WO2000055324A1 - Interferon alpha murin egalement appele zcyto13 - Google Patents

Interferon alpha murin egalement appele zcyto13 Download PDF

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Publication number
WO2000055324A1
WO2000055324A1 PCT/US2000/006993 US0006993W WO0055324A1 WO 2000055324 A1 WO2000055324 A1 WO 2000055324A1 US 0006993 W US0006993 W US 0006993W WO 0055324 A1 WO0055324 A1 WO 0055324A1
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zcyto
amino acid
seq
nucleic acid
sequence
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PCT/US2000/006993
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English (en)
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Scott R. Presnell
Andrew L. Feldhaus
Zeren Gao
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Zymogenetics, Inc.
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Priority to AU38916/00A priority Critical patent/AU3891600A/en
Publication of WO2000055324A1 publication Critical patent/WO2000055324A1/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
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to a new cytokine having diagnostic and therapeutic uses.
  • the present invention relates to a novel murine interferon- ⁇ , and to nucleic acid molecules encoding interferon- ⁇ .
  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form tissues and organs, and to repair and regenerate damaged tissue.
  • hormones and growth factors include the steroid hormones, parathyroid hormone, follicle stimulating hormone, the interferons, the interleukins, platelet derived growth factor, epidermal growth factor, and granulocyte-macrophage colony stimulating factor, among others.
  • Hormones and growth factors influence cellular metabolism by binding to receptor proteins. Certain receptors are integral membrane proteins that bind with the hormone or growth factor outside the cell, and that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble intracellular molecules. Of particular interest, from a therapeutic standpoint, are the interferons
  • Interferons exhibit a variety of biological activities, and are useful for the treatment of certain autoimmune diseases, particular cancers, and the enhancement of the immune response against infectious agents, including viruses, bacteria, fungi, and protozoa. To date, six forms of interferon have been identified, which have been classified into two major groups.
  • type I interferons include interferon- ⁇ , interferon- ⁇ , interferon- ⁇ . interferon- ⁇ , and interferon- ⁇ .
  • interferon- ⁇ and one subclass of interferon- ⁇ are the only type II interferons.
  • Type I interferons which are thought to be derived from the same ancestral gene, have retained sufficient similar structure to act by the same cell surface receptor.
  • the ⁇ -chain of the human interferon- ⁇ / ⁇ receptor comprises an extracellular N-terminal domain, which has the characteristics of a class II cytokine receptor.
  • Interferon- ⁇ does not share significant homology with the type I interferons or with the type II interferon- ⁇ subtype, but shares a number of biological activities with the type I interferons.
  • interferon- ⁇ In humans, at least 16 non-allelic genes code for different subtypes of interferon- ⁇ , while interferons ⁇ and ⁇ are encoded by single genes.
  • Type I interferon genes are clustered in the short arm of chromosome 9. Unlike typical structural human genes, interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ lack introns.
  • a single gene for human interferon- ⁇ is localized on chromosome 12 and contains three introns. To date, interferon- ⁇ has been described only in cattle and sheep, while interferon- ⁇ has been described only in pigs.
  • interferon- ⁇ has been approved for use in more than 50 countries for the treatment of medical conditions such as hairy cell leukemia, renal cell carcinoma, basal cell carcinoma, malignant melanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis, mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitis C, chronic hepatitis D, and chronic non-A, non-B/C hepatitis.
  • medical conditions such as hairy cell leukemia, renal cell carcinoma, basal cell carcinoma, malignant melanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis, mycos
  • Interferon- ⁇ is used to treat chronic granulomatous diseases, in which the interferon enhances the patient's immune response to destroy infectious bacterial, fungal, and protozoal pathogens.
  • Clinical studies also indicate that interferon- ⁇ may be useful in the treatment of AIDS, leishmaniasis, and lepromatous leprosy.
  • the present invention provides a novel murine interferon- ⁇ , designated "Zcytol3.”
  • Zcytol3 also provides Zcytol3 polypeptides and Zcytol3 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins, and methods for using these nucleic acid molecules and amino acid sequences.
  • a nucleic acid molecule containing a sequence that encodes murine interferon- ⁇ , initially designated as "Zcytol3,” has the following nucleotide sequence:
  • the encoded polypeptide has the following amino acid sequence: MTPKFLWLVA LVALYIPPIQ SLNCVYLDDS ILENVKLLGS TMTGFPLRCL KDITDFKFPK EILPYIQHMK REINAVSYRI SSLALTIFNL KGSIPPVTEE HWERIRSGLF KQVRQAQECF MDEEKENREH PHSEDFLTVY LELGKYFFRI KKFLINKKYS FCAWKIVTVE IRRCFIIFSK SRKLLKMISE SPTFKQELK (SEQ ID NO:2).
  • the Zcytol3 form of an interferon- ⁇ gene encodes a polypeptide of 199 amino acids, as shown in SEQ ID NO:2.
  • the signal sequence for Zcytol3 can be predicted as comprising Met 1 through Ser 21 of SEQ ID NO:2.
  • the mature peptide for Zcytol3 begins at Leu 22 . Additional structural features of Zcytol3 are summarized in Table 4.
  • Hybridization analyses indicate that the Zcytol3 gene is strongly expressed in murine heart and liver tissue, and to a lesser extent, in murine brain, kidney, uterine, spleen, and seven-day embryo tissues. Zcytol3 RNA was also detected in lung, skeletal muscle, and testis tissues, as well as in the tissues of 11-, 15-, and 17- day embryos. Typically, Zcytol3 mRNA appeared as bands of about 2.8 and 6.6 kilobases. These results show that the interferon- ⁇ sequences can be used differentiate among various tissues. Southern analyses revealed that a Zcytol3 probe bound with rat and mouse genomic DNA. When incubated with Ec ⁇ RI-digested DNA, the probe hybridized with fragments of 4.8 and 5.65 kilobases in murine genomic DNA, and 2.1 and 5.2 kilobases in rat genomic DNA.
  • the Zcytol3 gene has been mapped to mouse chromosome 4 (framework marker D4Mit94, located at 4.6 centimorgans).
  • the murine interferon- ⁇ /- ⁇ gene cluster is also located at chromosome 4.
  • the present invention provides isolated polypeptides having an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to either amino acid residues 22 to 199 of S ⁇ Q ID NO:2 or amino acid residues 1 to 199 of S ⁇ Q ID NO:2, wherein such isolated polypeptides can either (a) specifically bind with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of S ⁇ Q ID NO:2, or (b) exhibits anti-viral activity or anti- proliferative activity.
  • An illustrative polypeptide is a polypeptide that comprises a first amino acid sequence consisting of amino acid residues 22 to 199 of S ⁇ Q ID NO:2, or a polypeptide that further comprises a signal secretory sequence that resides in an amino- terminal position relative to the first amino acid sequence, wherein the signal secretory sequence comprises amino acid residues 1 to 21 of the amino acid sequence of S ⁇ Q ID NO:2.
  • polypeptides include polypeptides that comprise at least one of the following amino acid motifs: (a) the amino acid sequence [FSPN] [PFG] [LK] [RSI] [CN]L[KT] [D Y] [IR] [TQKA]DF[GK] [FI]P, wherein the sequence is further defined by at least one condition selected from the group consisting of: (i) the first residue is F, (ii) the fourth residue is R, (iii) the ninth residue is I, and (iv) the tenth residue is T, or (b) the amino acid sequence [KVR] [F Y]L[IRK] [N ⁇ KL] [KM]K[ YH] [SN] [FPLS] [C Y] AW[K ⁇ M] [IV] [IV] [TR]
  • sequence is further defined by at least one condition selected from the group consisting of: (i) the first residue is K, (ii) the fourth residue is I, (iii) the fifth residue is N, (iv) the tenth residue is F, (v) the fourteenth residue is K, (vi) the fifteenth residue is I, (vii) the seventeenth residue is T, and (viii) the eighteenth residue is V.
  • illustrative polypeptides comprise both motif (a) and motif (b).
  • Additional exemplary polypeptides include polypeptides comprising an amino acid sequence of 15, 20, or 30 contiguous amino acid residues of the following amino acid sequences within SEQ ID NO:2: amino acid residues 22 to 199, amino acid residues 22 to 188, amino acid residues 22 to 45, amino acid residues 46 to 64, amino acid residues 65 to 89, amino acid residues 99 to 124, amino acid residues 134 to 155, amino acid residues 22 to 89, and amino acid residues 161 to 181.
  • polypeptides comprise an amino acid sequence selected from the group consisting of: amino acid residues 22 to 199 of SEQ ID NO:2, amino acid residues 22 to 188 of SEQ ID NO:2, amino acid residues 22 to 45 of SEQ ID NO:2, amino acid residues 46 to 64 of SEQ ID NO:2, amino acid residues 65 to 89 of SEQ ID NO:2, amino acid residues 99 to 124 of SEQ ID NO:2, amino acid residues 134 to 155 of SEQ ID NO:2, amino acid residues 22 to 89 of SEQ ID NO:2, and amino acid residues 161 to 181 of SEQ ID NO:2.
  • polypeptides consisting of an amino acid sequence selected from the group consisting of: amino acid residues 22 to 199 of SEQ ID NO:2, amino acid residues 22 to 188 of SEQ ID NO:2, amino acid residues 22 to 45 of SEQ ID NO:2, amino acid residues 46 to 64 of SEQ ID NO:2, amino acid residues 65 to 89 of SEQ ID NO:2, amino acid residues 99 to 124 of SEQ ID NO:2, amino acid residues 134 to 155 of SEQ ID NO:2, amino acid residues 22 to 89 of SEQ ID NO:2, and amino acid residues 161 to 181 of SEQ ID NO:2.
  • the polypeptides described herein can further comprise an affinity tag.
  • the present invention further provides antibodies and antibody fragments that specifically bind with such polypeptides.
  • Exemplary antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
  • Illustrative antibody fragments include F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, and minimal recognition units.
  • the present invention also includes anti-idiotype antibodies that specifically bind with such antibodies or antibody fragments. Certain anti-idiotype antibodies, or anti- idiotype antibody fragments, possesses anti-viral activity or anti-proliferative activity.
  • the present invention further includes compositions comprising a carrier and a peptide, polypeptide, antibody, or anti-idiotype antibody described herein.
  • the composition can be a pharmaceutical composition
  • the carrier can be a pharmaceutically acceptable carrier.
  • the present invention also provides isolated nucleic acid molecules that encode a Zcytol3 polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, (b) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2, and (c) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 1 , or the complement of SEQ ID NO: 1.
  • Illustrative nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO:2 is due to a conservative amino acid substitution.
  • the present invention further contemplates isolated nucleic acid molecules that comprise a nucleotide sequence of nucleotides 86 to 619 of SEQ ID NO:l .
  • the present invention also includes vectors and expression vectors comprising such nucleic acid molecules.
  • Such expression vectors may comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.
  • the present invention further includes recombinant host cells comprising these vectors and expression vectors.
  • Illustrative host cells include bacterial, yeast, fungal, avian, insect, mammalian, and plant cells.
  • Recombinant host cells comprising such expression vectors can be used to prepare Zcytol3 polypeptides by culturing such recombinant host cells that comprise the expression vector and that produce the Zcytol3 protein, and, optionally, isolating the Zcytol3 protein from the cultured recombinant host cells.
  • the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and at least one of such an expression vector or recombinant virus comprising such expression vectors.
  • the present invention also contemplates methods for detecting the presence of Zcyto 13 RNA in a biological sample, comprising the steps of (a) contacting a Zcyto 13 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of SEQ ID NO: l, or its complement, and (b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of Zcyto 13 RNA in the biological sample.
  • the present invention further provides methods for detecting the presence of Zcyto 13 polypeptide in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody or an antibody fragment that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
  • an antibody or antibody fragment may further comprise a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold.
  • kits for performing these detection methods may comprise a container that comprises a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 86 to 619 of SEQ ID NO:l, (b) a nucleic acid molecule comprising the complement of nucleotides 86 to 619 of the nucleotide sequence of SEQ ID NO:l, (c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides, and (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides.
  • kit may also comprise a second container that comprises one or more reagents capable of indicating the presence of the nucleic acid molecule.
  • a kit for detection of Zcyto 13 protein may comprise a container that comprises an antibody, or an antibody fragment, that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
  • the present invention also contemplates anti-idiotype antibodies, or anti- idiotype antibody fragments, that specifically bind an antibody or antibody fragment that specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, wherein the anti-idiotype antibody, or anti-idiotype antibody fragment, possesses anti-viral activity or anti-proliferative activity.
  • the present invention also provides isolated nucleic acid molecules comprising a nucleotide sequence that encodes a Zcyto 13 secretion signal sequence and a nucleotide sequence that encodes a biologically active polypeptide, wherein the Zcyto 13 secretion signal sequence comprises an amino acid sequence of residues 1 to 21 of SEQ ID NO:2.
  • Illustrative biologically active polypeptides include Factor Vila, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukin, colony stimulating factor, interferon, erythropoietin, and thrombopoietin.
  • the present invention provides fusion proteins comprising a Zcyto 13 secretion signal sequence and a polypeptide, wherein the Zcyto 13 secretion signal sequence comprises an amino acid sequence of residues 1 to 21 of SEQ ID NO:2.
  • Additional fusion proteins comprise a Zcyto 13 moiety and an immunoglobulin moiety.
  • An illustrative immunoglobulin moiety is an immunoglobulin heavy chain constant region, such as a human F c fragment.
  • the present invention also includes isolated nucleic acid molecules that encode such fusion proteins.
  • the present invention further contemplates variant Zcyto 13 polypeptides, wherein the amino acid sequence of the variant is characterized by at least one amino acid substitution within SEQ ID NO:2 selected from the group consisting of: (a) an alanine for glycine 39 , (b) a valine for leucine 63 , (c) a threonine for serine 81 , (d) a valine for isoleucine 105 , and (e) a leucine for valine” 3 .
  • variant Zcyto 13 polypeptides have an amino acid sequence that shares an identity with the amino acid sequence of SEQ ID NO:2 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO:2 is due to one or more conservative amino acid substitutions.
  • the present invention also includes isolated polypeptides consisting of an amino acid sequence of amino acid residues 22-89 of SEQ ID NO:2.
  • the present invention further includes methods of inhibiting a viral infection of cells, comprising the step of administering a composition comprising Zcyto 13 to the cells.
  • the composition can be a pharmaceutical composition, which is administered in a therapeutically effective amount to a subject, which has a viral infection.
  • In vivo treatment of a viral infection can provide at least one physiological effect selected from the group consisting of decreased viral titer, decreased detectable viral antigen, and increased anti-viral antibody titer.
  • the present invention also includes methods of inhibiting the proliferation of tumor cells, comprising the step of administering a composition comprising Zcyto 13 to the tumor cells.
  • the composition is a pharmaceutical composition, administered in a therapeutically effective amount to a subject, which has a tumor.
  • Such in vivo administration can provide at least one physiological effect selected from the group consisting of decreased number of tumor cells, decreased metastasis, decreased size of a solid tumor, and increased necrosis of a tumor.
  • the present invention also provides fusion proteins comprising a Zcyto 13 polypeptide moiety.
  • Such fusion proteins can further comprise an immunoglobulin moiety.
  • An exemplary immunoglobulin moiety is a human immunoglobulin heavy chain constant region.
  • nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • nucleic acid molecule also includes so- called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • nucleic acid molecule refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a nucleic acid molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule. Contiguous sequences are said to "overlap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid 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).
  • structural gene refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • an "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • a nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
  • a "nucleic acid molecule construct " is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • Linear DNA denotes non-circular DNA molecules having free 5' and 3' ends.
  • Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
  • Codon DNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA refers to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • cDNA also refers to a clone of a cDNA molecule synthesized from an RNA template.
  • a “promoter” is a nucleotide sequence that directs the transcription of a structural gene.
  • a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al, Mol. Endocrinol 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol.
  • DSEs differentiation-specific elements
  • CREs cyclic AMP response elements
  • SREs serum response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O'Reilly et al, J. Biol. Chem. 267:19938 (1992)), AP2 (Ye et al, J. Biol Chem. 269:2512% (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr. 5:253 (1993)) and octamer factors (see, in general, Watson et al, eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J.
  • a promoter contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
  • a “regulatory element” is a nucleotide sequence that modulates the activity of a core promoter.
  • a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific,” “tissue-specific,” or “organelle-specific” manner.
  • the Zcyto 13 regulatory element preferentially induces gene expression in heart, liver, brain, kidney, and spleen tissues, as opposed to lung, skeletal muscle, and testis tissues.
  • Heterologous DNA refers to a DNA molecule, or a population of
  • DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA).
  • a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.
  • a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter.
  • a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.
  • 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.”
  • 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.
  • a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
  • an "integrated genetic element” is a segment of DNA that has been inco ⁇ orated into a chromosome of a host cell after that element is introduced into the cell through human manipulation.
  • integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its progeny.
  • a "cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • An "expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked to" the promoter.
  • a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
  • a “recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
  • a recombinant host is a cell that produces the Zcyto 13 form of interferon- ⁇ from an expression vector.
  • Zcyto 13 can be produced by a cell that is a "natural source" of Zcytol3, and that lacks an expression vector.
  • “Integrative transformants” are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
  • a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • a fusion protein can comprise at least part of a Zcyto 13 polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of Zcyto 13 using affinity chromatography.
  • Receptor denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • 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).
  • 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. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the 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, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often 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.
  • secretory signal sequence denotes a DNA sequence that encodes a peptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • secretory peptide a DNA sequence that encodes a 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.
  • isolated polypeptide is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • SDS sodium dodecyl sulfate
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • ammo-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.
  • expression refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence.
  • the term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
  • immunomodulator includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.
  • 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 less than 10 9 M "1 .
  • an "anti-idiotype antibody” is an antibody that binds with the variable region domain of an immunoglobulin.
  • an anti-idiotype antibody binds with the variable region of an anti-Zcytol3 antibody, and thus, an anti-idiotype antibody mimics an epitope of Zcytol3.
  • an “antibody fragment” is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-Zcytol3 monoclonal antibody fragment binds with an epitope of Zcytol3.
  • antibody fragment also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • scFv proteins peptide linker
  • a “chimeric antibody” is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
  • Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • a "therapeutic agent” is a molecule or atom which is conjugated to an antibody moiety to produce a conjugate which is useful for therapy.
  • therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
  • a "detectable label” is a molecule or atom, which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al, EMBO J. 4: 015 (1985); Nilsson et al, Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al, Proc.
  • naked antibody is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
  • antibody component includes both an entire antibody and an antibody fragment.
  • an “immunoconjugate” is a conjugate of an antibody component with a therapeutic agent or a detectable label.
  • antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a therapeutic agent.
  • therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein”) and toxins
  • antibody-toxin fusion protein (“antibody-toxin fusion protein”).
  • tumor associated antigen is a protein normally not expressed, or expressed at lower levels, by a normal counterpart cell.
  • tumor associated antigens include alpha-fetoprotein, carcinoembryonic antigen, and Her-2/neu. Many other illustrations of tumor associated antigens are known to those of skill in the art.
  • an "infectious agent” denotes both microbes and parasites.
  • a “microbe” includes viruses, bacteria, rickettsia, mycoplasma, protozoa, fungi and like microorganisms.
  • a “parasite” denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible to immune-mediated clearance or lytic or phagocytic destruction, such as malarial parasites, spirochetes, and the like.
  • infectious agent antigen is an antigen associated with an infectious agent.
  • a “target polypeptide” or a “target peptide” is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen.
  • T cells recognize peptide epitopes presented by a major histocompatibility complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.
  • antigenic peptide is a peptide, which will bind a major histocompatibility complex molecule to form an MHC -peptide complex which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell.
  • antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell which binds or expresses the antigen.
  • the antigenic peptide can be bound in the context of a class I or class II major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.
  • RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA.
  • a nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA.
  • the RNA transcript is termed an "anti- sense RNA” and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene.”
  • Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
  • an “anti-sense oligonucleotide specific for Zcyto 13" or a “Zcyto 13 anti- sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the Zcytol3 gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Zcyto 13 gene.
  • a "ribozyme” is a nucleic acid molecule that contains a catalytic center. The term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
  • a nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene.”
  • An "external guide sequence” is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P.
  • a nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene.”
  • variant murine Zcytol3 gene refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO:2. Such variants include naturally-occurring polymorphisms of Zcyto 13 genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO:2. Additional variant forms of Zcytol 3 genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein. A variant Zcytol3 gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: l, or its complement, under stringent conditions.
  • variant Zcytol3 genes can be identified by sequence comparison. Two amino acid sequences have "100% amino acid sequence identity” if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence. Similarly, two nucleotide sequences have "100% nucleotide sequence identity” if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin).
  • a variant gene or polypeptide encoded by a variant gene is functionally characterized by either its anti-viral or anti-proliferative activities, or by the ability to bind specifically to an anti-Zcytol3 antibody.
  • 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.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • the present invention includes functional fragments of Zcytol 3 genes.
  • a "functional fragment" of a Zcyto 13 gene refers to a nucleic acid molecule that encodes a portion of a Zcyto 13 polypeptide, which either
  • a functional fragment of a Zcytol 3 gene comprises a portion of the nucleotide sequence of SEQ ID NO:l, and encodes a polypeptide having either an anti-viral or anti-proliferative activity.
  • Nucleic acid molecules encoding a murine Zcytol 3 gene can be obtained by screening a murine cDNA or genomic library using polynucleotide probes based upon SEQ ID NO:l. These techniques are standard and well-established.
  • a nucleic acid molecule that encodes a murine Zcytol3 gene can be isolated from a murine cDNA library.
  • the first step would be to prepare the cDNA library by isolating RNA from a tissue, such as uterine, heart, or liver tissue, using methods well-known to those of skill in the art.
  • RNA isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants.
  • total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells, extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and separating RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et al (eds.), Short Protocols in Molecular Biology, ⁇ d Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)”]; Wu et al, Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)”]).
  • total RNA can be isolated from uterine tissue (or, heart tissue or liver tissue) by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation (see, for example, Chirgwin et al, Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages 33-41).
  • poly(A) + RNA In order to construct a cDNA library, poly(A) + RNA must be isolated from a total RNA preparation. Poly(A) + RNA can be isolated from total RNA using the standard technique of oligo(dT)-cellulose chromatography (see, for example, Aviv and Leder, Proc. Nat 'I Acad. Sci. USA -59. 1408 (1972); Ausubel (1995) at pages 4-11 to 4- 12). Double-stranded cDNA molecules are synthesized from poly(A) + RNA using techniques well-known to those in the art. (see, for example, Wu (1997) at pages 41-46). Moreover, commercially available kits can be used to synthesize double- stranded cDNA molecules.
  • kits are available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI) and STRATAGENE (La Jolla, CA).
  • a cDNA library can be prepared in a vector derived from bacteriophage, such as a ⁇ gtlO vector. See, for example, Huynh et al, "Constructing and Screening cDNA Libraries in ⁇ gtlO and ⁇ gtl l," in DNA Cloning: A Practical
  • double-stranded cDNA molecules can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a
  • LAMDAGEM-4 Promega Corp.
  • Suitable cloning vectors also can be obtained from the American Type Culture Collection
  • the cDNA library is inserted into a prokaryotic host, using standard techniques.
  • a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained, for example, from Life Technologies, Inc. (Gaithersburg, MD).
  • a murine genomic library can be prepared by means well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).
  • Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl, digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient.
  • Genomic DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases.
  • Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307- 327).
  • Nucleic acid molecules that encode a murine Zcytol 3 gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the Zcytol3 gene, as described herein.
  • PCR polymerase chain reaction
  • General methods for screening libraries with PCR are provided by, for example, Yu et al. , "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).
  • a library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon SEQ ID NO:l, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).
  • Anti-Zcytol3 antibodies produced as described below, can also be used to isolate DNA sequences that encode murine Zcyto 13 genes from cDNA libraries.
  • the antibodies can be used to screen ⁇ gtl 1 expression libraries, or the antibodies can be used for immunoscreening following hybrid selection and translation (see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al, "Screening ⁇ expression libraries with antibody and protein probes," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University Press 1995)).
  • a Zcytol3 gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).
  • the nucleic acid molecules of the present invention can also be synthesized with "gene machines” using protocols such as 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 base pairs is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 base pairs), however, special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • Zcytol 3 cDNA or Zcytol 3 genomic fragment can be determined using standard methods.
  • Zcyto 13 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a Zcyto 13 gene.
  • this gene region is expected to provide for preferential expression in heart, liver, brain, kidney, and spleen tissues.
  • Promoter elements from a Zcytol 3 gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy.
  • the identification of genomic fragments containing a Zcyto 13 promoter or regulatory element can be achieved using well-established techniques, such as deletion analysis (see, generally, Ausubel (1995)).
  • Cloning of 5' flanking sequences also facilitates production of Zcyto 13 proteins by "gene activation," as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous Zcyto 13 gene in a cell is altered by introducing into the Zcytol3 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a Zcyto 13 5' non-coding sequence that permits homologous recombination of the construct with the endogenous Zcytol3 locus, whereby the sequences within the construct become operably linked with the endogenous Zcyto 13 coding sequence.
  • an endogenous Zcyto 13 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • SEQ ID NO:3 is a degenerate nucleotide sequence that encompasses all nucleic acid molecules that encode the Zcyto 13 polypeptide of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:3 also provides all RNA sequences encoding SEQ ID NO:2, by substituting U for T. Thus, the present invention contemplates Zcyto 13 polypeptide-encoding nucleic acid molecules comprising nucleotide 86 to nucleotide 619 of SEQ ID NO:l, and their RNA equivalents.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO: 3 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter.
  • “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • degenerate codon representative of all possible codons encoding an amino acid.
  • WSN can, in some circumstances, encode arginine
  • MGN can, in some circumstances, encode serine
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2).
  • the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the present invention further provides variant polypeptides and nucleic acid molecules that represent counterparts from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are Zcyto 13 polypeptides from other mammalian species, including human, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of murine Zcyto 13 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a Zcyto 13 cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zcyto 13 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 Zcytol 3 -encoding cDNA can be isolated by a variety of methods, such as by probing with a complete or partial murine 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 with primers designed from the representative murine Zcyto 13 sequences disclosed herein.
  • a 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 Zcyto 13 polypeptide.
  • Kawade et al, Antiviral Res. 1:161 (1981) have shown that ⁇ types of human and mouse interferon share an antigenic homology.
  • anti- Zyctol3 antibodies can be used to isolate human Zcyto 13 from natural sources, and to isolate human Zcyto 13 sequences from a library that expresses cloned DNA.
  • SEQ ID NO:l represents a single allele of murine Zcytol3, 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 nucleotide sequence shown in SEQ ID NO:l, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2.
  • cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the Zcyto 13 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.
  • isolated nucleic acid molecules that encode murine Zcyto 13 can hybridize to nucleic acid molecules having the nucleotide sequence of SEQ ID NO:l, or a sequence complementary thereto, under "stringent conditions.”
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50%) of the target sequence hybridizes to a perfectly matched probe.
  • a nucleic acid molecule encoding a variant Zcyto 13 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) at 42°C overnight in a solution comprising 50%> formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2%> (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • 5xSSC lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution lOO
  • hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide.
  • a higher temperature such as about 65°C
  • premixed hybridization solutions are available (e.g., EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed according to the manufacturer's instructions.
  • nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions.
  • Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C.
  • nucleic acid molecules encoding a variant Zcyto 13 polypeptide remain hybridized with a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:l (or its complement) following stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • SSPE for SSC in the wash solution.
  • Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65°C.
  • SDS sodium dodecyl sulfate
  • nucleic acid molecules encoding a variant Zcyto 13 polypeptide remain hybridized with a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: l (or its complement) following highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 0.1% SDS at 50°C, or 0.2xSSC with 0.1% SDS at 65°C.
  • the present invention also provides isolated Zcyto 13 polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO:2, or their orthologs.
  • substantially similar sequence identity is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the sequences shown in SEQ ID NO:2, or their orthologs.
  • the present invention also contemplates Zcyto 13 variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ID NO:2, and a hybridization assay, as described above.
  • Such Zcyto 13 variants include nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 86 to 619 of SEQ ID NO:l (or its complement) following stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, and (2) that encode a polypeptide having at least 70%), at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • certain Zcyto 13 variants can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 86 to 619 of SEQ ID NO:l (or its complement) following highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1%o SDS at 50 - 65 °C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 59:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one- letter codes). The percent identity is then calculated as: ([Total number of identical matches]/ [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative Zcyto 13 variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat 'I Acad. Sci. USA #5:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAMJ. Appl. Math. 26:181 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from four to six.
  • the present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared with the amino acid sequence of SEQ ID NO:2. That is, variants can be obtained that contain one or more amino acid substitutions of SEQ ID NO:2, in which an alkyl amino acid is substituted for an alkyl amino acid in a Zcyto 13 amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in a Zcyto 13 amino acid sequence, a sulfur- containing amino acid is substituted for a sulfur-containing amino acid in a Zcyto 13 amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy- containing amino acid in a Zcyto 13 amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a Zcyto 13 amino acid sequence, a basic amino acid is substituted for a basic amino acid in a Zcyto 13 amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in a Zcyto
  • a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • variant Zcyto 13 polypeptides that have an amino acid sequence that differs from SEQ ID NO:2 can be obtained by substituting alanine for glycine 39 , valine for leucine 63 , threonine for serine 81 , valine for isoleucine 105 , or leucine for valine" 3 . Additional variants can be obtained by producing polypeptides having two or more of these amino acid substitutions.
  • 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. Natl Acad. Sci. USA 59:10915 (1992)).
  • 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 can refer to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1 , 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Particular variants of murine Zcyto 13 are characterized by having at least 70%), at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the corresponding amino acid sequence (i.e., SEQ ID NO:2), wherein the variation in amino acid sequence is due to one or more conservative amino acid substitutions.
  • Conservative amino acid changes in a Zcyto 13 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO:l .
  • Such "conservative amino acid” variants can be obtained, for example, by oligonucleotide- directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).
  • the ability of such variants to promote anti-viral or anti-proliferative activity can be determined using a standard method, such as the assay described herein.
  • a variant Zcyto 13 polypeptide can be identified by the ability to specifically bind anti-Zcytol3 antibodies.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tr ⁇ r ⁇ -3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ // -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).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine.
  • non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al, Biochem. 33:1410 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395 (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 Zcyto 13 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 (1989), Bass et al, Proc. Nat 'I Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site- Directed Mutagenesis and Protein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)).
  • Sequence analysis can also identify motifs that reside within murine interferon polypeptides.
  • murine interferon sequences were analyzed for common amino acid sequence motifs: nine murine interferon- ⁇ polypeptides, interferon- ⁇ , interferon- ⁇ , and Zcyto 13.
  • the results of this analysis revealed two motifs in the interferon sequences.
  • One motif occurs at amino acid residues 45 to 59 of Zcyto 13, and has the following sequence: [FSPN] [PFG] [LK] [RSI] [CN]L[KT] [D Y] [IR] [TQKA]DF [GK] [FI]P, wherein acceptable amino acids for a given position are indicated within square brackets ("murine interferon motif 1").
  • Zcyto 13 can be distinguished from other murine interferons when the motif is further defined by the following conditions: (1) the first residue of the sequence is F, or (2) the fourth residue is R, or (3) the ninth residue is I, or (4) the tenth residue is T.
  • the present invention includes polypeptides that comprise an amino acid motif having the sequence [FSPN] [PFG] [LK] [RSI] [CN]L[KT][DY][IR][TQKA]DF[GK][FI]P, wherein the sequence is further defined by at least one of the following conditions: the first residue is F, the fourth residue is R, the ninth residue is I, or the tenth residue is T.
  • a second motif occurs at amino acid residues 152 to 170 of Zcytol 3, and has the following amino acid sequence: [KVR][FY]L[IRK][NEKL] [KM]K[YH][SN][FPLS][CY]AW[KEM][IV][IV][TR][VA]E, wherein acceptable amino acids for a given position are indicated within square brackets ("murine interferon motif 2").
  • Zcyto 13 can be distinguished from other murine interferons when the motif is further defined by one of the following conditions: (1) the first residue is K, or (2) the fourth residue is I, or (3) the fifth residue is N, or (4) the tenth residue is F, or (5) the fourteenth residue is K, or (6) the fifteenth residue is I, or (7) the seventeenth residue is T, or (8) the eighteenth residue is V.
  • the present invention includes polypeptides that comprise an amino acid motif having the sequence: [KVR] [F Y]L [IRK] [NEKL] [KM]K[ YH] [SN] [FPLS] [C Y] A W[KEM] [IV] [IV][TR][VA]E, wherein the sequence is further defined by at least one of the following conditions: the first residue is K, the fourth residue is I, the fifth residue is N, the tenth residue is F, the fourteenth residue is K, the fifteenth residue is I, the seventeenth residue is T, or the eighteenth residue is V.
  • the present invention also includes polypeptides having both murine interferon motifs 1 and 2.
  • Zcyto 13 receptor binding domains 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 (1992), Smith et al, J. Mol. Biol. 224:899 (1992), and Wlodaver et al, FEBS Lett. 309:59 (1992). Moreover, Zcytol3 labeled with biotin or FITC can be used for expression cloning of Zcyto 13 receptors. To date, studies have identified two main receptor binding sites in Type
  • I interferons one at a high affinity responsible for the binding to the receptor, and another site at lower affinity involved in mediating signal transduction (see, for example, Viscomi, Biotherapy 10:59 (1997)).
  • the first site engages Helices A and B, and Loop AB, while the second site engages Helices A and C and Loop DE.
  • mutations can be introduced into Helix C or Loop DE to interfere with Zcyto 13 receptor signaling.
  • Such a mutant would be expected to bind a Zcyto 13 receptor without producing a biological effect, and therefore, would have the properties of a Zcyto 13 antagonist.
  • Helix C and Loop DE are represented by amino acids 99 to 124, and 156 to 160, respectively, of SEQ ID NO:2.
  • Another form of Zcyto 13 antagonist could consist of Helices A and B, and Loop AB of the Zcytol3 form described herein (i.e., amino acids 22 to 89 of SEQ ID NO:2).
  • variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes. Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind with anti-Zcytol3 antibodies, 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.
  • the present invention also includes "functional fragments" of Zcyto 13 polypeptides and nucleic acid molecules encoding such functional fragments.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a Zcyto 13 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ID NO:l can be digested with Bal31 nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for anti-viral or anti-proliferative activity, or for the ability to bind anti-Zcytol3 antibodies.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of a Zcyto 13 gene can be synthesized using the polymerase chain reaction.
  • the present invention also contemplates functional fragments of a Zcyto 13 gene that has amino acid changes, compared with the amino acid sequence of SEQ ID NO:2.
  • a variant Zcytol 3 gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequence of SEQ ID NO:2, as discussed above.
  • An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant Zcytol3 gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, as discussed above.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zcyto 13 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, Proc. Nat 'I Acad. Sci. USA 81:3998 (1983)).
  • polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind.
  • Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al, Science 219:660 (1983)). Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.
  • Antigenic epitope-bearing peptides and polypeptides can contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID NO:2. Such epitope-bearing peptides and polypeptides can be produced by fragmenting a Zcyto 13 polypeptide, or by chemical peptide synthesis, as described herein. Moreover, epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol 5:268 (1993), and Cortese et al, Curr. Opin. Biotechnol 7:616 (1996)).
  • variant Zcytol 3 gene encodes a polypeptide that is characterized by its anti-viral or anti- proliferative activity, or by the ability to bind specifically to an anti-Zcytol3 antibody. More specifically, variant murine Zcytol 3 genes encode polypeptides, which exhibit at least 50%, or, greater than 70, 80, or 90%>, of the activity of polypeptide encoded by the murine Zcytol 3 gene described herein.
  • any Zcyto 13 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.
  • those of skill in the art can use standard software to devise Zcyto 13 variants based upon the nucleotide and amino acid sequences described herein.
  • the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO:l l .
  • Suitable forms of computer- readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIP disk.
  • Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD- rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
  • compact discs e.g., CD-read only memory (ROM), CD- rewritable (RW), and CD-recordable
  • DVD digital versatile/video discs
  • Fusion proteins of Zcyto 13 can be used to express Zcyto 13 in a recombinant host, and to isolate expressed Zcyto 13. As described below, particular Zcyto 13 fusion proteins also have uses in diagnosis and therapy.
  • fusion protein comprises a peptide that guides a Zcyto 13 polypeptide from a recombinant host cell.
  • a secretory signal sequence also known as a signal peptide, a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence may be derived from Zcyto 13
  • a suitable signal sequence may also be derived from another secreted protein or synthesized de novo.
  • the secretory signal sequence is operably linked to a Zcyto 13- encoding sequence such that 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 nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide sequence of interest (see, e.g., Welch et ⁇ l, U.S. Patent No. 5,037,743; Holland et ⁇ l, U.S. Patent No. 5,143,830).
  • yeast signal sequence is preferred for expression in yeast cells.
  • suitable yeast signal sequences are those derived from yeast mating phermone ⁇ -factor (encoded by the MFal gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene).
  • Zcyto 13 can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide.
  • Glutathione S-transferease fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • a Zcyto 13 fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resin column, while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose.
  • Peptide tags that are useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaryotic cells include polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies). See, for example, Luo et al, Arch. Biochem. Biophys. 329:215 (1996), Morganti et al, Biotechnol Appl Biochem.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1 to 21 of SEQ ID NO:2 is operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor.
  • Such fusions may be used in a transgenic animal or in a cultured recombinant host to direct peptides through the secretory pathway.
  • exemplary polypeptides include pharmaceutically active molecules such as Factor Vila, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferons- ⁇ , - ⁇ , - ⁇ , - ⁇ , - ⁇ , and - ⁇ ), the stem cell growth factor designated "SI factor,” erythropoietin, and thrombo
  • interleukins e.
  • the Zcyto 13 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. Fusion proteins comprising a Zcyto 13 secretory signal sequence can be constructed using standard techniques.
  • fusion protein comprises a Zcyto 13 polypeptide and an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two or three constant region domains and a hinge region but lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • F c fragment an immunoglobulin heavy chain constant region
  • Chang et al, U.S. Patent No. 5,723,125 describe a fusion protein comprising a human interferon and a human immunoglobulin Fc fragment.
  • the C- terminal of the interferon is linked to the N-terminal of the Fc fragment by a peptide linker moiety.
  • An example of a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert.
  • An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO:4).
  • a preferred Fc moiety is a human ⁇ 4 chain, which is stable in solution and has little or no complement activating activity.
  • the present invention contemplates a Zcyto 13 fusion protein that comprises a Zcyto 13 moiety and a human Fc fragment, wherein the C-terminus of the Zcyto 13 moiety is attached to the N- terminus of the Fc fragment via a peptide linker, such as a peptide consisting of the amino acid sequence of SEQ ID NO:4.
  • the Zcyto 13 moiety can be a Zcyto 13 molecule or a fragment thereof.
  • a Zcyto 13 fusion protein comprises an IgG sequence, a Zcyto 13 moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently joined to the aminoterminal of the Zcyto 13 moiety, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH 2 domain, and a CH 3 domain. Accordingly, the IgG sequence lacks a CH, domain.
  • the Zcyto 13 moiety displays a Zcyto 13 activity, as described herein, such as the ability to bind with a Zcyto 13 receptor.
  • Fusion proteins comprising a Zcyto 13 moiety and an Fc moiety can be used, for example, as an in vitro assay tool.
  • the presence of an Zcyto 13 receptor in a biological sample can be detected using a Zcyto 13 -immunoglobulin fusion protein, in which the Zcyto 13 moiety is used to target the cognate receptor, and a macromolecule, such as Protein A or anti-Fc antibody, is used to detect the bound fusion protein-receptor complex.
  • fusion proteins can be used to identify agonists and antagonists that interfere with the binding of Zcyto 13 to its receptor.
  • antibody-Zcytol3 fusion proteins comprising antibody variable domains, are useful as therapeutic proteins, in which the antibody moiety binds with a target antigen, such as a tumor associated antigen.
  • a target antigen such as a tumor associated antigen.
  • Methods of making antibody- cytokine fusion proteins are known to those of skill in the art.
  • antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al, Ann. Oncol. 6:945 (1995), Nicolet et al, Cancer Gene Ther. 2:161 (1995), Becker et al, Proc. Nat Acad. Sci. USA 93:1826 (1996), Hank et al, Clin. Cancer Res. 2:1951 (1996), and Hu et al, Cancer Res. 56:4998 (1996).
  • cytokine-antibody fusion proteins include IL-8, IL-12, or interferon- ⁇ as the cytokine moiety (Holzer et al, Cytokine 8:214 (1996); Gillies et al, J. Immunol. 160:6195 (1998); Xiang et al, Hum. Antibodies Hybridomas 7:2 (1996)). Also see, Gillies, U.S. Patent No. 5,650,150.
  • hybrid Zcyto 13 proteins can be constructed using regions or domains of the inventive Zcyto 13 in combination with those of other interferon family proteins (i.e., interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ ), or heterologous proteins (see, for example, Picard, Cur. Opin. Biology 5:511 (1994)).
  • interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ or heterologous proteins.
  • 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.
  • 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.
  • part or all of a domain(s) conferring a biological function may be swapped between interferon- ⁇ of the present invention with the functionally equivalent domain(s) from another interferon- ⁇ , or from another family member, such as interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , or interferon- ⁇ .
  • Such domains include, but are not limited to, the secretory signal sequence, helices A, B, C, D, and E, and loops AB, BC, CD, and DE.
  • 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 interferon family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein. General methods for enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) at pages 16-19 to 16- 25.
  • the present invention also contemplates chemically modified Zcyto 13 compositions, in which a Zcyto 13 polypeptide is linked with a polymer.
  • the polymer is water soluble so that the Zcyto 13 conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled.
  • a reactive aldehyde is polyethylene glycol propionaldehyde, or mono-(Cl-ClO) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al, U.S. Patent No. 5,252,714).
  • the polymer may be branched or unbranched.
  • a mixture of polymers can be used to produce Zcyto 13 conjugates.
  • Zcyto 13 conjugates used for therapy can comprise pharmaceutically acceptable water-soluble polymer moieties. Conjugation of interferons with water- soluble polymers has been shown to enhance the circulating half-life of the interferon, and to reduce the immunogenicity of the polypeptide (see, for example, Nieforth et al, Clin. Pharmacol. Ther. 59:636 (1996), and Monkarsh et al, Anal. Biochem. 247:434 (1997)).
  • Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(Cl-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bw-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers.
  • Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000.
  • a Zcytol 3 conjugate can also comprise a mixture of such water-soluble polymers.
  • Zcyto 13 conjugate comprises a Zcyto 13 moiety and a polyalkyl oxide moiety attached to the N-terminus of the Zcyto 13 moiety.
  • PEG is one suitable polyalkyl oxide.
  • Zcyto 13 can be modified with PEG, a process known as "PEGylation.” PEGylation of Zcyto 13 can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316, Delgado et al, Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al, hit J Hematol 68: 1 (1998)).
  • PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule.
  • Zcyto 13 conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see. for example, Karasiewicz et al, U.S. Patent No. 5,382,657).
  • PEGylation by acylation typically requires reacting an active ester derivative of PEG with a Zcyto 13 polypeptide.
  • An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide.
  • acylation includes the following types of linkages between Zcyto 13 and a water soluble polymer: amide, carbamate, urethane, and the like.
  • Methods for preparing PEGylated Zcyto 13 by acylation will typically comprise the steps of (a) reacting a Zcyto 13 polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to Zcytol 3, and (b) obtaining the reaction product(s).
  • PEG such as a reactive ester of an aldehyde derivative of PEG
  • the optimal reaction conditions for acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG:Zcytol3, the greater the percentage of polyPEGylated Zcyto 13 product.
  • the product of PEGylation by acylation is typically a polyPEGylated
  • the resulting Zcyto 13 product wherein the lysine ⁇ -amino groups are PEGylated via an acyl linking group.
  • An example of a connecting linkage is an amide.
  • the resulting Zcyto 13 will be at least 95% mono-, di-, or tri-pegylated, although some species with higher degrees of PEGylation may be formed depending upon the reaction conditions.
  • PEGylated species can be separated from unconjugated Zcyto 13 polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, and the like.
  • PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with Zcyto 13 in the presence of a reducing agent.
  • PEG groups are preferably attached to the polypeptide via a -CH 2 -NH group.
  • Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization.
  • the reaction is performed at a pH that allows one to take advantage of the pKa differences between the ⁇ -amino groups of the lysine residues and the ⁇ -amino group of the N-terminal residue of the protein.
  • a water-soluble polymer that contains a reactive group such as an aldehyde
  • the conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups.
  • the present invention provides a substantially homogenous preparation of Zcyto 13 monopolymer conjugates.
  • Reductive alkylation to produce a substantially homogenous population of monopolymer Zcyto 13 conjugate molecule can comprise the steps of: (a) reacting a Zcyto 13 polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the ⁇ -amino group at the amino terminus of the Zcyto 13, and (b) obtaining the reaction product(s).
  • the reducing agent used for reductive alkylation should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation.
  • Preferred reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, and pyridine borane.
  • the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the N-terminus of Zcytol 3.
  • Such reaction conditions generally provide for pKa differences between the lysine amino groups and the ⁇ -amino group at the N-terminus.
  • the pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal ⁇ -group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer :Zcyto 13 need not be as large because more reactive groups are available. Typically, the pH will fall within the range of 3 - 9, or 3 - 6.
  • the molecular weight of the water-soluble polymer is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25 kDa.
  • the molar ratio of water-soluble polymer to Zcytol3 will generally be in the range of 1 : 1 to 100: 1.
  • the molar ratio of water-soluble polymer to Zcyto 13 will be 1 :1 to 20:1 for polyPEGylation, and 1 :1 to 5:1 for monoPEGylation.
  • compositions comprising a peptide or polypeptide described herein.
  • Such compositions can further comprise a carrier.
  • the carrier can be a conventional organic or inorganic carrier. Examples of carriers include water, buffer solution, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.
  • Peptides and polypeptides of the present invention comprise at least six, at least nine, or at least 15 contiguous amino acid residues of the following amino acid sequences within SEQ ID NO:2: amino acid residues 22 to 199, amino acid residues 22 to 188, amino acid residues 22 to 45, amino acid residues 46 to 64, amino acid residues 65 to 89, amino acid residues 90 to 98, amino acid residues 99 to 124, amino acid residues 125 to 133, amino acid residues 134 to 155, amino acid residues 22 to 89, and amino acid residues 161 to 181.
  • the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of these amino acid sequences. Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes. 6. Production of Zcytol 3 Polypeptides in Cultured Cells
  • polypeptides of the present invention can be produced in recombinant host cells following conventional techniques.
  • a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene, which is suitable for selection of cells that carry the expression vector.
  • Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
  • expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell.
  • a Zcyto 13 expression vector may comprise a Zcyto 13 gene and a secretory sequence derived from a Zcyto 13 gene or another secreted gene.
  • Zcyto 13 proteins of the present invention may be expressed in mammalian cells.
  • suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293- HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1 ; ATCC CCL61 ; CHO DG44 [Chasin et al, Som. Cell. Molec. Genet.
  • GH1 rat pituitary cells
  • H-4-II-E rat hepatoma cells
  • COS-1 SV40-transformed monkey kidney cells
  • NIH- 3T3 ATCC CRL 1658
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Molec. Appl Genet.
  • a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter
  • a prokaryotic promoter can be used to control Zcyto 13 gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al, Mol Cell. Biol. 10:4529 (1990), and Kaufman et al, Nucl Acids Res. 79:4485 (1991)).
  • An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like.
  • the transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).
  • one suitable selectable marker is a gene that provides 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 suitable amplifiable selectable marker is dihydrofolate reductase. which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • 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.
  • Zcyto 13 polypeptides can also be produced by cultured mammalian cells using a viral delivery system.
  • viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV).
  • Adenovirus a double- stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et al, Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)).
  • Advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters.
  • Adenovirus vector-infected human 293 cells ATCC Nos. CRL-1573, 45504, 45505
  • can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein see Gamier et al, Cytotechnol. 75: 145 (1994)).
  • Zcyto 13 may also be expressed in other higher eukaryotic cells, such as avian, fungal, insect, yeast, or plant cells.
  • the baculovirus system provides an efficient means to introduce cloned Zcytol3 genes into insect cells.
  • Suitable expression vectors are based upon the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, Autographa californica nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter.
  • hsp Drosophila heat shock protein
  • ie-1 Autographa californica nuclear polyhedrosis virus immediate-early gene promoter
  • baculovirus plO promoter the Drosophila metallothionein promoter.
  • a second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, et al, J. Virol. 67:4566 (1993)).
  • This system which utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Zcyto 13 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 77:971 (1990), Bonning, et al, J. Gen.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Zcyto 13 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al, Proc. Natl Acad. Sci. 82:1952 (1985)).
  • a transfer vector containing a Zcytol3 gene is transformed into E coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques.
  • the illustrative PFASTBAC vector can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as ⁇ cor, 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, for example, Hill-Perkins and Possee, J. Gen. Virol. 71:911 (1990), Bonning, et al, J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995).
  • a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed, which replace the native Zcyto 13 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from ⁇ cdysteroid Glucosyltransferase ( ⁇ GT), honey bee Melittin (Invitrogen Corporation; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native Zcyto 13 secretory signal sequence.
  • the recombinant virus or bacmid is used to transfect host cells.
  • suitable insect host cells include cell lines derived from YPU3-Sf-2l, a Spodoptera frugiperda pupal ovarian cell line, such as S ⁇ (ATCC CRL 171 1), S/21A ⁇ , and S 21 (Invitrogen Corporation; San Diego, CA), as well as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).
  • S ⁇ ATCC CRL 171 1
  • S/21A ⁇ S 21
  • S 21 Invitrogen Corporation
  • Drosophila Schneider-2 cells Drosophila Schneider-2 cells
  • HIGH FIVEO cell line Invitrogen
  • Commercially available serum-free media can be used to grow and to maintain the cells.
  • Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are typically grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • yeast cells can also be used to express the genes described herein.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), A OX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
  • GAL1 galactose
  • PGK phosphoglycerate kinase
  • ADH alcohol dehydrogenase
  • a OX1 alcohol oxidase
  • HIS4 histidinol dehydrogenase
  • These vectors include Yip-based vectors, such as YIp5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCp 19.
  • Methods for transforming S cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al, U.S. Patent No. 4,931,373, Brake, U.S. Patent No. 4,870,008, Welch et al, U.S. Patent No. 5,037,743, and Murray et al, U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a suitable vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Additional suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,31 1, Kingsman et al, U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454.
  • Transformation systems for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol 132:3459 (1986), and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349.
  • methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or A UG2).
  • Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes.
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a suitable 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), and which allows ade2 host cells to grow in the absence of adenine.
  • a suitable 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), and which allows ade2 host cells to grow in the absence of adenine.
  • a UG1 and A UG2 methanol utilization genes
  • host cells deficient in vacuolar protease genes PEP4 and PRB1 are preferred.
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells.
  • P. methanolica cells can be transformed 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.
  • Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells.
  • Methods for introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch et al, Science 227:1229 (1985), Klein et al, Biotechnology 10:268 (1992), and Miki et al, "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology. Glick et al. (eds.), pages 67-88 (CRC Press, 1993).
  • Zcytol3 genes can be expressed in prokaryotic host cells.
  • Suitable promoters that can be used to express Zcyto 13 polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promoters of E coli, promoters of B.
  • subtilis subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene.
  • Prokaryotic promoters have been reviewed by Glick, J Ind. Microbiol 1:211 (1987), Watson et al, Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995/
  • Illustrative prokaryotic hosts include E. coli and Bacillus subtilus. Suitable strains of E. coli include BL21(D ⁇ 3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH1 1 S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)).
  • Suitable strains of Bacillus subtilus include BR151, YB886, Mil 19, Ml 120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)).
  • the polypeptide When expressing a Zcyto 13 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Standard methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are provided, for example, by Ausubel (1995).
  • General methods for expressing and recovering foreign protein produced by a mammalian cell system are provided by, for example, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture,” in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
  • Standard techniques for recovering protein produced by a bacterial system is provided by, for example, Grisshammer et al. , "Purification of over-produced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
  • interferon polypeptides from cultured cells
  • recombinant interferons have been produced by bacteria, yeasts, plant cells, insect cells, vertebrate cells, as well as in cell-free systems (Horisberger and Di Marco, Pharmac. Ther. 66:501 (1995)).
  • Reviews of methods for producing recombinant interferon are provided, for example, by Edge and Camble, Biolechnol Genet. Eng. Rev. 2:215 (1984), Langer and Pestka, J. Invest. Dermatol 55: 128s (1984), Pestka, Semin. Hematol 23:21 (1986), Baron and Narula, Crit. Rev.
  • polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well-known to those of skill in the art (see, for example, Merrifield, J. Am. Chem. Soc. 55:2149 (1963), Stewart et al, "Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot.
  • polypeptides of the present invention can be purified to at least about 80%) purity, to at least about 90%> purity, to at least about 95% purity, or even greater than 95%) purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • the polypeptides of the present invention may also be purified to a pharmaceutically pure state, which is greater than 99.9% pure.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Fractionation and/or conventional purification methods can be used to obtain preparations of Zcyto 13 purified from natural sources (e.g., heart, liver, brain, kidney, or spleen), and recombinant Zcyto 13 polypeptides and fusion Zcyto 13 polypeptides purified from recombinant host cells.
  • natural sources e.g., heart, liver, brain, kidney, or spleen
  • ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used.
  • These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Beare et ⁇ l Biochim. Biophys. Act ⁇ 1310:81 (1996), describe the isolation of a murine interferon- ⁇ using a combination of Blue Sepharose chromatography, immunoaffinity exclusion, and Q Sepharose ion exchange fractionation.
  • Examples of coupling chemistries include cyanogen bromide activation,
  • N-hydroxysuccinimide activation N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • solid media are well known and widely used in the art, and are available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996).
  • Zcyto 13 isolation and purification can be devised by those of skill in the art.
  • anti-Zcytol3 antibodies obtained as described below, can be used to isolate large quantities of protein by immunoaffinity purification.
  • the use of monoclonal antibody columns to purify interferons from recombinant cells and from natural sources has been described, for example, by Staehelin et al, J. Biol. Chem. 256:9150 (1981), and by Adolf et al, J. Biol. Chem. 265:9290 (1990).
  • methods for binding ligands, such as Zcytol 3, to receptor polypeptides bound to support media are well known in the art.
  • the polypeptides of the present invention can also be isolated by exploitation of particular 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.
  • IMAC immobilized metal ion adsorption
  • Zcyto 13 polypeptides or fragments thereof may also be prepared through chemical synthesis, as described below.
  • Zcyto 13 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.
  • a Zcyto 13 variant may be functionally characterized by its ability to specifically bind with an anti-Zcytol3 antibody, or by a biological activity such as anti- viral activity, anti-proliferative activity, the ability to stimulate the expression of a gene known to be induced by murine interferon- ⁇ (e.g., a xanthine dehydrogenase gene of L929 fibroblastic cells, a 2'5'-oligoadenylate synthetase in quiescent BALB/c mouse 3T3 cells), or the ability to decrease the development of spontaneous diabetes and the passive transfer of diabetes in NOD mouse model of human IDDM.
  • murine interferon- ⁇ e.g., a xanthine dehydrogenase gene of L929 fibroblastic cells, a 2'5'-oligoadenylate synthetase in quiescent BALB/c mouse 3T3 cells
  • Illustrative activity assays are described below.
  • a Zcyto 13 variant gene product that lacks biological activity may be a Zcytol 3 antagonist.
  • These biologically-inactive Zcytol 3 variants can be initially identified on the basis of hybridization analysis, sequence identity determination, or by the ability to specifically bind anti-Zcytol3 antibody.
  • a Zcyto 13 antagonist can be further characterized by its ability to inhibit the biological response induced by Zcyto 13 or by a Zcyto 13 agonist. This inhibitory effect may result, for example, from the competitive or non-competitive binding of the antagonist to the Zcyto 13 receptor.
  • Zcytol 3, its agonists and antagonists are valuable in both in vivo and in vitro uses.
  • cytokines can be used as components of defined cell culture media, alone or in combination with other cytokines and hormones, to replace serum that is commonly used in cell culture.
  • interferons have been shown to stimulate the production of other biologically active polypeptides, such as interleukin-1, by cultured cells, which can be isolated from the culture (see, for example, Danis et al, Clin. Exp. Immunol. 80:435 (1990)).
  • Interferons have also been shown to induce the expression of antigens by cultured cells (see, for example, Auth et al, Hepatology 18:546 (1993), Guadagni et al, Int. J. Biol.
  • Antagonists are also useful as research reagents for characterizing sites of interaction between Zcyto 13 and its receptor.
  • pharmaceutical compositions comprising Zcyto 13 antagonists can be used to inhibit Zcyto 13 activity.
  • Zcyto 13 analogs are agonists or antagonists having an amino acid sequence that is a mutation of the amino acid sequences disclosed herein.
  • Another general class of Zcyto 13 analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below.
  • recombinant antibodies comprising anti-idiotype variable domains can be used as analogs (see, for example, Monfardini et al, Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domains of anti- idiotype Zcyto 13 antibodies mimic Zcyto 13, these domains can provide either Zcyto 13 agonist or antagonist activity.
  • Lim and Langer, J Interferon Res. 13:295 (1993) describe anti-idiotypic interferon- ⁇ antibodies that have the properties of either interferon- ⁇ agonists or antagonists.
  • a third approach to identifying Zcyto 13 analogs is provided by the use of combinatorial libraries.
  • Methods for constructing and screening phage display and other combinatorial libraries are provided, for example, by Kay et al, Phage Display of
  • One assay that can be used to measure Zcyto 13 biological activity is an interferon in vitro virus inhibition assay.
  • the anti-viral activity of variant Zcyto 13 polypeptides can be assessed by a cytopathic effect reduction assay, in which mouse L929 cells are challenged with Mengo virus or vesicular stomatitis virus (Stewart, 77ze Interferon System (Springer 1979); Zawatzky et al, J. Gen. Virol. 63:325 (1982); Van Heuvel et al, J. Gen. Virol. 67:22 5 (1986)).
  • the anti-viral activity of a variant Zcyto 13 can also be measured with murine cytomegalovirus-infected NIH 3T3 fibroblasts (Gribaudo et al, Virology 797:303 (1993)).
  • Example 5 illustrates a method for testing anti-viral activity using encephalomyocarditis virus and murine L929 cells, as well as other methods for determining Zcyto 13 activity.
  • Another approach to evaluating Zcyto 13 activity is to use an assay that measures the inhibition of the proliferation of cultured murine cells.
  • the anti-proliferative activity of a variant Zcyto 13 polypeptide can be determined with mouse B-16 melanoma cells (Fleischmann and Fleischmann, J Biol. Regul Homeost. Agents 2:113 (1988)).
  • Zcyto 13 activity is measured by the ability of the test polypeptide to stimulate the expression of a gene that is induced by murine interferon- ⁇ .
  • Illustrative genes include a xanthine dehydrogenase gene of L929 fibroblastic cells, and a 2'5'-oligoadenylate synthetase in quiescent BALB/c mouse 3T3 cells (Falciani et al, Biochem. J. 255:1001 (1992); Yan et al, Proc. Natl Acad. Sci USA 86:2243 (1989)).
  • the activity of a Zcyto 13 variant is determined using an in vivo assay.
  • interferon- ⁇ administration decreases the development of spontaneous diabetes and the passive transfer of diabetes in NOD mouse model of human IDDM (Sobel and Ahvazi, Diabetes 47:1861 (1998)).
  • the activity of Zcyto 13 can be measured by a silicon-based biosensor microphysiometer, which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent cellular responses.
  • An exemplary device is the CYTOSENSOR Microphysiometer manufactured by Molecular Devices Corp. (Sunnyvale, CA).
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method (see, for example, McConnell et al, Science 257: 1906 (1992), Pitchford et al, Meth. Enzymol. 228:84 (1997), Arimilli et al, J. Immunol. Meth.
  • microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells.
  • any event which alters cellular ATP levels such as receptor activation and the initiation of signal transduction, will cause a change in cellular acid section.
  • An early event in interferon signal transduction is protein phosphorylation, which requires ATP.
  • the microphysiometer directly measures cellular responses to various stimuli, including Zcyto 13, its agonists, or antagonists.
  • a microphysiometer can be used to measure responses of a Zcyto 13 responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to Zcytol3 polypeptide.
  • Zcytol3 responsive eukaryotic cells comprise cells into which a receptor for Zcyto 13 has been transfected to create a cell that is responsive to Zcyto 13, or cells that are naturally responsive to Zcytol 3.
  • murine cells that respond to murine interferon- ⁇ members include embryonic stem cells, B-16 melanoma cells, L 929 fibroblastic cells, and 3T3 cells (see, for example, Fleischmann and Fleischmann, J Biol. Regul Homeost. Agents 2:173 (1988); Yan et al, Proc. Natl Acad. Sci USA 86:2243 (1989); Falciani et al, Biochem. J. 255:1001 (1992); Whyatt et ⁇ /., Mol Cell Biol.
  • Zcyto 13 modulated cellular responses are measured by a change (e.g., an increase or decrease in extracellular acidification) in the response of cells exposed to Zcytol3, compared with control cells that have not been exposed to Zcytol 3.
  • a microphysiometer can be used to identify cells, tissues, or cell lines which respond to a Zcyto 13 stimulated pathway, and which express a functional Zcyto 13 receptor.
  • cells that express a functional Zcyto 13 receptor can be identified by (a) providing test cells, (b) incubating a first portion of the test cells in the absence of Zcytol 3, (c) incubating a second portion of the test cells in the presence of Zcyto 13, and (d) detecting a change (e.g., an increase or decrease in extracellular acidification rate, as measured by a microphysiometer) in a cellular response of the second portion of the test cells, as compared to the first portion of the test cells, wherein such a change in cellular response indicates that the test cells express a functional Zcyto 13 receptor.
  • An additional negative control may be included in which a portion of the test cells is incubated with Zcyto 13 and an anti-Zcytol3 antibody to inhibit the binding of Zcyto 13 with its cognate receptor.
  • the microphysiometer also provides one means to identify Zcyto 13 agonists.
  • agonists of Zcyto 13 can be identified by a method, comprising the steps of (a) providing cells responsive to Zcytol3, (b) incubating a first portion of the cells in the absence of a test compound, (c) incubating a second portion of the cells in the presence of a test compound, and (d) detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells, wherein such a change in cellular response indicates that the test compound is a Zcyto 13 agonist.
  • An illustrative change in cellular response is a measurable change in extracellular acidification rate, as measured by a microphysiometer.
  • incubating a third portion of the cells in the presence of Zcytol 3 and in the absence of a test compound can be used as a positive control for the Zcyto 13 responsive cells, and as a control to compare the agonist activity of a test compound with that of Zcytol 3.
  • An additional control may be included in which a portion of the cells is incubated with a test compound (or Zcyto 13) and an anti-Zcytol3 antibody to inhibit the binding of the test compound (or Zcytol 3) with the Zcyto 13 receptor.
  • the microphysiometer also provides a means to identify Zcyto 13 antagonists.
  • Zcyto 13 antagonists can be identified by a method, comprising the steps of (a) providing cells responsive to Zcyto 13, (b) incubating a first portion of the cells in the presence of Zcyto 13 and in the absence of a test compound, (c) incubating a second portion of the cells in the presence of both Zcyto 13 and the test compound, and (d) comparing the cellular responses of the first and second cell portions, wherein a decreased response by the second portion, compared with the response of the first portion, indicates that the test compound is a Zcytol3 antagonist.
  • An illustrative change in cellular response is a measurable change extracellular acidification rate, as measured by a microphysiometer.
  • Zcyto 13, its analogs, and anti-iodiotype Zcyto 13 antibodies can be used to identify and to isolate Zcyto 13 receptors.
  • proteins and peptides of the present invention can be immobilized on a column and used to bind receptor proteins from membrane preparations that are run over the column (Hermanson et al (eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)).
  • Radiolabeled or affinity labeled Zcyto 13 polypeptides can also be used to identify or to localize Zcytol 3 receptors in a biological sample (see, for example, Lieber (ed.), Methods in Enzymol, vol. 182, pages 721-37 (Academic Press 1990); Brunner et al, Ann.
  • Antibodies to Zcyto 13 can be obtained, for example, using the product of a Zcyto 13 expression vector or Zcyto 13 isolated from a natural source as an antigen. Particularly useful anti-Zcytol3 antibodies "bind specifically" with Zcytol 3. Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to Zcyto 13 with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to Zcytol 3.
  • antibodies specifically bind if they bind to a Zcyto 13 polypeptide, peptide or epitope with a binding affinity (K a ) of 10 6 M “ ' or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, and most preferably 10 9 M “1 or greater.
  • K a binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 57:660 (1949)).
  • antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zcytol3, but not known polypeptides using a standard Western blot analysis.
  • anti-Zcyto 13 antibodies bind with Zcyto 13, but not with polypeptides such as other interferon- ⁇ polypeptides, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , or interferon- ⁇ .
  • Suitable antibodies include antibodies that bind with Zcyto 13 in regions having a low sequence similarity with other interferons.
  • Anti-Zcytol3 antibodies can be produced using antigenic Zcyto 13 epitope-bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, or between 15 to about 30 amino acids contained within SEQ ID NO:2.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with Zcytol 3. It is desirable that 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, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.
  • a surface contour value designated as the "antigenic index.”
  • a peak broadening function was applied to the antigenic index, which broadens major surface peaks by adding 20, 40, 60, or 80% of the respective peak value to account for additional free energy derived from the mobility of surface regions relative to interior regions. This calculation was not applied, however, to any major peak that resides in a helical region, since helical regions tend to be less flexible.
  • amino acids 51 to 61 amino acids 51 to 61
  • amino acids 68 to 73 amino acids 90 to 96
  • amino acids 97 to 136 amino acids 104 to 110
  • amino acids 120 to 136 amino acids 156 to 161
  • amino acids 179 to 185 amino acids 190 to 199
  • antigenic peptide 9 amino acids 190 to 199
  • the present invention contemplates the use of any one of antigenic peptides 1 to 9 to generate antibodies to Zcytol 3.
  • the present invention also contemplates polypeptides comprising at least one of antigenic peptides 1 to 9.
  • Polyclonal antibodies to recombinant Zcyto 13 protein or to Zcyto 13 isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al, "Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al, "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995).
  • the immunogenicity of a Zcyto 13 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zcyto 13 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • polypeptide portion is "hapten-like,” such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • an anti- Zcyto 13 antibody of the present invention may also be derived from a subhuman primate antibody.
  • General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al, international patent publication No. WO 91/11465, and in Losman et al, Int. J. Cancer (5:310 (1990).
  • monoclonal anti-Zcytol3 antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al, Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) ["Coligan”], Picksley et al, "Production of monoclonal antibodies against proteins expressed in E. coli, ' " in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al (eds.), page 93 (Oxford University Press 1995)).
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising a Zcyto 13 gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • an anti-Zcytol3 antibody of the present invention may be derived from a human monoclonal antibody.
  • Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al, Nature Genet. 7:13 (1994), Lonberg et al, Nature 368:856 (1994), and Taylor et al, Int. Immun. (5:579 (1994).
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al, "Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol 10, pages 79-104 (The Humana Press, Inc. 1992)).
  • antibody fragments of anti- Zcyto 13 antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.
  • the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
  • These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al, Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem. J. 75:1 19 (1959), Edelman et al, in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
  • Fv fragments comprise an association of V H and V L chains.
  • variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:431 (1992)).
  • the Fv fragments may comprise V H and V L chains, which are connected by a peptide linker.
  • scFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli.
  • a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing scFvs are described, for example, by Whitlow et al, Methods: A Companion to Methods in Enzymology 2:91 (1991) (also see, Bird et al, Science 242:423 (1988), Ladner et al, U.S. Patent No. 4,946,778, Pack et al, Bio/Technology 77:1271 (1993), and Sandhu, supra).
  • an scFV can be obtained by exposing lymphocytes to lymphocytes.
  • Zcyto 13 polypeptide in vitro and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zcyto 13 protein or peptide).
  • Genes encoding polypeptides having potential Zcyto 13 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al, U.S. Patent No. 5,223,409, Ladner et al, U.S. Patent No. 4,946,778, Ladner et al, U.S. Patent No. 5,403,484, Ladner et al, U.S. Patent No. 5,571,698, and Kay et al, Phage Display of Peptides and Proteins (Academic Press, Inc.
  • Random peptide display libraries can be screened using the Zcyto 13 sequences disclosed herein to identify proteins which bind to Zcytol 3.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al, Methods: A Companion to Methods in Enzymology 2: 106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al.
  • an anti-Zcytol3 antibody may be derived from a
  • humanized monoclonal antibody Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat 'I Acad. Sci. USA 86:3833 (1989).
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zcytol3 antibodies or antibody fragments, using standard techniques. See, for example, Green et al, "Production of Polyclonal Antisera,” in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7.
  • monoclonal anti- idiotype antibodies can be prepared using anti-Zcytol3 antibodies or antibody fragments as immunogens with the techniques, described above.
  • humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques.
  • Nucleic acid molecules can be used to detect the expression of a Zcytol 3 gene in a biological sample.
  • Suitable probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: l, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:l, or a portion thereof. Probe molecules may be
  • portion refers to at least eight nucleotides to at least 20 or more nucleotides. Certain probes bind with regions of the Zcytol 3 gene that have a low sequence similarity to comparable regions in other interferons.
  • RNA isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target Zcyto 13 RNA species.
  • RNA detection includes northern analysis and dot/slot blot hybridization (see, for example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et ⁇ l. (eds.), "Analysis of Gene Expression at the RNA
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 33 S.
  • Zcyto 13 RNA can be detected with a nonradioactive hybridization method
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemilumineseent substrates.
  • Illustrative nonradioactive moieties include biotin, fluorescein, .and digoxigenin.
  • Zcyto 13 oligonucleotide probes are also useful for in vivo diagnosis.
  • 18 F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al, Nature Medicine 4:461 (1998)).
  • PCR polymerase chain reaction
  • Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).
  • PCR primers can be designed to amplify a portion of the Zcyto 13 gene that has a low sequence similarity to a comparable region in other interferons.
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with Zcyto 13 primers (see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)). PCR is then performed and the products are analyzed using standard techniques.
  • RNA is isolated from biological sample using, for example, the gunadinium-thiocyanate cell lysis procedure described above.
  • a solid-phase technique can be used to isolate mRNA from a cell lysate.
  • a reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or Zcyto 13 anti-sense oligomers.
  • Oligo- dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • Zcyto 13 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length.
  • PCR amplification products can be detected using a variety of approaches.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled Zcyto 13 probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • CPT cycling probe technology
  • Zcyto 13 sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA), cooperative amplification of templates by cross-hybridization (CATCH), and the ligase chain reaction (LCR) (see, for example, Marshall et al, U.S. Patent No. 5,686,272 (1997), Dyer et al, J. Virol. Methods 60:161 (1996), Ehricht et al, Eur. J. Biochem. 243:358 (1997), and Chadwick et al, J. Virol. Methods 70:59 (1998)).
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross-hybridization
  • LCR ligase chain reaction
  • Nucleic acid molecules comprising Zcyto 13 nucleotide sequences can also be used to determine whether a subject's chromosomes contain a mutation in the Zcytol3 gene. Detectable chromosomal aberrations at the Zcytol 3 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Of particular interest are genetic alterations that inactivate the Zcyto 13 gene.
  • Aberrations associated with the Zcyto 13 locus can be detected using nucleic acid molecules of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • molecular genetic techniques such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch
  • RNA is isolated from a biological sample, and used to synthesize cDNA. PCR is then used to amplify the Zcytol3 target sequence and to introduce an RNA polymerase promoter, a translation initiation sequence, and an in-frame ATG triplet. PCR products are transcribed using an RNA polymerase, and the transcripts are translated in vitro with a T7-coupled reticulocyte lysate system.
  • the translation products are then fractionated by SDS-PAGE to determine the lengths of the translation products.
  • the protein truncation test is described, for example, by Dracopoli et al. (eds.), Current Protocols in Human Genetics, pages 9.11.1 - 9.11.18 (John Wiley & Sons 1998).
  • the present invention also contemplates kits for performing a diagnostic assay for Zcytol 3 gene expression or to detect mutations in the Zcytol 3 gene.
  • kits comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: l, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:l, or a portion thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • Kits may comprise nucleic acid primers for performing PCR.
  • kits can contain all the necessary elements to perform a nucleic acid diagnostic assay described above.
  • a kit will comprise at least one container comprising a Zcyto 13 probe or primer.
  • the kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zcyto 13 sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like.
  • a kit may also comprise a means for conveying to the user that the Zcyto 13 probes and primers are used to detect Zcytol 3 gene expression.
  • written instructions may state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes Zcytol 3, or a nucleic acid molecule having a nucleotide sequence that is complementary to a Zcyto 13 -encoding nucleotide sequence.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • the present invention contemplates the use of anti-Zcyto 13 antibodies to screen biological samples in vitro for the presence of Zcyto 13.
  • anti-Zcyto 13 antibodies are used in liquid phase.
  • the presence of Zcyto 13 in a biological sample can be tested by mixing the biological sample with a trace amount of labeled Zcyto 13 and an anti-Zcyto 13 antibody under conditions that promote binding between Zcyto 13 and its antibody.
  • Complexes of Zcyto 13 and anti-Zcyto 13 in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein which binds with the antibody, such as an Fc antibody or Staphylococcus protein A.
  • the concentration of Zcyto 13 in the biological sample will be inversely proportional to the amount of labeled Zcyto 13 bound to the antibody and directly related to the amount of free labeled Zcytol 3.
  • in vitro assays can be performed in which anti- Zcyto 13 antibody is bound to a solid-phase carrier.
  • antibody can be attached to a polymer, such as aminodextran, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube.
  • polymer such as aminodextran
  • anti-Zcyto 13 antibodies can be used to detect Zcyto 13 in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of Zcyto 13 and to determine the distribution of Zcyto 13 in the examined tissue.
  • General immunochemistry techniques are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application,” in Mammalian Development: A Practical Approach, Monk (ed.), pages 1 15-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular Biology, Vol. 10: Immunochemical Protocols (The Humana Press, Inc. 1992)).
  • Immunochemical detection can be performed by contacting a biological sample with an anti-Zcyto 13 antibody, and then contacting the biological sample with a detectably labeled molecule which binds to the antibody.
  • the detectably labeled molecule can comprise an antibody moiety that binds to anti-Zcyto 13 antibody.
  • the anti-Zcyto 13 antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin). Numerous variations of this basic technique are well-known to those of skill in the art.
  • an anti-Zcyto 13 antibody can be conjugated with a detectable label to form an anti-Zcyto 13 immunoconjugate.
  • Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
  • the detectable label can be a radioisotope that is detected by autoradiography.
  • Isotopes that are particularly useful for the purpose of the present invention are 3 H, ,2 T, 131 1, 35 S and 14 C.
  • Anti-Zcyto 13 immunoconjugates can also be labeled with a fluorescent compound.
  • the presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence.
  • Fluorescent labeling compounds include fluorescein isothiocyanate, rhoda- mine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • anti-Zcyto 13 immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • Bioluminescent compound can be used to label anti-Zcyto 13 immunoconjugates of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-Zcyto 13 immunoconjugates can be detectably labeled by linking an anti-Zcytol3 antibody component to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galac- tosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • the convenience and versatility of immunochemical detection can be enhanced by using anti-Zcyto 13 antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al, "Immunochemical Applications of Avidin-Biotin Technology,” in Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).
  • biotin- or FITC-labeled Zcyto 13 can be used to identify cells that bind Zcytol 3. Such can binding can be detected, for example, using flow cytometry.
  • kits for performing an immunological diagnostic assay for Zcyto 13 gene expression comprise at least one container comprising an anti-Zcyto 13 antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zcyto 13 antibody or antibody fragments.
  • indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like.
  • a kit may also comprise a means for conveying to the user that Zcyto 13 antibodies or antibody fragments are used to detect Zcyto 13 protein.
  • written instructions may state that the enclosed antibody or antibody fragment can be used to detect Zcytol 3.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • the present invention includes the use of proteins, polypeptides, and peptides having Zcyto 13 activity (such as Zcyto 13 polypeptides, Zcyto 13 analogs, and Zcyto 13 fusion proteins) to provide antiviral, immunomodulatory, or anti-proliferative activity.
  • the present invention contemplates the use of these molecules for either veterinary or for human therapeutic uses.
  • Both recombinant interferons and interferons isolated from natural sources have been approved in the United States for treatment of autoimmune diseases, renal cell carcinoma, basal cell carcinoma, malignant melanoma, condylomata acuminata, chronic hepatitis B, hepatitis C, chronic hepatitis D, chronic non-A, non- B/C hepatitis, bladder carcinoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphoma, cervical carcinoma, laryngeal papilloma, fungoides mycosis, Kaposi's sarcoma in patients infected with human immunodeficiency virus, hairy cell leukemia, and multiple sclerosis.
  • Zcyto 13 may be used to treat forms of arteriosclerosis, such as atherosclerosis, by inhibiting cell proliferation, or to treat retinopathy. Accordingly, the present invention contemplates the use of proteins, polypeptides, and peptides having Zcyto 13 activity to treat such conditions.
  • the present invention includes the use of proteins, polypeptides and peptides having Zcyto 13 activity as an adjuvant for immunotherapy or immunoscintigraphy using anti-tumor antigen antibodies.
  • the dosage of administered Zcyto 13 will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of Zcyto 13, which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of subject), although a lower or higher dosage also may be administered as circumstances dictate.
  • Administration of a molecule having Zcyto 13 activity to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
  • the administration may be by continuous infusion or by single or multiple boluses.
  • Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous.
  • Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid- based systems (see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).
  • the feasibility of an intranasal delivery is exemplified by such a mode of insulin administration (see, for example, Hinchcliffe and Ilium, Adv. Drug Deliv. Rev. 55:199 (1999)).
  • Dry or liquid particles comprising Zcyto 13 can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al, Adv. Drug Deliv. Rev. 35:235 (1999)).
  • This approach is illustrated by the AERX diabetes management system, which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs.
  • proteins as large as 48,000 kDa have been delivered across skin at therapeutic concentrations with the aid of low-frequency ultrasound, which illustrates the feasibility of trascutaneous administration (Mitragotri et al, Science 269:850 (1995)).
  • Transdermal delivery using electroporation provides another means to administer a molecule having Zcyto 13 activity (Potts et al, Pharm. Biotechnol 10:213 (1997)).
  • a pharmaceutical composition comprising a protein, polypeptide, or peptide having Zcyto 13 activity can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient subject.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • molecules having Zcyto 13 activity and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount.
  • a combination of a protein, polypeptide, or peptide having Zcyto 13 activity and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject.
  • an agent is physiologically significant if its presence results in the inhibition of the growth of tumor cells or in the inhibition of a viral infection.
  • An inhibition of tumor growth may be indicated, for example, by a decrease in the number of tumor cells, decreased metastasis, a decrease in the size of a solid tumor, or increased necrosis of a tumor.
  • Indicators of viral infection inhibition include decreased viral titer, a decrease in detectable viral antigen, or an increase in anti-viral antibody titer.
  • a pharmaceutical composition comprising Zcyto 13 can be furnished in liquid form, in an aerosol, or in solid form.
  • Liquid forms are illustrated by injectable solutions and oral suspensions.
  • Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al, Pharm.
  • Liposomes provide one means to deliver therapeutic polypeptides to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation, or intranasal administration.
  • Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol Infect. Dis. 12 (Suppl.
  • Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
  • a variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy et al, Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al, American J. Hosp. Pharm. 46:1516 (1989)).
  • hydrophobic agents partition in the bilayers
  • hydrophilic agents partition within the inner aqueous space(s)
  • it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes. Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent.
  • an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof et al, Ann. N Y. Acad. Sci. 446:368 (1985)).
  • small liposomes 0.1 to 1.0 ⁇ m
  • liposomes larger than 3.0 ⁇ m are deposited in the lung. This preferential uptake of smaller liposomes by the cells of the reticuloendothelial system has been used to deliver chemotherapeutic agents to macrophages and to tumors of the liver.
  • the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means (Claassen et al, Biochim. Biophys. Acta 802:428 (1984)).
  • incorporation of glycolipid- or polyethelene glycol- derivatized phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Allen et al, Biochim. Biophys. Acta 1068:133 (1991); Allen et al, Biochim. Biophys. Acta 1150:9 (1993)).
  • Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes.
  • liposomes prepared with a high content of a nonionic surfactant, have been used to target the liver (Hayakawa et al, Japanese Patent 04- 244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)).
  • These formulations were prepared by mixing soybean phospatidylcholine, ⁇ -tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water.
  • DPPC dipalmitoylphosphatidylcholine
  • SG soybean-derived sterylglucoside mixture
  • Cho cholesterol
  • liposomes can be modified with branched type galactosyllipid derivatives to target asialoglycoprotein (galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:281 (1997); Murahashi et al, Biol. Pharm. Bull.20:259 (1997)).
  • galactose asialoglycoprotein
  • target cells are prelabeled with biotinylated antibodies specific for a ligand expressed by the target cell (Harasym et al, Adv. Drug Deliv. Rev. 32:99 (1998)). After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered. In another approach, targeting antibodies are directly attached to liposomes (Harasym et al, Adv. Drug Deliv. Rev. 32:99 (1998)).
  • Polypeptides having Zcyto 13 activity can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al, Infect. Immun. 57:1099 (1981), Anderson et al, Cancer Res. 50:1853 (1990), and Cohen et al, Biochim. Biophys. Acta 1063:95 (1991), Alving et al "Preparation and Use of Liposomes in Immunological Studies," in Liposome Technology, 2nd Edition, Vol. Ill, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al, Meth. Enzymol. 749:124 (1987)).
  • therapeutically useful liposomes may contain a variety of components.
  • liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allen et al, Biochim. Biophys. Acta 1150:9 (1993)).
  • Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins.
  • Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer (Gombotz and Pettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers in Drug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable Controlled Release Systems Useful for Protein Delivery,” in Protein Delivery: Physical Systems.
  • PLG poly(lactide-co-glycolide)
  • polyanhydrides poly (ortho esters)
  • nonbiodegradable ethylvinyl acetate polymers in which proteins are entrapped in
  • Polyethylene glycol (PEG)-coated nanospheres can also provide carriers for intravenous administration of therapeutic proteins (see, for example, Gref et al, Pharm. Biotechnol 10: 161 (1997)).
  • the present invention also contemplates chemically modified polypeptides having Zcyto 13 activity and Zcyto 13 antagonists, in which a polypeptide is linked with a polymer, as discussed above.
  • compositions may be supplied as a kit comprising a container that comprises a molecule having Zcyto 13 activity or a Zcyto 13 antagonist.
  • Therapeutic polypeptides can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic polypeptide.
  • Such a kit may further comprise written information on indications and usage of the pharmaceutical composition.
  • such information may include a statement that the Zcyto 13 composition is contraindicated in subjects with known hypersensitivity to Zcytol 3.
  • Immunomodulator genes can be introduced into a subject to enhance immunological responses.
  • immunomodulator gene therapy has been examined in model systems using vectors that express IL-2, IL-3, IL-4, IL-6, IL- 10, IL-12, IL-15, interferon- ⁇ , tumor necrosis factor- ⁇ , or granulocyte-macrophage colony-stimulating factor (see, for example, Cao et ⁇ l, J. G ⁇ stroenterol Hep ⁇ tol 77:1053 (1996), Tahara et ⁇ l, Ann. N Y. Ac ⁇ d. Sci. 795:215 (1996), Rakhmilevich et ⁇ l., Hum. Gene Ther.
  • the present invention includes the use of Zcyto 13 nucleotide sequences to augment an immunological response to a tumor or viral infection in a subject.
  • a therapeutic expression vector can be provided that inhibits Zcytol3 gene expression, such as an anti-sense molecule, a ribozyme, or an external guide sequence molecule.
  • an expression vector is constructed in which a nucleotide sequence encoding a Zcytol 3 gene is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription.
  • a core promoter and optionally a regulatory element
  • a Zcytol3 gene can be delivered using recombinant viral vectors, including for example, adenoviral vectors (e.g., Kass-Eisler et al, Proc. Natl Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Natl Acad. Sci. USA 97:215 (1994), Li et al, Hum. Gene Ther. 4:403 (1993), Vincent et al, Nat. Genet. 5: 130 (1993), and Zabner et al, Cell 75:201 (1993)), adenovirus-associated viral vectors (Flotte et al, Proc. Natl Acad. Sci.
  • adenoviral vectors e.g., Kass-Eisler et al, Proc. Natl Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Natl Acad. Sci. USA 97:215 (1994), Li et
  • alphaviruses such as Semliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:851 (1992), Raju and Huang, J Vir. 65:2501 (1991), and Xiong et al, Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Patent Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors (Koering et al, Hum. Gene Therap. 5:457 (1994)), pox virus vectors (Ozaki et al, Biochem. Biophys. Res. Comm.
  • pox viruses such as canary pox virus or vaccinia virus (Fisher-Hoch et al, Proc. Natl Acad. Sci. USA 86:311 (1989), and Flexner et al, Ann. N. Y. Acad. Sci. 569:86 (1989)), and retroviruses (e.g., Baba et al. J. Neurosurg 79:129 (1993), Ram et al, Cancer Res. 55:83 (1993), Takamiya et al, J. Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res.
  • pox viruses such as canary pox virus or vaccinia virus (Fisher-Hoch et al, Proc. Natl Acad. Sci. USA 86:311 (1989), and Flexner et al, Ann. N. Y. Acad. Sci. 569:86 (1989)
  • retroviruses e.g., Baba et al. J. Neuro
  • adenovirus a double-stranded DNA virus
  • a viral particle which contains the viral vector may be utilized in the methods and compositions described below.
  • adenovirus a double-stranded DNA virus
  • Douglas and Curiel Science & Medicine 4:44 (1997).
  • the adenovirus system offers several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii) the ability to be grown to high-titer, (iii) the ability to infect a broad range of mammalian cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters.
  • adeno viruses can be administered by intravenous injection, because the viruses are stable in the bloodstream.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell.
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. Although an adenoviral delivery system with an El gene deletion cannot replicate in the host cells, the host's tissue will express and process an encoded heterologous protein. Host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence. Secreted proteins will enter the circulation from tissue that expresses the heterologous gene (e.g., the highly vascularized liver).
  • adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El -deleted, and in addition, contain deletions of E2A or E4 (Lusky et al, J. Virol 72:2022 (1998); Raper et al, Human Gene Therapy 9:671 (1998)).
  • the deletion of E2b has also been reported to reduce immune responses (Amalfitano et al, J. Virol. 72:926 (1998)). By deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated.
  • High titer stocks of recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using standard methods.
  • recombinant HSV can be prepared in Vero cells, as described by Brandt et al, J. Gen. Virol 72:2043 (1991), Herold et al, J. Gen. Virol 75: 121 1 (1994), Visalli and Brandt, Virology 755:419 (1991), Grau et al, Invest. Ophthalmol Vis. Sci. 30:2414 (1989), Brandt et al, J. Virol. Meth. 36:209 (1992), and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).
  • an expression vector comprising a Zcytol 3 gene can be introduced into a subject's cells 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. Nat Acad. Sci. USA 54:7413 (1987); Mackey et al, Proc. Nat 'I Acad. Sci. USA 55:8027 (1988)).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Liposomes can be used to direct transfection to particular cell types, which is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas. liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • Electroporation is another alternative mode of administration. For example, Aihara and Miyazaki, Nature Biotechnology 16:861 (1998), have demonstrated the use of in vivo electroporation for gene transfer into muscle.
  • a therapeutic gene may encode a Zcyto 13 anti-sense RNA that inhibits the expression of Zcytol 3.
  • Suitable sequences for anti-sense molecules can be derived from the nucleotide sequences of Zcyto 13 disclosed herein.
  • an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
  • Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example, Draper and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337).
  • ribozymes include nucleotide sequences that bind with Zcyto 13 mRNA.
  • expression vectors can be constructed in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of mRNA molecules that encode a Zcytol 3 gene.
  • an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see, for example, Altman et al, U.S. Patent No. 5,168,053, Yuan et al, Science 263:1269 (1994), Pace et al, international publication No. WO 96/18733, George et al, international publication No.
  • the external guide sequence comprises a ten to fifteen nucleotide sequence complementary to Zcytol 3 mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine.
  • the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5'-side of the base-paired region.
  • the dosage of a composition comprising a therapeutic vector having a Zcyto 13 nucleotide acid sequence, such as a recombinant virus will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history.
  • Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into a cavity that contains a tumor.
  • Horton et al Proc. Natl Acad. Sci. USA 96:1553 (1999) demonstrated that intramuscular injection of plasmid DNA encoding interferon- ⁇ produces potent antitumor effects on primary and metastatic tumors in a murine model.
  • a composition comprising viral vectors, non-viral vectors, or a combination of viral and non-viral vectors of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby vectors or vimses are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition such as phosphate-buffered saline is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient subject.
  • suitable carriers are well-known to those in the art (see, for example, Remington 's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman 's the Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)).
  • a therapeutic gene expression vector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount.
  • a combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject. In the present context, an agent is physiologically significant if its presence inhibits the growth of tumor cells or inhibits viral infection.
  • An inhibition of tumor growth may be indicated, for example, by a decrease in the number of tumor cells, decreased metastasis, a decrease in the size of a solid tumor, or increased necrosis of a tumor.
  • Indicators of viral infection inhibition include decreased viral titer, a decrease in detectable viral antigen, or an increase in anti-viral antibody titer.
  • the therapy is preferably somatic cell gene therapy. That is, the preferred treatment of a human with a therapeutic gene expression vector or a recombinant virus does not entail introducing into cells a nucleic acid molecule that can form part of a human germ line and be passed onto successive generations (i.e., human germ line gene therapy). 14. Production of Transgenic Mice
  • Transgenic mice can be engineered to over-express the Zcytol 3 gene in all tissues or under the control of a tissue-specific or tissue-preferred regulatory element. These over-producers of Zcyto 13 can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serve as models for human disease caused by excess Zcytol 3. Transgenic mice that over-express Zcyto 13 also provide model bioreactors for production of Zcyto 13 in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses a Zcytol 3 gene can begin with adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2f 1 , 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl, 2-4 months, (Taconic Farms)).
  • the donors are acclimated for one week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, MO) I. P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) I. P. to induce superovulation.
  • Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating.
  • Fertilized eggs are collected under a surgical scope.
  • the oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma).
  • Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (described, for example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated with 5% CO 7 , 5% O,, and 90% N at 37°C.
  • the eggs are then stored in a 37°C/5% CO, incubator until microinjection.
  • Zcyto 13 encoding sequences Ten to twenty micrograms of plasmid DNA containing a Zcyto 13 encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinjection.
  • the Zcyto 13 encoding sequences can encode amino acid residues 22 to 199 of SEQ ID NO:2.
  • Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO -equilibrated mineral oil.
  • the DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei.
  • Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue-culture dish with pre-gassed W640 medium for storage overnight in a 37°C/5% CO 2 incubator.
  • two-cell embryos are transferred into pseudopregnant recipients.
  • the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
  • Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope.
  • a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
  • the reproductive organs are exteriorized onto a small surgical drape.
  • the fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
  • a baby serrefine Robot, Rockville, MD
  • the pipette is transferred into the nick in the oviduct, and the embryos are blown in, allowing the first air bubble to escape the pipette.
  • the fat pad is gently pushed into the peritoneum, and the reproductive organs allowed to slide in.
  • the peritoneal wall is closed with one suture and the skin closed with a wound clip.
  • the mice recuperate on a 37°C slide warmer for a minimum of four hours.
  • Genomic DNA is prepared from the tail snips using, for example, a QIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to amplify a Zcytol 3 gene or a selectable marker gene that was introduced in the same plasmid. Illustrative primers suitable for amplifying Zcyto 13 are described in the Examples, below.
  • mice After animals are confirmed to be transgenic, they are back-crossed into an inbred strain by placing a transgenic female with a wild-type male, or a transgenic male with one or two wild-type female(s). As pups are bom and weaned, the sexes are separated, and their tails snipped for genotyping. To check for expression of a transgene in a live animal, a partial hepatectomy is performed. A surgical prep is made of the upper abdomen directly below the zyphoid process. Using sterile technique, a small 1.5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized.
  • a tie is made around the lower lobe securing it outside the body cavity.
  • An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed proximal to the first tie.
  • a distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish.
  • the excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice.
  • the surgical site is closed with suture and wound clips, and the animal's cage placed on a 37°C heating pad for 24 hours post operatively.
  • the animal is checked daily post operatively and the wound clips removed 7-10 days after surgery.
  • the expression level of Zcyto 13 mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction. This general approach was used to produce transgenic mice comprising a
  • Zcytol 3 expression vector contained a Zcytol 3 gene that was synthesized with PCR primers ZC22243 (5' CGT ACG GGC CGG CCA CCA TGA CTC CAA AGT TT 3'; SEQ ID NO: 14) and ZC22244 (5' CGC GCG GGC GCG CCC TAT TTA AGT TCT TGC TT 3'; SEQ ID NO: 15).
  • ZC22243 5' CGT ACG GGC CGG CCA CCA TGA CTC CAA AGT TT 3'
  • ZC22244 5' CGC GCG GGC GCG CCC TAT TTA AGT TCT TGC TT 3'; SEQ ID NO: 15.
  • a preliminary study was performed with a male transgenic mouse, which expressed Zcyto 13 in its liver. Flow cytometry analysis of spleen- and bone marrow-derived cells indicated that Zcyto 13 may affect the development of the myeloid lineage in bone marrow.
  • transgenic mice that over-express Zcytol 3
  • Zcytol 3 gene expression can be inhibited using anti- sense genes, ribozyme genes, or external guide sequence genes.
  • inhibitory sequences are targeted to Zcyto 13 mRNA.
  • An alternative approach to producing transgenic mice that have little or no Zcyto 13 gene expression is to generate mice having at least one normal Zcyto 13 allele replaced by a nonfunctional Zcyto 13 gene.
  • One method of designing a nonfunctional Zcytol3 gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes Zcytol 3. Standard methods for producing these so-called “knockout mice” are known to those skilled in the art (see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice," in Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 1 1 1- 124 (Academic Press, Ltd. 1994), and Wu et al, "New Strategies for Gene Knockout,” in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)).
  • 5' RACE was performed to obtain a 5' sequence from an expressed sequence tag.
  • the 5' RACE was performed as follows: 3 ⁇ l of 1/100 diluted placenta marathon cDNA, 20 pmoles each of oligonucleotide primers ZC9739 (5' CCA TCC
  • New primers ZC21590 (5' ATA CTA AGC ACC AGG GTT GAG AAT G 3'; SEQ ID NO:9) and ZC21591 (5' AGG TAG CAT TAG CAG CAT CCT GGT A 3'; SEQ ID NO: 10) were designed according to the new 5' sequence information on the clone.
  • PCR was used to amplify entire coding sequence with the primer pair above, and RNA samples from placenta, testis and 7d embryo tissues were used as templates.
  • Three microliters of 1/100 diluted marathon cDNAs, 20 pmoles of each oligonucleotide primers, and 1 U of mixture of ExTaqlTaq antibody (1 :1) were used in 25 ⁇ l reactions.
  • PCR products were separated on an agarose gel and purified with QIAquick (QIAGEN Inc.). Purified PCR products were then ligated into pCR2.1 vector using TA cloning kit (Invitrogen) and inserts were sequenced.
  • Northern analyses were performed using a Mouse Multiple Tissue Northern Blot, a Mouse Embryo Multiple Tissue Northern Blot, and a Mouse RNA Master Blot (dot blot) (CLONTECH Laboratories, Inc., Palo Alto, CA).
  • the hybridization probe was generated from a gel purified PCR amplification product.
  • the probe was made using ZC20979 (5' CTG ACA GTC TAC CTG GAG TTG GG 3'; SEQ ID NO: 11) and ZC20980 as primers and mouse seven-day embryo Marathon Ready cDNA as template.
  • the probe length was 310 base pairs.
  • the probe was a radioactively labeled using the REDIPRIME II labeling kit (AMERSHAM PHARMACIA BIOTECH, Inc.; Piscataway, NJ) according to the manufacturer's protocol.
  • the probe was purified using a NUCTRAP push column (STRATAGENE, La Jolla, CA). EXPRESSHYB (CLONTECH) solution was used for the prehybridization and hybridization solutions for the Northern blots. Hybridization took place overnight at 65°C. Following hybridization, the blots were washed in 2xSSC, 0.1% SDS at room temperature, followed by a wash in O.lxSSC and 0.1% SDS at 50°C. The blots were exposed to Kodak BioMax film.
  • RNA Master Blot showed positive signals from heart, submaxillary gland, epididymus, and seven-day embryo.
  • Southern analysis was performed using a commercially prepared Interspecies Zoo-Blot from CLONTECH Laboratories, Inc.
  • the Southern blot contained Ec RI-digested DNA.
  • the hybridization probe was generated as described for Example 2.
  • the probe was a radioactively labeled using the REDIPRIME II labeling kit (AMERSHAM PHARMACIA BIOTECH, Inc.; Piscataway, NJ) according to the manufacturer's protocol, and the probe was purified using a NUCTRAP push column (STRATAGENE, La Jolla, CA).
  • EXPRESSHYB (CLONTECH) solution was used for the prehybridization and hybridization solutions for the Southern blots.
  • the blot was hybridized overnight at 65°C, and then, the blots were washed in 2x SSC, 0.1%) SDS at room temperature, followed by a wash in O.lx SSC and 0.1% SDS at 50°C. After washing, the blots were exposed to Kodak BioMax film.
  • the mouse genomic DNA sample contained hybridizing fragments at approximately 5.65 and 4.8 kilobases, while the rat sample contained hybridizing fragments at approximately 5.2 and 2.1 kilobases.
  • PCR primers ZC21924 (5' CAC ACA GGC CGG CCA CCA TGA CTC CAA AGT TTT TAT GGC 3'; SEQ ID NO: 12) and ZC21923 (5' CAC ACA GGC GCG CCT CTA TTT AAG TTC TTG CTT GAA GGT GGG 3'; SEQ ID NO: 13) were used with template pCR2.1 plasmid containing the full-length murine Zcytol 3 cDNA in a PCR reaction as follows: one cycle at 95°C for 5 minutes, followed by 15 cycles at 95°C for 0.5 minute, 58°C for 0.5 minute, and 72°C for 0.5 minute, followed by 72°C for 7 minutes, followed by a 4°C soak.
  • the PCR reaction product was loaded onto a 1.2% (low melt) SEAPLAQUE GTG (FMC BioProducts; Rockland, ME) gel in TAE buffer.
  • the Zcyto 13 PCR product was excised from the gel, melted at 65°, phenol extracted twice and then ethanol precipitated.
  • the PCR product was then digested with Fsel-Asc , phenol/chloroform extracted, ethanol precipitated, and rehydrated in 20 ⁇ l TE (Tris/EDTA, pH 8).
  • the 600 base pair Zcyto 13 fragment was then ligated into the Fsel-Ascl sites of a modified pAdTrack CMV (He et al, Proc. Natl Acad. Sci. USA 95:2509 (1998)).
  • This construct also contains the green fluorescent protein (GFP) marker gene.
  • GFP green fluorescent protein
  • the CMV promoter driving GFP expression was replaced with the SV40 promoter and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal.
  • the native polylinker was replaced with Fsel, Ec RV, and Ascl sites.
  • This modified form of pAdTrack CMV was named pZy Track.
  • the co-transformation was performed with a BIO-RAD GENE PULSER (BIO-RAD laboratories, Inc.; Hercules, CA) at 2.5kV, 200 ohms and 25mFa. The entire co-transformation was plated on four LB plates containing 25 ⁇ g/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin and recombinant adenovirus DNA identified by standard DNA miniprep procedures. Digestion of the recombinant adenovirus DNA with Fsel-Ascl confirmed the presence of Zcyto 13. The recombinant adenovirus miniprep DNA was transformed into DH10B competent cells and DNA prepared using a QIAGEN maxi prep kit as per kit instructions.
  • recombinant adenoviral DNA Approximately 5 ⁇ g of recombinant adenoviral DNA were digested with Pad enzyme for three hours at 37°C in a reaction volume of 100 ⁇ l containing 20-30 U of Pad. The digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol. The DNA pellet was resuspended in 5 ⁇ l distilled water. QBI-293A cells (Quantum Biotechnologies, Inc.; Montreal, Quebec, Canada), inoculated the day before and grown to 60-70% confluence in a T25 flask, were transfected with the Pad digested DNA.
  • the R ⁇ cl-digested DNA was diluted up to a total volume of 50 ⁇ l with sterile HBS (150 mM NaCl, 20 mM HEPES).
  • HBS sterile HBS
  • 25 ⁇ l DOTAP (1 mg/ml; Roche Molecular Biochemicals; Indianapolis, IN) were diluted to a total volume of 100 ⁇ l with HBS.
  • the DNA was added to the DOTAP, mixed gently by pipeting up and down, and left at room temperature for 15 minutes.
  • the medium was removed from the 293A cells and washed with 5 ml serum- free MEMalpha (LIFE TECHNOLOGIES, Inc; Rockville, MD) containing 1 mM sodium pyruvate (LIFE TECHNOLOGIES, Inc), 0.1 mM MEM non-essential amino acids (LIFE TECHNOLOGIES, Inc) and 25 mM HEPES buffer (LIFE TECHNOLOGIES, Inc).
  • 5 ml serum-free MEMalpha (LIFE TECHNOLOGIES, Inc; Rockville, MD) containing 1 mM sodium pyruvate (LIFE TECHNOLOGIES, Inc), 0.1 mM MEM non-essential amino acids (LIFE TECHNOLOGIES, Inc) and 25 mM HEPES buffer (LIFE TECHNOLOGIES, Inc).
  • Five milliliters of serum-free MEM were added to the 293A cells and held at 37°C.
  • the DNA/lipid mixture was added drop-wise to the T25 flask of 2
  • NP-40 detergent was added to a final concentration of 0.5%> to the bottles of cmde lysate to lyse all cells. Bottles were placed on a rotating platform for 10 minutes, agitating as fast as possible without displacing the bottles. The debris was pelleted by centrifugation at 20,000xg for 15 minutes. The supernatant was transferred to 250 ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 M NaCl solution was added. The bottles were shaken overnight on ice. The bottles were centrifuged at 20,000xg for 15 minutes and supernatant discarded into a bleach solution. The precipitated virus/PEG appeared as a white precipitate located in two vertical lines along the wall of the bottle on either side of the spin mark.
  • the precipitate from two bottles was resuspended in 2.5 ml PBS.
  • the vims solution was placed in 2 ml microcentrifuge tubes and centrifuged at 14,000xg in the micro fuge for 10 minutes to remove any additional cell debris.
  • the supernatant from the 2 ml microcentrifuge tubes was transferred into a 15 ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with cesium chloride (CsCl).
  • CsCl cesium chloride
  • the volume of the vims solution was estimated and 0.55 g/ml of was CsCl added.
  • the CsCl was dissolved and 1 ml of this solution weighed 1.34 g.
  • the solution was transferred to polycarbonate thick-walled centrifuge tubes, and centrifuged at 80,000 rpm (348,000xg) for 3-4 hours at 25°C in a Beckman Optima TLX micro-ultracentrifuge with the TLA- 100.4 rotor.
  • the vims formed a white band. Using wide-bore pipette tips, the virus band was collected.
  • the virus from the gradient has a large amount of CsCl which must be removed before it can be used with cells.
  • Pharmacia PD-10 columns prepacked with SEPHADEX G-25M (Amersham Pharmacia Biotech, Inc; Piscataway, NJ) were used to desalt the virus preparation.
  • the column was equilibrated with 20 ml of PBS.
  • the virus was loaded and allowed to run into the column.
  • Five milliliters of PBS were added to the column and fractions of 8-10 drops collected.
  • the optical densities of 1 :50 dilutions of each fraction were determined at 260 nm on a spectrophotometer. A clear absorbance peak was present between fractions 7-12.
  • TCID 50 tissue culture infectious dose at 50% cytopathic effect
  • TCID 50 tissue culture infectious dose at 50% cytopathic effect
  • the titer is determined from a plate in which vims is diluted from 10 " to 10 " , and read five days after the infection. At each dilution a ratio (R) of positive wells for cytopathic effect per the total number of wells is determined.
  • factor "F” was first calculated, as l+d(S-0.5), where "S” is the sum of the ratios (R), and “d” is log, 0 of the dilution series (e.g., "d” is equal to one for a ten-fold dilution series).
  • TCID 50 /ml To convert TCID 50 /ml to pfu/ml, 0.7 is subtracted from the exponent in the calculation for titer (T). Using this method, the Zcytol3 adenovims had a titer of 4.0x10 pfu/ml.
  • CM conditioned medium
  • Zcyto 13m protein was generated by infecting 293 A cells with recombinant adenovirus containing the cDNA for Zcyto 13m (AdZy-zcytol3m) at a multiplicity of infection of 400 particles per cell.
  • CM was harvested at time points between 40 hours post infection and stored at -20°C.
  • CM was also generated from an infection with a recombinant adenovirus lacking a cDNA (AdZy -parental).
  • CM Prior to use, a portion of the CM was concentrated 14 fold in a Millipore Ultrafree-15 (5,000 nominal molecular weight limit) centrifugal filter, and then, filtered through a Millipore Ultrafree-15 (100,000 nominal molecular weight limit) centrifugal filter to reduce the amount of viral particles present in the media, and finally filtered through a Millipore 0.2 ⁇ m syringe filter to sterilize the CM.
  • Concentrated CM samples were diluted 1 :2 in binding buffer and incubated with cells from a murine cell line for 5 hours at 37°C.
  • CM containing Zcyto 13 protein stimulated a 17-fold increase in gene expression from an interferon-responsive promoter, while control CM, appeared to stimulate gene expression by 1.4 fold.
  • the anti-viral assay was performed by plating L929 cells (ATCC No. CCL-1) in growth media RPMI medium 1640 containing 10% fetal bovine serum, penicillin, streptomycin, and L-glutamine in 96-well format at 50,000 cells per well.
  • Adenovirus CM from 293 A cells infected with either AdZy-zcytol3m or AdZy- parental, as described above, was incubated with cells overnight.
  • a positive control in the assay was provided by murine interferon- ⁇ serially diluted 1:10, starting at 100 ng/ml.
  • L929 cells with growth media alone provided the negative control. Treated cells were incubated for 24 hours.
  • encephalomyocarditis vims (ATCC No. vrl29b) was introduced at a multiplicity of infection of 0.1 (i.e., one vims particle for every ten L929 cells).
  • the cells were incubated in the presence of the vims for 24 hours, and then, the wells were scored for percent cytopathic effect (CPE). As shown in Table 5, conditioned medium containing Zcytol 3 effectively inhibited viral infection of the cells.
  • Baf3 an interleukin-3 dependent pre-B cell line, was transfected with two chimeric receptors containing the extracellular domains of human interferon receptor and the signaling domains of murine MPL.
  • Baby hamster kidney (BHK) cells were stably transfected with an expression vector containing the CMV promoter plus intron A upstream of either the murine Zcyto 13 cDNA, or an unrelated cDNA, encoding "Z ⁇ 30," using BRL lipofectamine.
  • Stably transfected cells were seeded in a cell factory with semm free medium and allowed to grow for three days before conditioned media were harvested and concentrated in a 5K filter to lOx. Concentrated conditioned medium was stored at 4°C.
  • the assay to test for proliferation of BaO cells via signaling through the chimeric receptor was performed as follows.
  • the final volume of each dilutions was lOO ⁇ l.
  • the BaO parental cell line and BaO cells transfected with human interferon/murine MPL receptor were washed three times in growth media (see above), pellets were resuspended in growth medium, and cells were counted and diluted in growth media to 5,000 cells/100 ⁇ l.
  • One hundred microliters of diluted cells were added to each dilution of samples.
  • the assay plate was incubated in a 37°C incubator for three to four days. A 20 ⁇ l aliquot of Alomar blue was added to each well and the plate was incubated overnight at 37°C.
  • the plates were read on the fluorescent plate reader at excitation wavelength of 544 and emission wavelength 590.
  • BaO was used to determine if Zcyto 13 has anti-proliferative properties.
  • Baby hamster kidney (BHK) cells were stably transfected with an expression vector containing the CMV promoter plus intron A upstream of the murine Zcyto 13 cDNA or an unrelated cDNA, called Z ⁇ 30, using BRL lipofectamine.
  • Stably transfected cells were seeded in a cell factory in serum free media and allowed to grow for three days before conditioned media was harvested and concentrated in a 5K filter to lOx. Concentrated conditioned medium samples were stored at 4°C. The following assay was used to test for anti-proliferation of BaO.
  • BHK Baby hamster kidney
  • 96 well plate eight 1 :2 serial dilutions were made of growth media alone (RPMI 1640, 10%) fetal bovine semm, ImM sodium pyruvate, 2mM L-glutamine), or murine IL-3 (starting at 50 pg/ml in growth medium) with final volume of lOO ⁇ l.
  • human interferon- ⁇ (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium), human interferon- ⁇ (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium), murine interferon- ⁇ (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium), murine interferon- ⁇ (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium), murine interferon- ⁇ (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium), murine Zcytol3 (at 2.5x, 0.5x, or O.lx), and murine Z ⁇ 30 (at 2.5x, 0.5x, or O.lx).
  • human interferon- ⁇ 100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium
  • the BaO cell line was washed three times in growth medium, pellets were resuspended in growth medium, cells were counted and diluted in growth medium to 5,000 cells/50 ⁇ l. Fifty microliters of diluted cells were then added to each dilution of samples. Assay plates were incubated in a 37°C incubator for three to four days. Twenty microliters of Alomar blue were then added to each well and the plate were incubated overnight at 37°C. The plates were read on the fluorescent plate reader at excitation wavelength of 544 and emission wavelength 590. The results demonstrated that ⁇ -interferon and Zcyto 13 inhibited proliferation of BaO cells.

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Abstract

Les interférons représentent une classe importante de produits biopharmaceutiques, qui ont fait leurs preuves en matière de traitement de diverses maladies, y compris le traitement de certaines maladies auto-immunitaires, de certains cancers et l'amélioration de la réponse immunitaire contre des agents infectieux. L'invention concerne une nouvelle forme d'interféron-α murin, qui possède des applications diagnostiques et thérapeutiques.
PCT/US2000/006993 1999-03-18 2000-03-17 Interferon alpha murin egalement appele zcyto13 WO2000055324A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042474A2 (fr) * 1999-12-08 2001-06-14 Amgen, Inc. Molecules de type interferon et utilisations
EP1501936A2 (fr) * 2001-04-20 2005-02-02 ZymoGenetics, Inc. Famille proteique de cytokine
WO2005023290A2 (fr) * 2003-05-23 2005-03-17 Pestka Biomedical Laboratories, Inc. Utilisation d'interferons pour traiter le syndrome respiratoire aigu severe et d'autres infections virales
US7445773B2 (en) 2001-04-20 2008-11-04 Zymogenetics, Inc. Cytokine protein family
US7495077B2 (en) 2003-08-07 2009-02-24 Zymogenetics, Inc. Homogeneous preparations of IL-28 and IL-29
US7910313B2 (en) 2001-04-20 2011-03-22 Zymogenetics, Inc. Cytokine protein family

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A. HIMMLER ET AL: "molecular cloning and expression in Escherichia coli of equine type I interferon", SWISSPROT DATABASE ENTRY INA2-HORSE, ACCESSION NUMBER P05004, 1 March 1989 (1989-03-01), XP002140884 *
DNA, vol. 5, no. 5, 1986, pages 345 - 356, XP000914990 *
M. MARRA ET AL: "The WashU-HHMI Mouse EST project", EMBL ADTABASE ENTRY AI155872, ACCESSION NUMBER AI155872, 30 September 1998 (1998-09-30), XP002140883 *
UNPUBLISHED *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042474A2 (fr) * 1999-12-08 2001-06-14 Amgen, Inc. Molecules de type interferon et utilisations
WO2001042474A3 (fr) * 1999-12-08 2002-02-28 Amgen Inc Molecules de type interferon et utilisations
US8148109B2 (en) 1999-12-08 2012-04-03 Amgen Inc. Interferon-like proteins and uses thereof
US7811791B2 (en) 1999-12-08 2010-10-12 Amgen Inc. Interferon-like molecules and uses thereof
US7462467B2 (en) 2001-04-20 2008-12-09 Zymogenetics, Inc. Cytokine protein family
US7655222B2 (en) 2001-04-20 2010-02-02 Zymogenetics, Llc Method of using interferon-like protein ZCYTO20 (IL-28A) or ZCYTO22 (IL-28B) to treat hepatitis C
EP1942187A1 (fr) * 2001-04-20 2008-07-09 ZymoGenetics, Inc. Famille de protéine de cytokine
US7445773B2 (en) 2001-04-20 2008-11-04 Zymogenetics, Inc. Cytokine protein family
US7446172B2 (en) 2001-04-20 2008-11-04 Zymogenetics, Inc. Cytokine protein family
US7455993B2 (en) 2001-04-20 2008-11-25 Zymogenetics, Inc. Cytokine protein family
EP1501936A4 (fr) * 2001-04-20 2005-07-20 Zymogenetics Inc Famille proteique de cytokine
US7485699B2 (en) 2001-04-20 2009-02-03 Zymogenetics, Inc. Cytokine protein family
US8231866B2 (en) 2001-04-20 2012-07-31 Zymogenetics, Inc. Cytokine protein family
EP1501936A2 (fr) * 2001-04-20 2005-02-02 ZymoGenetics, Inc. Famille proteique de cytokine
US7662927B2 (en) 2001-04-20 2010-02-16 Zymogenetics, Llc Antibodies to ZCYTO20 (IL-28A) and ZCYTO22 (IL-28B)
US8124065B2 (en) 2001-04-20 2012-02-28 Zymogenetics, Inc. Cytokine Protein Family
US7829076B2 (en) 2001-04-20 2010-11-09 Zymogenetics, Llc Method of using interferon-like potein ZCYTO20 (IL-28A) or ZCYTO22(IL-28B) to treat hepatitis B
US7910313B2 (en) 2001-04-20 2011-03-22 Zymogenetics, Inc. Cytokine protein family
WO2005023290A2 (fr) * 2003-05-23 2005-03-17 Pestka Biomedical Laboratories, Inc. Utilisation d'interferons pour traiter le syndrome respiratoire aigu severe et d'autres infections virales
WO2005023290A3 (fr) * 2003-05-23 2005-12-22 Pestka Biomedical Lab Inc Utilisation d'interferons pour traiter le syndrome respiratoire aigu severe et d'autres infections virales
US7495077B2 (en) 2003-08-07 2009-02-24 Zymogenetics, Inc. Homogeneous preparations of IL-28 and IL-29

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