis, pelvic inflammatory disease. (PID), psoriasis, arthritis, eczema, scleroderma, and other inflammatory diseases.
As such, zalpha31 polypeptide, agonists or its antagonists, have potential uses in inflammatory diseases such as asthma and arthritis. For example, if zalpha31 is proinflammatory, antagonists would be valuable in asthma therapy or other anti- inflammatory therapies where migration of lymphocytes is damaging. In addition, zalpha31 can serve other important roles in lung function, for instance, bronchodilation, tissue elasticity, recmitment of lymphocytes in lung infection and damage. Assays to assess the activity of zalpha31 in lung cells are discussed in Laberge, S. et al., Am. J. Respir. Cell Mol. Biol. 17:193-202, 1997; Rumsaeng, V. et al, J. Immunol., 159:2904- 2910, 1997; and Schluesener, H.J. et al., J. Neurosci. Res. 44:606-611, 1996. Methods to determine proinflammatory and antiinflammatory qualities of zalpha31 its agonists or its antagonists are known in the art. Moreover, other molecular biological, immunological, and biochemical techniques known in the art and disclosed herein can be used to determine zalpha31 activity and to isolate agonists and antagonists.
While the Northern blot (Example 2) for zalpha31 shows relatively ubiquitous distribution of the gene, the electronic northern is very informative. The large size of the accessible EST database is such that the incidence of a gene, such as zalpha31 , in the data is suggestive of the expression levels found in the respective library (i.e. rare, regulated genes are underrepresented in most libraries while highly abundant or inducible genes have high copy number).
The data for zalpha31 suggests a highly inducible gene that is prevalent in B-cells (tonsils) and those cells of the monocyte/macrophage/dendritic lineage, (consistent with a ubiquitous distribution in the "normal", non-induced state) and particularly following treatment with a proinflammatory stimulus such as PMA, TNF, or LPS. This conclusion is based on its presence at higher than expected frequency in the following tissues: germinal center B cell (tonsil) library, multiple sclerosis lesions; stimulated THP-1 cells (pro-monocyte line); and peripheral blood dendritic cells (stimulated). The gene was also found in a T-cell line (stimulated) and peripheral blood mononuclear cells (stimulated), fetal liver CD34+ progenitor cells and cartilage from an osteoarthritis patient showing a tendency to be associated with inflammatory events. Moreover, there was a significant number of ESTs found in libraries of neural origin, both tissues (normal and diseased) and differentiated cell lines (neurons). The disease association shows up with: multiple sclerosis, Huntingtons, gliosis, oligoastrocytoma, brain tumor (e.g., mets hypemephroma), and spinal cord w/lymphoma, of which zalpha31 may be associated with activated immune responses associated with those diseases. Other proinflammatory cytokines are produced by brain tumors, found in MS lesions, and exhibit other neuropathies (Fontana, A. et al., J. Immunol. 132:1837-1844, 1984; Suarez, GA et al., Neurology 46:559-561, 1996) Moreover, abnormalities in single cytokines can lead to neurological disease, such as by inducing immune cell infiltration into neurological tissues (Hanisch, UK et al., Synapse 24:104-114, 1996; Sugita, Y et al, J. Neuropathol. Exp. Neurol. 58:480-488, 1999); or both neurological and immune disease (Zhu, J. et al., J. Neurol. Sci. 125:132-137, 1994; Zhu, J. et al., J. Neurosci. Res. 54:373-381, 1998).
Moreover, an antagonist of zalpha31 would be predicted to be an anti- inflammatory agent. Agonists and antagonists may be useful for a broad range on neural diseases such as; MS or Huntington's Disease, while a cytokine-radionuclide (or similar) may be useful for neural tumors. Moreover, zalpha31 may act indirectly or in conjunction with other cytokines in exerting it's effects. For example, an interferon, IL-1 or IL-2 could exacerbate a disease in which there is an ir.ϋammation component. As such, zalpha31 antagonists that dissociate or block the combined effects of various cytokines, are also useful.
Moreover, there appears to be a "neurological disease cluster" present at the chromosomal localization site for zalpha31 as discussed below. This suggests that zalpha31 polypeptides, polynucleotides or antibodies can be used as diagnostics for neurologic diseases or to determine genetic susceptibility to such diseases.
The molecules of the present invention can be useful for proliferation of cardiac tissue cells, such as cardiac myocytes or myoblasts; skeletal myocytes or myoblasts and smooth muscle cells; chrondrocytes; endothelial cells; adipocytes and osteoblasts in vitro. For example, molecules of the present invention are useful as components of defined cell culture media, and can be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Molecules of the present invention are particularly useful in specifically promoting the growth and/or development of myocytes in culture, and may also prove useful in the study of cardiac myocyte hyperplasia and regeneration.
The polypeptides, nucleic acids and/or antibodies of the present invention can be used in treatment of disorders associated with myocardial infarction, congestive heart failure, hypertrophic cardiomyopathy and dilated cardiomyopathy. Molecules of the present invention may also be useful for limiting infarct size following a heart attack, aiding in recovery after heart transplantation, promoting angiogenesis and wound healing following angioplasty or endarterectomy, to develop coronary collateral circulation, for revascularization in the eye, for complications related to poor circulation such π - diabetic foot ulcers, for stroke, following coronary reperfusion using pharmacologic methods, and other indications where angiogenesis is of benefit. Molecules of the present invention may be useful for improving cardiac function, either by inducing cardiac myocyte neogenesis and/or hyperplasia, by inducing coronary collateral development, or by inducing remodeling of necrotic myocardial area. Other therapeutic uses for the present invention include induction of skeletal muscle neogenesis and/or hyperplasia, kidney regeneration and/or for treatment of systemic and pulmonary hypertension.
Zalpha31 induced coronary collateral development is measured in rabbits, dogs or pigs using models of chronic coronary occlusion (Landau et al., Amer.
Heart 29:924-931, 1995; Sellke et al., Surgery 120(2)182-188, 1996; and Lazarous et al., 1996, ibid.) Zalpha31 efficacy for treating stroke is tested in vivo in rats, utilizing bilateral carotid artery occlusion and measuring histological changes, as well as maze performance (Gage et al., Neurobiol. Aging 9:645-655, 1988). Zalpha31 efficacy in hypertension is tested in vivo utilizing spontaneously hypertensive rats (SHR) for systemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol. Suppl. 1 :S114- 116, 1995).
Moreover, based on high expression in thyriod, zalpha31 polypeptide may exhibit antiviral activity by inhibiting viral replication by specific signaling via it's receptor(s) on a host cell (e.g. T-cell). Zalpha31 can exhibit immune cell proliferative activity (See. example 8), can be assayed for this activity as disclosed herein, and may stimulate the immune system to fight viral infections. Moreover, zalpha31 may bind CD4 or another lymphocyte receptor and exhibit antiviral effects, for example, against human immunodeficiency virus (HIV) or human T-cell lymphotropic virus (HTLV). Alternatively, zalpha31 polypeptide may compete for a viral receptor or co-receptor to block viral infection. Zalpha31 may be given parentally to prevent viral infection or to reduce ongoing viral replication and re-infection (Gayowski, T. et al., Transplantation 64:422-426, 1997). Thus, zalpha31 may be used as an antiviral therapeutic, for example, for viral leukemias (HTLV), AIDS (HIV), or gastrointestinal viral infections caused by, for example, rotavirus, calicivirus (e.g., Norwalk Agent) and certain strains of pathogenic adenovirus.
Both zalpha31 modulated direct and indirect inflammation can be assayed by methods in the art. Foe example see, Hamada. T. et al. J. Exp. Med. 188:539-548, 1998; and Liu, L. et al., J. Immunol. 161 :3064-3070, 1998. For example, proinflammatory effects of zalpha31 polypeptide can be directly tested in assays using a Transwell™ (Costar), wherein endothelial cells are plated on a semi-permeable membrane and zalpha31 polypeptide is present in the lower chamber of the transwell and Cr51 or fluorescently-labeled neutrophils (PMNs), lymphocytes, HL60 cells, K562 cells, or the like are added on to the upper chamber of the transwell. Migration of the PMNs and the like to the lower chamber of the transwell in the presence of zalpha31 polypeptide, but not its absence (Negative control), would demonstrate zalpha31 polypeptide as a direct chemoattractant of the PMNs. Moreover, IL-8 could be employed in this assay as a positive control. To test zalpha31 as indirect stimulator of inflammatory response, a similar method can be employed. For example, an experiment can be set up as per above where in addition to the presence of zalpha31 on the lower chamber of the transwell, fibroblast or adipocytes are plated there. In this way, effects of zalpha31 polypeptide in inducing these cells to secrete factors that enhance migration of PMNs, i.e., inflammation, can be measured. The bFGF can be used as a positive control for indirect assay. Anti-inflammatory effects of zalpha31 polypeptide can also be measured when added on the upper chamber in the presence of PMN's using a similar transwell assay known in the art. The activity of molecules of the present invention may be measured using a variety of assays that, for example, measure neogenesis or hyperplasia (i.e., proliferation) of cardiac cells based on the potential effects of extrathyroidal activity of zalpha31. Additional activities likely associated with the polypeptides of the present invention include proliferation of endothelial cells, cardiomyocytes, fibroblasts, skeletal myocytes directly or indirectly through other growth factors; action as a chemotaxic factor for endothelial cells, fibroblasts and/or phagocytic cells; osteogenic factor; and factor for expanding mesenchymal stem cell and precursor populations.
Proliferation can be measured using cultured cardiac cells or in vivo by administering molecules of the present invention to the appropriate animal model. Generally, proliferative effects are seen as an increase in cell number, and may include inhibition of apoptosis as well as stimulation of mitogenesis. Cultured cells for use in these assays include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, and human umbilical vein endothelial cells from primary cultures. Suitable established cell lines include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No. CRL-1446), Shionogi mammary carcinoma cells (Tanaka et al., Proc. Natl. Acad. Sci. 89:8928-8932, 1992), and LNCap.FGC adenocarcinoma cells (ATCC No. CRL-1740.) Assays measuring cell proliferation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al.,
Investigational New Drugs 8:347-354, 1990), incorporation of radiolabeled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of 5-bromo-2'- deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988).
Differentiation is a progressive and dynamic process, σeginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made.
Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products, and receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al.. Ciba Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J. of Cell Sci. 87:731-738, 1987), so identification is usually made at the progenitor and mature cell stages. The existence of early stage cardiac myocyte progenitor cells (often referred to as cardiac myocyte stem cells) has been speculated, but not demonstrated, in adult cardiac tissue. The novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and cardiac myocyte progenitor cells, both in vivo and ex vivo. There is evidence to suggest that factors that stimulate specific cell types down a pathway towards terminal differentiation or dedifferentiation affect the entire cell population originating from a common precursor or stem cell. Thus, the present invention includes stimulating or inhibiting the proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chrondrocytes and endoth-,iιal cells. Molecules of the present invention may, while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of the affect on their common precursor/stem cells. Thus molecules of the present invention may have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
Assays measuring differentiation include, for example, measuring cell- surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991 ; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
In vivo assays for evaluating cardiac neogenesis or hyperplasia include treating neonatal and mature rats with the molecules of the present invention. The animals' cardiac function is measured as heart rate, blood pressure, and cardiac output to determine left ventricular function. Post-mortem methods for assessing cardiac decline or improvement include: increased or decreased cardiac weight, nuclei/cytoplasmic volume, and staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res. 75:1050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci. 93:8630- 8635, 1996.)
Proteins of the present invention are useful for stimulating proliferation, activation, differentiation and/or induction or inhibition of specialized cell function of cells of the involved homeostasis of the hematopoiesis and immune function. In particular, zalpha31 polypeptides are useful for stimulating proliferation, activation, differentiation, induction or inhibition of specialized cell functions of cells of the hematopoietic lineages, including, but not limited to, T cells, B cells, NK cells, dendritic cells, monocytes. and macrophages, as well as epithelial cells. Proliferation and/or differentiation of hematopoietic cells can be measured in vitro using cultured cells or in vivo b> administering molecules of the claimed invention to the appropriate animal model. Assays measuring cell proliferation or differentiation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990, incorporated herein by reference), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incorporated herein by reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporated herein by reference), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988; all incorporated herein by reference). Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
Alternatively, zalpha31 polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage-specific expression of a tissue. As such, direct measurement of zalpha31 polypeptide, or its loss of expression in a tissue as it differentiates, can serve as a marker for differentiation of tissues.
Similarly, direct measurement of zalpha31 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression. Increases in invasiveness and motility of cells, or the gain or loss of expression of zalpha31 in a pre-cancerous or cancerous condition, in comparison to normal tissue, can serve as a diagnostic for transformation, invasion and metastasis in tumor progression. As such, knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient. Methods of measuring gain and loss of expression (of either mRNA or protein) are well known in the art and described herein and can be applied to zalpha31 expression. For example, appearance or disappearance of polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of cell motility, zalpha31 gain or loss of expression may serve as a diagnostic for lymphoid, B-cell, endothelial, hematopoietic and other cancers.
Moreover, the activity and effect of zalpha31 on tumor progression and metastasis can be measured in vivo. Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL- 1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994). C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus. Three days following this treatment, 10 to 10 cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant adenovirus, such as one expressing zalpha31 , before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically. The mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a
3 period of up to 3 weeks, during which time they may reach a size of 1500 - 1800 mm in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment. At the time of sacrifice, the tumor is removed and weighed along with the lungs and the liver. The lung weight has been shown to correlate well with metastatic tumor burden. As an additional measure, lung surface metastases are counted. The resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e.g., zalpha31, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed. In addition, aside from using adenovirus, the implanted cells can be transiently transfected with zalpha31. Use of stable zalpha31 transfectants as well as use of induceable promoters to activate zalpha31 expression in vivo are known in the art and can be used in this system to assess zalpha31 induction of metastasis. Moreover, purified zalpha31 or zalpha31 conditioned media can be directly injected in to this mouse model, and hence be used in this system. For general reference see, O'Reilly MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis .14:349-361, 1995. The activity of zalpha31 and its derivatives (conjugates) on growth and dissemination of tumor cells derived from human hematologic malignancies can also be measured in vivo in a mouse Xenograft model Several mouse models have been developed in which human tumor cells are implanted into immunodefϊcient mice, collectively referred to as xenograft models. See Cattan, AR and Douglas, E Leuk. Res. 18:513-22, 1994; and Flavell, DJ, Hematological Oncology 14:67-82, 1996. The characteristics of the disease model vary with the type and quantity of cells delivered to the mouse. Typically, the tumor cells will proliferate rapidly and can be found circulating in the blood and populating numerous organ systems. Therapeutic strategies appropriate for testing in such a model include antibody induced toxicity, ligand-toxin conjugates or cell-based therapies. The latter method, commonly referred to adoptive immunotherapy, involves treatment of the animal with components of the human immune system (i.e. lymphocytes, NK cells) and may include ex vivo incubation of cells with zalpha31 or other immunomodulatory agents.
Polynucleotides encoding Zalpha31 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit Zalpha31 activity. If a mammal has a mutated or absent Zalpha31 gene, the Zalpha31 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a Zalpha31 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA vims, such as, but not limited to, herpes simplex vims (HSV), papillomavirus, Epstein Barr vims (EBV), adenovims, adeno-associated vims (AAV), and the like. Defective vimses, 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 vims 1 (HSV1) vector, Kaplitt et al, Molec. Cell. Neurosci. 2:320 (1991); an attenuated adenovims vector, such as the vector described by Stratford-Perricaudet et al, J. Clin. Invest. 90:626 (1992); and a defective adeno-associated vims vector, Samulski et al, J. Virol. 61:3096 (1987); Samulski et al, J Virol 63:3822 (1989).
In another embodiment, a Zalpha31 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al, U.S. Patent No. 5,399,346; Mann et al. Cell 55:153, 1983; Temin et al, U.S. Patent No. 4,650,764; Temin et al, U.S. Patent No. 4,980,289; Markowitz et al, J. Virol. 62:1120 (1988); Temin et al, U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published
March 16, 1995 by Dougherty et al; and Kuo et al. Blood 82:845 (1993). Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker, Feigner et al, Proc. Natl. Acad. Sci. USA 54:7413 (1987); Mackey et al, Proc. Natl. Acad. Sci. USA 55:8027 (1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. 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, or non-peptide molecules can be coupled to liposomes chemically.
It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked 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 α/., J. Biol Chem. 2(55:14621-4, 1988. Antisense methodology can be used to inhibit Zalpha31 gene transcription, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a Zalpha31 -encoding polynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:l) are designed to bind to Zalpha31 -encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of Zalpha31 polypeptide-encoding genes in cell culture or in a subject.
The present invention also provides reagents which will find use in diagnostic applications. For example, the zalpha31 gene, a probe comprising zalpha31 DNA or RNA or a subsequence thereof can be used to determine if the zalpha31 gene is present on human chromosome 10 or if a mutation has occurred. Zalpha31 is located at the 10q23-q24 region of chromosome 10 (See, Example 3). Detectable chromosomal aberrations at the zalpha31 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
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.
Sequence tagged sites (STSs) 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. Since 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.
The zalpha31 gene is located at the 10q23-q24 region of chromosome 10. Several disease related genes in a cluster in this region that are associated with neuropathies, brain cancer, and other neural effects. For example, phosphatase and tensin homolog (PTEN; loss linked to brain tumors; 10q23.3), Bannayan-Riley-
Ruvalcaba syndrome (10q23), glioma-inactivated leucine rich gene (LGI1; 10q24); macrocephaly, (10q23.3); partial epilepsy (10q23.3-q24.1); infant-onset spinocerebellar ataxia (IOSCA; 10q24); urofacial syndrome (Ochoa syndrome) (10q23-q24); and autosomal dominant spastic paraplegia 9 (10q23.3-q24.1) all map to this region of chromosome 10. In addition, several of these diseases are linked to large chromosomal rearrangements, such as chromosome loss or loss of heterogeneity in the 10q23-q24 region chromosome 10. Moreover, Loss of 1 copy of chromosome 10 is the most common genetic event in high grade glioma, wherein rearrangement and loss of at least some parts of the second copy of the chromosome, particularly in the 10q23-26 region, has been demonstrated in approximately 80% of glioblastoma tumors (Bigner, S. and Vogelstein, B. Brain Path. 1 : 12-18, 1990). Moreover, loss of heterogeneity at 10q23 occurs in about 70% of glioblastomas and 60% of advanced prostate cancers (Li, J et al. Science 275:1943-1946, 1997), as well as other cancers. Moreover, a significant percentage (e.g., 7%) of childhood T-cell acute leukemia is accompanied by translocation within the 10q24 locus (Dube, ID et al., Blood 78:2996-3003, 1991). As the zalpha31 gene is also located at the 10q23-q24 region zalpha31, polynucleotide probes can be used to detect chromosome 10q23-q24 loss or translocation associated with human diseases, such as glioblastoma, macrocephaly, and T-cell leukemia, or other cancers, neurologic or immune diseases.
Further, zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 10 trisomy. For example, split-hand/foot malformation, type 3 (SHFM3) appears to be a result of a trisomy at 10q24-q25 (Nunes, ME et al., Hum. Molec. Genet. 4:2165-2170, 1995). As the zalpha31 gene is also located at the 10q23-q24 region zalpha31, polynucleotide probes can be used to detect chromosome 10q23-q24 gain, or trisomy associated with such human diseases. Moreover, amongst other genetic loci, those for dilated cardiomyopathy (10q21-q23), autoimmune diseases associated with the FAS ligand which maps to 10q24.1, retinitis pigmentosa associated with retinal G-protein coupled receptor (10q23), Cytochrome P450-2C9 (CYP2C9) (10q24) all manifest themselves in human disease states as well as map to this region of the human genome. See the Online Mendellian Inheritance of Man (OMIM) gene map, and references therein, for this region of chromosome 10 on a publicly available WWW server (http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=10q23-q24). All of these serve as possible candidate genes for an inheritable disease that show linkage to the same chromosomal region as the zalpha31 gene.
Similarly, defects or over expression in the zalpha31 locus itself may result in a heritable human disease state. For example, zalha31 is located in a chromosomal region that is associated with neural and brain implications as well as several tumors. As discussed herein a significant number of ESTs for zalpha31 are found in libraries of neural origin with disease association with multiple sclerosis, Huntington's Disease, gliosis, oligoastrocytoma, brain tumors (e.g., mets hypemephroma), and spinal cord w/lymphoma. Moreover, proinflammatory cytokines are produced by brain tumors, found in MS lesions, and exhibit other neuropathies.
Molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zalpha31 genetic defect.
A diagnostic could assist physicians in determining the type of disease and appropriate associated therapy, or assistance in genetic counseling. As such, the inventive anti-zalpha31 antibodies, polynucleotides, and polypeptides can be used for the detection of zalpha31 polypeptide, mRNA or anti-zalpha3 i antibodies, thus serving as markers and be directly used for detecting or genetic diseases or cancers, as described herein, using methods known in the art and described herein. Further, zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 10q23-q24 deletions and translocations associated with human diseases, other translocations involved with malignant progression of tumors or other 10q23-q24 mutations, which are expected to be involved in chromosome rearrangements in malignancy; or in other cancers, or in spontaneous abortion. Similarly, zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 10q23-q24 trisomy and chromosome loss associated with human diseases or spontaneous abortion. Thus, zalpha31 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects. As discussed above, defects in the zalpha31 gene itself may result in a heritable human disease state. Molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zalpha31 genetic defect. In addition, zalpha31 polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zalpha31 chromosomal locus. As such, the zalpha31 sequences can be used as diagnostics in forensic DNA profiling.
In general, the diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient, are known in the art. Most diagnostic met c ds comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (iv) comparing the visualized first reaction product to a second control reaction product of a genetic sample from wild type patient. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre- implantation embryo. For example, a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the zalpha31 genetic locus, and the like, are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples. Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l, or an RNA equivalent thereof. Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press. Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).
Mutations associated with the zalpha31 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 705:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al. (eds.), Genome Analysis, Vol. 2:
Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998), and Richards and Ward, "Molecular Diagnostic Testing," in Principles of Molecular Medicine, pages 83- 88 (Humana Press, Inc. 1998)). Direct analysis of an zalpha31 gene for a mutation can be performed using a subject's genomic DNA. Methods for amplifying genomic DNA, obtained for example from peripheral blood lymphocytes, are well-known to those of skill in the art (see, for example, Dracopoli et al. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).
Mice engineered to express the zalpha31 gene, referred to as "transgenic mice," and mice that exhibit a complete absence of zalpha31 gene function, referred to as "knockout mice," may also be generated (Snouwaert et al.. Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986). For example, transgenic mice that over-express zalpha31, either ubiquitously or under a tissue- specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype. For example, over-expression of a wild-type zalpha31 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zalpha31 expression is functionally relevant and may indicate a therapeutic target for the zalpha31, its agonists or antagonists. For example, a preferred transgenic mouse to engineer is one that over- expresses the zalpha31 mature polypeptide (approximately amino acid residue 20 (Asp) to residue 142 (Arg) of SEQ ID NO:2). Moreover, such over-expression may result in a phenotype that shows similarity with human diseases. Similarly, knockout zalpha31 mice can be used to determine where zalpha31 is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a zalpha31 antagonist, such as those described herein, may have. The human zalpha31 cDNA can be used to isolate murine zalpha31 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the zalpha31 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases. Moreover, transgenic mice expression of zalpha31 antisense polynucleotides or ribozymes directed against zalpha31, described herein, can be used analogously to transgenic mice described above.
For pharmaceutical use, 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. In general, pharmaceutical formulations will include a Zalpha31 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.,(Mack Publishing Co., Easton, PA, 19th ed., 1995). Therapeutic doses will generally be in the range of 0.1 to 100 μg/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.
The invention is further illustrated by the following non-limiting examples.
Example 1
Identification of zalpha31 Using an EST Sequence to Obtain Full-length zalpha31 Scanning of translated DNA databases of predicted full-length assemblies using a signal sequence and alpha helices as a query resulted in identification of an assembly having a 5' EST sequence (EST362610; Image Consortium).
Confirmation of the EST sequence was made by sequence analyses of the cDNA from which the EST originated. This cDNA was contained in a plasmid, and was sequenced using the following primers to generate complete double stranded sequence of this clone: ZC694 (SEQ ID NO:4), ZC7625 (SEQ ID NO:5), ZC22487
(SEQ ID NO:6), ZC22488 (SEQ ID NO:7), ZC22249 (SEQ ID NO:8). The EST362610 sequence (SEQ ID NOT) encoded a full-length protein designated zalpha31, as described herein and in SEQ ID NO:2.
Example 2 Tissue Distribution
Northerns were performed using Human Multiple Tissue Blots(MTNl, MTN2 and MTN3) and Master Dot blot (Clontech, Palo Alto, CA). An cDNA probe was prepared using PCR. Oligo nucleotides ZC22,230 (SEQ ID NO:9) and ZC22,249 (SEQ ID NO:8), designed off the EST INC515639H2 (Incyte Pharmaceuticals, Palo Alto, CA), were used as primers. The template was human brain Marathon cDNA (Clontech) mad.- in house using manufacturer's instmctions. PCR thermocycler conditions were as follows: one cycle at 94°C for 1.5 min.; 35 cycles at 94°C for 10 sec, 62°C for 20 sec, 72°C for 30 se ; one cycle at 72°C for 10 min.; followed by a 4°C hold. The approximately 500bp bp probe was purified using a Gel Extraction Kit (Qiagen, Chatsworth, CA) according to manufacturer's instmctions. The probe was radioactively labeled using a Rediprime II DNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA). EXPRESSHYB (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 55°C, using 1.5 x 10-6 cpm/ml labeled probe. The blots were then washed in 2XSSC and 0.1% SDS at room temperature, then with 2XSSC and 0.1% SDS at 65 °C, followed by a wash in 0.1X SSC and 0.1% SDS at 65°C. Two transcript sizes were observed on the blots at ~1.35kb and ~2kb in all tissues with strongest expression in heart, thyroid, spinal cord, adrenal gland, brain, and testis.
Dot Blots were also performed using Human RNA Master Blots™ (Clontech). The methods and conditions for the Dot Blots are the same as for the Multiple Tissue Blots disclosed above. The Dot blots showed signals in all tissues with strongest in brain, liver and heart. Th<-. EST electronic northern data for zalpha31 suggests that it is a highly inducible gene that is prevalent in B-cells (tonsils) and those cells of the monocyte/macrophage/dendritic lineage, (consistent with a ubiquitous distribution in the "normal", non-induced state) and particularly following treatment with a proinflammatory stimulus such as PMA, TNF. or LPS. This conclusion is based on its presence at higher than expected frequency in the following tissues: germinal center B cell (tonsil) library, multiple sclerosis lesions; stimulated THP-1 cells (pro-monocyte line); and peripheral blood dendritic cells (stimulated). The gene was also found in a T- cell line (stimulated) and peripheral blood mononuclear cell s(stimulated), fetal liver CD34+ progenitor cells and cartilage from an osteoarthritis patient. Moreover, there was a significant number of ESTs found in libraries of neural origin, both tissues (normal and diseased) and differentiated cell lines (neurons).
Example 3 Chromosomal Assignment and Placement of Zalpha31 Zalpha31 was mapped to chromosome 10 using the commercially available "GeneBridge 4 Radiation Hybrid (RH) Mapping Panel"(Research Genetics, Inc., Huntsville, AL). The GeneBridge 4 RH panel contains DNA from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient). A publicly available WWW server (http://www-genome.wi.mit.edu/cgi- bin contig/rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR" radiation hybrid map) which was constmcted with the GeneBridge 4 RH panel.
For the mapping of Zalpha31 with the GeneBridge 4 RH panel, 20 μl reactions were set up in a 96-well microtiter plate compatible for PCR (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2 μl 1 OX KlenTaq PCR reaction buffer (CLONTECH
Laboratories, Inc., Palo Alto, CA), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl sense primer, ZC 23,469 (SEQ ID NO:10), 1 μl antisense primer, ZC 23,470 (SEQ ID NOT 1), 2 μl "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 μl 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and distilled water for a total volume of
20 μl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C,
35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 58°C and 1 minute and 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (EM Science, Gibbstown, NJ) and visualized by staining with ethidium bromide.
The results showed that Zalpha31 maps 0.90 cR_3000 distal from the framework marker WI-8488 on the chromosome 10 WICGR radiation hybrid map. The use of surrounding genes/markers positions Zalpha31 in the 10q23-q24 chromosomal region.
Example 4 Generation of untagged zalpha31 Recombinant Adenovims A. Generation of expression vector constmct for Adenovims expression
The protein coding region of human zalpha31 was amplified by PCR using primers that added Fsel and Ascl restriction sties at the 5' and 3' termini respectively. PCR primers ZC23457 (SEQ ID NO:12) and ZC23458 (SEQ ID NO:13) were used with template pT7T3D plasmid containing the full-length human zalpha31 cDNA in a PCR reaction as follows: one cycle at 95°C for 5 minutes; followed by 15 cycles at 95°C for 0.5 min., 58°C for 0.5 min., and 72°C for 0.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak. The PCR reaction product was loaded onto a 1.2 %
(low melt) SeaPlaque GTG (FMC, Rockland, ME) gel in TAE buffer. The zalpha31 PCR product was excised from the gel, melted at 65°C, phenol extracted twice and then ethanol precipitated. The PCR product was then digested with Fsel-Ascl, phenol/chloroform extracted, EtOH precipitated, and rehydrated in TE (Tris/EDTA pH 8). The 429bp /-alpha31 fragment was then ligated into the Fsel -Ascl sites of a modified pAdTrack CMV (He, T-C. et al., PNAS 95:2509-2514, 1998). This constmct also contains the GFP marker gene. The CMV promoter driving GFP expression was replaced with the SV40 promoter and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal. In addition, the native polylinker was replaced with Fsel, EcoRV, and Ascl sites. This modified form of pAdTrack CMV was named pZy Track. Ligation was performed using the Fast-Link™ DNA ligation and screening kit (Epicentre Technologies, Madison, WI). Clones containing the zalpha31 cDNA were identified by standard mini prep procedures. In order to linearize the plasmid, approximately 5 μg of the pZy Track zalpha31 plasmid was digested with Pmel. Approximately 1 μg of the linearized plasmid was cotransformed with 200ng of supercoiled pAdEasy (He et al., supra.) into BJ5183 cells. The co-transformation was done using a Bio-Rad Gene Pulser at 2.5kV, 200 ohms and 25mFa. The entire co-transformation was plated on 4 LB plates containing 25 μg/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin and recombinant adenovims DNA identified by standard DNA miniprep procedures. Digestion of the recombinant adenovims DNA with Fsel-Ascl confirmed the presence of zalpha31. The recombinant adenovims miniprep DNA was transformed into DH10B competent cells and DNA prepared using a Qiagen maxi prep kit as per kit instmctions.
B. Transfection of 293a Cells with Recombinant DNA Approximately 5 μg of recombinant adenoviral DNA was digested with
Pad enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 μl containing 20-30U of Pad. The digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol. i he DNA pellet was resuspended in 5 μl distilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada), inoculated the day before and grown to 60-70% confluence, were transfected with the Pad digested DNA. The Pad-digested DNA was diluted up to a total volume of 50 μl with sterile HBS (150mM NaCl, 20mM HEPES). In a separate tube, 25 μl DOTAP (Boehringer Mannheim, lmg/ml) was diluted to a total volume of 100 μl with HBS. The DNA was added to the DOTAP, mixed gently by pipeting up and down, and left at room temperature for 15 minutes.
The media was removed from the 293A cells and washed with 5 ml serum-free MEMalpha (Gibco BRL) containing ImM Sodium Pymvate (GibcoBRL), 0.1 mM MEM non-essential amino acids (GibcoBRL) and 25mM HEPES buffer (GibcoBRL). 5 ml of semm-free MEM was added to the 293A cells and held at 37°C. The DNA/lipid mixture was added drop-wise to the T25 flask of 293A cells, mixed gently and incubated at 37°C for 4 hours. After 4 hours the media containing the DNA/lipid mixture was aspirated off and replaced with 5 ml complete MEM containing 5% fetal bovine semm. The transfected cells were monitored for Green Fluorescent Protein (GFP) expression and formation of foci, i.e., viral plaques. Seven days after transfection of 293A cells with the recombinant adenoviral DNA, the cells expressed the GFP protein and started to form foci. These foci are viral "plaques" and the cmde viral lysate was collected by using a cell scraper to collect all of the 293A cells. The lysate was transferred to a 50ml conical tube. To release most of the virus particles from the cells, three freeze/thaw cycles were done in a dry ice/ethanol bath and a 37 waterbath.
C. Amplificati-'. of Recombinant Adenovims (rAdV)
The cmde lysate was amplified (Primary (1°) amplification) to obtain a working "stock" of zalpha31 rAdV lysate. Ten 10cm plates of nearly confluent (80- 90%) 293 A cells were set up 20 hours previously, 200ml of cmde rAdV lysate added to each 10cm plate and monitored for 48 to 72 hours looking for CPE under the white light microscope and expression of GFP under the fluorescent microscope. When all of the 293 A cells showed CPE (Cytopathic Effect) this 1° stock lysate was collected and freeze/thaw cycles performed as described under Crude rAdV Lysate.
Secondary (2°) Amplification of zalpha31 rAdV was obtained as follows: Twenty 15cm tissue culture dishes of 293 A cells were prepared so that the cells were 80-90% confluent. All but 20 mis of 5%MEM media was removed and each dish was inoculated with 300-500ml 1° amplified rAdv lysate. After 48 hours the 293A cells were lysed from vims production and this lysate was collected into 250ml polypropylene centrifuge bottles and the rAdV purified. D. Purification of recombinant Adenovirus
NP-40 detergent was added to a final concentration of 0.5% to the bottles of cmde lysate in order to lyse all cells. Bottles were placed on a rotating platform for 10 min. agitating as fast as possible without the bottles falling over. The debris was pelleted by centrifugation at 20,000 X G for 15 minutes. The supernatant was transferred to 250ml polycarbonate centrifuge bottles and 0.5 volumes of 20%PEG8000/2.5M NaCl solution added. The bottles were shaken overnight on ice. The bottles were - .ntrifuged at 20,000 X G for 15 minutes and supernatant discarded into a bleach solution. The white precipitate in two vertical lines along the wall of the bottle on either side of the spin mark is the precipitated vims/PEG. Using a sterile cell scraper, the precipitate from 2 bottles was resuspended in 2.5 ml PBS. The vims solution was placed in 2 ml microcentrifuge tubes and centrifuged at 14,000 X G in the microfuge for 10 minutes to remove any additional cell debris. The supernatant from the 2ml microcentrifuge tubes was transferred into a 15ml polypropylene snapcap tube and adjusted to a density of 1.34g/ml with cesium chloride (CsCl). The volume of the virus solution was estimated and 0.55 g/ml of CsCl added. The CsCl was dissolved and 1 ml of this solution weighed 1.34 g. The solution was transferred polycarbonate thick- walled centrifuge tubes 3.2ml (Beckman No. 362305) and spin at 80,000 rpm (348,000 X G) for 3-4 hours at 25°C in a Beckman Optima TLX microultracentrifuge with the TLA- 100.4 rotor. The vims formed a white band. Using wide-bore pipette tips, the vims band was collected.
The vims from the gradient has a large amount of CsCl which must be removed before it can be used on cells. Pharmacia PD-10 columns prepacked with Sephadex G-25M (Pharmacia) were used to desalt the vims preparation. The column was equilibrated with 20 ml of PBS. The vims was loaded and allowed to mn into the column. 5 ml of PBS was added to the column and fractions of 8-10 drops collected. The optical densities of 1 :50 dilutions of each fraction was determined at 260nm on a spectrophotometer. A clear absorbance peak was present between fractions 7-12. These fractions were pooled and the optical density (OD) of a 1:25 dilution determined. A formula is used to convert OD into vims concentration: (OD at 260nm)(25)(l.l x 1012) = virions/ml. The OD of a 1:25 dilution of the zalpha31 rAdV was 0.059, giving a vims concentration of 3.0 X 10 12 virions/ml.
To store the vims, glycerol was added to the purified vims to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80°C.
E. Tissue Culture Infectious Dose at 50% CPE (TCID 50) Viral Titration Assay A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Qc.
Canada) was followed to measure recombinant vims infectivity. Briefly, two 96-well
4 tissue culture plates were seeded with 1X10 293 A cells per well in MEM containing
2% fetal bovine semm for each recombinant vims to be assayed. After 24 hours 10- fold dilutions of each vims from 1X10 -2 to 1X10 -14 were made in MEM containing
2% fetal bovine semm. 100 μl of each dilution was placed in each of 20 wells. After 5 days at 37°C, wells were read either positive or negative for Cytopathic Effect (CPE) and a value for "Plaque Forming Units/ml" (PFU) is calculated.
TCID50 formulation used was as per Quantum Biotechnologies, Inc.,
_2 above. The titer (T) is determined from a plate where vims used is diluted from 10 to 10 -14 , and read 5 days after the infection. At each dilution a ratio (R) of positive wells for CPE per the total number of wells is determined.
To Calculate titer of the undiluted vims sample: the factor, "F" = l+d(S-
0.5); where "S" is the sum of the ratios (R); and "d" is Log 10 of the dilution series, for example, "d" is equal to 1 for a ten-fold dilution series. The titer of the undiluted sample is T = 10^1+F^ = TCID5θ/ml. To convert TCIDso/ml to pfu/ml, 0.7 is subtracted from the exponent in the calculation for titer (T).
The zalpha31 adenovims had a titer of 1.3 X 10 pfu ml.
Example 5
Constmct for generating human zalpha31 Transgenic Mice A. Constmct for expressing human zalpha31 from the MT-1 promoter
Approximately 10 °g Zytrack vector containing the sequence confirmed zalpha31 coding region (Example 4) was digested with Fsel and Ascl. The vector was then ethanol precipitated and the pellet was resuspended in TE. The released 429 bp zalpha31 fragment was isolated by running the digested vector on a 1.2% SeaPlaque gel and exicising the fragment. DNA was purified using the QiaQuick (Qiagen) gel extraction kit. The zalpha31 fragment was then ligated into our standard transgenic vector pTG12-8, which was previously digested with Fsel and Ascl. The pTG12-8 plasmid, designed for expression of a gene of interest in transgenic mice, contains an expression cassette flanked by 10 kb of MT-1 5' DNA and 7 kb of MT-1 3' DNA. The expression cassette comprises the MT-1 promoter, the rat insulin II intron, a polylinker for the insertion of the desired clone, and the human growth hormone poly A sequence. About one microliter of the ligation reaction was electroporated into DH10B ElectroMax® competent cells (GIBCO BRL, Gaithersburg, MD) according to manufacturer's direction, plated onto LB plates containing 100 μg/ml ampicillin, and incubated overnight at 37°C. Colonies were picked and grown in LB media containing 100 μg/ml ampicillin. Miniprep DNA was prepared from the picked clones and screened for the zalpha31 insert by restriction digestion with EcoRI, and subsequent agarose gel electrophoresis. Maxipreps of the correct pTG12-8 zalpha31 constmct were performed. A Sail fragment containing with 5' and 3' flanking sequences, the MT promoter, the rat insulin II intron, zalpha31 cDNA and the human growth hormone poly A sequence was prepared using standard techniques described herein, and used for microinjection into fertilized murine oocytes. Micro injection and production of transgenic mice were produced as described in Hogan, B. et al. Manipulating the Mouse Embryo, 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1994.
B. Construct for expressing human zalpha31 from the lymphoid-specific EμLCK promoter
The zalpha31 DNA fragment digested with Fsel and Ascl (Example 5A) is cloned into pKFO51 , a lymphoid-specific transgenic vector, previously digested with Fsel and Ascl as described above. The pKFO51 transgenic vector is derived from pl026X (Iritani, B.M., et al., EMBO J. 16:7019-31, 1997) and contains the T cell- specific lck proximal promoter, the B/T cell-specific immunoglobulin μ heavy chain enhancer, a polylinker for the insertion of the desired clone, and a mutated hGH gene that encodes an inactive growth hormone protein (providing 3' introns and a polyadenylation signal). About one microliter of each ligation reaction is electroporated, plated, clones picked and screened for the human zalpha31 insert by restriction digestion as described above. A correct clone of pKFO51-zalpha31 is verified by sequencing, and a maxiprep of this clone is performed. A Notl fragment, containing the lck proximal promoter and immunoglobulin μ enhancer (EμLCK), zalpha31 cDNA, and the mutated hGH gene is prepared to be used for microinjection into fertilized murine oocytes.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. An isolated polynucleotide encoding a polypeptide that is at least 90% identical to a sequence of amino acid residues selected from the group of:
(a) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 37 (He), to residue number 132 (Leu);
(b) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 20 (Asp), to residue number 142 (Arg); and
(c) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 1 (Met), to residue number 142 (Arg).
2. An isolated polynucleotide according to claim 1, wherein the polypeptide encoded by the polynucleotide further contains four alpha helices spaced apart from N- terminus to C-terminus in a configuration represented by the following:
Asp20-{16}-Hl-{ 13}-H2-{7}-H3-{ 16}-H4-{9}- Arg142, where Asp20is residue 20 (Asp) as shown in SEQ ID NO:2),
Arg142is .esidue 142 (Arg) as shown in SEQ ID NO:2),
HI is "helix A" (corresponding to amino acids 37 (He) to 51 (Tyr) of SEQ ID
NO:2);
H2 is "helix B" (corresponding to amino acids 65 (Leu) to 79 (Glu) of SEQ ID NO:2); H3 is "helix C" (corresponding to amino acids 87 (He) to 101 (Leu) of SEQ ID NO:2); and H4 is "helix D" (corresponding to amino acids 118 (Leu) to 132 (Leu) of SEQ ID NO:2); and
{#} denotes the approximate number of amino acid residues between the motifs, up to plus or minus 2 residues.
3. An isolated polynucleotide according to claim 1, wherein the polynucleotide comprises nucleotide 1 to nucleotide 426 of SEQ ID NO:3.
4. An isolated polynucleotide according to claim 1, encoding a polypeptide that includes a sequence of amino acid residues selected from the group of:
(a) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 37 (He), to residue number 132 (Leu);
(b) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 20 (Asp), to residue number 142 (Arg); and
(c) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 1 (Met), to residue number 142 (Arg).
5. An expression vector that includes the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1 ; and a transcription terminator.
6. An expression vector according to claim 5, further including a secretory signal sequence operably linked to the DNA segment.
7. A cultured cell into which has been introduced an expression vector according to claim 5, wherein the cell expresses a polypeptide encoded by the DNA segment.
8. A DNA constmct encoding a fusion protein, the DNA construct including: a first DNA segment encoding a polypeptide that includes a sequence of amino acid residues selected from the group of:
(a) the amino acid sequence of SEQ ID NO: 2 from residue number 1 (Met), to residue number 19 (Asp); (b) the amino acid sequence of SEQ ID NO: 2 from residue number 87 (He), to residue number 101 (Leu);
(c) the amino acid sequence of SEQ ID NO: 2 from residue number 118 (Leu), to residue number 132 (Leu);
(d) the amino acid sequence of SEQ ID NO: 2 from residue number 37 (He), to residue number 132 (Leu); and
(e) the amino acid sequence of SEQ ID NO: 2 from residue number 20 (Tyr), to residue number 142 (Leu); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in-frame; and encode the fusion protein.
9. A fusion protein produced by a method that includes: culturing a host cell into which has been introduced a vector that includes the following operably linked elements:
(a) a transcriptional promoter;
(b) a DNA constmct encoding a fusion protein according to claim 8; and
(c) a transcriptional terminator; and recovering the protein encoded by the DNA segment.
10. An isolated polypeptide that includes a sequence of amino acid residues that is at least 90% identical to a sequence of amino acid residues selected from the group of:
(a) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 37 (He), to residue number 132 (Leu);
(b) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 20 (Asp), to residue number 142 (Arg); and
(c) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 1 (Met), to residue number 142 (Arg).
11. An isolated polypeptide according to claim 10, wherein the polypeptide further contains four alpha helices spaced apart from N-terminus to C-terminus in a configuration represented by the following:
Asp20-{16}-Hl-{13}-H2-{7}-H3-{ 16}-H4-{9}- Arg142, where Asp20is the starting residue of the mature polypeptide (as shown in SEQ ID NO:2),
Arg142 is the ending residue of the mature polypeptide(as shown in SEQ ID NO:2),
HI is "helix A" (corresponding to amino acids 37 (He) to 51 (Tyr) of SEQ ID NO:2);
H2 is "i i lix B" (corresponding to amino acids 65 (Leu) to 79 (Glu) of SEQ ID NO:2);
H3 is "helix C" (corresponding to amino acids 87 (He) to 101 (Leu) of SEQ ID NO:2); and
H4 is "helix D" (corresponding to amino acids 118 (Leu) to 132 (Leu) of SEQ ID NO:2); and
{#} denotes the approximate number of amino acid residues between the motifs, up to plus or minus 2 residues.
12. An isolated polypeptide according to claim 10, that includes a sequence of amino acid residues selected from the group of:
(a) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 37 (He), to residue number 132 (Leu);
(b) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 20 (Asp), to residue number 142 (Arg); and
(c) the amino acid sequence as shown in SEQ ID NO: 2 from residue number 1 (Met), to residue number 142 (Arg).
13. A method of producing a zalpha31 polypeptide that includes: culturing a cell according to claim 7; and isolating the zalpha31 polypeptide produced by the cell.
14. A method of detecting, in a test sample, the presence of an antagonist of zalpha31 protein activity, that includes: culturing a cell that is responsive to a zalpha31 -stimulated cellular pathway; and producing a zalpha31 polypeptide by the method of claim 13; and exposing the zalpha31 polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the zalpha31 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the antagonist of zalpha31 activity in the test sample.
15. A method of detecting, in a test sample, the presence of an agonist of zalpha31 protein activity, that includes: culturing a cell that is responsive to a zalpha31 -stimulated cellular pathway; and adding a test sample; and comparing levels of response in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the agonist of zalpha31 activity in the test sample.
16. A method of producing an antibody to zalpha31 polypeptide that includes the following steps in order: inoculating an animal with a polypeptide selected from the group of:
(a) a polypeptide according to claim 10 or 12;
(b) a polypeptide that includes amino acid number 87 (He) to 101 (Leu) of SEQ ID NO: 2; (c) a polypeptide that includes amino acid number 118 (Leu) to 132 (Leu) of SEQ ID NO: 2;
(d) a polypeptide that includes amino acid number 14 (Asp) to 19 (Asp) of SEQ ID NO:2;
(e) a polypeptide that includes amino acid number 26 (Ala) to 31 (Glu) of SEQ ID NO:2;
(f) a polypeptide that includes amino acid number 27 (Glu) to 32 (Pro) of SEQ ID NO:2;
(g) a polypeptide that includes amino acid number 136 (Tyr) to 141 (Lys) of SEQ ID NO:2; and
(h) a polypeptide that includes amino acid number 137 (Lys) to 142 (Arg) of SEQ ID NO:2;
(i) a polypeptide that includes amino acid number 127 (Ala) to 135 (Lys) of SEQ ID NO:2; and
(j) a polypeptide that includes amino acid number 127 (Ala) to 139 (Leu) of SEQ ID NO:2; and wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
17. An antibody produced by the method of claim 16. which specifically binds to a zalpha31 polypeptide.
18. An antibody that specifically binds to a polypeptide of claim 10 or 12.
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WO2000055320A1 (en) * 1999-03-12 2000-09-21 Human Genome Sciences, Inc. Human pancreas and pancreatic cancer associated gene sequences and polypeptides

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AU2456397A (en) * 1996-04-12 1997-11-07 Muro Pharmaceutical, Inc. Isolated and cloned mast cell 78 kda phosphoprotein (mast cell degranulation inhibitory agent) and use thereof
AU6956698A (en) * 1997-04-10 1998-10-30 Genetics Institute Inc. Secreted expressed sequence tags (sests)

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2000055320A1 (en) * 1999-03-12 2000-09-21 Human Genome Sciences, Inc. Human pancreas and pancreatic cancer associated gene sequences and polypeptides

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See also references of WO0073458A1 *

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JP2003501033A (en) 2003-01-14
MXPA01012181A (en) 2002-07-22
WO2000073458A1 (en) 2000-12-07
AU5448700A (en) 2000-12-18

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