WO2000042070A1 - Proteine-12 a mammalienne helice alpha - Google Patents

Proteine-12 a mammalienne helice alpha Download PDF

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Publication number
WO2000042070A1
WO2000042070A1 PCT/US2000/001015 US0001015W WO0042070A1 WO 2000042070 A1 WO2000042070 A1 WO 2000042070A1 US 0001015 W US0001015 W US 0001015W WO 0042070 A1 WO0042070 A1 WO 0042070A1
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polypeptide
zalphal2
seq
cells
polypeptides
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PCT/US2000/001015
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English (en)
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Darrell C. Conklin
Emily Cooper
Stephen R. Jaspers
Joyce M. Lehner
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Zymogenetics, Inc.
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Priority to AU27282/00A priority Critical patent/AU2728200A/en
Priority to JP2000593636A priority patent/JP2002534964A/ja
Priority to CA002360584A priority patent/CA2360584A1/fr
Priority to EP00905629A priority patent/EP1144450A1/fr
Publication of WO2000042070A1 publication Critical patent/WO2000042070A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • Inflammation normally is a localized, protective response to trauma or microbial invasion that destroys, dilutes, or walls-off the injurious agent and the injured tissue. It is characterized in the acute form by the classic signs of pain, heat, redness, swelling, and loss of function. Microscopically it involves a complex series of events, including dilation of arterioles, capillaries, and venules, with increased permeability and blood flow, exudation of fluids, including plasma proteins, and leukocyte migration into the area of inflammation.
  • inflammation occurs as a defensive response to invasion of the host by foreign, particularly microbial, material.
  • Responses to mechanical trauma, toxins, and neoplasia also may results in inflammatory reactions.
  • the accumulation and subsequent activation of leukocytes are central events in the pathogenesis of most forms of inflammation. Deficiencies of inflammation compromise the host .
  • Excessive inflammation caused by abnormal recognition of host tissue as foreign or prolongation of the inflammatory process may lead to inflammatory diseases as diverse as diabetes, arteriosclerosis, cataracts, reperfusion injury, and cancer, to post-infectious syndromes such as in infectious meningitis, rheumatic fever, and to rheumatic diseases such as systemic lupus erythematosus and rheumatoid arthritis.
  • the centrality of the inflammatory response in these varied disease processes makes its regulation a major element in the prevention control or cure of human disease.
  • cytokines which contribute to inflammation and inflammatory related diseases so that antagonists such as antibodies can be administered to down-regulate the cytokine so as to ameliorate the inflammation.
  • the present invention addresses this need by providing novel polypeptides and related compositions and methods and their antagonists.
  • the present invention provides an isolated polynucleotide encoding a mammalian cytokine termed Zalphal2. The data show that the cytokine is involved in the inflammation response.
  • antagonists of Zalphal2 can be used to lessen inflammation especially inflammation associated with coronary heart disease, arteriosclerosis, Crohn's disease and inflammatory bowel disease.
  • Each Zalphal2 polypeptide has a signal sequence which extends from amino acid residue 1, a methionine to and including amino acid residue 34, a serine of SEQ ID NOs : 2 , 4 and 6.
  • the Zalphal2 polypeptide represented by SEQ ID NO: 2 has a mature sequence extending from amino acid residue 35, an alanine, to and including amino acid residue 202, an asparagine, also represented by SEQ ID NO: 8.
  • the Zalphal2 polypeptide represented by SEQ ID NO: 4 has a mature sequence extending from amino acid residue 35, an alanine to and including amino acid residue 288, an asparagine also represented by SEQ ID NO: 9.
  • the Zalphal2 polypeptide represented by SEQ ID NO: 6 has a mature sequence extending from amino acid residue 35, an alanine to and including amino acid residue 158, an asparagine, also represented by SEQ ID NO: 10.
  • an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding Zalphal2 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.
  • a cultured eukaryotic or prokaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a protein polypeptide encoded by the DNA segment.
  • a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond.
  • the first portion of the chimeric polypeptide consists essentially of (a) a Zalphal2 polypeptide (b) allelic variants of Zalphal2; and (c) protein polypeptides that are at least 80% identical to (a) or (b) .
  • the second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag.
  • the affinity tag is an immunoglobulin F c polypeptide.
  • the invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
  • an antibody that specifically binds to a Zalphal2 polypeptide as disclosed above, and also an anti- idiotypic antibody which neutralizes the antibody to a Zalphal2 polypeptide.
  • An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zalphal2 polypeptide having an amino acid sequence described above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zalphal2 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention.
  • epitope binding regions are SEQ ID NOs: 18, 19, 20, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • affinity tag any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly-histidine tract, protein A,
  • DNAs encoding affinity tags are available from commercial suppliers (e.gr., Pharmacia Biotech, Piscataway, NJ) .
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and “carboxyl- terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl- terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 9 M -1 .
  • polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5 ' ATGCACGGG 3 ' is complementary to 5 ' CCCGTGCAT 3' .
  • contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5 ' - ATGGCTTAGCTT-3 ' are 5 ' -TAGCTTgagtct-3 ' and 3 ' - gtcgacTACCGA-5' .
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
  • expression vector 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.
  • 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.
  • 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
  • an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e . g. , transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the . result of speciation. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, b- globin, and myoglobin are paralogs of each other.
  • 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 vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt”), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs”.
  • 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
  • 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 mR ⁇ As transcribed from the same gene . Splice variants may encode polypeptides having altered amino acid sequence.
  • splice variant is also used herein to denote a protein encoded by a splice variant of an mR ⁇ A transcribed from a gene .
  • the Zalphal2 protein is an alpha helical protein.
  • SEQ ID NO: 2 has four alpha helices, A, B, C and D.
  • Helix A of SEQ ID NO: 2 extends from amino acid residue 56, a leucine, to and including amino acid residue 70, an isoleucine also defined by SEQ ID NO: 21.
  • Helix B of SEQ ID NO: 2 extends from amino acid residue 96, a leucine to and including amino acid residue 110, a tyrosine, also defined by SEQ ID NO: 22.
  • Helix C of SEQ ID NO: 2 extends from amino acid residue 130, a leucine, to and including amino acid residue 144, a leucine, also defined by SEQ ID NO: 23.
  • Helix D of SEQ ID NO: 2 extends from amino acid residue 162, a methionine, to and including amino acid residue 176, a serine, also defined by SEQ ID NO: 24.
  • SEQ ID NO: 25 contains helices A + B;
  • SEQ ID NO: 26 contains helices A + B + C;
  • SEQ ID NO: 27 contains helices A + B + C + D;
  • SEQ ID NO:28 contains helices B + C + D;
  • SEQ ID NO:29 contains helices B + C;
  • SEQ ID NO: 30 contains helices C + D of SEQ ID NO:2.
  • the polypeptide of SEQ ID NO: 4 also contains four helices A, B, C and D.
  • Helix A of SEQ ID NO: 4 extends from amino acid residue 45, a histidine, to and including amino acid residue 59, a leucine, also defined by SEQ ID NO: 31.
  • Helix B of SEQ ID NO: 4 extends from amino acid residue 116, a valine, to and including amino acid residue 130, a lysine, also defined by SEQ ID NO: 32.
  • Helix C of SEQ ID NO: 4 extends from amino acid residue 142, a leucine, to and including amino acid residue 156, an isoleucine, also defined by SEQ ID NO: 33.
  • Helix D of SEQ ID NO: 4 extends from amino acid residue 182, a leucine, to and including amino acid residue 196, a tyrosine, also defined by SEQ ID NO: 34.
  • SEQ ID NO: 35 contains helices A + B of SEQ ID NO : 4.
  • SEQ ID NO: 36 contains helices A + B + C of SEQ ID NO:4.
  • SEQ ID NO: 37 contains helices A + B + C + D of SEQ ID NO:
  • SEQ ID NO: 38 contains helices B + C + D of SEQ ID NO:4.
  • SEQ ID NO: 39 contains helices B + C of SEQ ID NO:4.
  • SEQ ID NO: 40 contains helices C + D of SEQ ID NO: 4.
  • Zalphal2 may play a general role in development and exert important regulatory control of testicular differentiation, of the hypothalamic-pituitary- gonadal axis, and of gonadal steroidogenesis and spermatogenesis .
  • testicular hormone production can be divided into early and late steps, with the latter dependent on the activation of functionally-determined Leydig cell precursors by luteinizing hormone (LH) .
  • LH luteinizing hormone
  • the factors that control the early steps in this process remain unknown, Huhtaniemi , Reprod. Fertil . Dev. 7: 1025-1035 (1995) suggesting that zalphal2 might be responsible for activation of a non-steroidogenic, non-LH responsive precursor cell.
  • Sertoli cell derived tumor necrosis factor-a, fibroblast growth factor, interleukin-1, transforming growth factor-B, epidermal growth factor/transforming growth factor-a, activin, inhibin, insulin-like growth factor-1, platelet derived growth factor, endothelin, and ariginine-vasopressin have all been shown to regulate Leydig cell function, Saez , Endocrin . Rev. 15 : 574-626 (1994) .
  • zalphal2 might control or modulate the activities of one or more of these genes .
  • aging is associated with a progressive decline in testicular function.
  • POLYNUCLEOTIDES The present invention also provides polynucleotide molecules, including D ⁇ A and RNA molecules, that encode the Zalphal2 polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.
  • Polynucleotides generally a cD ⁇ A sequence, of the present invention encode the described polypeptides herein.
  • a cD ⁇ A sequence which encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides.
  • the amino acid residues are encoded by their respective codons as follows.
  • Alanine (Ala) is encoded by GCA, GCC, GCG or
  • Cysteine is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT;
  • Glutamic acid (Glu) is encoded by GAA or GAG;
  • Phenylalanine (Phe) is encoded by TTC or TTT;
  • Glycine is encoded by GGA, GGC, GGG or GGT; Histidine (His) is encoded by CAC or CAT;
  • Isoleucine (lie) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA, or AAG;
  • Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;
  • Methionine (Met) is encoded by ATG; Asparagine (Asn) is encoded by AAC or AAT;
  • Proline is encoded by CCA, CCC, CCG or CCT;
  • Glutamine (Gin) is encoded by CAA or CAG;
  • Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;
  • Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;
  • Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
  • Valine (Val) is encoded by GTA, GTC, GTG or GTT;
  • Trp Tryptophan
  • Tyrosine (Tyr) is encoded by TAC or TAT.
  • mRNA messenger RNA
  • T thymine nucleotide
  • U uracil nucleotide
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) .
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant D ⁇ A 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 ⁇ 0s:l, 3 or 5 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.
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • RNA is isolated from a tissue or cell that produces large amounts of Zalphal2 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 a CsCl gradient, Chirgwin et al . , Biochemistry 18:52-94
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl . Acad. Sci . USA
  • cDNA Complementary DNA
  • poly (A) + RNA using known methods .
  • genomic DNA can be isolated.
  • Polynucleotides encoding Zalphal2 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding Zalphal2 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 Zalphal2, receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesizers.
  • the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • synthetic genes double-stranded are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs) .
  • species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species.
  • Zalphal2 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides .
  • Orthologs of human Zalphal2 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 Zalphal2 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 Zalphal2-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to Zalphal2 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 NOs:l, 3 or 5 represent specific alleles of human Zalphal2 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 are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs : 2 , 4 or 6.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the Zalphal2 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
  • Zalphal2 polypeptides that are substantially identical to the polypeptides of SEQ ID NOs : 2 , 4, 6, 8, 9 or 10 and their orthologs.
  • the term "substantially identical” is used herein to denote polypeptides having 50%, 60%, 70%, 80% and most preferably at least 90%, 95% or 99% sequence identity to the sequences shown in SEQ ID NOs: 2, 4, 6, 8, 9 or 10 or their orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math . Bio . 48 : 603-616 (1986) and
  • 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 and the amino acid sequence of a putative variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat 'l Acad. Sci . USA 85:2444 (1988), and by Pearson, Meth . Enzymol . 183 : 63 (1990).
  • the ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • 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, SIAM J. Appl . Math . 26: 181 (1974), which allows for amino acid insertions and deletions.
  • SMATRIX scoring matrix file
  • 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 form four to six.
  • 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 NOs:2, 4, 6, 8, 9 or 10.
  • 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. Na t ' l Acad. Sci . USA 89:10915 (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.
  • 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) .
  • Variant Zalphal2 polypeptides or substantially homologous Zalphal2 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from 20 to 30 amino acid residues that comprise a sequence that is at least 90%, preferably at least 95%, and more preferably 99% or more identical to the corresponding region of SEQ ID NOs:2, 4, 6, 8, 9 or 10.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the Zalphal2 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • Polar glutamine asparagine Table 2 cont .
  • Hydrophobic leucine isoleucine valine 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] .
  • a Zalphal2 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-Zalphal2 polypeptide fusions can be expressed in genetically engineered cells [to produce a variety of multimeric Zalphal2 analogs] .
  • Auxiliary domains can be fused to Zalphal2 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a Zalphal2 polypeptide or protein could be targeted to a predetermined cell type by fusing a Zalphal2 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 Zalphal2 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al . , Connective Tissue Research
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2, 4-methanoproline, cis-4-hydroxyproline, tra ⁇ s-4-hydroxyproline, N- methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine , hydroxyethylhomocysteine , nitroglutamine, homoglutamine , pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4- methylproline, 3 , 3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine .
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) .
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 33:7470-7476 (1994).
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci . 2:395-403 (1993) .
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zalphal2 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 . ,
  • 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 .
  • Variants of the disclosed Zalphal2 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391,
  • variant D ⁇ As are generated by in vi tro homologous recombination by random fragmentation of a parent D ⁇ A followed by reassembly using PCR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent D ⁇ As, such as allelic variants or D ⁇ As from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment . These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure .
  • any Zalphal2 polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
  • the Zalphal2 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 Zalphal2 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 Zalphal2, 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 Zalphal2 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.,
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al . , Cell
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen . Virol . 36: 59 (1977) and
  • Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 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
  • 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 MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci . (Bangalore) 11 : 41 (1987).
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) .
  • DNA encoding the Zalphal2 polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods. The first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the Zalphal2 flanked by AcNPV sequences.
  • Suitable insect cells e.g.
  • SF9 cells are infected with wild-type AcNPV and transfected with a transfer vector comprising a Zalphal2 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • a transfer vector comprising a Zalphal2 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • Recombinant viral stocks are made by methods commonly used in the art .
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow, V.A, et al . , J Virol 57:4566 (1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD) . This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Zalphal2 polypeptide into a baculovirus genome maintained in E.
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case Zalphal2.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen
  • transfer vector constructs a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native Zalphal2 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 Zalphal2 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 Zalphal2 polypeptide, for example, a Glu-Glu epitope tag, Grussenmeyer, T. et al . ,
  • bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g.
  • Recombinant virus that expresses Zalphal2 is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art .
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall army worm, Spodoptera frugiperda . See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni
  • Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex- cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 s cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • the recombinant virus-infected cells typically produce the recombinant Zalphal2 polypeptide at 12-72 hours post- infection and secrete it with varying efficiency into the medium.
  • the culture is usually harvested 48 hours post- infection. Centrifugation is used to separate the cells from the medium (supernatant) .
  • the supernatant containing the Zalphal2 polypeptide is filtered through micropore filters, usually 0.45 ⁇ m pore size. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid. ; O'Reilly, D.R. et al . , ibid. ; Richardson, C. D., ibid. ) . Subsequent purification of the Zalphal2 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 .
  • 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 PCTl vector system disclosed by Kawasaki et al . (U.S. Patent
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S.
  • Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida mal tosa are known in the art.
  • 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
  • a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art, see, e.g., Sambrook et al . , ibid. ) .
  • the polypeptide When expressing a Zalphal2 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,
  • Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a Zalphal2 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, Geysen, H.M. et al . , Proc . Na tl . Acad Sci . USA 81:3998-4002 (1984).
  • 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. All of the polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zalphal2 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 preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant Zalphal2 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, Montgomeryvilie, 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, Affini ty
  • the polypeptides of the present invention can be isolated by exploitation of their properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3 : 1 (1985) . Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography. Methods in Enzymol . , Vol.
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
  • polypeptide fusions, or hybrid Zalphal2 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. For example, part or all of a domain (s) conferring a biological function may be swapped between Zalphal2 of the present invention with the functionally equivalent domain (s) from another family member. Such domains include, but are not limited to, the secretory signal sequence, conserved, and significant domains or regions in this family. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • Zalphal2 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • Zalphal2 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.
  • Chemical Synthesis of Polypeptides Polypeptides, especially polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Merrifield, J. Am . Chem . Soc . 85:2149 (1963).
  • 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 .
  • Zalphal2 can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model.
  • Zalphal2 transfected (or co- transfected) expression host cells may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers have been described as a means to entrap transfected mammalian cells or primary mammalian cells.
  • non-immunogenic "encapsulations” or microenvironments permit the transfer of nutrients into the microenvironment , and also permit the diffusion of proteins and other macromolecules secreted or released by the captured cells across the environmental barrier to the recipient animal. Most importantly, the capsules or microenvironments mask and shield the foreign, embedded cells from the recipient animal's immune response. Such microenvironments can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells) .
  • Alginate threads provide a simple and quick means for generating embedded cells.
  • the materials needed to generate the alginate threads are readily available and relatively inexpensive. Once made, the alginate threads are relatively strong and durable, both in vi tro and, based on data obtained using the threads, in vivo.
  • the alginate threads are easily manipulable and the methodology is scalable for preparation of numerous threads. In an exemplary procedure, 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C. Becker et al . , Meth . Cell Biol . 43:161 (1994); and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44
  • adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary) .
  • the host cell the human 293 cell line is exemplary
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells.
  • the host's tissue e.g., liver
  • the host's tissue will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein.
  • Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • the adenovirus system can also be used for protein production in vi tro.
  • the cells By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al . , Cytotechnol . 15:145 (1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant . Within the infected 293S cell production protocol, non- secreted proteins may also be effectively obtained.
  • agonists and antagonists In view of the tissue distribution observed for Zalphal2, agonists (including the natural ligand/ substrate/ cofactor/ etc.) and antagonists have enormous potential in both in vi tro and in vivo applications.
  • Zalphal2 agonists are useful for stimulating the immune system or spermatogenesis.
  • Zalphal2 and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction.
  • Antagonists of Zalphal2 can also be used to down-regulate inflammation as discussed in more further detail below.
  • Zalphal2 antagonists include anti-Zalphal2 antibodies and soluble Zalphal2 receptors, as well as other peptidic and non-peptidic agents (including ribozymes) .
  • Zalphal2 can also be used to identify inhibitors
  • Zalphal2 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a Zalphal2-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a Zalphal2-DNA response element operably linked to a gene encoding a protein which can be assayed, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE) , hormone response elements (HRE) insulin response element (IRE), Nasrin et al . , Proc . Natl . Acad.
  • compounds or other samples can be tested for direct blocking of Zalphal2 binding to receptor using Zalphal2 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like) .
  • a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like.
  • the ability of a test sample to inhibit the binding of labeled Zalphal2 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 Zalphal2 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 Zalphal2 ligand-binding polypeptide can also be used for purification of ligand.
  • the polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
  • the ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl) , or pH to disrupt ligand-receptor binding.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complement/ anti-complement pair
  • a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ)
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol . Methods 145:229 (1991) and Cunningham and Wells, J " . Mol . Biol .
  • 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
  • Zalphal2 polypeptides can also be used to prepare antibodies that specifically bind to Zalphal2 epitopes, peptides or polypeptides.
  • the Zalphal2 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • Suitable antigens would be the Zalphal2 polypeptides encoded by SEQ ID NOs: 2, 4, 6, 8- 10, 18- 20, 25-30 and 35-44.
  • Antibodies generated from this immune response can be isolated and purified as described herein.
  • 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 Zalphal2 polypeptide or a fragment thereof.
  • the immunogenicity of a Zalphal2 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 Zalphal2 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 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.
  • 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.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to Zalphal2 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zalphal2 protein or peptide) .
  • Genes encoding polypeptides having potential Zalphal2 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
  • Random peptide display libraries can be screened using the Zalphal2 sequences disclosed herein to identify proteins which bind to Zalphal2.
  • binding proteins which interact with Zalphal2 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 proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as Zalphal2 "antagonists" to block Zalphal2 binding and signal transduction in vi tro and in vivo.
  • Antibodies are determined 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.
  • antibodies herein specifically bind if they bind to a Zalphal2 polypeptide, peptide or epitope with a binding affinity 6 -1 7 —1 (K a ) of 10 M or greater, preferably 10 M or greater,
  • antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zalphal2 but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid. ) . Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family (e.g. IL-16) ,
  • Zalphal2 polypeptides and non-human Zalphal2.
  • antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides. For example, antibodies raised to Zalphal2 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to Zalphal2 will flow through the matrix under the proper buffer conditions. Such screening allows isolation of polyclonal and monoclonal antibodies non- crossreactive to closely related polypeptides, Antibodies :
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • dot blot or Western blot assay inhibition or competition assay
  • sandwich assay antibodies can be screened for binding to wild-type versus mutant Zalphal2 protein or polypeptide.
  • Antibodies to Zalphal2 may be used for tagging cells that express Zalphal2 ; for isolating Zalphal2 by affinity purification; for diagnostic assays for determining circulating levels of Zalphal2 polypeptides; for detecting or quantitating soluble Zalphal2 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 Zalphal2 in vi tro and in vivo.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to Zalphal2 or fragments thereof may be used in vi tro to detect denatured Zalphal2 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) .
  • Zalphal2 polypeptides or anti-Zalphal2 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule .
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like) , as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance) .
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues) .
  • a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
  • the domain only fusion protein includes a complementary molecule
  • the anti- complementary molecule can be conjugated to a detectable or cytotoxic molecule.
  • Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti- complementary-detectable/ cytotoxic molecule conjugates.
  • Zalphal2-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 Zalphal2 polypeptide or anti-Zalphal2 antibody targets the hyperproliferative blood or bone marrow cell.
  • target tissues for example, blood and bone marrow cancers
  • Zalphal2 polypeptide or anti-Zalphal2 antibody targets the hyperproliferative blood or bone marrow cell.
  • the described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine.
  • Suitable Zalphal2 polypeptides or anti-Zalphal2 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 Zalphal2 polypeptide or anti-Zalphal2 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.
  • a radionuclide and particularly with a beta-emitting radionuclide
  • 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.
  • 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.
  • USES OF POLYNUCLEOTIDE/POLYPEPTIDE 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 or receptors of the immune system.
  • proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column, Immobilized
  • GENE THERAPY Polynucleotides encoding Zalphal2 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit Zalphal2 activity. If a mammal has a mutated or absent Zalphal2 gene, the Zalphal2 gene can be introduced into the cells of the mammal . In one embodiment, a gene encoding a Zalphal2 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector, Kaplitt et al . , Molec. Cell. Neurosci.
  • an attenuated adenovirus vector such as the vector described by Stratford-Perricaudet et al., J. Clin.
  • a Zalphal2 gene can be introduced in a retroviral vector, e.g., as described in
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker, Feigner et al., Proc. Natl. Acad.
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g. , hormones or neurotransmitters
  • proteins such as antibodies
  • non-peptide molecules can be coupled to liposomes chemically.
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al . , J.
  • Antisense methodology can be used to inhibit
  • Zalphal2 gene transcription such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a Zalphal2 -encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ID NO:l
  • Such antisense polynucleotides are used to inhibit expression of Zalphal2 polypeptide-encoding genes in cell culture or in a subject .
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the Zalphal2 gene a probe comprising Zalphal2 DNA or RNA or a subsequence thereof can be used to determine if the Zalphal2 gene is present on chromosome 22ql3.1 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the Zalphal2 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 (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 (1995).
  • Transgenic mice engineered to express the Zalphal2 gene, and mice that exhibit a complete absence of Zalphal2 gene function, referred to as "knockout mice", Snouwaert et al . , Science 257:1083 (1992), may also be generated, Lowell et al . , Nature 356:740-42 (1993). These mice may be employed to study the Zalphal2 gene and the protein encoded thereby in an in vivo system.
  • CHROMOSOMAL LOCALIZATION Zalphal2 has been mapped to chromosome 22ql3.1.
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al . ,
  • Radiomación 250 : 245 (1990) Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels.
  • Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) . These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs) , and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers .
  • the precise knowledge of a gene's position can be useful 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
  • 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 Zalphal2 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 . Administration of the protein can be subcutaneous, intraperitoneal or rectal depending on the disease to be treated.
  • Tissue Expression and Use Zalphal2 represents a novel polypeptide with a putative signal peptide leader sequence and alpha helical structure.
  • This gene may encode a secreted polypeptide with secondary structure indicating it is a member of the four helix bundle cytokine family.
  • 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.
  • Northern blot analysis detects transcripts for zalphal2 in spleen, thymus, testis, small intestine, colon, PBL, stomach, lymph node, trachea, and bone marrow. Many of these organs have important immunological function or contain cells which play a role in the immune system. Zalphal2 is also expressed in CD4+ enriched peripheral T cells and to a lesser degree in CD8+ T cells. There is very weak expression in CD19+ B cells suggesting that Zalphal2 has expression in T lymphoid lineage cells and little if any in B lymphoid lineage cells. There is elevated expression of zalphal2 in RNA derived from a 7 day human mixed lymphocyte reaction. This suggests that zalphal2 expression is regulated and increases after T cell activation.
  • Zalphal2 Zalphal2 can be administered to an immunocompromised mammal, preferably a human, such as cancer patients who have undergone chemotherapy, AIDS patients and the elderly. This will stimulate their immune systems.
  • Zalphal2 can also be used as a vaccine adjuvant to be administered before, with or after the administration of a vaccine. Zalphal2 may also be administered to stimulate the immune system to attack tumors . Use of Antagonists of Zalphal2
  • An antagonist to Zalphal2 such as an antibody, soluble receptor or small molecule antagonist can be administered to a mammal, preferably a human, to alleviate an inflammatory response.
  • Antagonists, such as antibodies, to Zalphal2 can be used to treat patients having inflammatory related diseases such arteriosclerotic heart disease [see Paulsson,G. et al . , Arterioscler Thromb.
  • Sequence Tag of SEQ ID NO: 7 as a probe in a spleen cDNA library was constructed using 1 microgram ( ⁇ g) of polyA RNA isolated from the spleen tissue of a 2- year-old Hispanic male, who died from cerebral anoxia. cDNA synthesis was initiated using a Notl-oligo (dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with Notl, size-selected, and cloned into the Notl and EcoRI sites of a vector. The sequenced clone resulted in the full-length DNA and polypeptide sequences of SEQ ID NOs: 1 and 2.
  • the EST of SEQ ID NO: 17 resulted in the discovery of a second clone.
  • the clone was sequence and found to have an unspliced intron.
  • Oligonucleotide primers SEQ ID NOs: 11 and 12 were designed to amplify the full- length sequence without the intron sequence .
  • the PCR was performed using lymph node MARATHON READY ® cDNA (Clontech, Palo Alto, CA) as template.
  • An 823 bp was cut out and gel purified using the QIAQUICK ® gel extraction kit (Qiagen, Santa Clarita, CA) .
  • the excised PCR fragment was sequenced and did not contain the intron sequence .
  • the next step was to see if the clone of SEQ ID NO: 17 had another initiation codon upstream.
  • a RACE ® reaction using lymph node cDNA was performed to obtain 5' sequence.
  • Oligonucleotide primers SEQ ID NOs : 13 and 14 were designed for 5 ' RACE ® reactions and nested RACE ® .
  • the RACE ® reactions revealed an additional initiation codon upstream.
  • the actual full- length was obtained through PCR using primers SEQ ID NOs: 15 and 16 which flanked the coding sequence.
  • a northern blot analysis was carried out by standard techniques using the polynucleotides of SEQ ID NOs : 1 and 3. The results indicate that Zalphal2 is expressed in lymph nodes, activated mixed lymphocytes, spleen, thymus, testis, small intestines, human aortic endothelial cells, smooth muscle, kidney, mast cells, eosinophils, tonsils, pancreas, colon, peripheral blood lymphocytes (PBL) , stomach, trachea, T-cells including CD4 + and CD8 + cells, and bone marrow.
  • PBL peripheral blood lymphocytes
  • Zalphal2 is also expressed by the following cell lines: HL60 (ATCC 45500), an acute promyelocytic leukemia cell line; Jurkat cells (ATCC TIB-152) a T lymphocyte from acute T cell leukemia; MOLT-4 cells (ATCC CRL-1582)a T lymphoblast from acute lymphoblastic leukemia; and HuT 78 cells (ATCC TIB-161) a cutaneous T lymphocyte from a lymphoma . From these data, it can be concluded that Zalphal2 is a cytokine involved in the inflammation cascade . Antagonists of Zalphal2 can be used to alleviate inflammation related to a number of diseases such as arteriosclerotic heart disease, cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease.
  • Polyclonal anti-peptide antibodies were prepared by immunizing two female New Zealand white rabbits with the peptide huzalphal2Xl-l
  • CEGLPPSTSSGQPPLQDMLCLGGVAVSLSHIRN SEQ ID NO: 43
  • SEQ ID NO: 4 FMRYRSSSVLSHEEC SEQ ID NO: 44
  • the peptides were synthesized using an Applied Biosystems Model 431A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) according to manufacturer's instructions. The peptides were then conjugated to the carrier protein maleimide-activated keyhole limpet hemocyanin (KLH) through cysteine residues according to manufacturer's instructions. (Pierce, Rockford, IL) .
  • KLH keyhole limpet hemocyanin
  • the rabbits were each given an initial intraperitoneal (IP) injection of 200 ⁇ g of the conjugated peptide in Complete Freund's Adjuvant (Pierce, Rockford, IL) followed by booster IP injections of 100 ⁇ g conjugated peptide in Incomplete Freund's Adjuvant every three weeks. Seven to ten days after the administration of the third booster injection, the animals were bled and the serum was collected. The rabbits were then boosted and bled every three weeks .
  • IP intraperitoneal
  • the zalphal2 peptide-specific Rabbit seras were characterized by an ELISA titer check using 1 ⁇ g/ml of the peptide used to make the antibody as an antibody target.
  • the 2 rabbit seras to the SEQ ID NO: 41 peptide have titer to their specific peptide at a dilution of 1:5E5 (1:100000).
  • the 2 rabbit seras to the SEQ ID N0:42 peptide had titer to their specific peptide at a dilution of 1:5E4 (1:10,000).
  • the 2 rabbit seras to the SEQ ID NO: 43 peptide had titer to their specific peptide at a dilution of 1:5E4.
  • the 2 rabbit seras to the SEQ ID NO:44 peptide had titer to their specific peptide at a dilution of 1:5E5.

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Abstract

La présente invention concerne des molécules polynucléotidiques et polypeptidiques de la protéine-12 mammalienne à hélice alpha (Zalpha-12). Les polypeptides et les polynucléotides codant ces polypeptides sont hormonaux et peuvent être utilisés pour réguler le fonctionnement du système immunitaire. L'invention concerne en outre des anticorps anti-polypeptides Zalpha-12. Des antagonistes sont utilisés pour traiter des inflammations et des maladies apparentées à des inflammations.
PCT/US2000/001015 1999-01-15 2000-01-14 Proteine-12 a mammalienne helice alpha WO2000042070A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU27282/00A AU2728200A (en) 1999-01-15 2000-01-14 Mammalian alpha-helical protein - 12
JP2000593636A JP2002534964A (ja) 1999-01-15 2000-01-14 哺乳類α−ヘリックスタンパク質
CA002360584A CA2360584A1 (fr) 1999-01-15 2000-01-14 Proteine-12 a mammalienne helice alpha
EP00905629A EP1144450A1 (fr) 1999-01-15 2000-01-14 Proteine-12 a mammalienne helice alpha

Applications Claiming Priority (2)

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US23242799A 1999-01-15 1999-01-15
US09/232,427 1999-01-15

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WO2000042070A1 true WO2000042070A1 (fr) 2000-07-20

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EP (1) EP1144450A1 (fr)
JP (1) JP2002534964A (fr)
AU (1) AU2728200A (fr)
CA (1) CA2360584A1 (fr)
WO (1) WO2000042070A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009626A1 (fr) * 2002-07-22 2004-01-29 Yamanouchi Pharmaceutical Co., Ltd. Nouveau gene associe a l'arthrite rhumatoide

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021308A1 (fr) * 1992-04-17 1993-10-28 The University Hospital Cytokines mutantes ayant une affinite d'un recepteur amelioree
WO1995013393A1 (fr) * 1993-11-08 1995-05-18 Fred Hutchinson Cancer Research Center Cytokines hybrides
WO1999029720A2 (fr) * 1997-12-10 1999-06-17 Zymogenetics, Inc. Proteine 1 mammalienne a helice alpha

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021308A1 (fr) * 1992-04-17 1993-10-28 The University Hospital Cytokines mutantes ayant une affinite d'un recepteur amelioree
WO1995013393A1 (fr) * 1993-11-08 1995-05-18 Fred Hutchinson Cancer Research Center Cytokines hybrides
WO1999029720A2 (fr) * 1997-12-10 1999-06-17 Zymogenetics, Inc. Proteine 1 mammalienne a helice alpha

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES, 1 May 1999 (1999-05-01), HINXTON, GB, XP002138913 *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES, 14 May 1997 (1997-05-14), HINXTON, GB, XP002138914 *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES, 2 August 1999 (1999-08-02), HINXTON, GB, XP002138916 *
DATABASE EMBL NUCLEOTIDE AND PROTEIN SEQUENCES, 30 November 1999 (1999-11-30), HINXTON, GB, XP002138915 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009626A1 (fr) * 2002-07-22 2004-01-29 Yamanouchi Pharmaceutical Co., Ltd. Nouveau gene associe a l'arthrite rhumatoide

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CA2360584A1 (fr) 2000-07-20
AU2728200A (en) 2000-08-01
EP1144450A1 (fr) 2001-10-17
JP2002534964A (ja) 2002-10-22

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