EP0669981A1 - Interferon tau compositions and methods of use - Google Patents

Interferon tau compositions and methods of use

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Publication number
EP0669981A1
EP0669981A1 EP93924958A EP93924958A EP0669981A1 EP 0669981 A1 EP0669981 A1 EP 0669981A1 EP 93924958 A EP93924958 A EP 93924958A EP 93924958 A EP93924958 A EP 93924958A EP 0669981 A1 EP0669981 A1 EP 0669981A1
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European Patent Office
Prior art keywords
seq
interferon
ifnt
cells
polypeptide
Prior art date
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EP93924958A
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German (de)
French (fr)
Inventor
Fuller Warren Center For Animal Biotechn. Bazer
Howard Marcellus Dept. Of Microbiology Johnson
Carol Hanlon Department Of Microbiology Pontzer
Troy Lee Department Of Animal Science Ott
Gino Van Heeke
Kazuhiko The Women's Research Institute Imakawa
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University of Florida
Women's Research Institute
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University of Florida
Women's Research Institute
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Priority to EP03006878A priority Critical patent/EP1360962A3/en
Publication of EP0669981A1 publication Critical patent/EP0669981A1/en
Withdrawn legal-status Critical Current

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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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to interferon- ⁇ compo- sitions and methods of use.
  • Mullis, K.B. U. S. Patent No. 4,683,202, issued 28 July 1987.
  • Mullis, K.B. et al., U. S. Patent No. 4,683,195, issued 28 July 1987.
  • ovine trophoblast protein- one (oTP-1)
  • the protein oTP-1 was shown to inhibit uterine secretion of prostaglandin F 2 -alpha, which causes the corpus luteum on the ovary to undergo physiological and endocrinological demise in nonpregnant sheep (Bazer, et al., 1986).
  • oTP-1 has antiluteolytic bio- logical activity.
  • the primary role of oTP-1 was assumed to be associated with the establishment of pregnancy.
  • oTP-1 was subsequently found to (i) exhibit limited homology (50-70%) with interferon alphas (IFN ⁇ ) of vari- ous species (Imakawa, et al . , 1987), and (ii) bind to a Type I interferon receptor (Stewart, et al . , 1987).
  • oTP-1 has several features that distinguish it from IFN ⁇ including the following: oTP-l's role in reproductive biochemistry (other interferons are not known to have any role in the biochemical regulation of reproductive cycles), oTP-l's cellular source — trophoblast cells (IFN ⁇ is derived from lymphocytes cells) , oTP-l's size — 172 amino acids (IFN ⁇ is typically about 166 amino acids) , and oTP-1 is weakly inducible by viruses (IFN ⁇ is highly inducible by viruses) .
  • interferon-tau interferon-tau
  • T trophoblast
  • the interferons have been classified into two dis ⁇ tinct groups: type I interferons, including IFN ⁇ , IFN/3, and IFN ⁇ (also known as IFN ⁇ ll) ; and type II interferons, represented by IFN7 (reviewed by DeMaeyer, et al . ) .
  • IFN ⁇ interferon-tau
  • IFN ⁇ 's has been shown to inhibit various types of cellular proliferation. IFN ⁇ 's are especially useful against hematologic malignancies such as hairy-cell leukemia (Quesada, et al . , 1984). Further, these pro ⁇ teins have also shown activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal- cell carcinoma, urinary bladder tumors and ovarian can- cers (Bonnem, et al . , 1984; Oldham, 1985).
  • interferons and interferon receptors have also been investigated (Benoit, et al . , 1993). IFN ⁇ 's are also useful against various types of viral infections (Finter, et al . , 1991).
  • Alpha interfe ⁇ rons have shown activity against human papillomavirus infection, Hepatitis B, and Hepatitis C infections (Finter, et al., 1991; Kashima, et al . , 1988; Dusheiko, et al., 1986; Davis, et al . , 1989).
  • IFN ⁇ 's have been limited by their toxicity: use of interferons in the treatment of cancer and viral disease has resulted in serious side effects, such as fever, chills, anorexia, weight loss, and fatigue (Pontzer, et al., 1991; Oldham, 1985). These side effects often require (i) the interferon dosage to be reduced to levels that limit the effectiveness of treatment, or (ii) the removal of the patient from treat ⁇ ment. Such toxicity has reduced the usefulness of these potent antiviral and antiproliferative proteins in the treatment of debilitating human and animal diseases.
  • the present invention includes a method of inhibiting tumor cell growth.
  • cells are contacted with interferon-T (IFN ⁇ ) at a concen ⁇ tration effective to inhibit growth of the tumor cells.
  • IFN ⁇ interferon-T
  • IFNT can be obtained from a number of sources including cows, sheep, and humans. Two embodiments include the IFNT presented as either SEQ ID NO:2 or SEQ ID NO:4.
  • a number of tumor cells can be targeted for growth inhibi ⁇ tion by IFNr, including but not limited to the following: human carcinoma cells and steroid-affected tumor cells (e .g. , mammary tumor cells).
  • the present invention also includes a method of inhibiting viral replication in a cell.
  • RNA e.g., feline leukemia virus, human immunodeficiency virus, or Hepatitis C Virus
  • DNA e.g., Hepatitis B Virus
  • the human IFNT molecules of the present invention can also be used in a method of enhancing fertility in a female mammal.
  • an amount of human IFNT effective to enhance fertility of a female mammal is administered, typically in a pharmaceutically acceptable carrier.
  • Exemplary of such human IFNT molecules are the protein sequences presented as SEQ ID NO:4 and SEQ ID NO:12.
  • the present invention also includes an isolated nucleic acid which encodes a human interferon- ⁇ . Exem ⁇ plary of such nucleic acid molecules are SEQ ID NO:3 and SEQ ID NO:11. Further, the invention includes expression vectors for the expression of human IFNT.
  • the expression vector includes (a) a nucleic acid containing an open reading frame that encodes human interferon- ⁇ ; and (b) regulatory sequences effective to express said open reading frame in a host cell.
  • the regulatory se ⁇ quence may include sequences useful for targeting or secretion of the IFNT polypeptide: such sequences may be endogenous (such as the normally occurring IFNT leader sequences, see SEQ ID NO:11) or heterologous (such as a secretory signal recognized in yeast or bacterial expres ⁇ sion systems) .
  • regulatory sequences may also include, 5' to said nucleic acid sequence, a promoter region and an ATG start codon in- frame with the interferon- ⁇ coding sequence, and 3' to said coding sequence, a translation termination signal followed by a transcription termination signal.
  • the nucleic acid in the expression vector may be selected from, for example, SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:ll.
  • the present invention in ⁇ cludes a recombinantly produced human interferon- ⁇ pro- tein.
  • One exemplary sequence for such a protein is given as SEQ ID NO:4.
  • Human IFNT may also contain a carboxy terminal extension (such as the sequence presented as SEQ ID NO:12) .
  • the invention includes a method of recombinantly producing interferon- ⁇ .
  • a recombinant expression system containing an open reading frame (ORF) having a polynucleotide sequence which encodes a human interferon- ⁇ polypeptide, where the vector is designed to express the ORF in the host is introduced into suitable host cells. The host is then cultured under conditions resulting in the expression of the ORF sequence.
  • ORF open reading frame
  • the human IFNT sequences discussed above are examples of suitable human IFNT polypeptides. Examples of polynucle ⁇ otide coding sequences are SEQ ID NO:3 and SEQ ID NO:11.
  • the invention further includes expression systems useful for the expression of interferon- ⁇ polypeptides. Typically these systems include a host capable of sup ⁇ porting expression of an open reading frame in a selected expression vector, and the selected expression vector containing an open reading frame (ORF) having a polynu- cleotide sequence which encodes a human interferon-T polypeptide.
  • ORF open reading frame
  • Exemplary of sequences that can be utilized in such expression systems are SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.
  • the invention also includes isolated interferon- ⁇ polypeptides. These polypeptides are derived from the interferon-T amino acid coding sequence and are between about 15 and 172 amino acids in length. Such polypep- tides can be selected, for example, from the sequences presented as SEQ ID NO:2 and SEQ ID NO:4. Exemplary IFNT-derived polypeptides include, but are not limited to, the following: SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:20. In another embodiment, the invention includes a method of blocking the binding of alpha-interferon to a cell having an alpha-interferon receptor.
  • the cell is contacted with an interferon-T polypeptide at a concentration effective to allow the binding of inter- feron-T to each alpha-interferon receptor.
  • the cells, having the IFNT polypeptide bound to the receptor, are then exposed to alpha-interferon (IFN ⁇ ) .
  • IFN ⁇ alpha-interferon
  • the IFNT poly ⁇ peptides and IFN ⁇ -derived polypeptides described above are examples of IFNT polypeptides useful in this method.
  • the invention includes a method of blocking the binding of interferon-T to a cell having a interfer ⁇ on-T receptor. In this method, the cell is contacted with an interferon-T-derived polypeptide (e .g.
  • polypeptide is at a concentration effective to allow the binding of the polypeptide to each interferon- -receptor.
  • the cells are then exposed to interferon-T.
  • the invention also includes purified antibodies that are immunoreactive with human interferon-T.
  • the antibod- ies may be polyclonal or monoclonal.
  • Exemplary IFNr polypeptide antigens include, but are not limited, to the following: SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:20.
  • the present invention also includes the following: interferon- ⁇ -derived polypeptides that have anti-tumor (i .e . , anti-proliferative) activity; interferon-T-derived polypeptides that have anti-viral activity; and hybrid ⁇ - interferon molecules in which the toxicity portion of native IFN ⁇ has been replaced by analogous sequences from IFNT.
  • Figure 1 presents the nucleic acid coding sequence of a synthetic gene of OVIFNT designed to include 19 unique restriction enzyme sites spaced evenly throughout the coding sequence.
  • Figure 2 shows the cloning strategy used for making a synthetic gene encoding OVIFNT.
  • Figure 3 shows a comparison of the predicted protein sequences of a human interferon-T gene and an ovine interferon-T gene. Divergent amino acids are indicated by presentation of the alternative amino acid on the line below the nucleic acid sequences.
  • Figure 4 presents data demonstrating that both
  • Figure 5 presents data demonstrating that rHuIFN ⁇ is cytotoxic and OVIFNT is not.
  • results of one of three replicate experiments are presented as mean % viability ⁇ SD.
  • Figure 6 presents the sequences of polypeptides derived from the IFNT sequence.
  • Figure 7 presents the complete nucleic acid and amino acid sequence of an OVIFNT sequence.
  • Figure 8 presents data supporting the lack of cyto- toxicity, relative to IFN ⁇ , when IFNT is used to treat peripheral blood mononuclear cells.
  • Figure 9 shows the results of treatment of a human cutaneous T cell lymphoma line, HUT 78, with IFNT.
  • Figure 10 shows the results of treatment of a human T cell lymphoma line, H9, with IFNT.
  • Figure 11A presents data for the peptide inhibition, relative to FIV (feline immunodeficiency virus) replica ⁇ tion, of polypeptides derived from OVIFNT with whole OVIFNT.
  • Figure 11B presents data for the peptide inhibi ⁇ tion, relative to HIV (human immunodeficiency virus) replication, of polypeptides derived from OVIFNT with whole OVIFNT.
  • Figure 12 presents data demonstrating the inhibition of the antiviral activity of IFNT by IFN -derived pep ⁇ tides.
  • Figure 13 presents data demonstrating the inhibition by IFNT-derived peptides of OVIFNT antiviral activity.
  • Figure 14 presents data demonstrating the inhibition by IFNT-derived peptides of bovine IFN ⁇ antiviral activi ⁇ ty.
  • Figure 15 presents data demonstrating the inhibition by IFNT-derived peptides of human IFN ⁇ antiviral activi ⁇ ty.
  • Figure 16 presents data evaluating the lack of inhibition by IFNT-derived peptides of bovine IFN7 anti ⁇ viral activity.
  • Figure 17 presents data demonstrating the anti-IFN ⁇ - derived peptide antisera inhibition of the antiviral activity of IFNT.
  • Figure 18 presents data demonstrating the anti-IFN ⁇ - derived peptide antisera inhibition of the binding of radiolabeled IFNT to cells.
  • SEQ ID N0:1 is the nucleotide sequence of a synthet- ic gene encoding ovine interferon-T (OVIFNT) . Also shown is the encoded amino acid sequence.
  • OVIFNT ovine interferon-T
  • SEQ ID NO:2 is an amino acid sequence of a mature OVIFNT protein.
  • SEQ ID NO:3 is a synthetic nucleotide sequence encoding a mature human interferon-T (HUIFNT) protein.
  • SEQ ID NO:4 is an amino acid sequence for a mature HUIFNT protein.
  • SEQ ID NO:5 is the amino acid sequence of fragment 1-37 Of SEQ ID NO:2.
  • SEQ ID NO:6 is the amino acid sequence of fragment 34-64 Of SEQ ID NO:2.
  • SEQ ID NO:7 is the amino acid sequence of fragment 62-92 of SEQ ID NO:2.
  • SEQ ID NO:8 is the amino acid sequence of fragment 90-122 Of SEQ ID NO:2.
  • SEQ ID NO:9 is the amino acid sequence of fragment 119-150 of SEQ ID NO:2.
  • SEQ ID NO:10 is the amino acid sequence of fragment 139-172 of SEQ ID NO:2.
  • SEQ ID NO:11 is the nucleotide sequence of a natural HUIFNT gene with a leader sequence.
  • SEQ ID NO:12 is the predicted amino acid coding sequence of the SEQ ID NO:11.
  • SEQ ID NO:13 is a 25-mer synthetic oligonucleotide according to the subject invention.
  • SEQ ID NO:14 is a 25-mer synthetic oligonucleotide according the subject invention.
  • SEQ ID NO:15 is the amino acid sequence of fragment 1-37 of SEQ ID NO:4.
  • SEQ ID NO:16 is the amino acid sequence of fragment 34-64 of SEQ ID NO:4.
  • SEQ ID NO:17 is the amino acid sequence of fragment 62-92 of SEQ ID NO:4.
  • SEQ ID NO:18 is the amino acid sequence of fragment 90-122 of SEQ ID NO:4.
  • SEQ ID NO:19 is the amino acid sequence of fragment 119-150 of SEQ ID NO:4.
  • SEQ ID NO:20 is the amino acid sequence of fragment 139-172 of SEQ ID NO:4.
  • Interferon-T refers to any one of a family of inter ⁇ feron proteins having the following characteristics: (i) anti-luteolytic properties; (ii) anti-viral properties; (iii) anticellular proliferation properties; (iv) 45-68% amino acid homology with ⁇ -Interferons and greater than 70% amino acid homology to the sequence presented as SEQ ID NO:2. Interferon-T can be isolated from a number of mammalian sources as described below.
  • An interferon-T polypeptide is a polypeptide having between about 15 and 172 amino acids derived from an interferon-T amino acid coding sequence, where said 15 to 172 amino acids are contiguous in native interferon- . Such 15-172 amino acid regions can also be assembled into polypeptides where two or more such interferon-T regions are joined that are normally discontinuous in the native protein.
  • Ovine interferon- ⁇ (OVIFNT) is a major conceptus secretory protein produced by the embryonic trophectoderm during the critical period of maternal recognition in sheep.
  • One isolate of mature OVIFNT is 172 amino acids in length (SEQ ID NO:2).
  • the cDNA coding sequence con ⁇ tains an additional 23 amino acids at the amino-terminal end of the mature protein (Imakawa, et al . , 1987).
  • the coding sequence of this OVIFNT isolate is presented as Figure 7.
  • OVIFNT Conceptuses were collected from pregnant sheep and cultured in vitro in a modified Minimum Essential Medium as described previously (Godkin, et al . , 1982). Conceptuses were collected on various days of pregnancy with the first day of mating being described as Day 0. IFNT was purified from conceptus culture medium essentially as described by Vallet, et al . , (1987) and Godkin, et al. (1982).
  • IFNT The homogeneity of IFNT was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE; Maniatis, et al . ; Ausubel, et al . ) . Determination of protein concentration in purified IFNT samples was performed using the bicinchoninic (BCA) assay (Pierce Chemical Co., Rockford, IL; Smith, et al . , 1985).
  • BCA bicinchoninic
  • a homologous protein to OVIFNT was isolated from cows (bIFN ⁇ ; Helmer, et al., 1987; Imakawa, et al . , 1989) .
  • OVIFNT and BOIFNT (i) have similar functions in maternal recognition of pregnancy, and (ii) share a high degree of amino acid and nucleotide sequence homology between mature proteins.
  • the nucleic acid sequence homology between OVIFNT and BOIFNT is 76.3% for the 5' non-coding region, 89.7% for the coding region, and 91.9% for the 3' non-coding region.
  • the amino acid sequence homology is 80.4%.
  • Example 1 describes the reproductive functions of OVIFNT.
  • OVIFNT and recombinant human ⁇ -2-Interferon (rHuIFN ⁇ 2 ) were infused into uterine lumen of ewes at a variety of concentrations.
  • the life span of the corpus luteum was assessed by examination of interestrous inter ⁇ vals, maintenance of progesterone secretion, and inhibi ⁇ tion of prostaglandin secretion (Davis, et al . , 1992 ) .
  • Comparison of the data resulting from these examinations demonstrated a considerable lengthening of the inter ⁇ estrous interval when IFNT is administered at lOO ⁇ g/day and no meaningful effect when rHuIFN ⁇ is administered.
  • Example 2 The antiviral properties of interferon-T at various stages of the reproductive cycle were also examined (Example 2) .
  • Conceptus cultures were established using conceptus obtained from sheep at days 12 through 16 of the estrus cycle. Antiviral activity of supernatant from each conceptus culture was assessed. Culture superna ⁇ tants had increasing antiviral activity associated with advancing development of the conceptus up to Day 16 post estrus.
  • IFNT Recombinant Production of IFNT.
  • Recombinant IFNT was produced using bacterial and yeast cells.
  • the amino acid coding sequence for OVIFNT was used to generate a corresponding DNA coding sequence with codon usage optimized for expression in E. coli
  • Example 3 The DNA coding sequence was synthetically constructed by sequential addition of oligonucleotides. Cloned oligonucleotides were fused into a single polynu- cleotide using the restriction digestions and ligations outlined in Figure 2. The polynucleotide coding sequence had the sequence presented as SEQ ID NO:l.
  • this synthetic coding sequence can be placed in a number of bacterial expression vectors: for example, lambda gtll (Promega, Madison WI) ; pGEX (Smith, et al . ) ; pNH vectors (Strata- gene, La Jolla CA) .
  • Cloning of the IFNT synthetic poly ⁇ nucleotide into a modified pIN III omp-A expression vector is described in Example 3. Production of the IFNT protein was induced by the addition of IPTG. Soluble recombinant IFNT was liberated from the cells by sonica- tion or osmotic fractionation.
  • the protein can be further purified by standard methods, including size fractionation (column chromatog- raphy or preoperative gel electrophoresis) or affinity chromatography (using, for example, anti-IFN ⁇ antibodies (solid support available from Pharmacia, Piscataway NJ) . Protein preparations can also be concentrated by, for example, filtration (A icon, Danvers, Mass.).
  • the synthetic IFNT gene was also cloned into the yeast cloning vector pBS24Ub (Example 4; Sabin, et al . ; Ecker, et al . ) . Synthetic linkers were constructed to permit in-frame fusion of the IFNT coding sequences with the ubiquitin coding sequences in the vector. The re- suiting junction allowed in vivo cleavage of the ubiqui ⁇ tin sequences from the IFNT sequences.
  • the recombinant plasmid pBS24Ub-IFN ⁇ was transformed into the yeast S. cerevisiae .
  • Transformed yeast cells were cultured, lysed and the recombinant IFNT (r-IFN ⁇ ) protein isolated from the cell lysates.
  • the amount of r-IFN ⁇ was quantified by radioimmuno- assay. Microsequencing of the purified r-IFN ⁇ was car ⁇ ried out. The results demonstrated identity with native IFNT through the first 15 amino acids. The results also confirmed that the ubiquitin/IFN ⁇ fusion protein was correctly processed in vivo.
  • Recombinant IFNT obtained by this method exhibited antiviral activity similar to the antiviral activity of IFNT purified from conceptus-conditioned culture medium.
  • yeast vectors can be used in the practice of the present invention including, but are not limited to, vectors with regulatable expression (Hitzeman, et al . ; Rutter, et a . ; Oeda, et al.) .
  • the yeast transformation host is typically Saccharomyces cerevisiae, however, other yeast suitable for transformation can be used as well (e .g. , Schiz ⁇ saccharomyce ⁇ pombe) .
  • the DNA encoding the IFNT polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system.
  • These systems include the above described bacterial and yeast expression systems as well as the following: baculovirus expression (Reilly, et al . ; Beames, et al . ; Clontech, Palo Alto CA) ; and expression in mammalian cells (Clontech, Palo Alto CA; Gibco-BRL,
  • polypeptides can be expressed as fusion proteins or as native proteins.
  • a number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium.
  • the recombinantly produced polypeptides are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed, as described above, using antibodies generated based on the IFNT polypeptides.
  • B. Human Interferon-T can be employed, as described above, using antibodies generated based on the IFNT polypeptides.
  • DNA was screened for sequences homologous to inter- feron-T (Example 5) .
  • Several sequences that hybridized with the OVIFNT CDNA probe were identified.
  • Several clones containing partial sequences of human interferon- ⁇ were then isolated (Example 6) .
  • Two synthetic 25-mer oligonucleotides, corresponding to sequences from the OVIFNT CDNA (Imakawa, et al . , 1987) were synthesized. These primers were employed in amplification reactions using DNA derived from the following two cDNA libraries: human term placenta and human term cytotrophoblast.
  • the resulting amplified DNA fragments were electrophoreti- cally separated and a band containing an IFNT amplifica ⁇ tion product was isolated.
  • the product was subcloned and the inserted amplification product sequenced using the dideoxy termination method.
  • Example 7 describes the isolation of a full-length human IFNT gene.
  • High molecular weight DNA was isolated from peripheral blood mononuclear cells (PBMCs) and size- fractionated. Fractions were tested for the presence of IFNT sequences using polymerase chain reaction: DNA molecules from fractions that tested amplification posi- tive were used to generate a subgenomic library in ⁇ gtll.
  • This subgenomic library was plated and hybridized with an OVIFNT CDNA probe (Example 7A) . Approximately 20 clones were identified that hybridized to the probe. Plaques corresponding to the positive clones were pas- saged, DNA isolated and analyzed by amplification reac- tions using OVIFNT primers. Of these twenty plaques, six plaques generated positive PCR signals. The phage from these six clones were purified and the inserts sequenced. One of the inserts from one of these six clones was used as a hybridization probe in the following screening.
  • Recombinant phage from the ⁇ gtll subgenomic library were screened using the hybridization probe just de ⁇ scribed (Example 7B) .
  • Three clones giving positive hybridization signals were isolated and the inserts sequenced.
  • the resulting nucleic acid sequence informa ⁇ tion is presented as SEQ ID NO:11 and the predicted protein coding sequence is presented as SEQ ID NO:12.
  • the predicted mature protein coding sequence is presented as SEQ ID NO:4.
  • Comparison of the predicted protein sequences (Fig ⁇ ure 3) of the human interferon-T gene (SEQ ID NO:4) and the ovine interferon-T gene demonstrates the levels of sequence homology and divergence at the amino acid level.
  • the human IFNT sequences presented as SEQ ID NO:12 and SEQ ID NO:11, and primers and probes derived there ⁇ from, can be used as specific probes to detect isolates of further human IFNT coding sequences and/or pseudo- genes. Further, there may be more than one isofor of the IFNT protein and more than one coding sequence per species.
  • the specific nucleic acid probes used in the practice of the present invention and antibodies reactive with the IFNT polypeptides of the present invention may be useful to isolate unidentified variants of interferon- T in mammals, according to the methods of the invention disclosed herein. 2. Characterization of the Expression of Interfe- ron- ⁇ in Human Tissues.
  • Human placental cDNA libraries and an ovine cDNA library were analyzed by hybridization to the OVIFNT CDNA probe (Example 8) .
  • This DNA hybridization analysis sug ⁇ gested that the IFN ⁇ -signals from human cDNA libraries were approximately 1/100 of the signal obtained using the ovine cDNA library.
  • OVIFNT CDNAS constitute around 0.4% of the ovine cDNA library. Accordingly, the abundance of human cDNAs responding to the OVIFNT probe appears to be low, at least in the term placenta from which the cDNA libraries were generated.
  • HUIFNT mRNA The presence of HUIFNT mRNA in human term placenta and amniocytes were also analyzed. The results suggest the presence of human IFNT mRNA in the feto-placental annex. The aminocytes also expressed the messages corre ⁇ sponding to OVIFNT primers and human probe, suggesting that the expression of IFNT mRNA is not limited to the term placenta.
  • a RT-PCR analysis for the presence of HUIFNT was applied to the total cellular RNA isolated from human adult lymphocytes: the results suggest that IFNT mRNA exists in lymphocytes.
  • interferon-T The expression of interferon-T in human tissue was also examined using in situ hybridization (Example 9) .
  • Sections from four healthy, different term and first tri ⁇ mester human placentas were examined. This analysis em ⁇ ployed a cDNA probe derived from the OVIFNT CDNA sequen ⁇ ces (Example 9B) . In situ hybridization was performed using an anti-sense RNA probe. In three separate experi ⁇ ments, specific hybridization was observed in all term and first trimester placental tissues.
  • First trimester placental villi (composed of an outer layer of syncytiotrophoblast, an underlying layer of cytotrophoblast, and a central stromal region with various types of mesenchymal cells) displayed the highest transcript level of IFNT in the cytotrophoblast cells. Less intense but detectable levels were present in both the syncytiotrophoblast and stromal cells. A similar pattern of transcript expression was demonstrated in the placental villi of term tissue but the level of signal detection was low. First trimester extravillous tropho- blasts displayed the highest amount of message and stained positive when present in the maternal blood spaces.
  • the present results demonstrate that the human IFNT gene is highly expressed in early placental tissues by migrating extravillous trophoblasts, but is also ex ⁇ pressed in villous syncytiotrophoblasts, cytotropho- blasts, and various stromal cells. These results demon ⁇ strate the detection of IFNT transcripts in human preg ⁇ nancy tissues, and IFNT expression in the villous cyto- trophoblasts as well as the extravillous trophoblast of first trimester placenta.
  • IFNT Antiviral Properties of Interferon-T.
  • the antiviral activity of IFNT has been evaluated against a number of viruses, including both RNA and DNA viruses.
  • IFNT had a higher specific antivi ⁇ ral activity than either rBoIFN ⁇ or rBoIFN ⁇ (Example 10, Table 3) .
  • IFNT has potent antiviral activity with limited cytotoxic effects.
  • Highly purified OVIFNT was tested for anti- retroviral and cytotoxic effects on peripheral blood lymphocytes exposed to feline AIDS and human AIDS retro- viruses (Bazer, F.W., et al., (1989)).
  • This feline AIDS lentivirus produces a chronic AIDS-like syndrome in cats and is a model for human AIDS (Pederson, et al . , 1987).
  • Replication of either virus in peripheral blood lympho ⁇ cytes (PBL) was monitored by reverse transcriptase (RT) activity in culture supernatants over time.
  • RT reverse transcriptase
  • RNA-dependent DNA polymerase RT activity was assayed in FIV- and HIV-infected feline and human PBL cultures treated with IFNT (Example 11) . Replication of FIV was reduced to about one-third of control values when cells were cultured in the presence of IFNT. Addition of OVIFNT produced a rapid, dose-dependent decrease in reverse transcriptase (RT) activity (Example 11, Table 4). While concentrations as low as 0.62 ng/ml of IFNT inhibited viral replication, much higher concentrations (40 ng/ml) having greater effects on RT-activity were without toxic effects on the cells.
  • RT reverse transcriptase
  • IFNT appeared to exert no cytotoxic effect on the cell ⁇ hosting the retrovirus. This was true even when IFNT was present at 40 ng per ml of culture medium. This concentration of IFNT is equivalent to about 8,000 anti ⁇ viral units of alpha interferon, when IFNT is assayed for its ability to protect Madin-Darby bovine kidney cells from lysis by vesicular stomatitis virus as described by Pontzer, et al . (1988).
  • IFNT was also tested for activity against HIV repli ⁇ cation in human cells.
  • Human peripheral lymphocytes, which had been infected with HIV were treated with vary ⁇ ing concentrations of IFNT (Example 12) .
  • Replication of HIV in peripheral blood lymphocytes was monitored by reverse transcriptase activity in culture supernatants over time. Over a range of concentrations of IFNT pro- prised significant anti-HIV effects (Example 12, Table 5).
  • a concentration of only 10 ng/ml resulted in over a 50% reduction in RT activity after only six days;
  • a concen ⁇ tration of 500 ng/ml resulted in a 90% reduction in RT activity within 10 days.
  • there was no evidence of any cytotoxic effects attributable to the administra ⁇ tion of IFNT (Example 12, Table 5).
  • IFNT antiviral effects of IFNT against HIV were evaluated by treating human PBMC cells with various amounts of either recombinant IFNT or recombinant human IFN ⁇ 2 at the time of infection with HIV (Example 18) .
  • the data from these experiments (Example 18, Table 12) sup ⁇ port the conclusion that, at similar concentrations, IFN ⁇ and IFNT are effective in reducing the replication of HIV in human lymphocytes.
  • treatment of cells with IFN ⁇ 2 resulted in cytoxicity, whereas no such cytotoxity was observed with treatment using IFNT, even when IFNT was used at much higher concentrations. No cytotoxicity was observed using IFNT, even when IFNT was used at 200 times the dosage of interferon-alpha II.
  • the hepatocyte was examined for the effects of IFN ⁇ and IFNT on hepatospeci- fic mRNA production (Example 18) .
  • IFNT is an effective antiviral agent against a wide variety of viruses, in ⁇ cluding both RNA and DNA viruses.
  • IFNT D. Antiproliferative Properties of IFNT.
  • the effects of IFNT on cellular growth have also been examined.
  • anti-cellular growth activity was examined using a colony inhibition assay (Example 13) .
  • Human amnion (WISH) or MDBK cells were plated at low cell densities to form colonies originating from single cells. Dilutions of interferons were added to triplicate wells and the plates were incubated to allow colony formation.
  • IFNT inhibited both colony size and number in these assays.
  • IFNT was more effective at inhibiting cell proliferation of the human cell line (WISH) than human IFN ⁇ .
  • the antiproliferative activity of IFNT was dose-dependent. High concentrations of IFNT stopped proliferation, while cell viability was not impaired.
  • IFNT appears to inhibit progress of cells through S phase. These results demonstrate the antiproliferative effect of IFNT, and underscore its low cytotoxicity.
  • the antiproliferative effects of IFNT were also studied for rat and bovine cell lines (Example 14) .
  • the rate of 3 H-thymidine incorporation was used to assess the rate of cellular proliferation.
  • IFNT The antiproliferative activity and lack of toxicity of IFNT was further examined using a series of human tumor cell lines (Example 15) .
  • a variety of human tumor cell lines were selected from the standard lines used in NIH screening procedure for antineoplastic agents (Pontzer, C.H., et al., (1991)). At least one cell line from each major neoplastic category, was examined.
  • HL-60 human promyelocytic leukemia
  • H9 human T cell lymphoma HUT 78 human cutaneous T cell lymphoma;
  • MCF7 human breast adenocarcinoma As above, the antiproliferative activity was evalu ⁇ ated by measuring the rate of 3 H-thymidine incorporation into cells which havt* been treated with IFNT. Signifi ⁇ cant differences between treatments were assessed by an analysis of variance followed by Scheffe's F-test. Cell cycle analysis was performed by flow cytometry.
  • IFNT inhibition of MCF7 (breast adenocarcinoma) proliferation demonstrated that IFNT reduced MCF7 proliferation in a dose-dependent manner.
  • a 50% reduction in 3 H-thymidine was observed with 10,000 units/ml of IFNT (Example 15, Table 8) . This cell line had previously been found to be unresponsive to anti- estrogen treatment.
  • IFNT and IFN ⁇ were equally efficacious antitumor agents.
  • SK-MEL-28 inhibition of proliferation by IFN ⁇ was accomplished by a 13% drop in viability, while IFNT was not cytotoxic.
  • IFNT is equal or preferable to IFN ⁇ as an antineoplastic agent against human tumors.
  • IFNT exhibits antiproliferative activity against human tumor cells without toxicity and is as potent or more potent than human IFN ⁇ .
  • Clinical trials of the IFN ⁇ 2s have shown them to be effective antitumor agents (Dianzani, F., 1992; Krown, 1987).
  • One therapeutic advantage of IFNT as a therapeutic is the elimination of toxic effects seen with high doses IFN ⁇ s.
  • IFNT IFNT-induced tumors like Kaposi's sarcoma (associated with HIV infec- tion) where the antineoplastic effects of IFNT are cou ⁇ pled with IFNT ability to inhibit retroviral growth.
  • B16-F10 is a syngeneic mouse transplantable tumor selected because of its high incidence of pulmonary metastases (Poste, et al . , 1981). Interferon treatment was initiated 3 days after the introduction of the tumor cells. The in vivo administration of IFNT dramatically reduced B16-F10 pulmonary tumors. Thus, IFNT appears to be an effica- cious antineoplastic agent in vivo as well as in vitro . III. Interferon-T Polypeptide Fragments. Protein Modeling and Protein Modifications.
  • IFNT activity has been examined using six overlapping synthet ⁇ ic peptides corresponding to the entire OVIFNT sequence ( Figure 6) .
  • the corresponding polypeptides derived from the ovine IFNT sequence are presented as SEQ ID NO:15 to SEQ ID NO:20.
  • Three peptides representing amino acids l- 37, 62-92 and 139-172 have been shown to inhibit IFNT antiviral activity (Example 17) .
  • the peptides were effective competitors at concentrations of 300 ⁇ M and above.
  • the two synthetic peptides OVIFNT(1-37) and OVIFNT(139-172) also blocked OVIFNT anti-FIV and anti-HIV activity (Example 17; Figures 11A and 11B) . While both peptides blocked FIV RT activity, only the C-terminal peptide, OVIFNT(139-172) , appeared to be an efficient inhibitor of vesicular stomatitis virus activity on the feline cell line, Fc9.
  • the above data taken together suggest that the C- terminal regions of type I interferons may bind to common site on the type I interferon receptor, while the N- terminal region may be involved in the elicitation of unique functions.
  • portions of the IFNT interferon molecule may be used to substitute regions of interferon alpha molecules.
  • the region of an interferon alpha molecule that is responsi ⁇ ble for increased cytotoxicity, relative to IFNT treat- ment can be identified by substituting polypeptide regions derived from IFNT for regions of an interferon alpha molecule.
  • substitutions can be carried out by manipulation of synthetic genes (see below) encoding the selected IFNT and interferon alpha molecules, coupled to the functional assays described herein (such as, antivi ⁇ ral, antiproliferative and cytoxicity assays) .
  • IFNT The antiproliferative activity of IFNT (Example 17, Table 11) involved a further region of the molecule, since IFNT(119-150) was the most effective inhibitor of OvIFNT-induced reduction of cell proliferation. This results suggests that the region of the molecule primari ⁇ ly responsible for inhibition of cell growth is the IFNT(119-150) region.
  • This region of the IFNT molecule may be useful alone or fused to other proteins (such as serum albumin, an antibody or an interferon alpha poly- peptide) as an antineoplastic agent.
  • a conjugated pro ⁇ tein between an N-terminal peptide derived from human interferon- ⁇ and serum albumin was shown to have anticel ⁇ lular proliferation activity (Ruegg, et al .
  • CD circular dichroism
  • the final modeling step was to apply the IFN/3 x-ray crystallographic coordinates of the IFN/3 carbon backbone to the IFNT sequence.
  • the functionally active domains of IFNT, identified above, were localized to one side of the molecule and found to be in close spatial proximity. This is consistent with multiple binding sites on IFNT interacting simultaneously with the type I IFN receptor.
  • the three dimensional modeling data coupled with the function data described above, provides the ability to introduce sequence variations into specific regions of IFNT to generate enhancement of selected functions (e.g., antiviral or anticellular proliferation) or the ability to substitute a region(s) of selected function into other interferon molecules (e .g. , antiviral, antineoplastic, or reduced cytotoxicity) .
  • Example 3 The construction of a synthetic gene for OVIFNT is described in Example 3. Briefly, a consensus amino sequence was back-translated using optimal codon usage for E. coli . The sequence was edited to include 20, unique, restriction sites spaced throughout the length of the construct. This 540 base pair synthetic gene se ⁇ quence was divided into 11 oligonucleotide fragments. Individual fragments were synthesized and cloned, either single or double stranded, into either pTZ 19R, pTZ 18R or pBluescript, amplified and fused. The synthetic OVIFNT construct was then cloned into a modified pIN-IIl- ompA expression vector for expression in bacteria and also cloned into a yeast expression plasmid. A similarly constructed human IFNT synthetic gene (SEQ ID NO:3) has been designed, constructed and expressed in yeast cells.
  • SEQ ID NO:3 A similarly constructed human IFNT synthetic gene (SEQ ID NO:3) has been designed, constructed and expressed in yeast cells.
  • OVIFNT synthetic gene in yeast allowed over production of recombinant IFNT in S. cerevisiae : large quantities (5-20 mg/1) of recom ⁇ binant IFNT can be purified from soluble yeast extract . using sequential ion exchange and molecular sieve chroma ⁇ tography. Recombinant IFNT purified in this fashion exhibited potent antiviral activity (2 to 3 X 10 8 units/mg) similar to native OVIFNT.
  • the synthetic gene construct facilitates introduc ⁇ tion of mutations for possible enhancement of antitumor (anticellular proliferative) and antiviral activities. Further, the disparate regions of the molecule responsi- ble for different functions allows for separate manipula ⁇ tion of different functions. For example, two deletion mutants, OVIFN (1-155) and OVIFNT(1-166) , have been constructed to examine the role of carboxy terminal sequences in IFNr molecules. Additional mutant IFNT molecules have been con ⁇ structed to identify residues critical for antiprolifera ⁇ tive activity. For example, one particular residue, Tyr 123 has been implicated in the anticellular proliferative activity of IFN ⁇ (Mclnnes, et al . , 1989). The equivalent of Tyr 123 in IFNT is contained within peptide
  • OVIFNT(119-150) this polypeptide inhibits OVIFNT and human IFN ⁇ antiproliferative activity. Mutations con ⁇ verting Tyr 123 to conservative (Trp) and nonconservative (Asp) substitutions have been generated, as well as mutant sequences having deletion of this residue. The codon for Tyr 123 is located within an Sspl site; elimi ⁇ nation of this site has been used for screening. The antiproliferative activity of these mutant IFNT is evalu- ated as described herein.
  • Synthetic peptides can be generated which correspond to the IFNT polypeptides of the present invention. Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art (Applied Biosystems, Foster City CA) .
  • oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of clon- ing steps involving a tandem array of multiple oligonu ⁇ cleotide fragments corresponding to the coding sequence (Crea; Yoshio et al . ; Eaton et al . ) . Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al . ; Ausubel et al . ) . The biological activities of the interferon- ⁇ poly ⁇ peptides described above can be exploited using either the interferon-T polypeptides alone or conjugated with other proteins (see below) .
  • the present invention includes interferon-T or interferon-T-derived polypeptides cova ⁇ lently attached to a second polypeptide to form a fused, or hybrid, protein.
  • the interferon-T sequences making up such fused proteins can be recombinantly produced inter ⁇ feron-T or a bioactive portion thereof, as described above.
  • the polypeptides presented as SEQ ID NO:10 and SEQ ID NO:20 may be advantageously fused with a soluble peptide, such as, serum albumin, an antibody (e .g. , specific against an virus-specific cell surface antigen) , or an interferon alpha polypeptide.
  • a soluble peptide such as, serum albumin, an antibody (e .g. , specific against an virus-specific cell surface antigen)
  • interferon alpha polypeptide include (i) replacing toxici- ty-associated regions of interferon- ⁇ with the interfer ⁇ on-T regions SEQ ID NO:5 and SEQ ID NO:15, and (ii) fusion proteins containing the interferon-T regions SEQ ID NO:9 and SEQ ID NO:19 as anticellular proliferation agents.
  • the fused proteins of the present invention may be formed by chemical conjugation or by recombinant tech ⁇ niques.
  • the interferon- ⁇ and second selected polypeptide are modified by conventional coupling agents for covalent attachment.
  • serum albumin is derivatized with N- succinimidyl-S-acetyl thioacetate (Duncan) , yielding thiolated serum albumin.
  • the activated serum albumin polypeptide is then reacted with interferon-T derivatized with N-succinimidyl 3-(2-pyridyldithio) propionate (Cum ⁇ ber) , to produce the fused protein joined through a disulfide linkage.
  • recombinant interferon-T may be prepared with a cysteine residue to allow disul- fide coupling of the interferon-T to an activated ligand, thus simplifying the coupling reaction.
  • An interferon- ⁇ expression vector, used for production of recombinant interferon-T can be modified for insertion of an inter ⁇ nal or a terminal cysteine codon according to standard methods of site-directed mutagenesis (Ausubel, et al . ) .
  • a fused protein is prepared recombi ⁇ nantly using an expression vector in which the coding sequence of a second selected polypeptide is joined to the interferon-T coding sequence.
  • human serum albumin coding sequences can be fused in-frame to the coding sequence of an interferon-T polypeptide, such as, SEQ ID NO:9.
  • the fused protein is then expressed using a suitable host cell.
  • the fusion protein may be purified by molecular-sieve and ion-exchange chromatogra- phy methods, with additional purification by polyacryl- amide gel electrophoretic separation and/or HPLC chroma ⁇ tography, if necessary.
  • interfer- on-T-containing fusion proteins may be prepared.
  • One variation on the above fusion is to exchange positions of the interferon-T and selected second protein molecules in the fusion protein (e.g., carboxy terminal versus amino terminal fusions) .
  • internal portions of a native interferon-T polypeptide for example, amino acid regions of between 15 and 172 amino acids
  • two or more such interferon- T portions are contiguous that are normally discontinuous in the native protein.
  • Fusion proteins containing the polypeptide antigens of the present invention fused with the glutathione-S- transferase (Sj26) protein can be expressed using the pGEX-GLI vector system in E. coli JM101 cells.
  • the fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography (Smith) . Expression and partial purification of IFNT proteins is described in (Example 20) , and is applicable to any of the other soluble, induced polypeptides coded by sequences de- scribed by the present invention.
  • Insoluble GST (sj26) fusion proteins can be puri ⁇ fied by preparative gel electrophoresis.
  • IFN ⁇ -?-galactosidase fusion proteins can be isolated as described in Example 19.
  • an expression vector such as the lambda gtll or pGEX vectors described above, containing IFNT coding sequences and expression control elements which allow expression of the coding regions in a suitable host.
  • the control elements gener ⁇ ally include a promoter, translation initiation codon, and translation and transcription termination sequences, and an insertion site for introducing the insert into the vector.
  • the DNA encoding the desired polypeptide can be cloned into any number of vectors (discussed above) to generate expression of the polypeptide in the appropriate host system. These recombinant polypeptides can be expressed as fusion proteins or as native proteins.
  • a number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium.
  • Recombinantly produced IFNT, and polypeptides derived therefrom are typically isolated from lysed cells or culture media. Purification can be carried out by meth ⁇ ods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated against selected IFNT antigens.
  • the invention includes specific antibodies directed against the polypeptides of the present invention. Typically, to prepare antibodies, a host animal, such as a rabbit, is immunized with the purified antigen or fused protein antigen.
  • Hybrid, or fused, proteins may be generated using a variety of coding sequences derived from other proteins, such as ⁇ - galactosidase or glutathione-S-transferase.
  • the host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen.
  • Example 20 describes the production of rabbit serum antibodies which are specific against the IFNT antigens in a Sj26/IFN ⁇ hybrid protein. These techniques can be applied to the all of the IFNT molecules and polypeptides derived therefrom.
  • the gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art for produ ⁇ cing polyclonal antibodies.
  • purified protein or fused protein may be used for producing monoclonal antibodies.
  • the spleen or lymphocytes from a animal immunized with the selected polypeptide antigen are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art (Harlow, et al . ) . Lym ⁇ phocytes can be isolated from a peripheral blood sample.
  • Epstein-Barr virus can be used to immortalize human lymphocytes or a fusion partner can be used to produce hybridomas.
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et al . ) .
  • Experi ⁇ ments performed in support of the present invention have yielded four hybridomas producing monoclonal antibodies specific for ovine IFNT have been isolated.
  • Antigenic regions of polypeptides are generally relatively small, typically 7 to 10 amino acids in length. Smaller fragments have been identified as anti- genie regions. Interferon-T polypeptide antigens are identified as described above. The resulting DNA coding regions can be expressed recombinantly either as fusion proteins or isolated polypeptides.
  • amino acid sequences can be conve- niently chemically synthesized (Applied Biosystems, Foster City CA) .
  • Antigens obtained by any of these methods may be directly used for the generation of anti ⁇ bodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e .g. , Pierce, Rockford IL) .
  • Antibodies reactive with IFNT are useful, for exam ⁇ ple, in the analysis of structure/function relationships.
  • IFNT bears some similarity to the IFN ⁇ family based on structure and its potent antiviral prop ⁇ erties, the IFN ⁇ s do not possess the reproductive proper ⁇ ties associated with IFNT. Also, recombinant bovine IFN ⁇ has little or no effect on interestrous interval compared to IFNT (Davis, et al., 1992).
  • IFNT has some structural simi ⁇ larities to other interferons, it has very distinctive properties of its own: for example, the capability of significantly influencing the biochemical events of the estrous cycle.
  • the human IFNT of the present invention can be used in methods of enhancing fertility and prolonging the life span of the corpus luteum in female mammals as generally described in Hansen, et al . , herein incorporated by reference. Further, the human interferon-T of the pres ⁇ ent invention could be used to regulate growth and devel ⁇ opment of uterine and/or fetal-placental tissues. The human IFNT is particularly useful for treatment of hu- mans, since potential antigenic responses are less likely using such a same-species protein.
  • IFNT The antiviral activity of IFNT has broad therapeutic applications without the toxic effects that are usually 40 associated with IFN ⁇ s. Although the presence of IFNr in culture medium inhibited reverse transcriptase activity of the feline immunodeficiency virus (Example 11) , this is not due to a direct effect of IFNT on the FIV. Rath- 5 er, IFNT appears to induce the host cell to produce a factor(s) which is inhibitory to the reverse transcrip ⁇ tase of the virus.
  • Formulations comprising the IFNT compounds of the present invention can be used to inhibit viral replica ⁇ tion.
  • the human IFNT of the present invention can be any suitable IFNT of the present invention.
  • the human interferon-T is particularly useful for treatment of humans, since potential antigenic
  • IFNT exhibits potent anticellular activity. IFNT can also be used to inhibit cellular growth without the 30 negative side effects associated with other interferons which are currently known. Formulations comprising the IFNT compounds of the subject invention can be used to inhibit, prevent, or slow tumor growth.
  • IFNT can suppress estrogen receptor numbers. Therefore, IFNT can be used in the treatment or prevention of estrogen-dependent tumors.
  • IFNT appears to interact with the Type I IFN recep ⁇ tor via several epitopes on the molecule, and these regions either separately or in combination may affect distinct functions of IFNT differently.
  • polypeptides of the present invention are useful for the selective inhibition of binding of interferons to the interferon receptor. Specifically, as described herein, certain of the disclosed peptides selectively inhibit the antiviral activity of IFNT while others inhibit the antiproliferative activity. Combinations of these peptides could be used to inhibit both activities.
  • these peptides despite binding to the interferon recep- tor and blocking IFNT activity, these peptides do not, themselves, elicit the antiviral or antiproliferative activity.
  • polypeptides can be used as immuno- regulatory molecules when it is desired to prevent immune responses triggered by interferon molecules.
  • These peptides could be used as immunosuppressants to prevent, for example, interferon-mediated immune responses to tissue transplants.
  • Other types of interferon mediated responses may also be blocked, such as the cytotoxic effects of alpha interferon.
  • IFNT proteins can be formulated according to known methods for preparing pharmaceutically useful composi- tions. Formulations comprising interferons or interfer- on-like compounds have been previously described (for example, Martin, 1976) . In general, the compositions of the subject invention will be formulated such that an effective amount of the IFNT is combined with a suitable carrier in order to facilitate effective administration of the composition.
  • compositions used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills,, powders, liquid solutions or suspensions, liposomes, suppositories, injectable, and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions also preferably include conventional pharma- ceutically acceptable carriers and adjuvants which are known to those of skill in the art.
  • the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day.
  • IFNT IFNT, or related polypeptides
  • IFNT may be administered to a patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intrale- sional, or subcutaneous injection.
  • compo ⁇ sitions and methods used for other interferon compounds can be used for the delivery of these compounds.
  • IFNT interferon-containing interferon
  • IFNT can be administered at rates from about 5 x 10 4 to 20 x 10 6 units/day to about 500 x 10 6 units/day or more. In a preferred embodiment, the dosage is about 10 6 units/day. High doses are preferred for systemic admin ⁇ istration. It should, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administra ⁇ tion, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • compositions of the subject invention can be administered through standard procedures to treat a variety of cancers and viral diseases including those for which other interferons have previously shown activity. See, for example, Finter, et al . (1991); Dianzani, et al . (1992); Francis, et al . (1992) and U.S. Patent Nos. 4,885,166 and 4,975,276.
  • the compositions of the subject invention have unique features and advantages, including their ability to treat these conditions without toxicity.
  • disorders of the skin can be treated intralesionally using IFNT, wherein formulation and dose will depend on the method of administration and on the size and severity of the lesion to be treated.
  • Preferred methods include intradermal and subcutaneous injection. Multiple injec ⁇ tions into large lesions may be possible, and several lesions on the skin of a single patient may be treated at one time.
  • the schedule for administration can be deter ⁇ mined by a person skilled in the art. Formulations designed for sustained release can reduce the frequency of administration.
  • Systemic Treatment is essentially equivalent for all applications. Multiple intravenous or subcutaneous doses are possible, and in the case of implantable methods for treatment, formulations designed for sustained release are particularly useful. Patients may also be treated using implantable subcutaneous portals, reservoirs, or pumps.
  • Regional treatment with the IFNT polypeptides of the present invention is useful for treatment of cancers in specific organs.
  • Treatment can be accomplished by intra- arterial infusion.
  • a catheter can be surgically or angiographically implanted to direct treatment to the affected organ.
  • a subcutaneous portal, connected to the catheter, can be used for chronic treatment, or an im ⁇ plantable, refillable pump may also be employed.
  • T4 DNA Iigase, T4 polynu- cleotide kinase, Tag DNA polymerase, and calf intestinal phosphatase were purchased from New England Biolabs (Beverly, MA) or Promega Biotech (Madison, WI) : these reagents were used according to the manufacturer's in ⁇ struction.
  • a "SEQUENASE DNA II" sequencing kit was used (United States Biochemical Corporation, Cleveland OH) .
  • Immunoblotting and other reagents were from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Needham, MA) . Nitrocellulose filters are obtained from Schleicher and Schuell (Keene, NH) .
  • Synthetic oligonucleotide linkers and primers are prepared using commercially available automated oligonu- cleotide synthesizers (e .g. , an ABI model 380B-02 DNA synthesizer (Applied Biosystems, Foster City, CA)) .
  • custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
  • cDNA synthesis kit and random priming labeling kits are obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN) .
  • Oligonucleotide sequences encoding polypeptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleo ⁇ tide fragments corresponding to the coding sequence (Crea; Yoshio et al.; Eaton et al.). Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al.; Ausubel et al.).
  • peptides can be synthesized directly by standard in vitro techniques (Applied Biosystems, Foster City CA) .
  • Recombinant human IFN ⁇ (rHuIFN ⁇ ) and rBoIFN7 was obtained from Genentech Inc. (South San Francisco, CA) .
  • the reference preparation of recombinant human IFN ⁇ (rHuIFN ⁇ ) was obtained from the National Institutes of Health: rHuIFN ⁇ is commercially available from Lee Biomolecular (San Diego, CA) . All tissue culture media, sera and IFNs used in this study were negative for endotoxin, as determined by assay with Limulus amebocyte lysate (Associates of Cape Cod, Woods Hole, MA) at a sensitivity level of 0.07 ng/ml.
  • HRP horseradish peroxidase conjugated goat anti- human antiserum
  • the reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate was added to each well, then incubated for 30 min at room temperature. The reaction was stopped with the addition of 0.050 mL 5% SDS ( /v) . The relative absorbance is determined at 410 nm.
  • EXAMPLE 1 Reproductive Functions of IFNT The effect of interferon-T on the lifespan of the corpus lutem was examined. IFNT was infused into uterine lumen of ewes at the concentrations given in Table 1. Recombinant human IFN ⁇ (rHuIFN ⁇ ) was infused at similar concentrations. In addition, control animals, which received co .trol pro ⁇ teins, were also used. The life span of the corpus luteum was assessed by examination of interestrous inter ⁇ vals, maintenance of progesterone secretion, and inhibi ⁇ tion of prostaglandin secretion (Davis, et al . , 1992 ) .
  • VSV vesicular stoma ⁇ titis virus
  • One antiviral unit caused a 50% reduction in de ⁇ struction of the monolayer, relative to untreated MDBK cells infected with VSV (control plates) .
  • Specific activities were further evaluated using normal ovine fibroblasts (Shnf) in a plaque inhibition assay (Lang- ford, et al . , 1981). A minimum of three samples were examined at each time point, and each sample was assayed in triplicate. The results presented in Table 2 are expressed as mean units/ml.
  • the amino acid coding sequence for OVIFNT (Imakawa, et al . , 1987) was used to generate a corresponding DNA coding sequence with codon usage optimized for expression in E. coli .
  • Linker sequences were added to the 5' and 3' ends to facilitate cloning in bacterial expression vec ⁇ tors.
  • the nucleotide sequence was designed to include 19 unique restriction enzyme sites spaced evenly throughout the coding sequence ( Figure 1) .
  • the nucleotide sequence was divided into eleven oligonucleotide fragments ranging in sizes of 33 to 75 bases.
  • oligonucleotides were synthe ⁇ sized on a 380-B 2-column DNA synthesizer (Applied Bio- sytems) and cloned single- or double-stranded into one of the following vectors: "pBLUESCRIPT + (KS)" (Stratagene, LaJolla, CA) , pTZ18R (Pharmacia, Piscataway, NJ) , or pTZ19R (Pharmacia, Piscataway, NJ) cloning vectors.
  • KS SK
  • the vectors were transformed into E. coli K. strain "XLl-BLUE" (recAl endAl gyrA96 thi hsdR17 (r , m ⁇ +) supE44 relAl ⁇ - (lac), ⁇ F', proAB, lac'Z ⁇ Ml ⁇ , Tnl0(tet R ⁇ ) which is commercially available from Stratagene (LaJolla, CA) .
  • Transformed cells were grown in L broth supplemented with ampicillin (50 ⁇ g/ml) . Oligonucleotide cloning and fusion was performed using standard recombinant DNA techniques.
  • Cloning vectors were cut with the appropriate re ⁇ striction enzymes to insert the synthetic oligonucleo ⁇ tides.
  • the vectors were treated with calf intestine alkaline phosphatase (CIP) to remove terminal phosphate groups.
  • Oligonucleotides were phosphorylated and cloned, as either single- or double-stranded molecules, into the appropriate vector using T4 DNA Iigase. When single- strands were introduced into cloning vectors, the second strand was completed by the bacterial host following transfection.
  • CIP calf intestine alkaline phosphatase
  • oligonucleotides were first annealed with their synthetic complementary strand then ligated into the cloning vector. E. coli K12 strains SB221 or NM522 were then transformed with the ligation. E. coli strain GM119 was used for cloning when the methylation-sensitive Stul and Clal restriction sites were involved. Restriction analyses were performed on isolated DNA at each stage of the cloning procedure. Cloned oligonucleotides were fused into a single polynucleotide using the restriction digestions and ligations outlined in Figure 2.
  • Oligonucleotide-contain- ing-DNA fragments were typically isolated after electro- phoretic size fractionation on low-melting point agarose gels (Maniatis, et a .; Sambrook, et al . ; Ausubel, et al . ) .
  • the resulting IFNT polynucleotide coding sequence spans position 16 through 531: a coding sequence of 172 amino acids.
  • the nucleotide sequence of the. final polynucleotide was confirmed by DNA sequencing using the dideoxy chain termination method.
  • the full length Stul/Sstl fragment (540 bp; Figure 2) was cloned into a modified pIN III omp-A expression vector and transformed into a competent SB221 strain of E. coli .
  • cells carrying the expression vector were grown in L-broth containing ampicillin to an OD (550 nm) of 0.1-1, induced with IPTG for 3 hours and harvested by centrifugation. Soluble recombinant IFNT was liberated from the cells by sonication or osmotic fractionation.
  • EXAMPLE 4 Expression of IFNT in Yeast The synthetic IFNT gene, synthesized in Example 3, was flanked at the 5' end by an Stul restriction site and at the 3' end by a Sacl restriction site.
  • Two oligonucleotide primers (SEQ ID NO:13 and SEQ ID NO:14) were used to attach linkers to the synthetic IFNr gene using polymerase chain reaction.
  • the linker at the 5' end allowed the placement of the synthetic IFNr gene in correct reading with the ubiquitin coding sequence present in the yeast cloning vector pBS24Ub (Chiron Corp., Emeryville, CA) .
  • the linker also constructed a ubiquitin-IFN ⁇ junction region that allowed in vivo cleavage of the ubiquitin sequences from the IFNT se ⁇ quences.
  • the 5' oligonucleotide also encoded a Sa ⁇ ll restriction endonuclease cleavage site.
  • the 3' oligonu ⁇ cleotide contained a Stul cleavage site.
  • the vector carrying the synthetic IFNT gene (Example 3) was isolated from E. coli strain "XLl-BLUE" by the alkaline lysis method. Isolated vector was diluted 500- fold in 10 mM Tris, pH 8.0/1 mM EDTA/10 mM NaCl.
  • the PCR reaction was performed in a 100 ⁇ l volume using Tag DNA polymerase and primers SEQ ID NO:13/SEQ ID NO:14.
  • the amplified fragments were digested with -Stul and -Sacll. These digested fragments were ligated into the Sacll and Smal sites of "pBLUESCRIPT+(KS) .”
  • the resulting plasmid was named pBSY-IFN ⁇ .
  • the DNA sequence was verified using double stranded DNA as the template.
  • Plasmid pBSY-IFN ⁇ was digested with Sacll and -BcoRV and the fragment containing the synthetic IFNT gene was isolated.
  • the yeast expression vector pBS24Ub (Sabin, et al . ; Ecker, et al . ) was digested with Sail . Blunt ends were generated using T4 DNA polymerase.
  • the vector DNA was extracted with phenol and ethanol precipitated (Sam- brook, et al . , 1989).
  • the recovered linearized plasmid was digested with Sacll, purified by agarose gel electro- phoresis, and ligated to the Sa ⁇ II--EcoRV fragment isolat ⁇ ed from pBSY-IFN ⁇ .
  • the resulting recombinant plasmid was designated pBS24Ub-IFN ⁇ .
  • the recombinant plasmid pBS24Ub-IFN ⁇ was transformed into E. coli .
  • Recombinant clones containing the IFNT insert were isolated and identified by restriction enzyme analysis. Plasmid DNA from clones containing IFNr coding sequences was used for transformation of S. cerevisiae (Rothstein, 1986) . Transformation mixtures were plated on uracil omission medium and incubated for 3-5 days at 30°C. Colonies were then streaked and maintained on uracil and leucine omission medium (Rothstein, 1986) .
  • IFNT Recombinant IFNT was visualized specifically by immunoblotting with monoclonal antibody or polyclonal antiserum against ovine IFNT upon electrotransfer of the fractionated cell extract to "NYTRAN" paper (Rothstein, 1986) .
  • pBS24-IFNr was grown for 24 hours at 30°C in 5 x uracil and leucine omission medium containing 8% glucose. This culture was then diluted 20-fold in YEP medium containing 1% glucose and further incubated for another 24-36 hours.
  • Cells were harvested by centrifugation, washed in 50 mM Tris, pH 7.6,/l mM EDTA and resuspended in wash buffer containing 1 mM PMSF. The cells were lysed using a Bead- beater apparatus (Biospec Products, Bartlesville, OK) . The lysate was spun at 43,000 x g for 20 minutes. The supernatant fraction was recovered and subjected to the purification protocol described below.
  • HMW DNA High molecular weight DNA was isolated from these cells (Sa brook, et al . , 1989). Two 10 ⁇ g samples of HMW DNA were digested with the restriction endonucleases Ifindlll or Pstl (Promega) for 2 hours at 37°C, and the DNA fragments electrophoretically separated in a 0.8% agarose gel (Bio-Rad, Richmond, CA) at 75 volts for 8 hours.
  • the DNA fragments were trans- ferred onto a nylon membrane (IBI-International Biotech ⁇ nologies, Inc., New Haven, CT) .
  • the membrane was baked at 80°C for 2 hours and incubated at 42°C for 4 hours in the following prehybridization solution: 5 x SSC (l x SSC is 0.15 M NaCl and 0.15 M sodium citrate), 50% vol/- vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 0.5 mg/ml single stranded herring sperm DNA (Promega) .
  • the filter was then incubated in a hybridization solution (5 x SSC, 20% vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 2 x 10 8 cpm/ml 32 P-labelled OVIFNT cDNA (Imakawa, et al., 1987)) for 18 hours at 42°C.
  • a hybridization solution 5 x SSC, 20% vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 2 x 10 8 cpm/ml 32 P-labelled OVIFNT cDNA (Imakawa, et al., 1987)
  • the filter was washed at 42°C for 15 minutes with 2 x SSC and 0.1% (wt/vol) SDS and exposed to X-ray film (XAR, Eastman Kodak, Rochester, NY) at -80°C for 48 hours in the pres ⁇ ence of an intensifying screen.
  • X-ray film XAR, Eastman Kodak, Rochester, NY
  • Two synthetic oligonucleotides (each 25-mer) , corre ⁇ sponding to the sequence 231 to 255 (contained in SEQ ID NO:13) and 566 to 590 (contained in SEQ ID NO:14) of OVIFNT CDNA (numbering relative to the cap site, Imakawa, et al . , 1987) were synthesized. These primers contained, respectively, cleavage sites for the restriction endonu ⁇ cleases Pstl and -EcoRI. SEQ ID NO:13 was modified to contain the EcoRI site, which begins at position 569.
  • DNA was isolated from approximately 1 x 10 5 plaque forming units (pfu) of the following two cDNA libraries: human term placenta (Clontech, Inc. , Palo Alto, CA) and human term cytotrophoblast (Dr. J.F. Strauss, University of Pennsylvania, Philadelphia PA) .
  • the DNA was employed in polymerase chain reaction (PCR) amplifications (Mul ⁇ lis; Mullis, et al . ; Pekin Elmer Cetus Corp. Norwalk CT) .
  • PCR polymerase chain reaction
  • Amplification reactions were carried out for 30 cycles (45°C, lm; 72°C, 2m; 94°C, lm) (thermal cycler and re ⁇ agents, Perkin Elmer Cetus) using primers SEQ ID NO:13/SEQ ID NO:14.
  • Amplification products were electrophoretically separated (100 volts in a 1.5% agarose gel (Bio-Rad)) and transferred onto a nylon membrane (IBI) .
  • the membrane was baked at 80°C for 2 hours and prehybridized and hybridized with 32 P-labelled OVIFNT CDNA as described above.
  • the membrane was washed in 5 x SSC/0.1% (wt/vol) SDS for 5 minutes at 42°C and in 2 x SSC/0.1% (wt/vol) SDS for 2 minutes at 42°C. It was then exposed at -80°C to "XAR" (Eastman Kodak) X-ray film for 24 hours in the presence of an intensifying screen. An amplification product that hybridized with the labelled probe DNA was detected.
  • PCR was performed again as directed above. Ampli ⁇ fied products were digested with the restriction endonu- cleases .EcoRI and Pstl (Promega) for 90 minutes at 37°C. The resulting DNA fragments were electrophoretically separated as described above and the band containing the IFNT amplification product was excised from the gel. DNA fragments were recovered by electroelution, subcloned into EcoRI /Pstl digested-dephosphorylated plasmid pUCl9 and transformed into E. coli strain JM101 (Promega) by calcium chloride method (Sambrook, et al . , 1989). The plasmids were isolated and the inserted amplification product sequenced using the dideoxy termination method (Sanger, et al . , 1977; "SEQUENASE" reactions. United
  • Plaques that hybridized to the probe were further analyzed by PCR using the OVIFNT primers described above. Six plaques which generated positive PCR signals were purified. The phage DNA from these clones was isolated and digested with .EcoRI restriction endonuclease. The DNA inserts were subcloned into pUC19 vectors and their nucleotide sequences determined by dideoxy nucleotide sequencings.
  • Recombinant phage from the ⁇ gtll subgenomic library were propagated in E. coli Y1080 and plated with E. coli
  • the plates were overlaid with duplicate nitrocellulose filters, which were hybridized with a 32 P-labelled probe from one of the six human IFNT CDNA clones isolated above. Three clones giving positive hybridization sig ⁇ nals were further screened and purified.
  • the phage DNAs were isolated, digested with -EcoRI, subcloned into pUC19 vector and sequenced. The three clones yielded sequence information for over 800 bases relative to cap site (clones were sequenced in both orientations) .
  • the nucle ⁇ ic acid sequence information is presented as SEQ ID NO:11 and the predicted protein coding sequence is presented as SEQ ID NO:12. Comparison of the predicted mature protein sequence (SEQ ID NO:12) of this gene to the predicted protein sequence of OVIFNT is shown in Figure 3.
  • HUIFNT mRNA The presence of HUIFNT mRNA in human term placenta and amniocytes (26 weeks, 2 million cells) was analyzed by using reverse transcriptase-PCR (RT-PCR) method (Clon ⁇ tech Laboratories, Palo Alto CA) .
  • RT-PCR reverse transcriptase-PCR
  • tcRNA Total cellular RNA isolated from human placenta, amniocytes and ovine conceptuses were reverse transcribed using the primer SEQ ID NO:14.
  • the primer SEQ ID NO:13 was then added to the reaction and polymer ⁇ ase chain reaction carried out for 40 cycles.
  • the PCR products were size fractionated on agarose gels and transferred to filters.
  • the DNA on the filters was hybridized with.
  • the results of these analyses demonstrate the presence of human IFNT mRNA in the feto-placental annex. •
  • the amino- cytes also expressed the messages corresponding to OVIFNT primers and human probe.
  • HUIFNT was applied to the tcRNA isolated from human adult lymphocytes. A densitometric analysis revealed that IFNT mRNA exists in lymphocytes.
  • OVIFNT CDNA is base #81-665; Figure 7
  • pBS New England Biolabs
  • Several pBS clones were isolated, subcloned, and their nucleotides sequenced. From this clone a 3' fragment (bases #425-736) was excised using the restriction endon ⁇ ucleases NlalV and -EcoRI and subcloned into the tran ⁇ scription vector pBS. This vector was designated pBS/OVIFNT.
  • an antisense cRNA probe was synthesized by in vitro tran ⁇ scription (Sambrook, et al., 1989) using T 7 RNA polymerase (Stratagene) .
  • a trace amount of 3 H-CTP was used in the transcription reaction.
  • dUTP labeled with digoxigenin was incorporated into the cRNA and yield was estimated through TCA precipitation and scin ⁇ tillation counting.
  • In situ hybridization was performed using the anti- sense RNA probe, as described by Lawrence, et al . (1985) with the following modifications.
  • Deparaffinized and hydrated sections were prehybridized for 10 minutes at room temperature in phosphate buffered saline (PBS) containing 5 mM MgCl 2 .
  • Nucleic acids in the sections were denatured for 10 minutes at 65°C in 50% formamide/2 x SSC.
  • Sections were incubated overnight at 37°C with a hybridization cocktail (30 ⁇ l/slide) containing 0.3 ⁇ g/ml digoxigenin-labelled cRNA probe and then washed for 30 minutes each at 37°C in 50 formamide/1 x SSC. Final washes were performed for 30 minutes each at room temper ⁇ ature in 1 x SSC and 0.1 x SSC.
  • the sections were blocked for 30 minutes with 0.5% Triton X-100 (Sigma) and 0.5% non-fat dry milk.
  • Hybridization signal was detected using purified sheep antidioxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim) . After unbound anti ⁇ body was removed, nitroblue tetrazolium/5-bromo-4-chloro- 3-indolyl-phosphate substrate (Promega) and levamisole (Bector Laboratories, Burlingame, CA) were added for signal detection via colorimetric substrate generation. The tissues were counterstained in methyl green (Sigma) , dehydrated, and mounted.
  • tissue sections were pretreated with 100 ⁇ g/ml of pancreatic RNaseA (Sigma) for 30 min ⁇ utes at 37°C.
  • the RNase was inactivated on the slide with 400 units of RNase inhibitor (Promega) .
  • the slides were then washed twice in 250 ml of PBS/5 mM MgCl 2 .
  • tRNA (Sigma) was substituted for the digoxigenin probes.
  • First trimester placental villi composed of an outer layer of syncytiotrophoblast, an underlying layer of cytotrophoblast, and a central stromal region with vari- ous types of mesenchymal cells, displayed the highest transcript level of IFNT in the cytotrophoblast cells. Less intense but detectable levels were present in both the syncytiotrophoblast and stromal cells. A similar pattern of transcript expression was demonstrated in the placental villi of term tissue but the level of signal detection was low. First trimester extravillous tropho ⁇ blast displayed the highest amount of message and stained positive when present in the maternal blood spaces.
  • Antiviral Activity of IFNr The relative specific activity of OVIFNT, purified to homogeneity, was evaluated in antiviral assays.
  • the antiviral assays were performed essentially as described above in Example 2. Specific activities are expressed in antiviral units/mg protein obtained from antiviral assays using either Madin-Darby bovine kidney (MDBK) cells or sheep normal fibroblasts (Shnf) . All samples were as ⁇ sayed simultaneously to eliminate interassay variability.
  • MDBK Madin-Darby bovine kidney
  • Scnf sheep normal fibroblasts
  • IFNT had a higher specific activity than either rBoIFN ⁇ or rBoIFN7 (Table 3) .
  • the NIH standard prepara ⁇ tion of rHuIFN ⁇ had a similar specific activity, while a commercial preparation of rHuIFN ⁇ exhibited low specific antiviral activity. Comparable relative antiviral activ ⁇ ity was demonstrated using either bovine or ovine cells.
  • OVIFNT Highly purified OVIFNT was tested for anti-retrovi- ral and cytotoxic effects on feline peripheral blood lymphocytes exposed to the feline immunodeficiency retro- virus.
  • This lentivirus produces a chronic AIDS-like syndrome in cats and is a model for human AIDS (Pederson, et al . , 1987). Replication of the virus in peripheral blood lymphocytes is monitored by reverse transcriptase activity in culture supernatants over time. The data from these assays are presented in Table 4.
  • IFNT HIV Infected Human Peripheral Lymphocytes
  • Human peripheral blood lymphocytes which had been infected with HIV (Crowe, et al . ) , were treated with varying concentrations of OVIFNT.
  • Replica ⁇ tion of HIV in peripheral blood lymphocytes was monitored by reverse transcriptase activity in culture supernatants over time. Reverse transcriptase activity was measured essentially by the method of Hoffman, et al . The data from these assays are presented in Table 5.
  • concentrations of OVIFNT pro ⁇ resulted significant antiviral effects.
  • a concentration of only 10 ng/ml resulted in over a 50% reduction in RT activity after only six days.
  • a concentration of 500 ng/ml resulted in a 90% reduction in RT activity within 10 days.
  • IFNT IFNT-induced cellular growth
  • WISH Human amnion
  • MDBK MDBK cells
  • HMEM fetal bovine serum
  • FBS fetal bovine serum
  • Various dilu ⁇ tions of interferons were added to triplicate wells, and the plates were incubated for 8 days to allow colony formation. Colonies were visualized after staining with crystal violet, and counted.
  • Cell cycle analysis was performed with HMEM containing 0.5% "spent" media for an additional 7 days. WISH cells were used without being synchronized.
  • IFNT activity For examination of IFNT activity, cells were re-plated at 2.5 x 10 5 cells/well in HMEM with 10% FBS in 6 well plates. Various dilutions of IFNT alone or in combination with peptides were added to achieve a final volume of 1 ml. Plates were incubated at 37°C in 5% Co 2 for 12, 15, 18, 24, or 48 hours. Cells were treated with trypsin, collected by low speed centrifugation and washed. The cell pellet was blotted dry and 250 ⁇ l of nuclear staining solution (5 mg propidium iodide, 0.3 ml NP40 and 0.1 gm sodium citrate i 100 ml distilled H 2 0) was added to each tube.
  • nuclear staining solution 5 mg propidium iodide, 0.3 ml NP40 and 0.1 gm sodium citrate i 100 ml distilled H 2 0
  • the tubes were incubated at room temperature. After 10 minutes, 250 ⁇ l of RNase (500 units/ml in 1.12% sodium citrate) was added per tube and incubated an additional 20 minutes. Nuclei were filtered through 44 ⁇ m mesh, and analyzed on a FACStar (Becton Dickinson, Mountain View, CA) using, the DNA Star 2.0 software.
  • RNase 500 units/ml in 1.12% sodium citrate
  • Ovine IFNT inhibited both colony size and number.
  • Ovine IFNT was more effective than human IFN ⁇ on the human cell line; thus, it is very potent in cross-species activity. Its activity was dose-dependent, and inhibition of proliferation could be observed at concentrations as low as l unit/ml. Concentrations as high as 50,000 units/ml (units of antiviral activity/ml) stopped proliferation, while cell viability was not impaired.
  • Rat (MtBr7 .c5) or bovine kidney (MDBK) cells were seeded in phenol red-free DME-F12 medium supplemented with 3% dextran-coated charcoal stripped Controlled Process Serum Replacement 2 (CPSR 2, Sigma) and 5% dex ⁇ tran-coated charcoal stripped fetal, bovine serum (FBS) . After attaching for approximately 15-18 hours, the cells were washed once with serum-free DME-F12 medium.
  • CPSR 2 Controlled Process Serum Replacement 2
  • FBS bovine serum
  • the medium was replaced with phenol red-free DME-F12 medium supplemented with 3% stripped CPSR2, 1% stripped FBS ("3/1" medium) or 3/1 medium containing OVIFNT at various units of antiviral activity as determined in the vesicu ⁇ lar stomatitis virus challenge assay for interferons (Example 2) .
  • Media containing a similar dilution of buffer undiluted buffer - 10 mM Tris, 330 mM NaCl, [TS]), in which the OVIFNT was dissolved was used for controls.
  • the antiproliferative activity of OVIFNT on human tumor cell lines was evaluated by measuring the rate of 3 H-thymidine incorporation into cells which have been treated with OVIFNT.
  • Adherent tumor lines were plated at 2.5 X 10 5 cells/well in 1 ml in 6-well plates. They received interferon treatments as just described, but were tryp- sinized prior to counting. Significant differences between treatments were assessed by an analysis of variance followed by Scheffe's F-test. Cell cycle analysis was performed by flow cyto ⁇ metry using propidium iodide.
  • Human MCF7 breast adenocarcinoma cells were seeded from logarithmically growing cultures in phenol red-free DME-F12 medium supplemented with 3% dextran-coated char ⁇ coal stripped CPSR and 5% dextran-coated FBS. After attaching for approximately 15-18 hours, the cells were washed once with serum-free DME-F12 medium. The medium was replaced with phenol red-free DME-F12 medium supple ⁇ mented with 3% stripped CPSR2, 1% stripped FBS ("3/1" medium) or 3/1 medium containing OVIFNT at the indicated number of units of antiviral activity as determined in the vesicular stomatitis virus challenge assay for inter ⁇ ferons.
  • TCA trichloroacetic acid
  • OVIFNT was able to substantially reduce the rate of 3 H- thymidine incorporation in the human carcinoma cell line. This demonstrates the efficacy of OVIFNT in inhibiting tumor cell proliferation, in particular, mammary tumor cell proliferation.
  • Figure 4 shows that both OVIFNT and IFN ⁇ were able to drastically reduce growth of HL-60 cells.
  • the growth reduction for each compound exceeded 60% for each concentration tested.
  • OVIFNT caused an approximately 80% reduction in growth while IFN ⁇ caused a 100% reduction in growth.
  • the data presented in Figure 4 reveal, that a substantial factor in the ability of IFN ⁇ to reduce growth was its toxic effect on the cells.
  • the toxicity of IFN ⁇ resulted in less than 25% of the cells remaining viable.
  • nearly 100% of the cells remained viable when OVIFNT was applied at 10,000 units/ml.
  • Figure 5 presents data demonstrating that rHuIFN ⁇ is cytotoxic.
  • results of one of three repli ⁇ cate experiments are presented as mean % viability ⁇ SD.
  • the T cell lymphoma cell line H9 was slightly less sensitive to the antiproliferative effects of the IFNs than the tumor cell lines described above. Results of one of three replicate experiments are presented in Figure 10 as mean % growth reduction ⁇ SD. While rHuIFN ⁇ was not toxic to the H9 cells, it failed to inhibit cell division significantly at any of the concentrations examined. In contrast, OVIFNT was observed to reduce H9 growth by approximately 60% ( Figure 10) . Thus, only OVIFNT is an effective growth inhibitor of this T cell lymphoma.
  • B16-F10 is a syngeneic mouse transplantable tumor selected because of its high incidence of pulmonary metastases (Poste, et al . , 1981). Interferon treatment was initiated 3 days after the introduction of the tumor cells. Each mouse received 100 ⁇ l of either PBS alone, PBS containing l X 10 s units of OVIFNT, or PBS containing 1 X 10 s units of recombinant murine IFN ⁇ (MuIFN ⁇ ) , i.v. per day for 3 consecutive days.
  • mice were sacrificed at 21 days and the lungs were preserved in 10% buffered formalin.
  • the frequency of pulmonary metastases were compared between control mice (PBS) , OVIFNT-treated mice, and MuIFN ⁇ -treated mice.
  • the results of these in vivo administrations demonstrated that OVIFNT dramatically reduced B16-F10 pulmonary tu ⁇ mors. These results support the use of IFNT as an effi ⁇ cacious antineoplastic agent in vivo.
  • Overlapping synthetic peptides were synthesized corresponding to the entire IFNT sequence ( Figure 6) . Average hydropathicity values were calculated by taking the sum of the hydropathy values for each amino acid divided by the total number of amino acids in each se ⁇ quence. Hydropathy values were taken from Kyte, et al . (1982) . These peptides were of approximately the same molec ⁇ ular weight but differed slightly in overall hydrophili- city. Despite this difference, all peptides were anti ⁇ genic as demonstrated by the production of rabbit anti ⁇ sera with titers greater than 1:3,000 as assessed by ELISA (Harlow, et al . ) .
  • the peptides were used to inhibit the antiviral activity (Example 2) of OVIFNT and rBoIFN ⁇ .
  • the results of this analysis are presented in Figure 12: 1 mM N- and C-terminal peptides both effectively blocked the antivi- ral activity of OVIFNT using MDBK cells.
  • the peptide OVIFNT (119-150) showed minimal inhibitory activity.
  • the OVIFNT (34-64) and (90-122) peptides had no apparent inhibitory activity.
  • Figure 13 are expressed as the percent of control antivi ⁇ ral activity: that is, in the absence of any competing peptide.
  • Data presented are the means of 6 replicate experiments. The data demonstrate that inhibition by OVIFNT (1-37), (62-92), (119-150), and (139-172) were significantly different than OVIFNT (34-64) and (90-122) at 10- 3 M and 3 x 10' 3 M. OVIFNT (139-172) was signifi ⁇ cantly different than all other peptides at 10' 3 M. Sig ⁇ nificance was assessed by analysis of variance followed by Scheffe's F test at p ⁇ 0.05. Thus, OVIFNT (1-37) (62-92), (119-150), and (139-172), in particular (139- 172), may represent receptor binding regions for IFNr.
  • OVIFNT ⁇ bovine IFN ⁇ antiviral activity
  • Monolayers of Madin Darby bovine kidney cells were incu ⁇ bated with 40 units/ml bovine IFN ⁇ in the presence or absence of various concentrations of OVIFNT peptides.
  • the results are presented in Figure 14 and are expressed as the percent of control antiviral activity in the absence of OVIFNT peptides.
  • the data presented are the means of 4 replicate experiments.
  • the results indicate that inhibition by OVIFNT (62-92), (119-150), and (139- 172) were significantly different from OVIFNT (1-37) , (34-64) and (90-122) at 10' 3 M.
  • OVIFNT (139-172) was significantly different than OVIFNT (1-37), (34-64) and (90-122) at 3 x 10" 3 M. Significance was assessed by analysis of variance followed by Scheffe's F test at p ⁇ 0.05. Thus, OVIFNT (62-92), (119-150), and (139-172), in particular (139-172) , may represent common receptor binding regions for IFNT and bovine IFN ⁇ .
  • OVIFNT may represent a common receptor binding region for IFNT and various IFN ⁇ (s).
  • the OVIFNT peptides described above appear to have no effect on the antiviral activity of IFN7.
  • Peptide inhibition of bovine IFN7 antiviral activity was evaluat ⁇ ed as follows. Monolayers of Madin Darby bovine kidney cells were incubated with 40 units/ml bovine IFN gamma in the presence or absence of various concentrations of OVIFNT peptides. Results are expressed as the percent of control antiviral activity in the absence of OVIFNT peptides. The data are presented in Figure 16 and are the means of 3 replicate experiments. There were no significant differences among peptides as assessed by analysis of variance followed by Scheffe's F test at p ⁇ 0.05.
  • RT Reverse transcriptase activity
  • FIV- infected FET-1 cells (1 X 10 6 /ml)
  • HIV-infected HPBL l X 10 6 /ml
  • Control cultures received no OVIFNT.
  • OVIFNT was used at 100 ng/ml
  • peptides were used at 200 ⁇ M.
  • Data from a representative experiment are expressed as cpm/ml culture supernatant and are presented for FIV infected cells, Figure 11A, and HIV infected cells, Figure 11B.
  • Antipep ⁇ tide antisera inhibition of OVIFNT antiviral activity was evaluated as follows. Monolayers of MDBK cells were incubated with 20 units/ml of OVIFNT in the presence a 1:30 dilution of either preimmune sera or antisera to each of the OVIFNT peptides described above. In Figure 17 the data from duplicate experiments are presented as the mean percent inhibition of OVIFNT antiviral activity produced by antipeptide antisera relative to the appro ⁇ priate preimmune sera ⁇ standard error. Significant differences were assessed by analysis of variance fol ⁇ lowed by Scheffe's F test at p ⁇ 0.05.
  • the IFNT binding assay was carried out as follows.
  • IFNT Five ⁇ g of IFNT was iodinated for 2 minutes with 500 ⁇ Ci of Na 125 I (15 mCi/ ⁇ g; Amersham Corporation, Arlington Heights, IL) in 25 ⁇ l of 0.5 M potassium phosphate buff ⁇ er, pH 7.4, and 10 ⁇ l of chloramine-T (5 mg/ml) (Griggs, et al . , 1992). The specific activity of the iodinated protein was 137 ⁇ Ci/ ⁇ g. For binding assays, monolayers of MDBK cells were fixed with paraformaldehyde and blocked with 5% nonfat dry milk.
  • Antisera to IFNT (119-150) , which inhibited binding of OVIFNT to receptor, also reversed the OVIFNT antipro- liferative effect.
  • IFNT 139-172
  • Several other peptides notably IFNT (139-172) , reversed the OVIFNT antiproliferative effect, but to a lesser extent.
  • EXAMPLE 18 Further Analysis of the Cellular and Anti-Viral Effects of IFNT A. HIV Anti-Viral Effects.
  • the antiviral effects of IFNT against HIV were evaluated by treating human PBMC cells with various amounts of either recombinant ovine IFNT (r-OvIFN ⁇ ) or recombinant human IFN ⁇ 2 at the time of infection with HIV. Drug was present throughout the experiment. At day 7 and day 14, p24 production was determined (by ELISA (Wang, et al . , 1988, 1989) and compared to a zero drug control. The results of this analysis are presented in Table 12.
  • IFNT are effective in reducing the replication of HIV in human lymphocytes.
  • B. In vitro Cytotoxicity Test in PBMCs Human PBMCs were seeded at 5 x 10 5 cells/ml. Cells were stimulated at day 0 with 3 ⁇ g/ml PHA. Cells were treated with recombinant human IFN ⁇ 2A (at concentrations of 10, 100, 1,000 and 10,000 units/ml) and IFNT (at concentrations of 2.6, 26, 260, 2,600, 26,000, 260,000, and 2,600,000 units/ml) in 200 ⁇ l/wells (4 replicates of each concentration using 96 well flat bottom plates) . Control cultures were given no interferons. After 4 days of incubation, cells were pulsed for 9 hours using 3 H- thymidine at 1 uCi/well. The cells were harvested and the incorporation of labeled thymidine into DNA was determined ( Figure 8) .
  • HepG2-T14 is a human cell that was derived from liver cells transfected with Hepatitis B
  • HBV Virus The cell line semi-stably produces HBV virus: over time the cell line's production of HBV intracellular DNA and secreted virus decreases.
  • the cells are pre-treated with deAZA-C (5-azacytidine; Miyoshi, et al . ) to induce production of the virus. Treatment was for 2-3 days and the amount of induction was about a factor of two.
  • the cells were then treated with either the IFN ⁇ and IFNT at levels of 0, 5,000, 10,000, 20,000 and 40,000 units per ml. All levels of either IFN ⁇ or IFNT reduced DNA pro ⁇ duction by about a factor of 2 compared to the no drug control.
  • the hepatocyte cell line HepG2-T14 (described above) was examined for the effects of IFN ⁇ and IFNT on hepato ⁇ specific mRNA production.
  • Cells were incubated in con ⁇ centrations of IFN ⁇ or IFNT at 0, 5,000, 10,000, 20,000, and 40,000 units per ml.
  • the messenger RNAs for the hepatocyte specific proteins Apo E and Apo Al were de ⁇ tected by hybridization analysis (Sambrook, et al . ; Maniatis, et al . ) using probes specific for these two mRNA's (Shoulders, et al . , and Wallis, et al . ) .
  • the IFNT coding sequence (e .g. , Figure 7) is cloned into the polylinker site of lambda gtll.
  • the IFNT coding sequence is placed in-frame with the amino terminal ⁇ - galactosidase coding sequences in lambda gtll.
  • Lysogens infected with gtll/IFN ⁇ are used to inoculate 500 ml of NZYDT broth.
  • the culture is incubated at 32°C with aeration to an O.D. of about 0.2 to 0.4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gtll peptide synthesis, and incubated further at 37°C for 1 hour.
  • the cells are pelleted by centrifuga ⁇ tion, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use.
  • the resuspended cells are frozen in liquid nitrogen then thawed, resulting in substantially complete cell lysis.
  • Non-solubilized material is removed by centrifugation.
  • the clarified lysate material is loaded on the Sepharose column, the ends of the column closed, and the column placed on a rotary shaker for 2 hrs. at room temperature and 16 hours at 4°C. After the column set ⁇ tles, it is washed with 10 ml of TX buffer.
  • the fused protein i ⁇ eluted with 0.1 M carbonate/bicarbonate buff ⁇ er, pHlO. Typically, 14 ml of the elution buffer is passed through the column, and the fusion protein is eluted in the first 4-6 ml of eluate.
  • the eluate containing the fusion protein is concen ⁇ trated in "CENTRICON-30" cartridges (Amicon, Danvers, Mass.).
  • the final protein concentrate is resuspended in, for example, 400 ⁇ l PBS buffer. Protein purity is ana ⁇ lyzed by SDS-PAGE.
  • the purified fused pro ⁇ tein is injected subcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum is typically collected at 6 and 8 weeks.
  • the IFNT coding sequence (e . g. , Figure 7) is cloned into the pGEX vector (Boyer, et al . ; Frangioni, et al . ;
  • the pGEX vector (Smith, et al . ) was modified by in ⁇ ertion of a thrombin cleavage sequence in-frame with the glutathi- one-S-transferase protein (GST — sj26 coding sequence) .
  • This vector is designated pGEXthr.
  • the IFNT coding sequence is placed in-frame with the ⁇ j26-thrombin coding sequences (Guan, et a . ; Hakes, et al . ) .
  • the IFNT coding sequence insert can be generated by the polymerase chain reaction using PCR primers specific for the insert.
  • the IFNT fragment is ligated to the linearized pGEXthr vector.
  • the ligation mixture is transformed into E. coli and ampicillin resistant colonies are selected. Plasmids are isolated from the ampicillin resistant colonies and analyzed by restriction enzyme digestion to identify clones containing the IFNT insert (vector desig ⁇ nated pGEXthr-IFNT) .
  • E. coli strain XL-I Blue is transformed with pGEXthr-IFNT and is grown at 37°C overnight. DNA i ⁇ prepared from randomly-picked colonies.
  • the presence of the insert coding sequence is typically confirmed by (i) restriction digest mapping, (ii) hybridization screening u ⁇ ing labelled IFNT probes (i.e.. Southern analysis), or (iii) direct DNA sequence analysis.
  • a pGEXthr-IFNT clone is grown overnight. The over- night culture is diluted 1:10 with LB medium containing ampicillin and grown for one hour at 37°C. Alternative ⁇ ly, the overnight culture is diluted 1:100 and gro-- to OD of 0.5-1.0 before addition of IPTG (isopropylth - ⁇ - galacto ⁇ ide) . IPTG (GIBCO-BRL, Gaithersburg MD) is added to a final concentration of 0.2-0.5 mM for the induction of protein expre ⁇ ion and the incubation i ⁇ typically continued for 2-5 hour ⁇ , preferably 3.5 hours.
  • Bacterial cells are harvested by centrifugation and resuspended in 1/100 culture volume of MTPBS (150 mM NaCl, 16 mM Na 2 HP0 4 , 4 mM NaH 2 P0 4 ) . Cells are lysed by lysozyme, sonication or French press, and lysate ⁇ cleared of cellular debri ⁇ by centrifugation.
  • MTPBS 150 mM NaCl, 16 mM Na 2 HP0 4 , 4 mM NaH 2 P0 4
  • the fusion proteins are partially purified over a glutathione agarose affinity column as de ⁇ cribed in detail by Smith, et a .
  • 100 ml culture ⁇ are grown overnight.
  • the culture ⁇ are dilut ⁇ ed to 1 liter, and the cell ⁇ grown another hour at 37°C.
  • Expression of the fusion proteins is induced using IPTG.
  • the induced cultures are grown at 37°C for 3.5 hours.
  • Cells are harvested and a ⁇ onicator u ⁇ ed to lyse the cells.
  • Cellular debris is pelleted and the clear lysate loaded onto a glutathione "SEPHAROSE" column. The column is washed with several column volumes.
  • the fusion pro ⁇ tein is eluted from the affinity column with reduced glutathione and dialyzed.
  • the IFNT can be liberated from the hybrid protein by treatment with thrombin.
  • the ⁇ j26 and IFNT fragment ⁇ of the hybrid protein can then be ⁇ eparated by ⁇ ize fractionation over column ⁇ or on gel ⁇ .
  • the IFNT portion of the hybrid pro- tein is released from the column by treatment with throm ⁇ bin (Guan, et al . ; Hakes, et al . ) .
  • the purified Sj26/IFN ⁇ fused protein i ⁇ injected subcutaneously in Freund's adjuvant in a rabbit. Approx ⁇ imately 1 mg of fused protein is injected at day ⁇ 0 and 21, and rabbit ⁇ erum is typically collected at 6 and 8 weeks.
  • a second rabbit is similarly immunized with purified Sj26 protein obtained from control bacterial lysate. Minilysates from the following bacterial cultures are prepared: (1) KM392 cells infected with pGEXthr and pGEXthr containing the IFNT insert; and (2) cells infect ⁇ ed with lambda gtll containing the IFNT insert.
  • the minilysates and a commercial source 8-galactosidase are fractionated by SDS-PAGE, and the bands transferred to nitrocellulose filters for Western blotting (Sambrook, et al . ; Ausubel, et al . ) .
  • serum from control (Sj26) rabbits is immunoreactive with each of the Sj.26 and Sj26 fused protein antigens.
  • Serum from the animal immunized with Sj26/IFN ⁇ fused protein is reactive with all Sj-26 and beta-gal fusion proteins containing IFNT coding ⁇ equence ⁇ , indicating the presence of specific immunoreaction with the IFNT antigen. None of the sera are expected to be immunoreactive with beta-galactosi- dase.
  • Anti-IFNT antibody present in the sera from the ani ⁇ mal immunized with the Sj26/IFN ⁇ is purified by affinity chromatography (u ⁇ ing immobilized recombinantly produced IFNT a ⁇ ligand, e ⁇ sentially as described above in Example 12 for the anti-beta-galactosidase antibody) .
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  • TTGTTGGAAC CATGTAGAAC CGGTTTGCAC CAACAATTGG ACAACTTGGA TGCATGTTTG 30 GGTCAAGTTA TGGGTGAAGA AGACTCTGCT CTCGGGAGAA CCGGTCCAAC GCTAGCTTTG
  • Gin Glu Ala Gin Ala lie Ser Val Leu His Glu Met Leu Gin Gin Ser 50 55 60
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)

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Abstract

The present invention describes the production of interferon-τ proteins and polypeptides derived therefrom. The antiviral and anticellular proliferation properties of these proteins and polypeptides are disclosed. One advantage of the proteins of the present invention is that they do not have cytotoxic side-effects when used to treat cells. Structure/function relationships for the interferon-τ protein are also described.

Description

INTERFERON TAϋ COMPOSITIONS AND METHODS OF USE
Field of the Invention
The present invention relates to interferon- τ compo- sitions and methods of use.
References
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Background of the Invention
Conceptus membranes, or trophectoderm, of various mammals produce biochemical signals that allow for the establishment and maintenance of pregnancy (Bazer, et al . , 1983). One such protein, ovine trophoblast protein- one (oTP-1) , was identified as a low molecular weight protein secreted by sheep conceptuses between days 10 and 21 of pregnancy (Wilson, et al., 1979; Bazer, et al . , 1986) . The protein oTP-1 was shown to inhibit uterine secretion of prostaglandin F2-alpha, which causes the corpus luteum on the ovary to undergo physiological and endocrinological demise in nonpregnant sheep (Bazer, et al., 1986). Accordingly, oTP-1 has antiluteolytic bio- logical activity. The primary role of oTP-1 was assumed to be associated with the establishment of pregnancy. oTP-1 was subsequently found to (i) exhibit limited homology (50-70%) with interferon alphas (IFNα) of vari- ous species (Imakawa, et al . , 1987), and (ii) bind to a Type I interferon receptor (Stewart, et al . , 1987). Despite some similarities with IFNα, oTP-1 has several features that distinguish it from IFNσ including the following: oTP-l's role in reproductive biochemistry (other interferons are not known to have any role in the biochemical regulation of reproductive cycles), oTP-l's cellular source — trophoblast cells (IFNσ is derived from lymphocytes cells) , oTP-l's size — 172 amino acids (IFNσ is typically about 166 amino acids) , and oTP-1 is weakly inducible by viruses (IFNσ is highly inducible by viruses) . The International Interferon Society recog¬ nizes oTP-1 as belonging to an entirely new class of interferons which have been named interferon-tau (IFNτ) . The Greek letter T stands for trophoblast. The interferons have been classified into two dis¬ tinct groups: type I interferons, including IFNα, IFN/3, and IFNω (also known as IFNαll) ; and type II interferons, represented by IFN7 (reviewed by DeMaeyer, et al . ) . In humans, it is estimated that there are at least 17 IFNα non-allelic genes, at least about 2 or 3 IFN/3 non-allelic genes, and a single IFN7 gene.
IFNα's has been shown to inhibit various types of cellular proliferation. IFNα's are especially useful against hematologic malignancies such as hairy-cell leukemia (Quesada, et al . , 1984). Further, these pro¬ teins have also shown activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal- cell carcinoma, urinary bladder tumors and ovarian can- cers (Bonnem, et al . , 1984; Oldham, 1985). The role of interferons and interferon receptors in the pathogenesis of certain autoimmune and inflammatory diseases has also been investigated (Benoit, et al . , 1993). IFNα's are also useful against various types of viral infections (Finter, et al . , 1991). Alpha interfe¬ rons have shown activity against human papillomavirus infection, Hepatitis B, and Hepatitis C infections (Finter, et al., 1991; Kashima, et al . , 1988; Dusheiko, et al., 1986; Davis, et al . , 1989). Significantly, however, the usefulness of IFNα's has been limited by their toxicity: use of interferons in the treatment of cancer and viral disease has resulted in serious side effects, such as fever, chills, anorexia, weight loss, and fatigue (Pontzer, et al., 1991; Oldham, 1985). These side effects often require (i) the interferon dosage to be reduced to levels that limit the effectiveness of treatment, or (ii) the removal of the patient from treat¬ ment. Such toxicity has reduced the usefulness of these potent antiviral and antiproliferative proteins in the treatment of debilitating human and animal diseases.
Summary of the Invention
In one aspect the present invention includes a method of inhibiting tumor cell growth. In the method, cells are contacted with interferon-T (IFNτ) at a concen¬ tration effective to inhibit growth of the tumor cells. IFNT can be obtained from a number of sources including cows, sheep, and humans. Two embodiments include the IFNT presented as either SEQ ID NO:2 or SEQ ID NO:4. A number of tumor cells can be targeted for growth inhibi¬ tion by IFNr, including but not limited to the following: human carcinoma cells and steroid-affected tumor cells (e .g. , mammary tumor cells). The present invention also includes a method of inhibiting viral replication in a cell. In this method cells infected with a virus are contacted with interfer¬ on-T at a concentration effective to inhibit viral repli- cation within the cells. The IFNT molecules described above are also useful in this method of the present invention. The replication of a number of viruses can be inhibited in cells, these viruses include RNA (e.g., feline leukemia virus, human immunodeficiency virus, or Hepatitis C Virus) and DNA (e.g., Hepatitis B Virus) viruses.
The human IFNT molecules of the present invention can also be used in a method of enhancing fertility in a female mammal. In this method an amount of human IFNT effective to enhance fertility of a female mammal is administered, typically in a pharmaceutically acceptable carrier. Exemplary of such human IFNT molecules are the protein sequences presented as SEQ ID NO:4 and SEQ ID NO:12. The present invention also includes an isolated nucleic acid which encodes a human interferon-τ. Exem¬ plary of such nucleic acid molecules are SEQ ID NO:3 and SEQ ID NO:11. Further, the invention includes expression vectors for the expression of human IFNT. Typically the expression vector includes (a) a nucleic acid containing an open reading frame that encodes human interferon-τ; and (b) regulatory sequences effective to express said open reading frame in a host cell. The regulatory se¬ quence may include sequences useful for targeting or secretion of the IFNT polypeptide: such sequences may be endogenous (such as the normally occurring IFNT leader sequences, see SEQ ID NO:11) or heterologous (such as a secretory signal recognized in yeast or bacterial expres¬ sion systems) . In the expression vector, regulatory sequences may also include, 5' to said nucleic acid sequence, a promoter region and an ATG start codon in- frame with the interferon-τ coding sequence, and 3' to said coding sequence, a translation termination signal followed by a transcription termination signal. The nucleic acid in the expression vector may be selected from, for example, SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:ll.
In another embodiment, the present invention in¬ cludes a recombinantly produced human interferon-τ pro- tein. One exemplary sequence for such a protein is given as SEQ ID NO:4. Human IFNT may also contain a carboxy terminal extension (such as the sequence presented as SEQ ID NO:12) .
The invention includes a method of recombinantly producing interferon-τ. In the method, a recombinant expression system containing an open reading frame (ORF) having a polynucleotide sequence which encodes a human interferon-τ polypeptide, where the vector is designed to express the ORF in the host, is introduced into suitable host cells. The host is then cultured under conditions resulting in the expression of the ORF sequence. The human IFNT sequences discussed above are examples of suitable human IFNT polypeptides. Examples of polynucle¬ otide coding sequences are SEQ ID NO:3 and SEQ ID NO:11. Numerous vectors and their corresponding hosts are useful in the practice of this method of the invention, includ¬ ing, lambda gtll phage vector and E. coli cells. Other host cells include, yeast and insect cell expression systems. The invention further includes expression systems useful for the expression of interferon-τ polypeptides. Typically these systems include a host capable of sup¬ porting expression of an open reading frame in a selected expression vector, and the selected expression vector containing an open reading frame (ORF) having a polynu- cleotide sequence which encodes a human interferon-T polypeptide. Exemplary of sequences that can be utilized in such expression systems are SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.
The invention also includes isolated interferon-τ polypeptides. These polypeptides are derived from the interferon-T amino acid coding sequence and are between about 15 and 172 amino acids in length. Such polypep- tides can be selected, for example, from the sequences presented as SEQ ID NO:2 and SEQ ID NO:4. Exemplary IFNT-derived polypeptides include, but are not limited to, the following: SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:20. In another embodiment, the invention includes a method of blocking the binding of alpha-interferon to a cell having an alpha-interferon receptor. In this method the cell is contacted with an interferon-T polypeptide at a concentration effective to allow the binding of inter- feron-T to each alpha-interferon receptor. The cells, having the IFNT polypeptide bound to the receptor, are then exposed to alpha-interferon (IFNα) . The IFNT poly¬ peptides and IFNτ-derived polypeptides described above are examples of IFNT polypeptides useful in this method. Further, the invention includes a method of blocking the binding of interferon-T to a cell having a interfer¬ on-T receptor. In this method, the cell is contacted with an interferon-T-derived polypeptide (e .g. , SEQ ID N0:5, SEQ ID NO:7, SEQ ID NO:10, SEQ ID N0:15, SEQ ID NO:17 or SEQ ID NO:20), where the polypeptide is at a concentration effective to allow the binding of the polypeptide to each interferon- -receptor. The cells are then exposed to interferon-T.
The invention also includes purified antibodies that are immunoreactive with human interferon-T. The antibod- ies may be polyclonal or monoclonal. Exemplary IFNr polypeptide antigens include, but are not limited, to the following: SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:20. The present invention also includes the following: interferon-τ-derived polypeptides that have anti-tumor (i .e . , anti-proliferative) activity; interferon-T-derived polypeptides that have anti-viral activity; and hybrid α- interferon molecules in which the toxicity portion of native IFNα has been replaced by analogous sequences from IFNT.
These and other objects and features of the inven¬ tion will be more fully appreciated when the following detailed description of the invention is read in conjunc- tion with the accompanying drawings.
Brief Description of the Figures
Figure 1 presents the nucleic acid coding sequence of a synthetic gene of OVIFNT designed to include 19 unique restriction enzyme sites spaced evenly throughout the coding sequence.
Figure 2 shows the cloning strategy used for making a synthetic gene encoding OVIFNT. Figure 3 shows a comparison of the predicted protein sequences of a human interferon-T gene and an ovine interferon-T gene. Divergent amino acids are indicated by presentation of the alternative amino acid on the line below the nucleic acid sequences. Figure 4 presents data demonstrating that both
OVIFNT and IFNα were able to drastically reduce growth of
HL-60 cells.
Figure 5 presents data demonstrating that rHuIFNα is cytotoxic and OVIFNT is not. In the figure, results of one of three replicate experiments are presented as mean % viability ± SD.
Figure 6 presents the sequences of polypeptides derived from the IFNT sequence. Figure 7 presents the complete nucleic acid and amino acid sequence of an OVIFNT sequence.
Figure 8 presents data supporting the lack of cyto- toxicity, relative to IFNα, when IFNT is used to treat peripheral blood mononuclear cells. Figure 9 shows the results of treatment of a human cutaneous T cell lymphoma line, HUT 78, with IFNT.
Figure 10 shows the results of treatment of a human T cell lymphoma line, H9, with IFNT.
Figure 11A presents data for the peptide inhibition, relative to FIV (feline immunodeficiency virus) replica¬ tion, of polypeptides derived from OVIFNT with whole OVIFNT. Figure 11B presents data for the peptide inhibi¬ tion, relative to HIV (human immunodeficiency virus) replication, of polypeptides derived from OVIFNT with whole OVIFNT.
Figure 12 presents data demonstrating the inhibition of the antiviral activity of IFNT by IFN -derived pep¬ tides.
Figure 13 presents data demonstrating the inhibition by IFNT-derived peptides of OVIFNT antiviral activity.
Figure 14 presents data demonstrating the inhibition by IFNT-derived peptides of bovine IFNα antiviral activi¬ ty.
Figure 15 presents data demonstrating the inhibition by IFNT-derived peptides of human IFNα antiviral activi¬ ty.
Figure 16 presents data evaluating the lack of inhibition by IFNT-derived peptides of bovine IFN7 anti¬ viral activity. Figure 17 presents data demonstrating the anti-IFNτ- derived peptide antisera inhibition of the antiviral activity of IFNT.
Figure 18 presents data demonstrating the anti-IFNτ- derived peptide antisera inhibition of the binding of radiolabeled IFNT to cells.
Brief Description of the Sequences
SEQ ID N0:1 is the nucleotide sequence of a synthet- ic gene encoding ovine interferon-T (OVIFNT) . Also shown is the encoded amino acid sequence.
SEQ ID NO:2 is an amino acid sequence of a mature OVIFNT protein.
SEQ ID NO:3 is a synthetic nucleotide sequence encoding a mature human interferon-T (HUIFNT) protein. SEQ ID NO:4 is an amino acid sequence for a mature HUIFNT protein.
SEQ ID NO:5 is the amino acid sequence of fragment 1-37 Of SEQ ID NO:2. SEQ ID NO:6 is the amino acid sequence of fragment 34-64 Of SEQ ID NO:2.
SEQ ID NO:7 is the amino acid sequence of fragment 62-92 of SEQ ID NO:2.
SEQ ID NO:8 is the amino acid sequence of fragment 90-122 Of SEQ ID NO:2.
SEQ ID NO:9 is the amino acid sequence of fragment 119-150 of SEQ ID NO:2.
SEQ ID NO:10 is the amino acid sequence of fragment 139-172 of SEQ ID NO:2. SEQ ID NO:11 is the nucleotide sequence of a natural HUIFNT gene with a leader sequence.
SEQ ID NO:12 is the predicted amino acid coding sequence of the SEQ ID NO:11.
SEQ ID NO:13 is a 25-mer synthetic oligonucleotide according to the subject invention. SEQ ID NO:14 is a 25-mer synthetic oligonucleotide according the subject invention.
SEQ ID NO:15 is the amino acid sequence of fragment 1-37 of SEQ ID NO:4. SEQ ID NO:16 is the amino acid sequence of fragment 34-64 of SEQ ID NO:4.
SEQ ID NO:17 is the amino acid sequence of fragment 62-92 of SEQ ID NO:4.
SEQ ID NO:18 is the amino acid sequence of fragment 90-122 of SEQ ID NO:4.
SEQ ID NO:19 is the amino acid sequence of fragment 119-150 of SEQ ID NO:4.
SEQ ID NO:20 is the amino acid sequence of fragment 139-172 of SEQ ID NO:4.
Detailed Description of the Invention I. Definitions.
Interferon-T refers to any one of a family of inter¬ feron proteins having the following characteristics: (i) anti-luteolytic properties; (ii) anti-viral properties; (iii) anticellular proliferation properties; (iv) 45-68% amino acid homology with α-Interferons and greater than 70% amino acid homology to the sequence presented as SEQ ID NO:2. Interferon-T can be isolated from a number of mammalian sources as described below.
An interferon-T polypeptide is a polypeptide having between about 15 and 172 amino acids derived from an interferon-T amino acid coding sequence, where said 15 to 172 amino acids are contiguous in native interferon- . Such 15-172 amino acid regions can also be assembled into polypeptides where two or more such interferon-T regions are joined that are normally discontinuous in the native protein. II. Isolation and Characterization of Interferon- . A. Ovine and Bovine Interferon-T. 1. Interferon-T Coding Sequences. Ovine interferon-τ (OVIFNT) is a major conceptus secretory protein produced by the embryonic trophectoderm during the critical period of maternal recognition in sheep. One isolate of mature OVIFNT is 172 amino acids in length (SEQ ID NO:2). The cDNA coding sequence con¬ tains an additional 23 amino acids at the amino-terminal end of the mature protein (Imakawa, et al . , 1987). The coding sequence of this OVIFNT isolate is presented as Figure 7.
For the isolation of OVIFNT protein, conceptuses were collected from pregnant sheep and cultured in vitro in a modified Minimum Essential Medium as described previously (Godkin, et al . , 1982). Conceptuses were collected on various days of pregnancy with the first day of mating being described as Day 0. IFNT was purified from conceptus culture medium essentially as described by Vallet, et al . , (1987) and Godkin, et al. (1982).
The homogeneity of IFNT was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE; Maniatis, et al . ; Ausubel, et al . ) . Determination of protein concentration in purified IFNT samples was performed using the bicinchoninic (BCA) assay (Pierce Chemical Co., Rockford, IL; Smith, et al . , 1985).
A homologous protein to OVIFNT was isolated from cows (bIFNτ; Helmer, et al., 1987; Imakawa, et al . , 1989) . OVIFNT and BOIFNT (i) have similar functions in maternal recognition of pregnancy, and (ii) share a high degree of amino acid and nucleotide sequence homology between mature proteins. The nucleic acid sequence homology between OVIFNT and BOIFNT is 76.3% for the 5' non-coding region, 89.7% for the coding region, and 91.9% for the 3' non-coding region. The amino acid sequence homology is 80.4%.
Example 1 describes the reproductive functions of OVIFNT. OVIFNT and recombinant human α-2-Interferon (rHuIFNα2) were infused into uterine lumen of ewes at a variety of concentrations. The life span of the corpus luteum was assessed by examination of interestrous inter¬ vals, maintenance of progesterone secretion, and inhibi¬ tion of prostaglandin secretion (Davis, et al . , 1992 ) . Comparison of the data resulting from these examinations demonstrated a considerable lengthening of the inter¬ estrous interval when IFNT is administered at lOOμg/day and no meaningful effect when rHuIFNα is administered. These data support the conclusion that IFNT significantly influences the biochemical events of the estrous cycle. The antiviral properties of interferon-T at various stages of the reproductive cycle were also examined (Example 2) . Conceptus cultures were established using conceptus obtained from sheep at days 12 through 16 of the estrus cycle. Antiviral activity of supernatant from each conceptus culture was assessed. Culture superna¬ tants had increasing antiviral activity associated with advancing development of the conceptus up to Day 16 post estrus.
2. Recombinant Production of IFNT. Recombinant IFNT was produced using bacterial and yeast cells. The amino acid coding sequence for OVIFNT was used to generate a corresponding DNA coding sequence with codon usage optimized for expression in E. coli
(Example 3) . The DNA coding sequence was synthetically constructed by sequential addition of oligonucleotides. Cloned oligonucleotides were fused into a single polynu- cleotide using the restriction digestions and ligations outlined in Figure 2. The polynucleotide coding sequence had the sequence presented as SEQ ID NO:l.
For expression of recombinant IFNT, this synthetic coding sequence can be placed in a number of bacterial expression vectors: for example, lambda gtll (Promega, Madison WI) ; pGEX (Smith, et al . ) ; pNH vectors (Strata- gene, La Jolla CA) . Cloning of the IFNT synthetic poly¬ nucleotide into a modified pIN III omp-A expression vector is described in Example 3. Production of the IFNT protein was induced by the addition of IPTG. Soluble recombinant IFNT was liberated from the cells by sonica- tion or osmotic fractionation.
The protein can be further purified by standard methods, including size fractionation (column chromatog- raphy or preoperative gel electrophoresis) or affinity chromatography (using, for example, anti-IFNτ antibodies (solid support available from Pharmacia, Piscataway NJ) . Protein preparations can also be concentrated by, for example, filtration (A icon, Danvers, Mass.). The synthetic IFNT gene was also cloned into the yeast cloning vector pBS24Ub (Example 4; Sabin, et al . ; Ecker, et al . ) . Synthetic linkers were constructed to permit in-frame fusion of the IFNT coding sequences with the ubiquitin coding sequences in the vector. The re- suiting junction allowed in vivo cleavage of the ubiqui¬ tin sequences from the IFNT sequences.
The recombinant plasmid pBS24Ub-IFNτ was transformed into the yeast S. cerevisiae . Transformed yeast cells were cultured, lysed and the recombinant IFNT (r-IFNτ) protein isolated from the cell lysates.
The amount of r-IFNτ was quantified by radioimmuno- assay. Microsequencing of the purified r-IFNτ was car¬ ried out. The results demonstrated identity with native IFNT through the first 15 amino acids. The results also confirmed that the ubiquitin/IFNτ fusion protein was correctly processed in vivo.
Recombinant IFNT obtained by this method exhibited antiviral activity similar to the antiviral activity of IFNT purified from conceptus-conditioned culture medium.
Other yeast vectors can be used in the practice of the present invention including, but are not limited to, vectors with regulatable expression (Hitzeman, et al . ; Rutter, et a . ; Oeda, et al.) . The yeast transformation host is typically Saccharomyces cerevisiae, however, other yeast suitable for transformation can be used as well (e .g. , Schizόsaccharomyceε pombe) .
The DNA encoding the IFNT polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system. These systems include the above described bacterial and yeast expression systems as well as the following: baculovirus expression (Reilly, et al . ; Beames, et al . ; Clontech, Palo Alto CA) ; and expression in mammalian cells (Clontech, Palo Alto CA; Gibco-BRL,
Gaithersburg MD) . These recombinant polypeptides can be expressed as fusion proteins or as native proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium. The recombinantly produced polypeptides are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed, as described above, using antibodies generated based on the IFNT polypeptides. B. Human Interferon-T.
1. Identification and Cloning of Human Genomic Sequences Encoding an Interferon-T Protein.
DNA was screened for sequences homologous to inter- feron-T (Example 5) . Several sequences that hybridized with the OVIFNT CDNA probe were identified. Several clones containing partial sequences of human interferon-τ were then isolated (Example 6) . Two synthetic 25-mer oligonucleotides, corresponding to sequences from the OVIFNT CDNA (Imakawa, et al . , 1987) were synthesized. These primers were employed in amplification reactions using DNA derived from the following two cDNA libraries: human term placenta and human term cytotrophoblast. The resulting amplified DNA fragments were electrophoreti- cally separated and a band containing an IFNT amplifica¬ tion product was isolated. The product was subcloned and the inserted amplification product sequenced using the dideoxy termination method.
Comparison of sequences from three of the clones revealed a high degree of sequence homology between the isolates, but the sequences were not identical. This result suggests the existence of multiple variants of human interferon-T genes.
Example 7 describes the isolation of a full-length human IFNT gene. High molecular weight DNA was isolated from peripheral blood mononuclear cells (PBMCs) and size- fractionated. Fractions were tested for the presence of IFNT sequences using polymerase chain reaction: DNA molecules from fractions that tested amplification posi- tive were used to generate a subgenomic library in λgtll. This subgenomic library was plated and hybridized with an OVIFNT CDNA probe (Example 7A) . Approximately 20 clones were identified that hybridized to the probe. Plaques corresponding to the positive clones were pas- saged, DNA isolated and analyzed by amplification reac- tions using OVIFNT primers. Of these twenty plaques, six plaques generated positive PCR signals. The phage from these six clones were purified and the inserts sequenced. One of the inserts from one of these six clones was used as a hybridization probe in the following screening.
Recombinant phage from the λgtll subgenomic library were screened using the hybridization probe just de¬ scribed (Example 7B) . Three clones giving positive hybridization signals were isolated and the inserts sequenced. The resulting nucleic acid sequence informa¬ tion is presented as SEQ ID NO:11 and the predicted protein coding sequence is presented as SEQ ID NO:12. The predicted mature protein coding sequence is presented as SEQ ID NO:4. Comparison of the predicted protein sequences (Fig¬ ure 3) of the human interferon-T gene (SEQ ID NO:4) and the ovine interferon-T gene demonstrates the levels of sequence homology and divergence at the amino acid level. The human IFNT sequences presented as SEQ ID NO:12 and SEQ ID NO:11, and primers and probes derived there¬ from, can be used as specific probes to detect isolates of further human IFNT coding sequences and/or pseudo- genes. Further, there may be more than one isofor of the IFNT protein and more than one coding sequence per species. The specific nucleic acid probes used in the practice of the present invention and antibodies reactive with the IFNT polypeptides of the present invention may be useful to isolate unidentified variants of interferon- T in mammals, according to the methods of the invention disclosed herein. 2. Characterization of the Expression of Interfe- ron-τ in Human Tissues.
Human placental cDNA libraries and an ovine cDNA library were analyzed by hybridization to the OVIFNT CDNA probe (Example 8) . This DNA hybridization analysis sug¬ gested that the IFNτ-signals from human cDNA libraries were approximately 1/100 of the signal obtained using the ovine cDNA library. OVIFNT CDNAS constitute around 0.4% of the ovine cDNA library. Accordingly, the abundance of human cDNAs responding to the OVIFNT probe appears to be low, at least in the term placenta from which the cDNA libraries were generated.
The presence of HUIFNT mRNA in human term placenta and amniocytes were also analyzed. The results suggest the presence of human IFNT mRNA in the feto-placental annex. The aminocytes also expressed the messages corre¬ sponding to OVIFNT primers and human probe, suggesting that the expression of IFNT mRNA is not limited to the term placenta. In addition, a RT-PCR analysis for the presence of HUIFNT was applied to the total cellular RNA isolated from human adult lymphocytes: the results suggest that IFNT mRNA exists in lymphocytes.
The expression of interferon-T in human tissue was also examined using in situ hybridization (Example 9) .
Sections from four healthy, different term and first tri¬ mester human placentas were examined. This analysis em¬ ployed a cDNA probe derived from the OVIFNT CDNA sequen¬ ces (Example 9B) . In situ hybridization was performed using an anti-sense RNA probe. In three separate experi¬ ments, specific hybridization was observed in all term and first trimester placental tissues.
First trimester placental villi (composed of an outer layer of syncytiotrophoblast, an underlying layer of cytotrophoblast, and a central stromal region with various types of mesenchymal cells) displayed the highest transcript level of IFNT in the cytotrophoblast cells. Less intense but detectable levels were present in both the syncytiotrophoblast and stromal cells. A similar pattern of transcript expression was demonstrated in the placental villi of term tissue but the level of signal detection was low. First trimester extravillous tropho- blasts displayed the highest amount of message and stained positive when present in the maternal blood spaces.
Howatson, et al . , (1988) noted IFNα production in the syncytiotrophoblast of chorionic villi in both first trimester and term tissues. Also, Paulesu, et al . (1991) observed IFNα in extravillous trophoblast as well as syncytiotrophoblasts, noting more intense and abundant reactivity in first trimester placental tissue when compared to those taken at term. These investigators employed antibodies raised against human IFNα subtypes, and none observed any IFNα in the villous cytotropho- blasts.
The present results demonstrate that the human IFNT gene is highly expressed in early placental tissues by migrating extravillous trophoblasts, but is also ex¬ pressed in villous syncytiotrophoblasts, cytotropho- blasts, and various stromal cells. These results demon¬ strate the detection of IFNT transcripts in human preg¬ nancy tissues, and IFNT expression in the villous cyto- trophoblasts as well as the extravillous trophoblast of first trimester placenta.
C. Antiviral Properties of Interferon-T. The antiviral activity of IFNT has been evaluated against a number of viruses, including both RNA and DNA viruses. The relative specific activity of OVIFNT, purified to homogeneity, was evaluated in antiviral assays (Example 10) . IFNT had a higher specific antivi¬ ral activity than either rBoIFNα or rBoIFNγ (Example 10, Table 3) .
One advantage of the present invention is that IFNT has potent antiviral activity with limited cytotoxic effects. Highly purified OVIFNT was tested for anti- retroviral and cytotoxic effects on peripheral blood lymphocytes exposed to feline AIDS and human AIDS retro- viruses (Bazer, F.W., et al., (1989)). This feline AIDS lentivirus produces a chronic AIDS-like syndrome in cats and is a model for human AIDS (Pederson, et al . , 1987). Replication of either virus in peripheral blood lympho¬ cytes (PBL) was monitored by reverse transcriptase (RT) activity in culture supernatants over time. To determine IFNT antiviral activity against FIV and HIV, RNA-dependent DNA polymerase RT activity was assayed in FIV- and HIV-infected feline and human PBL cultures treated with IFNT (Example 11) . Replication of FIV was reduced to about one-third of control values when cells were cultured in the presence of IFNT. Addition of OVIFNT produced a rapid, dose-dependent decrease in reverse transcriptase (RT) activity (Example 11, Table 4). While concentrations as low as 0.62 ng/ml of IFNT inhibited viral replication, much higher concentrations (40 ng/ml) having greater effects on RT-activity were without toxic effects on the cells. The results suggest that replication of the feline immunodeficiency virus was reduced significantly compared to control values when cells were cultured in the presence of OVIFNT. IFNT appeared to exert no cytotoxic effect on the cellε hosting the retrovirus. This was true even when IFNT was present at 40 ng per ml of culture medium. This concentration of IFNT is equivalent to about 8,000 anti¬ viral units of alpha interferon, when IFNT is assayed for its ability to protect Madin-Darby bovine kidney cells from lysis by vesicular stomatitis virus as described by Pontzer, et al . (1988).
IFNT was also tested for activity against HIV repli¬ cation in human cells. Human peripheral lymphocytes, which had been infected with HIV were treated with vary¬ ing concentrations of IFNT (Example 12) . Replication of HIV in peripheral blood lymphocytes was monitored by reverse transcriptase activity in culture supernatants over time. Over a range of concentrations of IFNT pro- duced significant anti-HIV effects (Example 12, Table 5). A concentration of only 10 ng/ml resulted in over a 50% reduction in RT activity after only six days; A concen¬ tration of 500 ng/ml resulted in a 90% reduction in RT activity within 10 days. Further, there was no evidence of any cytotoxic effects attributable to the administra¬ tion of IFNT (Example 12, Table 5).
Further, the antiviral effects of IFNT against HIV were evaluated by treating human PBMC cells with various amounts of either recombinant IFNT or recombinant human IFNα2 at the time of infection with HIV (Example 18) . The data from these experiments (Example 18, Table 12) sup¬ port the conclusion that, at similar concentrations, IFNα and IFNT are effective in reducing the replication of HIV in human lymphocytes. However, treatment of cells with IFNα2 resulted in cytoxicity, whereas no such cytotoxity was observed with treatment using IFNT, even when IFNT was used at much higher concentrations. No cytotoxicity was observed using IFNT, even when IFNT was used at 200 times the dosage of interferon-alpha II. Both FIV and HIV reverse transcriptase themselves were unaffected by IFNT in the absence of PBL. There¬ fore, the antiviral activity is not due to a direct effect on the viral RT. Interferon-τ has also been shown to inhibit Hepati¬ tis B Virus DNA replication in hepatocytes (Example 18) . A human cell derived from liver cells transfected with Hepatitis B Virus (HBV) was used to test the antiviral effects of IFNT. The cells were treated with both the IFNα and IFNT over a range of concentrations. Both IFNα and IFNT reduced DNA production by approximately two-fold compared to the no interferon control.
To demonstrate that the effect of the interferons was specific to the in±ecting virus and not the result of an effect on general cell metabolism, the hepatocyte was examined for the effects of IFNα and IFNT on hepatospeci- fic mRNA production (Example 18) . Two hepatocyte specif¬ ic proteins, Apo E and Apo Al, were detected by hybrid- ization analysis. There was no apparent reduction of mRNA production for either hepatospecific mRNA at concen¬ trations up to 40,000 units/ml of either IFNα or IFNT. Further, no evidence for hepatotoxicity with IFNT was seen in this assay. These results suggest that IFNT is an effective antiviral agent against a wide variety of viruses, in¬ cluding both RNA and DNA viruses. One advantage of IFNT over other interferons, such as IFNα, is that treatment with IFNT does not appear to be associated with any cytotoxicity.
D. Antiproliferative Properties of IFNT. The effects of IFNT on cellular growth have also been examined. In one analysis, anti-cellular growth activity was examined using a colony inhibition assay (Example 13) . Human amnion (WISH) or MDBK cells were plated at low cell densities to form colonies originating from single cells. Dilutions of interferons were added to triplicate wells and the plates were incubated to allow colony formation. IFNT inhibited both colony size and number in these assays. IFNT was more effective at inhibiting cell proliferation of the human cell line (WISH) than human IFNα. The antiproliferative activity of IFNT was dose-dependent. High concentrations of IFNT stopped proliferation, while cell viability was not impaired.
Based on cell cycle analysis, using flow cytometry, IFNT appears to inhibit progress of cells through S phase. These results demonstrate the antiproliferative effect of IFNT, and underscore its low cytotoxicity. The antiproliferative effects of IFNT were also studied for rat and bovine cell lines (Example 14) . The rate of 3H-thymidine incorporation was used to assess the rate of cellular proliferation. The data obtained demon- strate that IFNT drastically reduced the rate of cellular proliferation (Example 14, Table 7) for each tested cell line.
The antiproliferative activity and lack of toxicity of IFNT was further examined using a series of human tumor cell lines (Example 15) . A variety of human tumor cell lines were selected from the standard lines used in NIH screening procedure for antineoplastic agents (Pontzer, C.H., et al., (1991)). At least one cell line from each major neoplastic category, was examined.
The following cell lines were obtained from American Type Culture Collection (12301 Parklawn Dr. , Rockville MD 20852) :
NCI-H460 human lung large cell carcinoma; DLD-1 human colon adenocarcinoma;
SK-MEL-28 human malignant melanoma;
ACHN human renal adenocarcinoma;
HL-60 human promyelocytic leukemia;
H9 human T cell lymphoma; HUT 78 human cutaneous T cell lymphoma; and
MCF7 human breast adenocarcinoma. As above, the antiproliferative activity was evalu¬ ated by measuring the rate of 3H-thymidine incorporation into cells which havt* been treated with IFNT. Signifi¬ cant differences between treatments were assessed by an analysis of variance followed by Scheffe's F-test. Cell cycle analysis was performed by flow cytometry.
Examination of IFNT inhibition of MCF7 (breast adenocarcinoma) proliferation demonstrated that IFNT reduced MCF7 proliferation in a dose-dependent manner. A 50% reduction in 3H-thymidine was observed with 10,000 units/ml of IFNT (Example 15, Table 8) . This cell line had previously been found to be unresponsive to anti- estrogen treatment.
A comparison of the antiproliferative effects of IFNT and IFNα was conducted using HL-60 (human promyelo- cytic leukemia) cells. Results with the promyelocytic leukemia HL-60 are typical of those obtained comparing IFNT with human IFNα (Example 15) . Concentrations as low as 100 units/ml of both IFNs produced signixicant (> 60%) growth reduction. Increasing amounts of IFNs further decreased tumor cell proliferation (Figure 4) . At high doses of IFNα, but not IFNT, was cytotoxic (Figure 5). Cell viability was reduced by approximately 80% by IFNα. By contrast, nearly 100% of the IFNT-treated cells re- mained viable when IFNT was applied at 10,000 units/ml. Thus, while both interferons inhibit proliferation, only IFNT is without cytotoxicity. This lack of toxicity provides an advantage of IFNT for use in vivo therapies. The human cutaneous T cell lymphoma, HUT 78, re- sponded similarly to HL-60 when treated with IFNT (Exam¬ ple 15, Figure 9) . Both OVIFNT and rHuIFNα reduce HUT 78 cell growth, but IFNα demonstrated adverse effects on cell viability. The T cell lymphoma H9 was less sensitive to the antiproliferative effects of IFNα than the tumor cell lines described above. While IFNα was not toxic to the H9 cells, it failed to inhibit cell division significant- ly at any of the concentrations examined (Example 15,
Figure 10) . In contrast, IFNT was observed to reduce H9 growth by approximately 60%. Thus, only OVIFNT is an effective growth inhibitor of this T cell lymphoma.
In three additional tumor cell lines (NCI-H460, DLD- 1 and SK-MEL-28) IFNT and IFNα were equally efficacious antitumor agents. In the melanoma, SK-MEL-28, inhibition of proliferation by IFNα was accomplished by a 13% drop in viability, while IFNT was not cytotoxic. In the majority of tumors examined, IFNT is equal or preferable to IFNα as an antineoplastic agent against human tumors. IFNT exhibits antiproliferative activity against human tumor cells without toxicity and is as potent or more potent than human IFNα. Clinical trials of the IFNα2s have shown them to be effective antitumor agents (Dianzani, F., 1992; Krown, 1987). One therapeutic advantage of IFNT as a therapeutic is the elimination of toxic effects seen with high doses IFNαs.
An additional application of the IFNT is against tumors like Kaposi's sarcoma (associated with HIV infec- tion) where the antineoplastic effects of IFNT are cou¬ pled with IFNT ability to inhibit retroviral growth.
The in vivo efficacy of interferon-T treatment was examined in a mouse system (Example 16) . B16-F10 is a syngeneic mouse transplantable tumor selected because of its high incidence of pulmonary metastases (Poste, et al . , 1981). Interferon treatment was initiated 3 days after the introduction of the tumor cells. The in vivo administration of IFNT dramatically reduced B16-F10 pulmonary tumors. Thus, IFNT appears to be an effica- cious antineoplastic agent in vivo as well as in vitro . III. Interferon-T Polypeptide Fragments. Protein Modeling and Protein Modifications.
The variety of IFNT activities, its potency and lack of cytotoxicity, as taught by the present specification, suggest the importance of structure/function analysis for this novel interferon. The structural basis for OVIFNT function has been examined using six overlapping synthet¬ ic peptides corresponding to the entire OVIFNT sequence (Figure 6) . The corresponding polypeptides derived from the ovine IFNT sequence are presented as SEQ ID NO:15 to SEQ ID NO:20. Three peptides representing amino acids l- 37, 62-92 and 139-172 have been shown to inhibit IFNT antiviral activity (Example 17) . The peptides were effective competitors at concentrations of 300 μM and above.
The C-terminal peptide of IFNT, OVIFNT (139-172), and the internal peptide OVIFNT (62-92) , inhibited IFNT and rBoIFNαπ antiviral activity to the same extent, while the N-terminal peptide OVIFNT (1-37) was more effective in inhibiting OVIFNT antiviral activity. Dose-response data indicated that IFNT (62-92) and IFNT (139-172) inhibited IFNT antiviral activity to similar extents. The same peptides that blocked IFNT antiviral activity also blocked the antiviral activity of recombinant bovine IFNα (rBoIFNα) ; recombinant bovine IFN7 was unaffected by the peptides. These two IFNT peptides may represent common receptor binding regions for IFNT and various IFNαs.
The two synthetic peptides OVIFNT(1-37) and OVIFNT(139-172) also blocked OVIFNT anti-FIV and anti-HIV activity (Example 17; Figures 11A and 11B) . While both peptides blocked FIV RT activity, only the C-terminal peptide, OVIFNT(139-172) , appeared to be an efficient inhibitor of vesicular stomatitis virus activity on the feline cell line, Fc9. The above data taken together suggest that the C- terminal regions of type I interferons may bind to common site on the type I interferon receptor, while the N- terminal region may be involved in the elicitation of unique functions. These results suggest that portions of the IFNT interferon molecule may be used to substitute regions of interferon alpha molecules. For example, the region of an interferon alpha molecule that is responsi¬ ble for increased cytotoxicity, relative to IFNT treat- ment, can be identified by substituting polypeptide regions derived from IFNT for regions of an interferon alpha molecule. Such substitutions can be carried out by manipulation of synthetic genes (see below) encoding the selected IFNT and interferon alpha molecules, coupled to the functional assays described herein (such as, antivi¬ ral, antiproliferative and cytoxicity assays) .
Polyclonal anti-peptide antisera against the IFNT peptides yielded similar results as the polypeptide inhibition studies, described above. Antibodies directed against the same three regions (OVIFNT (1-37) , IFNT (62- 92) and IFNT (139-172)) blocked OVIFNT function, confirm¬ ing the importance of these three domains in antiviral activity (Example 17) . These peptides, although appar¬ ently binding to the interferon receptor, did not in and of themselves elicit interferon-like effects in the cells.
The antiproliferative activity of IFNT (Example 17, Table 11) involved a further region of the molecule, since IFNT(119-150) was the most effective inhibitor of OvIFNT-induced reduction of cell proliferation. This results suggests that the region of the molecule primari¬ ly responsible for inhibition of cell growth is the IFNT(119-150) region. This region of the IFNT molecule may be useful alone or fused to other proteins (such as serum albumin, an antibody or an interferon alpha poly- peptide) as an antineoplastic agent. A conjugated pro¬ tein between an N-terminal peptide derived from human interferon-α and serum albumin was shown to have anticel¬ lular proliferation activity (Ruegg, et al . , 1990). Finally, binding of 15I-OvIFNτ to its receptor on MDBK cells could be blocked by antisera to 4 of the 6 peptides; the 4 polypeptides representing amino acids 1- 37, 62-92, 119-150 and 139-172 of OVIFNT. This reflects the multiple binding domains as well as the functional significance of. these regions. Since different regions of IFNT are involved in elicitation of different func¬ tions, modification of selected amino acids could poten¬ tially result in IFNT-like interferons with selective biological activity. The above data demonstrate the identification of synthetic peptides having four discontinuous sites on the OVIFNT protein that are involved in receptor interaction and biological activity. In order to elucidate the structural relationship of these regions, modeling of the three dimensional structure of IFNT was undertaken. A three dimensional model would be useful in interpretation of existing data and the design of future struc¬ ture/function studies.
Combining circular dichroism (CD) of both the full length recombinant OVIFNT to IFN/8 (a protein of known three dimensional structure (Senda, et al. , 1992)), a model of OVIFNT has been constructed. The most striking feature of this model is that IFNT falls into a class of proteins with a four-helix bundle motif. The CD spectra of IFNT was taken on an AVIV 60 S spectropolarimeter.
Two different methods were employed for secondary struc¬ ture estimations, the algorithm of Perczel, et al . , (1991) and variable selection by W.C. Johnson, Jr. (1992). Secondary structure estimations of the spectra indicate 70-75% alpha helix (characterized by minima at 222 and 208 nm and maximum at 190 nm) . The variable selection algorithm estimates the remainder of the mole- cule to be 20% beta sheet and 10% turn. The Chang method estimates the remainder to be 30% random coil. Alignment of IFNT and IFN/3 sequences revealed homology between the two molecules, specifically in the regions of known helical structure in IFN/3. Sequence analysis of IFNT also showed that proposed helical regions possess an apolar periodicity indicative of a four-helix bundle motif.
The final modeling step was to apply the IFN/3 x-ray crystallographic coordinates of the IFN/3 carbon backbone to the IFNT sequence. The functionally active domains of IFNT, identified above, were localized to one side of the molecule and found to be in close spatial proximity. This is consistent with multiple binding sites on IFNT interacting simultaneously with the type I IFN receptor. The three dimensional modeling data coupled with the function data described above, provides the ability to introduce sequence variations into specific regions of IFNT to generate enhancement of selected functions (e.g., antiviral or anticellular proliferation) or the ability to substitute a region(s) of selected function into other interferon molecules (e .g. , antiviral, antineoplastic, or reduced cytotoxicity) .
The construction of a synthetic gene for OVIFNT is described in Example 3. Briefly, a consensus amino sequence was back-translated using optimal codon usage for E. coli . The sequence was edited to include 20, unique, restriction sites spaced throughout the length of the construct. This 540 base pair synthetic gene se¬ quence was divided into 11 oligonucleotide fragments. Individual fragments were synthesized and cloned, either single or double stranded, into either pTZ 19R, pTZ 18R or pBluescript, amplified and fused. The synthetic OVIFNT construct was then cloned into a modified pIN-IIl- ompA expression vector for expression in bacteria and also cloned into a yeast expression plasmid. A similarly constructed human IFNT synthetic gene (SEQ ID NO:3) has been designed, constructed and expressed in yeast cells.
Expression of the OVIFNT synthetic gene in yeast (Example 4) allowed over production of recombinant IFNT in S. cerevisiae : large quantities (5-20 mg/1) of recom¬ binant IFNT can be purified from soluble yeast extract . using sequential ion exchange and molecular sieve chroma¬ tography. Recombinant IFNT purified in this fashion exhibited potent antiviral activity (2 to 3 X 108 units/mg) similar to native OVIFNT.
The synthetic gene construct facilitates introduc¬ tion of mutations for possible enhancement of antitumor (anticellular proliferative) and antiviral activities. Further, the disparate regions of the molecule responsi- ble for different functions allows for separate manipula¬ tion of different functions. For example, two deletion mutants, OVIFN (1-155) and OVIFNT(1-166) , have been constructed to examine the role of carboxy terminal sequences in IFNr molecules. Additional mutant IFNT molecules have been con¬ structed to identify residues critical for antiprolifera¬ tive activity. For example, one particular residue, Tyr 123 has been implicated in the anticellular proliferative activity of IFNα (Mclnnes, et al . , 1989). The equivalent of Tyr 123 in IFNT is contained within peptide
OVIFNT(119-150) : this polypeptide inhibits OVIFNT and human IFNα antiproliferative activity. Mutations con¬ verting Tyr 123 to conservative (Trp) and nonconservative (Asp) substitutions have been generated, as well as mutant sequences having deletion of this residue. The codon for Tyr 123 is located within an Sspl site; elimi¬ nation of this site has been used for screening. The antiproliferative activity of these mutant IFNT is evalu- ated as described herein.
Synthetic peptides can be generated which correspond to the IFNT polypeptides of the present invention. Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art (Applied Biosystems, Foster City CA) .
Alternatively, oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of clon- ing steps involving a tandem array of multiple oligonu¬ cleotide fragments corresponding to the coding sequence (Crea; Yoshio et al . ; Eaton et al . ) . Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al . ; Ausubel et al . ) . The biological activities of the interferon-τ poly¬ peptides described above can be exploited using either the interferon-T polypeptides alone or conjugated with other proteins (see below) .
IV. Production of Fusion Proteins.
In another aspect, the present invention includes interferon-T or interferon-T-derived polypeptides cova¬ lently attached to a second polypeptide to form a fused, or hybrid, protein. The interferon-T sequences making up such fused proteins can be recombinantly produced inter¬ feron-T or a bioactive portion thereof, as described above.
For example, where interferon-T is used to inhibit viral expression, the polypeptides presented as SEQ ID NO:10 and SEQ ID NO:20 may be advantageously fused with a soluble peptide, such as, serum albumin, an antibody (e .g. , specific against an virus-specific cell surface antigen) , or an interferon alpha polypeptide. Other examples of fusion proteins include (i) replacing toxici- ty-associated regions of interferon-α with the interfer¬ on-T regions SEQ ID NO:5 and SEQ ID NO:15, and (ii) fusion proteins containing the interferon-T regions SEQ ID NO:9 and SEQ ID NO:19 as anticellular proliferation agents. The fused proteins of the present invention may be formed by chemical conjugation or by recombinant tech¬ niques. In the former method, the interferon-τ and second selected polypeptide are modified by conventional coupling agents for covalent attachment. In one exempla- ry method for coupling soluble serum albumin to an inter¬ feron-T polypeptide, serum albumin is derivatized with N- succinimidyl-S-acetyl thioacetate (Duncan) , yielding thiolated serum albumin. The activated serum albumin polypeptide is then reacted with interferon-T derivatized with N-succinimidyl 3-(2-pyridyldithio) propionate (Cum¬ ber) , to produce the fused protein joined through a disulfide linkage.
As an alternative method, recombinant interferon-T may be prepared with a cysteine residue to allow disul- fide coupling of the interferon-T to an activated ligand, thus simplifying the coupling reaction. An interferon-τ expression vector, used for production of recombinant interferon-T, can be modified for insertion of an inter¬ nal or a terminal cysteine codon according to standard methods of site-directed mutagenesis (Ausubel, et al . ) . In one method, a fused protein is prepared recombi¬ nantly using an expression vector in which the coding sequence of a second selected polypeptide is joined to the interferon-T coding sequence. For example, human serum albumin coding sequences can be fused in-frame to the coding sequence of an interferon-T polypeptide, such as, SEQ ID NO:9. The fused protein is then expressed using a suitable host cell. The fusion protein may be purified by molecular-sieve and ion-exchange chromatogra- phy methods, with additional purification by polyacryl- amide gel electrophoretic separation and/or HPLC chroma¬ tography, if necessary.
It will be appreciated from the above how interfer- on-T-containing fusion proteins may be prepared. One variation on the above fusion is to exchange positions of the interferon-T and selected second protein molecules in the fusion protein (e.g., carboxy terminal versus amino terminal fusions) . Further, internal portions of a native interferon-T polypeptide (for example, amino acid regions of between 15 and 172 amino acids) can be assem¬ bled into polypeptides where two or more such interferon- T portions are contiguous that are normally discontinuous in the native protein.
V. Antibodies Reactive with Interferon-T.
Fusion proteins containing the polypeptide antigens of the present invention fused with the glutathione-S- transferase (Sj26) protein can be expressed using the pGEX-GLI vector system in E. coli JM101 cells. The fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography (Smith) . Expression and partial purification of IFNT proteins is described in (Example 20) , and is applicable to any of the other soluble, induced polypeptides coded by sequences de- scribed by the present invention.
Insoluble GST (sj26) fusion proteins can be puri¬ fied by preparative gel electrophoresis.
Alternatively, IFNτ-?-galactosidase fusion proteins can be isolated as described in Example 19. Also included in the invention is an expression vector, such as the lambda gtll or pGEX vectors described above, containing IFNT coding sequences and expression control elements which allow expression of the coding regions in a suitable host. The control elements gener¬ ally include a promoter, translation initiation codon, and translation and transcription termination sequences, and an insertion site for introducing the insert into the vector. The DNA encoding the desired polypeptide can be cloned into any number of vectors (discussed above) to generate expression of the polypeptide in the appropriate host system. These recombinant polypeptides can be expressed as fusion proteins or as native proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium. Recombinantly produced IFNT, and polypeptides derived therefrom, are typically isolated from lysed cells or culture media. Purification can be carried out by meth¬ ods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated against selected IFNT antigens. In another aspect, the invention includes specific antibodies directed against the polypeptides of the present invention. Typically, to prepare antibodies, a host animal, such as a rabbit, is immunized with the purified antigen or fused protein antigen. Hybrid, or fused, proteins may be generated using a variety of coding sequences derived from other proteins, such as β- galactosidase or glutathione-S-transferase. The host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen. Example 20 describes the production of rabbit serum antibodies which are specific against the IFNT antigens in a Sj26/IFNτ hybrid protein. These techniques can be applied to the all of the IFNT molecules and polypeptides derived therefrom. The gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art for produ¬ cing polyclonal antibodies. Alternatively, purified protein or fused protein may be used for producing monoclonal antibodies. Here the spleen or lymphocytes from a animal immunized with the selected polypeptide antigen are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art (Harlow, et al . ) . Lym¬ phocytes can be isolated from a peripheral blood sample. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a fusion partner can be used to produce hybridomas. Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et al . ) . Experi¬ ments performed in support of the present invention have yielded four hybridomas producing monoclonal antibodies specific for ovine IFNT have been isolated.
Antigenic regions of polypeptides are generally relatively small, typically 7 to 10 amino acids in length. Smaller fragments have been identified as anti- genie regions. Interferon-T polypeptide antigens are identified as described above. The resulting DNA coding regions can be expressed recombinantly either as fusion proteins or isolated polypeptides.
In addition, some amino acid sequences can be conve- niently chemically synthesized (Applied Biosystems, Foster City CA) . Antigens obtained by any of these methods may be directly used for the generation of anti¬ bodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e .g. , Pierce, Rockford IL) . Antibodies reactive with IFNT are useful, for exam¬ ple, in the analysis of structure/function relationships.
VI. Utility A. Reproductive.
Although IFNT bears some similarity to the IFNα family based on structure and its potent antiviral prop¬ erties, the IFNαs do not possess the reproductive proper¬ ties associated with IFNT. Also, recombinant bovine IFNα has little or no effect on interestrous interval compared to IFNT (Davis, et al., 1992).
Therefore, although IFNT has some structural simi¬ larities to other interferons, it has very distinctive properties of its own: for example, the capability of significantly influencing the biochemical events of the estrous cycle.
The human IFNT of the present invention can be used in methods of enhancing fertility and prolonging the life span of the corpus luteum in female mammals as generally described in Hansen, et al . , herein incorporated by reference. Further, the human interferon-T of the pres¬ ent invention could be used to regulate growth and devel¬ opment of uterine and/or fetal-placental tissues. The human IFNT is particularly useful for treatment of hu- mans, since potential antigenic responses are less likely using such a same-species protein.
B. Antiviral Properties.
The antiviral activity of IFNT has broad therapeutic applications without the toxic effects that are usually 40 associated with IFNαs. Although the presence of IFNr in culture medium inhibited reverse transcriptase activity of the feline immunodeficiency virus (Example 11) , this is not due to a direct effect of IFNT on the FIV. Rath- 5 er, IFNT appears to induce the host cell to produce a factor(s) which is inhibitory to the reverse transcrip¬ tase of the virus.
IFNT was found to exert its antiviral activity without adverse effects on the cells: no evidence of
10 cytotoxic effects attributable to the administration of IFNT was observed. It is the lack of cytotoxicity of IFNT which makes it extremely valuable as an in vivo therapeutic agent. This lack of cytotoxicity sets IFNT apart from most other known antiviral agents and all
15 other known interferons.
Formulations comprising the IFNT compounds of the present invention can be used to inhibit viral replica¬ tion.
The human IFNT of the present invention can be
20 employed in methods for affecting the immune relationship between fetus and mother, for example, in preventing transmission of maternal viruses (e . g. , HIV) to the developing fetus. The human interferon-T is particularly useful for treatment of humans, since potential antigenic
25 responses are less likely using a homologous protein.
C. Anticellular Proliferation Properties. IFNT exhibits potent anticellular activity. IFNT can also be used to inhibit cellular growth without the 30 negative side effects associated with other interferons which are currently known. Formulations comprising the IFNT compounds of the subject invention can be used to inhibit, prevent, or slow tumor growth.
The development of certain tumors is mediated by 35 estrogen. Experiments performed in support of the pres- ent invention indicate that IFNT can suppress estrogen receptor numbers. Therefore, IFNT can be used in the treatment or prevention of estrogen-dependent tumors.
D. Interfering with the Binding of Interferons to Receptors.
IFNT appears to interact with the Type I IFN recep¬ tor via several epitopes on the molecule, and these regions either separately or in combination may affect distinct functions of IFNT differently.
The polypeptides of the present invention are useful for the selective inhibition of binding of interferons to the interferon receptor. Specifically, as described herein, certain of the disclosed peptides selectively inhibit the antiviral activity of IFNT while others inhibit the antiproliferative activity. Combinations of these peptides could be used to inhibit both activities. Advantageously, despite binding to the interferon recep- tor and blocking IFNT activity, these peptides do not, themselves, elicit the antiviral or antiproliferative activity.
Therefore, such polypeptides can be used as immuno- regulatory molecules when it is desired to prevent immune responses triggered by interferon molecules. These peptides could be used as immunosuppressants to prevent, for example, interferon-mediated immune responses to tissue transplants. Other types of interferon mediated responses may also be blocked, such as the cytotoxic effects of alpha interferon.
E. Pharmaceutical Compositions.
IFNT proteins can be formulated according to known methods for preparing pharmaceutically useful composi- tions. Formulations comprising interferons or interfer- on-like compounds have been previously described (for example, Martin, 1976) . In general, the compositions of the subject invention will be formulated such that an effective amount of the IFNT is combined with a suitable carrier in order to facilitate effective administration of the composition.
The compositions used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills,, powders, liquid solutions or suspensions, liposomes, suppositories, injectable, and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharma- ceutically acceptable carriers and adjuvants which are known to those of skill in the art. Preferably, the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day. IFNT, or related polypeptides, may be administered to a patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intrale- sional, or subcutaneous injection. Specifically, compo¬ sitions and methods used for other interferon compounds can be used for the delivery of these compounds.
One primary advantage of the compounds of the sub¬ ject invention, however, is the extremely low cytotoxici¬ ty of the IFNT proteins. Because of this low cytotoxici¬ ty, it is possible to administer the IFNT in concentra- tions which are greater than those which can generally be utilized for other interferon (e .g. , IFNα) compounds. Thus, IFNT can be administered at rates from about 5 x 104 to 20 x 106 units/day to about 500 x 106 units/day or more. In a preferred embodiment, the dosage is about 106 units/day. High doses are preferred for systemic admin¬ istration. It should, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies. Once improvement of a patient's condition has oc¬ curred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administra¬ tion, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
The compositions of the subject invention can be administered through standard procedures to treat a variety of cancers and viral diseases including those for which other interferons have previously shown activity. See, for example, Finter, et al . (1991); Dianzani, et al . (1992); Francis, et al . (1992) and U.S. Patent Nos. 4,885,166 and 4,975,276. However, as discussed above, the compositions of the subject invention have unique features and advantages, including their ability to treat these conditions without toxicity.
F. Treatment of Skin Disorders.
Disorders of the skin can be treated intralesionally using IFNT, wherein formulation and dose will depend on the method of administration and on the size and severity of the lesion to be treated. Preferred methods include intradermal and subcutaneous injection. Multiple injec¬ tions into large lesions may be possible, and several lesions on the skin of a single patient may be treated at one time. The schedule for administration can be deter¬ mined by a person skilled in the art. Formulations designed for sustained release can reduce the frequency of administration.
G. Systemic Treatment. Systemic treatment is essentially equivalent for all applications. Multiple intravenous or subcutaneous doses are possible, and in the case of implantable methods for treatment, formulations designed for sustained release are particularly useful. Patients may also be treated using implantable subcutaneous portals, reservoirs, or pumps.
H. Regional Treatment.
Regional treatment with the IFNT polypeptides of the present invention is useful for treatment of cancers in specific organs. Treatment can be accomplished by intra- arterial infusion. A catheter can be surgically or angiographically implanted to direct treatment to the affected organ. A subcutaneous portal, connected to the catheter, can be used for chronic treatment, or an im¬ plantable, refillable pump may also be employed.
The following examples illustrate, but in no way are intended to limit the present invention.
Materials and Methods Restriction endonucleases, T4 DNA Iigase, T4 polynu- cleotide kinase, Tag DNA polymerase, and calf intestinal phosphatase were purchased from New England Biolabs (Beverly, MA) or Promega Biotech (Madison, WI) : these reagents were used according to the manufacturer's in¬ struction. For sequencing reactions, a "SEQUENASE DNA II" sequencing kit was used (United States Biochemical Corporation, Cleveland OH) . Immunoblotting and other reagents were from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Needham, MA) . Nitrocellulose filters are obtained from Schleicher and Schuell (Keene, NH) .
Synthetic oligonucleotide linkers and primers are prepared using commercially available automated oligonu- cleotide synthesizers (e .g. , an ABI model 380B-02 DNA synthesizer (Applied Biosystems, Foster City, CA)) . Alternatively, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) . cDNA synthesis kit and random priming labeling kits are obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN) .
Oligonucleotide sequences encoding polypeptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleo¬ tide fragments corresponding to the coding sequence (Crea; Yoshio et al.; Eaton et al.). Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al.; Ausubel et al.).
Alternatively, peptides can be synthesized directly by standard in vitro techniques (Applied Biosystems, Foster City CA) .
Common manipulations involved in polyclonal and monoclonal antibody work, including antibody purification from sera, are performed by standard procedures (Harlow et al.). Pierce (Rockford, IL) is a source of many antibody reagents.
Recombinant human IFNα (rHuIFNα) and rBoIFN7 was obtained from Genentech Inc. (South San Francisco, CA) . The reference preparation of recombinant human IFNα (rHuIFNα) was obtained from the National Institutes of Health: rHuIFNα is commercially available from Lee Biomolecular (San Diego, CA) . All tissue culture media, sera and IFNs used in this study were negative for endotoxin, as determined by assay with Limulus amebocyte lysate (Associates of Cape Cod, Woods Hole, MA) at a sensitivity level of 0.07 ng/ml.
General ELISA Protocol for Detection of Antibodies. Polystyrene 96 well plates Immulon II (PGC) were coated with 5 μg/mL (100 μL per well) antigen in 0.1 M carb/bicarbonate buffer, pH 9.5. Plates were sealed with parafilm and stored at 4°C overnight.
Plates were aspirated and blocked with 300 uL 10% NGS and incubated at 37°C for 1 hr.
Plates were washed 5 times with PBS 0.5% "TWEEN-20". Antisera were diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) were added to each well and the plate incubated 1 hours at 37°C. The plates was then washed 5 times with PBS 0.5% "TWEEN-20". Horseradish peroxidase (HRP) conjugated goat anti- human antiserum (Cappel) was diluted 1/5,000 in PBS. 0.1 mL of this solution was added to each well. The plate was incubated 30 min at 37°C, then washed 5 times with PBS.
Sigma ABTS (substrate) was prepared just prior to addition to the plate. The reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate was added to each well, then incubated for 30 min at room temperature. The reaction was stopped with the addition of 0.050 mL 5% SDS ( /v) . The relative absorbance is determined at 410 nm.
EXAMPLE 1 Reproductive Functions of IFNT The effect of interferon-T on the lifespan of the corpus lutem was examined. IFNT was infused into uterine lumen of ewes at the concentrations given in Table 1. Recombinant human IFNα (rHuIFNα) was infused at similar concentrations. In addition, control animals, which received co .trol pro¬ teins, were also used. The life span of the corpus luteum was assessed by examination of interestrous inter¬ vals, maintenance of progesterone secretion, and inhibi¬ tion of prostaglandin secretion (Davis, et al . , 1992 ) .
Taiole 1 Effect of Interferons on Reproductive Physiology
Comparison of the interestrous intervals for the control animals and for animals receiving OVIFNT demon¬ strate a considerable lengthening of the interval, when IFNT is administered at lOOμg/day. On the other hand, comparison of the interestrous interval for the control animal and for animals receiving recombinant human IFNα, demonstrated that rHuIFNα had no meaningful effect.
These results demonstrate that interferon-T has the capability of significantly influencing the biochemical events of the reproductive cycle.
EXAMPLE 2
Antiviral Properties of Interferon-T at Various Stages of the Reproductive Cycle
Conceptus cultures were established using conceptus obtained from sheep at days 12 through 16 of the estrous cycle. Antiviral activity of supernatant from each conceptus culture was assessed using a cytopathic effect assay (Familetti, et al., 1981). Briefly, dilutions of IFNT or other IFNs were incubated with Madin-Darby bovine kidney (MDBK) cells for 16-18 hours at 37°C. Following incubation, inhibition of viral replication was deter¬ mined in a cytopathic effect assay using vesicular stoma¬ titis virus (VSV) as the challenge virus.
One antiviral unit caused a 50% reduction in de¬ struction of the monolayer, relative to untreated MDBK cells infected with VSV (control plates) . Specific activities were further evaluated using normal ovine fibroblasts (Shnf) in a plaque inhibition assay (Lang- ford, et al . , 1981). A minimum of three samples were examined at each time point, and each sample was assayed in triplicate. The results presented in Table 2 are expressed as mean units/ml.
Table 2
IFNr Antiviral Activity of Conceptus Cultures and Allantoic and Amniotic Fluids
Culture supernatants had increasing antiviral activ¬ ity associated with advancing development of the concep¬ tus (Table 2) .
EXAMPLE 3
Expression of IFNT in Bacteria The amino acid coding sequence for OVIFNT (Imakawa, et al . , 1987) was used to generate a corresponding DNA coding sequence with codon usage optimized for expression in E. coli . Linker sequences were added to the 5' and 3' ends to facilitate cloning in bacterial expression vec¬ tors. The nucleotide sequence was designed to include 19 unique restriction enzyme sites spaced evenly throughout the coding sequence (Figure 1) . The nucleotide sequence was divided into eleven oligonucleotide fragments ranging in sizes of 33 to 75 bases. Each of the eleven oligonucleotides were synthe¬ sized on a 380-B 2-column DNA synthesizer (Applied Bio- sytems) and cloned single- or double-stranded into one of the following vectors: "pBLUESCRIPT+(KS)" (Stratagene, LaJolla, CA) , pTZ18R (Pharmacia, Piscataway, NJ) , or pTZ19R (Pharmacia, Piscataway, NJ) cloning vectors.
The vectors were transformed into E. coli K. strain "XLl-BLUE" (recAl endAl gyrA96 thi hsdR17 (r , mκ+) supE44 relAl λ - (lac), {F', proAB, lac'ZΔMlδ, Tnl0(tetR}) which is commercially available from Stratagene (LaJolla, CA) . Transformed cells were grown in L broth supplemented with ampicillin (50 μg/ml) . Oligonucleotide cloning and fusion was performed using standard recombinant DNA techniques.
Cloning vectors were cut with the appropriate re¬ striction enzymes to insert the synthetic oligonucleo¬ tides. The vectors were treated with calf intestine alkaline phosphatase (CIP) to remove terminal phosphate groups. Oligonucleotides were phosphorylated and cloned, as either single- or double-stranded molecules, into the appropriate vector using T4 DNA Iigase. When single- strands were introduced into cloning vectors, the second strand was completed by the bacterial host following transfection.
For double-stranded cloning, oligonucleotides were first annealed with their synthetic complementary strand then ligated into the cloning vector. E. coli K12 strains SB221 or NM522 were then transformed with the ligation. E. coli strain GM119 was used for cloning when the methylation-sensitive Stul and Clal restriction sites were involved. Restriction analyses were performed on isolated DNA at each stage of the cloning procedure. Cloned oligonucleotides were fused into a single polynucleotide using the restriction digestions and ligations outlined in Figure 2. Oligonucleotide-contain- ing-DNA fragments were typically isolated after electro- phoretic size fractionation on low-melting point agarose gels (Maniatis, et a .; Sambrook, et al . ; Ausubel, et al . ) . The resulting IFNT polynucleotide coding sequence spans position 16 through 531: a coding sequence of 172 amino acids.
The nucleotide sequence of the. final polynucleotide was confirmed by DNA sequencing using the dideoxy chain termination method.
The full length Stul/Sstl fragment (540 bp; Figure 2) was cloned into a modified pIN III omp-A expression vector and transformed into a competent SB221 strain of E. coli . For expression of the IFNT protein, cells carrying the expression vector were grown in L-broth containing ampicillin to an OD (550 nm) of 0.1-1, induced with IPTG for 3 hours and harvested by centrifugation. Soluble recombinant IFNT was liberated from the cells by sonication or osmotic fractionation. EXAMPLE 4 Expression of IFNT in Yeast The synthetic IFNT gene, synthesized in Example 3, was flanked at the 5' end by an Stul restriction site and at the 3' end by a Sacl restriction site.
A. Isolation of the Synthetic IFNT Gene. Two oligonucleotide primers (SEQ ID NO:13 and SEQ ID NO:14) were used to attach linkers to the synthetic IFNr gene using polymerase chain reaction. The linker at the 5' end allowed the placement of the synthetic IFNr gene in correct reading with the ubiquitin coding sequence present in the yeast cloning vector pBS24Ub (Chiron Corp., Emeryville, CA) . The linker also constructed a ubiquitin-IFNτ junction region that allowed in vivo cleavage of the ubiquitin sequences from the IFNT se¬ quences. The 5' oligonucleotide also encoded a Saσll restriction endonuclease cleavage site. The 3' oligonu¬ cleotide contained a Stul cleavage site. The vector carrying the synthetic IFNT gene (Example 3) was isolated from E. coli strain "XLl-BLUE" by the alkaline lysis method. Isolated vector was diluted 500- fold in 10 mM Tris, pH 8.0/1 mM EDTA/10 mM NaCl. The PCR reaction was performed in a 100 μl volume using Tag DNA polymerase and primers SEQ ID NO:13/SEQ ID NO:14. The amplified fragments were digested with -Stul and -Sacll. These digested fragments were ligated into the Sacll and Smal sites of "pBLUESCRIPT+(KS) ."
The resulting plasmid was named pBSY-IFNτ. The DNA sequence was verified using double stranded DNA as the template.
B. Construction of the Expression Plasmid. Plasmid pBSY-IFNτ was digested with Sacll and -BcoRV and the fragment containing the synthetic IFNT gene was isolated. The yeast expression vector pBS24Ub (Sabin, et al . ; Ecker, et al . ) was digested with Sail . Blunt ends were generated using T4 DNA polymerase. The vector DNA was extracted with phenol and ethanol precipitated (Sam- brook, et al . , 1989). The recovered linearized plasmid was digested with Sacll, purified by agarose gel electro- phoresis, and ligated to the SaσII--EcoRV fragment isolat¬ ed from pBSY-IFNτ. The resulting recombinant plasmid was designated pBS24Ub-IFNτ. The recombinant plasmid pBS24Ub-IFNτ was transformed into E. coli . Recombinant clones containing the IFNT insert were isolated and identified by restriction enzyme analysis. Plasmid DNA from clones containing IFNr coding sequences was used for transformation of S. cerevisiae (Rothstein, 1986) . Transformation mixtures were plated on uracil omission medium and incubated for 3-5 days at 30°C. Colonies were then streaked and maintained on uracil and leucine omission medium (Rothstein, 1986) .
C. Expression Experiments.
For small-scale expression, a single colony of S. cerevisiae AB116 containing pBS24Ub-IFNτ was picked from a leucine and uracil omission plate and grown at 30°C in YEP medium (1% yeast extract, 2% peptone) containing 1% glucose for inducing conditions or 8% glucose for non- inducing conditions. Cell lysates were recovered and subjected to SDS-PAGE in 15% acrylamide, 0.4% bisacryla- mide (Sambrook, et al . , 1989). The fractionated proteins were visualized by Coomassie blue staining. Recombinant IFNT was visualized specifically by immunoblotting with monoclonal antibody or polyclonal antiserum against ovine IFNT upon electrotransfer of the fractionated cell extract to "NYTRAN" paper (Rothstein, 1986) . For large-scale expression, pBS24-IFNr was grown for 24 hours at 30°C in 5 x uracil and leucine omission medium containing 8% glucose. This culture was then diluted 20-fold in YEP medium containing 1% glucose and further incubated for another 24-36 hours.
Cells were harvested by centrifugation, washed in 50 mM Tris, pH 7.6,/l mM EDTA and resuspended in wash buffer containing 1 mM PMSF. The cells were lysed using a Bead- beater apparatus (Biospec Products, Bartlesville, OK) . The lysate was spun at 43,000 x g for 20 minutes. The supernatant fraction was recovered and subjected to the purification protocol described below.
D. Purification of r-IFNτ from Yeast Cell Lvsate. The supernatant was loaded on a 1 x 10 cm DEAE column and washed with 10 mM Tris, pH 8.0. Retained proteins were eluted with a 300 ml, 0 to 0.5 M NaCl gradient in 10 mM Tris, pH 8.0. Three-milliliter frac¬ tions were collected. en-microliter samples of frac- tions 17-26 containing the recombinant (r-IFNτ) were electrophorectically separated on 15% SDS-polyacrylamide gels. The gels were stained with Coomassie blue.
Fractions 18, 19, and 20 contained largest amount of r-IFNτ. These fractions were loaded individually on a 1.5 x 90 cm Sephadex S-200 column and proteins were resolved in two peaks. Aliquots of each protein peak (25 μl) were electrophoretically separated on 15% SDS-poly¬ acrylamide gels and the proteins visualized with Coomas¬ sie staining. Purified r-IFNτ-containing fractions were combined and the amount of r-IFNτ quantified by radioimmunoassay (Vallet, et al . , 1988). Total protein concentration was determined by using the Lowry protein assay (Lowry, et al . , 1951). Microsequencing of purified r-IFNτ demonstrated identity with native IFNT through the first 15 amino acids, confirming that the ubiquitin/r-IFNτ fusion pro¬ tein was correctly processed in vivo .
Purified r-IFNτ exhibited 2 to 3 x 108 units of antiviral activity per milligram of protein (n = 3 repli¬ cate plates) which is similar to the antiviral activity of IFNT purified from conceptus-conditioned culture medium (2 x 108 U/mg) .
EXAMPLE 5
Southern Blot Analysis of Human High Molecular Weight DNA
Human venous blood samples from healthy donors were collected in heparinized tubes and peripheral blood lymphocytes were isolated by density-gradient centrifuga¬ tion using a Ficoll-Isopaque gradient (1.077 g/ml) (Sigma Chemical Co.). High molecular weight (HMW) DNA was isolated from these cells (Sa brook, et al . , 1989). Two 10 μg samples of HMW DNA were digested with the restriction endonucleases Ifindlll or Pstl (Promega) for 2 hours at 37°C, and the DNA fragments electrophoretically separated in a 0.8% agarose gel (Bio-Rad, Richmond, CA) at 75 volts for 8 hours. The DNA fragments were trans- ferred onto a nylon membrane (IBI-International Biotech¬ nologies, Inc., New Haven, CT) . The membrane was baked at 80°C for 2 hours and incubated at 42°C for 4 hours in the following prehybridization solution: 5 x SSC (l x SSC is 0.15 M NaCl and 0.15 M sodium citrate), 50% vol/- vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 0.5 mg/ml single stranded herring sperm DNA (Promega) .
The filter was then incubated in a hybridization solution (5 x SSC, 20% vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 2 x 108 cpm/ml 32P-labelled OVIFNT cDNA (Imakawa, et al., 1987)) for 18 hours at 42°C. The filter was washed at 42°C for 15 minutes with 2 x SSC and 0.1% (wt/vol) SDS and exposed to X-ray film (XAR, Eastman Kodak, Rochester, NY) at -80°C for 48 hours in the pres¬ ence of an intensifying screen.
Autoradiography detected a hybridization signal at approximately 3.4 kb in DNA digested with Pstl and a slightly smaller (« 3.0 kb) fragment in the Hindlll digested DNA. These results indicate the presence of human DNA sequences complementary to the OVIFNT cDNA probe.
EXAMPLE 6 Isolation of Partial Sequence of
Human IFN cDNA bv PCR
Two synthetic oligonucleotides (each 25-mer) , corre¬ sponding to the sequence 231 to 255 (contained in SEQ ID NO:13) and 566 to 590 (contained in SEQ ID NO:14) of OVIFNT CDNA (numbering relative to the cap site, Imakawa, et al . , 1987) were synthesized. These primers contained, respectively, cleavage sites for the restriction endonu¬ cleases Pstl and -EcoRI. SEQ ID NO:13 was modified to contain the EcoRI site, which begins at position 569. DNA was isolated from approximately 1 x 105 plaque forming units (pfu) of the following two cDNA libraries: human term placenta (Clontech, Inc. , Palo Alto, CA) and human term cytotrophoblast (Dr. J.F. Strauss, University of Pennsylvania, Philadelphia PA) . The DNA was employed in polymerase chain reaction (PCR) amplifications (Mul¬ lis; Mullis, et al . ; Pekin Elmer Cetus Corp. Norwalk CT) . Amplification reactions were carried out for 30 cycles (45°C, lm; 72°C, 2m; 94°C, lm) (thermal cycler and re¬ agents, Perkin Elmer Cetus) using primers SEQ ID NO:13/SEQ ID NO:14. Amplification products were electrophoretically separated (100 volts in a 1.5% agarose gel (Bio-Rad)) and transferred onto a nylon membrane (IBI) . The membrane was baked at 80°C for 2 hours and prehybridized and hybridized with 32P-labelled OVIFNT CDNA as described above. The membrane was washed in 5 x SSC/0.1% (wt/vol) SDS for 5 minutes at 42°C and in 2 x SSC/0.1% (wt/vol) SDS for 2 minutes at 42°C. It was then exposed at -80°C to "XAR" (Eastman Kodak) X-ray film for 24 hours in the presence of an intensifying screen. An amplification product that hybridized with the labelled probe DNA was detected.
PCR was performed again as directed above. Ampli¬ fied products were digested with the restriction endonu- cleases .EcoRI and Pstl (Promega) for 90 minutes at 37°C. The resulting DNA fragments were electrophoretically separated as described above and the band containing the IFNT amplification product was excised from the gel. DNA fragments were recovered by electroelution, subcloned into EcoRI /Pstl digested-dephosphorylated plasmid pUCl9 and transformed into E. coli strain JM101 (Promega) by calcium chloride method (Sambrook, et al . , 1989). The plasmids were isolated and the inserted amplification product sequenced using the dideoxy termination method (Sanger, et al . , 1977; "SEQUENASE" reactions. United
States Biochemical, Cleveland, OH) . Nucleotide sequences were determined, and comparison of these as well as the deduced amino acid sequences to other IFN sequences were performed using DNA Star Software (Madison, WI) . Comparison of the sequences of these clones revealed three different clones: from the human placental li¬ brary, Clone 15 (306 bp) and Clone 21 (315 bp) , which exhibit 95% identity in their nucleotide sequences; from the cytotrophoblast library clone CTB 35 (301 basepairs) , which shares 95% and 98% identity with Clone 15 and Clone 21, respectively.
EXAMPLE 7
Isolation of Full-Length Human IFNT Gene Ten micrograms PBMC HMW DNA was digested with re¬ striction endonuclease JEσoRI and subjected to electropho- retic analysis in a 0.8% agarose gel. A series of sam- pies containing ranges of DNA fragments sized 1.5 to 10 kb (e .g. , 1.5 to 2.5 kb, 2.5 kb to 3 kb) were excised from the gel. The DNAs were electroeluted and purified. Each DNA sample was amplified as described above using the OVIFNT primers. The DNA molecules of any sample that yielded a positive PCR signal were cloned into λgtll (the subgenomic λgtll library) .
A. PCR Identification of Clones Containing Se¬ quences Complementary to OVIFNT. The λgtll phage were then plated for plaques and plaque-lift hybridization performed using the 3P-labelled OVIFNT CDNA probe. Approximately 20 clones were identi¬ fied that hybridized to the probe.
Plaques that hybridized to the probe were further analyzed by PCR using the OVIFNT primers described above. Six plaques which generated positive PCR signals were purified. The phage DNA from these clones was isolated and digested with .EcoRI restriction endonuclease. The DNA inserts were subcloned into pUC19 vectors and their nucleotide sequences determined by dideoxy nucleotide sequencings. B. Hybridization Identification of Clones Contain¬ ing Sequences Complementary to PCR-Positive Phage.
Recombinant phage from the λgtll subgenomic library were propagated in E. coli Y1080 and plated with E. coli
Y1090 at a density of about 20,000 plaques/150 mm plate.
The plates were overlaid with duplicate nitrocellulose filters, which were hybridized with a 32P-labelled probe from one of the six human IFNT CDNA clones isolated above. Three clones giving positive hybridization sig¬ nals were further screened and purified. The phage DNAs were isolated, digested with -EcoRI, subcloned into pUC19 vector and sequenced. The three clones yielded sequence information for over 800 bases relative to cap site (clones were sequenced in both orientations) . The nucle¬ ic acid sequence information is presented as SEQ ID NO:11 and the predicted protein coding sequence is presented as SEQ ID NO:12. Comparison of the predicted mature protein sequence (SEQ ID NO:12) of this gene to the predicted protein sequence of OVIFNT is shown in Figure 3.
EXAMPLE 8
Analysis of the Presence of HUIFNT mRNA bv RT-PCR Human placental cDNA libraries and an ovine cDNA library, constructed from day 15-16 conceptuses, were analyzed by hybridization to the OVIFNT CDNA probe, described above. cDNAs were size-fractionated on agarose gels and transferred to filters (Maniatis, et al . ; Sam- brook, et al . ) . Southern blot analysis with OvIFNr probe showed that the autoradiographic signals from human cDNA libraries were approximately 1/100 of the signal obtained using the OVIFNT CDNA library.
The presence of HUIFNT mRNA in human term placenta and amniocytes (26 weeks, 2 million cells) was analyzed by using reverse transcriptase-PCR (RT-PCR) method (Clon¬ tech Laboratories, Palo Alto CA) .
Total cellular RNA (tcRNA) isolated from human placenta, amniocytes and ovine conceptuses were reverse transcribed using the primer SEQ ID NO:14. The primer SEQ ID NO:13 was then added to the reaction and polymer¬ ase chain reaction carried out for 40 cycles. The PCR products were size fractionated on agarose gels and transferred to filters. The DNA on the filters was hybridized with.32P-labelled OVIFNT and HUIFNT CDNAS. The results of these analyses demonstrate the presence of human IFNT mRNA in the feto-placental annex. • The amino- cytes also expressed the messages corresponding to OVIFNT primers and human probe. In addition, a RT-PCR analysis for the presence of
HUIFNT was applied to the tcRNA isolated from human adult lymphocytes. A densitometric analysis revealed that IFNT mRNA exists in lymphocytes.
EXAMPLE 9
In Situ Hybridization
A. Tissue
Slides of semiserial 5-μ paraffin embedded sections from four healthy, different term and first trimester human placentas were examined.
B. cRNA Probe Preparation
From the cDNA clone isolated from OVIFNT amplified library a fragment corresponding to the OVIFNT CDNA bases #77-736 (base #1 is cap site; open reading frame of
OVIFNT CDNA is base #81-665; Figure 7) was subcloned into the transcription vector, pBS (New England Biolabs) . Several pBS clones were isolated, subcloned, and their nucleotides sequenced. From this clone a 3' fragment (bases #425-736) was excised using the restriction endon¬ ucleases NlalV and -EcoRI and subcloned into the tran¬ scription vector pBS. This vector was designated pBS/OVIFNT. After linearization of the pBS/OvIFNT plasmid, an antisense cRNA probe was synthesized by in vitro tran¬ scription (Sambrook, et al., 1989) using T7 RNA polymerase (Stratagene) . A trace amount of 3H-CTP (NEN-DuPont, Cambridge, MA) was used in the transcription reaction. dUTP labeled with digoxigenin (Boehringer-Mannheim, Indianapolis, IN) was incorporated into the cRNA and yield was estimated through TCA precipitation and scin¬ tillation counting.
\ C. Hybridization
In situ hybridization was performed using the anti- sense RNA probe, as described by Lawrence, et al . (1985) with the following modifications. Deparaffinized and hydrated sections were prehybridized for 10 minutes at room temperature in phosphate buffered saline (PBS) containing 5 mM MgCl2. Nucleic acids in the sections were denatured for 10 minutes at 65°C in 50% formamide/2 x SSC. Sections were incubated overnight at 37°C with a hybridization cocktail (30 μl/slide) containing 0.3 μg/ml digoxigenin-labelled cRNA probe and then washed for 30 minutes each at 37°C in 50 formamide/1 x SSC. Final washes were performed for 30 minutes each at room temper¬ ature in 1 x SSC and 0.1 x SSC. The sections were blocked for 30 minutes with 0.5% Triton X-100 (Sigma) and 0.5% non-fat dry milk.
Hybridization signal was detected using purified sheep antidioxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim) . After unbound anti¬ body was removed, nitroblue tetrazolium/5-bromo-4-chloro- 3-indolyl-phosphate substrate (Promega) and levamisole (Bector Laboratories, Burlingame, CA) were added for signal detection via colorimetric substrate generation. The tissues were counterstained in methyl green (Sigma) , dehydrated, and mounted.
As a control, some tissue sections were pretreated with 100 μg/ml of pancreatic RNaseA (Sigma) for 30 min¬ utes at 37°C. The RNase was inactivated on the slide with 400 units of RNase inhibitor (Promega) . The slides were then washed twice in 250 ml of PBS/5 mM MgCl2. In other control experiments, tRNA (Sigma) was substituted for the digoxigenin probes.
Specific hybridization was observed in all term and first trimester placental tissues in three separate experiments with various OVIFNT cRNA probe concentrations and blocking reagents.
First trimester placental villi composed of an outer layer of syncytiotrophoblast, an underlying layer of cytotrophoblast, and a central stromal region with vari- ous types of mesenchymal cells, displayed the highest transcript level of IFNT in the cytotrophoblast cells. Less intense but detectable levels were present in both the syncytiotrophoblast and stromal cells. A similar pattern of transcript expression was demonstrated in the placental villi of term tissue but the level of signal detection was low. First trimester extravillous tropho¬ blast displayed the highest amount of message and stained positive when present in the maternal blood spaces.
EXAMPLE 10
Antiviral Activity of IFNr The relative specific activity of OVIFNT, purified to homogeneity, was evaluated in antiviral assays. The antiviral assays were performed essentially as described above in Example 2. Specific activities are expressed in antiviral units/mg protein obtained from antiviral assays using either Madin-Darby bovine kidney (MDBK) cells or sheep normal fibroblasts (Shnf) . All samples were as¬ sayed simultaneously to eliminate interassay variability. The results, presented in Table 3, are the means of four determinations where the standard deviation was less than 10% of the mean.
Table 3 Antiviral Activity of IFNT and Known IFNs
IFNT had a higher specific activity than either rBoIFNα or rBoIFN7 (Table 3) . The NIH standard prepara¬ tion of rHuIFNα had a similar specific activity, while a commercial preparation of rHuIFNα exhibited low specific antiviral activity. Comparable relative antiviral activ¬ ity was demonstrated using either bovine or ovine cells.
EXAMPLE 11
Anti-Retroviral Activity and Cytotoxic Effects of IFNT
Highly purified OVIFNT was tested for anti-retrovi- ral and cytotoxic effects on feline peripheral blood lymphocytes exposed to the feline immunodeficiency retro- virus. This lentivirus produces a chronic AIDS-like syndrome in cats and is a model for human AIDS (Pederson, et al . , 1987). Replication of the virus in peripheral blood lymphocytes is monitored by reverse transcriptase activity in culture supernatants over time. The data from these assays are presented in Table 4.
Table 4 Effect of OVIFNT on FIV Replication
Addition of OVIFNT produced a rapid, dose-dependent decrease in reverse transcriptase (RT) activity (Table 4). While concentrations as low as 0.62 ng/ml of IFNT inhibited viral replication, much higher concentrations (40 ng/ml) having greater effects on RT-activity were without toxic effects on the cells. The results suggest that replication of the feline immunodeficiency virus was reduced significantly compared to control values when cells were cultured in the presence of OVIFNT.
IFNT appeared to exert no cytotoxic effect on the cells hosting the retrovirus. This was true even when IFNT was present at 40 ng per ml of culture medium.
EXAMPLE 12
Effects of IFNT on HIV Infected Human Peripheral Lymphocytes IFNT was also tested for activity against HIV infec¬ tion in human cells. Human peripheral blood lymphocytes, which had been infected with HIV (Crowe, et al . ) , were treated with varying concentrations of OVIFNT. Replica¬ tion of HIV in peripheral blood lymphocytes was monitored by reverse transcriptase activity in culture supernatants over time. Reverse transcriptase activity was measured essentially by the method of Hoffman, et al . The data from these assays are presented in Table 5.
Table 5
Effect of OVIFNT on HIV Replication in Human Peripheral Lymphocytes
As shown in Table 5, concentrations of OVIFNT pro¬ duced significant antiviral effects. A concentration of only 10 ng/ml resulted in over a 50% reduction in RT activity after only six days. A concentration of 500 ng/ml resulted in a 90% reduction in RT activity within 10 days.
The viability of human peripheral blood lymphocytes after treatment with IFNT, over a range of concentrations for 3-13 days, was evaluated by trypan blue exclusion. The results of this viability analysis are presented in Table 6.
Table 6
Effect of OVIFNT on Viability of HIV Infected Human Peripheral Lymphocytes
The data presented in Table 6 show no evidence of cytotoxic effects attributable to the administration of IFNT.
EXAMPLE 13 Inhibition of Cellular Growth
The effects of IFNT on cellular growth were also examined. Anti-cellular growth activity was examined using a colony inhibition assay. Human amnion (WISH) or MDBK cells were plated at low cell densities to form colonies originating from single cells. Cells were cultured at 200 or 400 cells/well in 24 well plates in HMEM supplemented with 2% fetal bovine serum (FBS) and essential and nonessential amino acids. Various dilu¬ tions of interferons were added to triplicate wells, and the plates were incubated for 8 days to allow colony formation. Colonies were visualized after staining with crystal violet, and counted. Cell cycle analysis was performed with HMEM containing 0.5% "spent" media for an additional 7 days. WISH cells were used without being synchronized.
For examination of IFNT activity, cells were re- plated at 2.5 x 105 cells/well in HMEM with 10% FBS in 6 well plates. Various dilutions of IFNT alone or in combination with peptides were added to achieve a final volume of 1 ml. Plates were incubated at 37°C in 5% Co2 for 12, 15, 18, 24, or 48 hours. Cells were treated with trypsin, collected by low speed centrifugation and washed. The cell pellet was blotted dry and 250 μl of nuclear staining solution (5 mg propidium iodide, 0.3 ml NP40 and 0.1 gm sodium citrate i 100 ml distilled H20) was added to each tube. The tubes were incubated at room temperature. After 10 minutes, 250 μl of RNase (500 units/ml in 1.12% sodium citrate) was added per tube and incubated an additional 20 minutes. Nuclei were filtered through 44 μm mesh, and analyzed on a FACStar (Becton Dickinson, Mountain View, CA) using, the DNA Star 2.0 software.
In the cellular growth assay using colony formation of both the bovine epithelial line, MDBK, and the human amniotic line, WISH, OVIFNT inhibited both colony size and number. Ovine IFNT was more effective than human IFNα on the human cell line; thus, it is very potent in cross-species activity. Its activity was dose-dependent, and inhibition of proliferation could be observed at concentrations as low as l unit/ml. Concentrations as high as 50,000 units/ml (units of antiviral activity/ml) stopped proliferation, while cell viability was not impaired.
Cell cycle analysis by flow cytometry with propidiu iodide-stained WISH cells revealed an increased propor- tion of cells in G2/M after 48 hours of OVIFNT treatment. IFNT, therefore, appears to inhibit progress of cells through S phase. Ovine IFNT antiproliferative effects can be observed as early as 12 hours after the initiation of culture and are maintained through 6 days. The results presented above demonstrate both the antiproliferative effect of IFNT as well as its low cytotoxicity.
EXAMPLE 14 Further Antiproliferative Effects of IFNT
The antiproliferative effects of OVIFNT were studied for a rat cell line and a bovine cell line. The rate of 3H-thymidine incorporation was used to assess the rate of cellular proliferation. Rat (MtBr7 .c5) or bovine kidney (MDBK) cells were seeded in phenol red-free DME-F12 medium supplemented with 3% dextran-coated charcoal stripped Controlled Process Serum Replacement 2 (CPSR 2, Sigma) and 5% dex¬ tran-coated charcoal stripped fetal, bovine serum (FBS) . After attaching for approximately 15-18 hours, the cells were washed once with serum-free DME-F12 medium. The medium was replaced with phenol red-free DME-F12 medium supplemented with 3% stripped CPSR2, 1% stripped FBS ("3/1" medium) or 3/1 medium containing OVIFNT at various units of antiviral activity as determined in the vesicu¬ lar stomatitis virus challenge assay for interferons (Example 2) . Media containing a similar dilution of buffer (undiluted buffer - 10 mM Tris, 330 mM NaCl, [TS]), in which the OVIFNT was dissolved was used for controls.
Cells were pulse labeled with 3H-thymidine for 2 hours at approximately 48 hours post-treatment. The trichloroacetic acid (TCA) precipitable incorporated counts were determined by scintillation counting. Three replicates were included per treatment. Mean values for OVIFNT treatments were compared to samples containing comparable dilutions of carrier TS buffer. Results of . these experiments are shown in Table 7.
Table 7 3H-Thvmidine Incorporation
As can be seen from Table 7, OVIFNT drastically reduced the rate of cellular proliferation (based on thymidine incorporation) for each of the cell lines tested EXAMPLE 15
Antiproliferative Effects of IFNT on Human Tumor Cell Lines
The antiproliferative activity of OVIFNT on human tumor cell lines was evaluated by measuring the rate of 3H-thymidine incorporation into cells which have been treated with OVIFNT.
For experiments on tumor lines that grow in suspen¬ sion, 1 ml of cells were plated at from 2.5 - 5 X 105 cells/well in 24-well plates. Triplicate wells received either the appropriate media, 100, 1,000 or 10,000 units/ml of OVIFNT or equivalent antiviral concentrations of rHuIFNα2A (Lee Biomolecular) . After 48 hours of incubation, cells were counted and viability assessed by trypan blue exclusion.
Adherent tumor lines were plated at 2.5 X 105 cells/well in 1 ml in 6-well plates. They received interferon treatments as just described, but were tryp- sinized prior to counting. Significant differences between treatments were assessed by an analysis of variance followed by Scheffe's F-test. Cell cycle analysis was performed by flow cyto¬ metry using propidium iodide.
A. Breast Adenocarcinoma Cells.
Human MCF7 breast adenocarcinoma cells were seeded from logarithmically growing cultures in phenol red-free DME-F12 medium supplemented with 3% dextran-coated char¬ coal stripped CPSR and 5% dextran-coated FBS. After attaching for approximately 15-18 hours, the cells were washed once with serum-free DME-F12 medium. The medium was replaced with phenol red-free DME-F12 medium supple¬ mented with 3% stripped CPSR2, 1% stripped FBS ("3/1" medium) or 3/1 medium containing OVIFNT at the indicated number of units of antiviral activity as determined in the vesicular stomatitis virus challenge assay for inter¬ ferons. Media containing a similar dilution of buffer (undiluted buffer = 10 mM Tris, 330 mM NaCl [TS]) was used for controls. Cells were pulse labeled with 3H- thymidine for 2 hours at approximately 48 hours post- treatment.
The trichloroacetic acid (TCA) precipitable incorpo¬ rated counts were determined by scintillation counting. Three replicates were included per treatment. Mean values for OVIFNT treatments were compared to samples containing comparable dilutions of carrier TS buffer. The results of these analyses are shown in Table 8.
Table 8 3H-Thvmidine Incorporation
As can be seen from the results shown in Table 8, OVIFNT was able to substantially reduce the rate of 3H- thymidine incorporation in the human carcinoma cell line. This demonstrates the efficacy of OVIFNT in inhibiting tumor cell proliferation, in particular, mammary tumor cell proliferation. B. Human Promvelocvtic Leukemia. A comparison of the antiproliferative effects of OVIFNT and IFNα was conducted using HL-60 (human leuke¬ mia) cells (Foa, et al.; Todd, et al . ) essentially as described above for MDBK cells. Both OVIFNT and rHuIFNα inhibit HL-60 cell proliferation. Results of one of three replicate experiments are presented as mean % growth reduction ± SD in Figure 4. Figure 4 shows that both OVIFNT and IFNα were able to drastically reduce growth of HL-60 cells. The growth reduction for each compound exceeded 60% for each concentration tested. At 10,000 units/ml, OVIFNT caused an approximately 80% reduction in growth while IFNα caused a 100% reduction in growth. However, the data presented in Figure 4 reveal, that a substantial factor in the ability of IFNα to reduce growth was its toxic effect on the cells. At 10,000 units/ml, the toxicity of IFNα resulted in less than 25% of the cells remaining viable. By contrast, nearly 100% of the cells remained viable when OVIFNT was applied at 10,000 units/ml.
Figure 5 presents data demonstrating that rHuIFNα is cytotoxic. In the figure, results of one of three repli¬ cate experiments are presented as mean % viability ± SD.
C. Human Cutaneous T Cell Lymphoma. The cutaneous T cell lymphoma, HUT 78, responded similarly to HL-60 when treated with IFNT (Figure 9) . Both OVIFNT and rHuIFNα reduce HUT 78 cell growth, but 10,000 units/ml of rHuIFNα decreased the cell number below that originally plated (5X105) . This is indicative of a reduction in cell viability to approximately 60%.
Cell cycle analysis (performed by cell flow cytome¬ try) revealed an increased proportion of cells in G2/M phase of the cell cycle upon 48 hours of treatment with both interferons (Table 10) . In Table 10 the results from one of three replicate experiments are presented as the percentage of cells in each phase of the cell cycle. 10,000 events were analyzed per sample.
This result is likely due to the slower progress of cells through the cell cycle. In the sample treated with 10,000 units/ml of rHuIFNα, a large percentage of events with low forward and high side scatter, identifying dead cells, were present. This is consistent with the data obtained from proliferation experiments, where only OVIFNT inhibited HUT 78 proliferation without toxicity.
Table 10. HUT 78 Cell Cycle Analysis.
D. Human T Cell Lymphoma.
The T cell lymphoma cell line H9 was slightly less sensitive to the antiproliferative effects of the IFNs than the tumor cell lines described above. Results of one of three replicate experiments are presented in Figure 10 as mean % growth reduction ± SD. While rHuIFNα was not toxic to the H9 cells, it failed to inhibit cell division significantly at any of the concentrations examined. In contrast, OVIFNT was observed to reduce H9 growth by approximately 60% (Figure 10) . Thus, only OVIFNT is an effective growth inhibitor of this T cell lymphoma.
The results presented above demonstrate both the antiproliferative effect of IFNT as well as its low cytotoxicity.
EXAMPLE 16 Preliminary In Vivo Treatment with OVIFNT Three groups of 4 C57B1/6 mice per group were given 2.5 X 104 B16-F10 cells via the tail vein: B16-F10 is a syngeneic mouse transplantable tumor selected because of its high incidence of pulmonary metastases (Poste, et al . , 1981). Interferon treatment was initiated 3 days after the introduction of the tumor cells. Each mouse received 100 μl of either PBS alone, PBS containing l X 10s units of OVIFNT, or PBS containing 1 X 10s units of recombinant murine IFNα (MuIFNα) , i.v. per day for 3 consecutive days. Mice were sacrificed at 21 days and the lungs were preserved in 10% buffered formalin. The frequency of pulmonary metastases were compared between control mice (PBS) , OVIFNT-treated mice, and MuIFNα-treated mice. The results of these in vivo administrations demonstrated that OVIFNT dramatically reduced B16-F10 pulmonary tu¬ mors. These results support the use of IFNT as an effi¬ cacious antineoplastic agent in vivo.
EXAMPLE 17 Competitive Binding of IFNT Peptide Fragments
A. The Ability of IFNτ-Based Peptides to Block IFNT and IFN-α Antiviral Activity.
Overlapping synthetic peptides were synthesized corresponding to the entire IFNT sequence (Figure 6) . Average hydropathicity values were calculated by taking the sum of the hydropathy values for each amino acid divided by the total number of amino acids in each se¬ quence. Hydropathy values were taken from Kyte, et al . (1982) . These peptides were of approximately the same molec¬ ular weight but differed slightly in overall hydrophili- city. Despite this difference, all peptides were anti¬ genic as demonstrated by the production of rabbit anti¬ sera with titers greater than 1:3,000 as assessed by ELISA (Harlow, et al . ) .
The peptides were used to inhibit the antiviral activity (Example 2) of OVIFNT and rBoIFNα. The results of this analysis are presented in Figure 12: 1 mM N- and C-terminal peptides both effectively blocked the antivi- ral activity of OVIFNT using MDBK cells. A third pep¬ tide, representing amino acids 62-92, also reduced IFNT antiviral activity (70% inhibition) . The peptide OVIFNT (119-150) showed minimal inhibitory activity. The OVIFNT (34-64) and (90-122) peptides had no apparent inhibitory activity.
Peptide inhibition of OVIFNT antiviral activity was also examined as follows. Monolayers of Madin Darby bovine kidney cells were incubated with 40 units/ml OVIFNT in the presence or absence of various concentra- tions of OVIFNT peptides (see Figure 13) . Results in
Figure 13 are expressed as the percent of control antivi¬ ral activity: that is, in the absence of any competing peptide. Data presented are the means of 6 replicate experiments. The data demonstrate that inhibition by OVIFNT (1-37), (62-92), (119-150), and (139-172) were significantly different than OVIFNT (34-64) and (90-122) at 10-3 M and 3 x 10'3 M. OVIFNT (139-172) was signifi¬ cantly different than all other peptides at 10'3 M. Sig¬ nificance was assessed by analysis of variance followed by Scheffe's F test at p < 0.05. Thus, OVIFNT (1-37) (62-92), (119-150), and (139-172), in particular (139- 172), may represent receptor binding regions for IFNr.
The ability of the OVIFNT peptides to inhibit bovine IFNα (BoIFNα) antiviral activity was examined as follows. Monolayers of Madin Darby bovine kidney cells were incu¬ bated with 40 units/ml bovine IFNα in the presence or absence of various concentrations of OVIFNT peptides. The results are presented in Figure 14 and are expressed as the percent of control antiviral activity in the absence of OVIFNT peptides. The data presented are the means of 4 replicate experiments. The results indicate that inhibition by OVIFNT (62-92), (119-150), and (139- 172) were significantly different from OVIFNT (1-37) , (34-64) and (90-122) at 10'3 M. OVIFNT (139-172) was significantly different than OVIFNT (1-37), (34-64) and (90-122) at 3 x 10"3 M. Significance was assessed by analysis of variance followed by Scheffe's F test at p < 0.05. Thus, OVIFNT (62-92), (119-150), and (139-172), in particular (139-172) , may represent common receptor binding regions for IFNT and bovine IFNα.
Peptide inhibition by OVIFNT peptides of human IFNα antiviral activity was also examined. Monolayers of Madin Darby bovine kidney cells were incubated with 40 units/ml human IFNα in the presence or absence of various concentrations of OVIFNT peptides. The results are expressed as the percent of control antiviral activity in the absence of OVIFNT peptides. The data are presented in Figure 15 and are the means of 3 replicate experi- ments. OVIFNT (139-172) was significantly different from all other peptides at 10"3 M. Significance was assessed by analysis of variance followed by Scheffe's F test at p < 0.05. Thus, OVIFNT (139-172) may represent a common receptor binding region for IFNT and various IFNα(s). The OVIFNT peptides described above appear to have no effect on the antiviral activity of IFN7. Peptide inhibition of bovine IFN7 antiviral activity was evaluat¬ ed as follows. Monolayers of Madin Darby bovine kidney cells were incubated with 40 units/ml bovine IFN gamma in the presence or absence of various concentrations of OVIFNT peptides. Results are expressed as the percent of control antiviral activity in the absence of OVIFNT peptides. The data are presented in Figure 16 and are the means of 3 replicate experiments. There were no significant differences among peptides as assessed by analysis of variance followed by Scheffe's F test at p < 0.05.
The two synthetic peptides OVIFNT(1-37) and OVIFNT(139-172) also blocked OVIFNT anti-FIV and anti-HIV activity. Reverse transcriptase (RT) activity (Exampleε 12 and 13) was monitored over a 14 day period in FIV- infected FET-1 cells (1 X 106/ml) and HIV-infected HPBL (l X 106/ml) . Control cultures received no OVIFNT. OVIFNT was used at 100 ng/ml, and peptides were used at 200 μM. Data from a representative experiment are expressed as cpm/ml culture supernatant and are presented for FIV infected cells, Figure 11A, and HIV infected cells, Figure 11B. Both the N- and C-terminus of OVIFNT appear to be involved in its anti-retroviral activity. While both peptides blocked FIV RT activity, only the C-termi¬ nal peptide, OVIFNT(139-172) , was an efficient inhibitor of vesicular stomatitis virus activity on the feline cell line, Fc9. Thus the C-terminal regions of type I IFNs may bind to common site on the type I IFN receptor, while the N-terminal region may be involved in the elicitation of unique functions. B. Anti-Peptide Sera.
The ability of anti-peptide antisera to inhibit OVIFNT antiviral activity was also determined. Antipep¬ tide antisera inhibition of OVIFNT antiviral activity was evaluated as follows. Monolayers of MDBK cells were incubated with 20 units/ml of OVIFNT in the presence a 1:30 dilution of either preimmune sera or antisera to each of the OVIFNT peptides described above. In Figure 17 the data from duplicate experiments are presented as the mean percent inhibition of OVIFNT antiviral activity produced by antipeptide antisera relative to the appro¬ priate preimmune sera ± standard error. Significant differences were assessed by analysis of variance fol¬ lowed by Scheffe's F test at p < 0.05. Consistent with peptide inhibition of antiviral activities, sera contain¬ ing antibodies immunoreactive to OVIFNT (1-37) , OVIFNT (62-92) , and OVIFNT (139-172) were also the most effec¬ tive inhibitors of OVIFNT antiviral activity, with anti¬ bodies directed against the N-terminal and C-terminal peptides being the most efficacious.
The same sera were also used to examine their effect on the binding of IFNT to its receptor.
The IFNT binding assay was carried out as follows.
Five μg of IFNT was iodinated for 2 minutes with 500 μCi of Na125I (15 mCi/μg; Amersham Corporation, Arlington Heights, IL) in 25 μl of 0.5 M potassium phosphate buff¬ er, pH 7.4, and 10 μl of chloramine-T (5 mg/ml) (Griggs, et al . , 1992). The specific activity of the iodinated protein was 137 μCi/μg. For binding assays, monolayers of MDBK cells were fixed with paraformaldehyde and blocked with 5% nonfat dry milk. Cells were incubated with 5 nM 12SI-IFNτ in phosphate buffered saline with 1% BSA for 2 hours at 4°C in the presence or absence of a 1:30 dilution of sera containing antibodies raised against IFNT peptides or the appropriate preimmune sera. Specific binding was assessed by incubation with a 100- fold molar excess of unlabeled IFNT. Specific binding of 36% was determined by competition with 500 nM unlabeled IFNT. For example, total counts bound were 6850 ± 133, and a 100-fold molar excess of OVIFNT produced 4398 ± 158 counts per minute. After incubation, the monolayers were washed three times, solubilized with 1% sodium dodecyl sulfate, and the radioactivity counted. Data from three replicate experiments are presented in Figure 18 as the mean percent reduction of OVIFNT specific binding pro¬ duced by antipeptide antisera relative to the appropriate preimmune sera ± standard deviation. Significant differ¬ ences were assessed by analysis of variance followed by Scheffe's F test.
The same sera (containing antibodies immunoreactive to OVIFNT (1-37), OVIFNT (62-92), and OvIFNT (139-172)) were the most effective inhibitors of 125I-IFNτ binding to its receptor on MDBK cells. The lack of effect of sera immunoreactive with other IFNT-derived peptides was not a function of titer against OVIFNT, since each sera had equal or greater titer to their respective peptide rela- tive to the three inhibiting sera: similar results were obtained when sera reactivity against the whole OVIFNT molecule was assessed by ELISA for each sera.
These peptides, although apparently binding to the interferon receptor, did not in and of themselves elicit interferon-like effects in the cells.
C. Anti-Proliferative Activity.
Functionally important sites for the antiprolifera¬ tive activity of IFNT were also examined using synthetic peptides (Table 11) . Cellular proliferation was assayed as described above using MDBK cells. MDBK cells were cultured at 5 x lθ5 cells/well in experiments l and 2 or 10 x 10s cells in experiment 3 and treated with medium alone, IFNT at a concentration of 300 units/ml and pep¬ tides at 1 mM for 48 hours. Duplicate wells were counted in each of three replicate experiments. For statistical analysis, data were normalized based on medium alone and assessed by analysis of variance followed by Least Sig¬ nificant Difference multiplate comparison test (p > 0.05) .
Table 11
Peptide Inhibition of IFNT Antiproliferative Activity
When proliferation of MDBK cells was monitored over a two-day period, cell number increased roughly 2-fold with greater than 95% viability. Addition of 300 units/ml of OVIFNT entirely eliminated cell proliferation without a decrease in cell viability. Ovine IFNT (119- 150) was the most effective inhibitor of IFNT antiproli¬ ferative activity.
Antisera to IFNT (119-150) , which inhibited binding of OVIFNT to receptor, also reversed the OVIFNT antipro- liferative effect. Several other peptides, notably IFNT (139-172) , reversed the OVIFNT antiproliferative effect, but to a lesser extent.
EXAMPLE 18 Further Analysis of the Cellular and Anti-Viral Effects of IFNT A. HIV Anti-Viral Effects. The antiviral effects of IFNT against HIV were evaluated by treating human PBMC cells with various amounts of either recombinant ovine IFNT (r-OvIFNτ) or recombinant human IFNα2 at the time of infection with HIV. Drug was present throughout the experiment. At day 7 and day 14, p24 production was determined (by ELISA (Wang, et al . , 1988, 1989) and compared to a zero drug control. The results of this analysis are presented in Table 12.
Table 12
The data from these experiments support the conclu- sion that, at relatively low concentrations, IFNα and
IFNT are effective in reducing the replication of HIV in human lymphocytes. B. In vitro Cytotoxicity Test in PBMCs Human PBMCs were seeded at 5 x 105 cells/ml. Cells were stimulated at day 0 with 3 μg/ml PHA. Cells were treated with recombinant human IFNα2A (at concentrations of 10, 100, 1,000 and 10,000 units/ml) and IFNT (at concentrations of 2.6, 26, 260, 2,600, 26,000, 260,000, and 2,600,000 units/ml) in 200 μl/wells (4 replicates of each concentration using 96 well flat bottom plates) . Control cultures were given no interferons. After 4 days of incubation, cells were pulsed for 9 hours using 3H- thymidine at 1 uCi/well. The cells were harvested and the incorporation of labeled thymidine into DNA was determined (Figure 8) .
No cytotoxicity was observed by measuring the uptake of thymidine at any concentration of IFNT. However, rHuIFNα2 was toxic to cells at 1,000 units/ml.
In a second experiment, the same human PBMCs were treated with either IFNT or human IFNα2A at concentra¬ tions of 100 units/ml or 10,000 units/ml. After 3 days or 8 days of incubation, viable cells were counted by flow cytometry. The results of this analysis are pre¬ sented in Table 13.
TABLE 13
No cytotoxicity was observed in the cells treated with IFNT. However, there was 10% cell death in IFNα treated cells at Day 3 and 49% cell death at Day 8.
C. Inhibition of Hepatitis B Virus DNA Replication in Hepatocytes
The cell line used, HepG2-T14, is a human cell that was derived from liver cells transfected with Hepatitis B
Virus (HBV) . The cell line semi-stably produces HBV virus: over time the cell line's production of HBV intracellular DNA and secreted virus decreases. In order to maximize production of HBV DNA and virus, the cells are pre-treated with deAZA-C (5-azacytidine; Miyoshi, et al . ) to induce production of the virus. Treatment was for 2-3 days and the amount of induction was about a factor of two.
The cells were then treated with either the IFNα and IFNT at levels of 0, 5,000, 10,000, 20,000 and 40,000 units per ml. All levels of either IFNα or IFNT reduced DNA pro¬ duction by about a factor of 2 compared to the no drug control.
D. Inhibition of Hepatospecific Messenger RNA Production in Hepatocytes
The hepatocyte cell line HepG2-T14 (described above) was examined for the effects of IFNα and IFNT on hepato¬ specific mRNA production. Cells were incubated in con¬ centrations of IFNα or IFNT at 0, 5,000, 10,000, 20,000, and 40,000 units per ml. The messenger RNAs for the hepatocyte specific proteins Apo E and Apo Al were de¬ tected by hybridization analysis (Sambrook, et al . ; Maniatis, et al . ) using probes specific for these two mRNA's (Shoulders, et al . , and Wallis, et al . ) . No reduction of mRNA production was seen for Apo E or Apo Al mRNA production with up to 40,000 units of either IFNα or IFNT. This result suggests that the reduction of viral DNA replication in previous experi- ments was not due to the effects of IFNs on cellular house-keeping activities; rather the reduction was likely due to specific inhibition of viral replication in the host cells.
EXAMPLE 19
Isolation of Interferon-T Fusion Protein Sepharose 4B beads conjugated with anti-beta galac- tosidase is purchased from Promega. The beads are packed in 2 ml column and washed successively with phosphate- buffered saline with 0.02% sodium azide and 10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin) .
The IFNT coding sequence (e .g. , Figure 7) is cloned into the polylinker site of lambda gtll. The IFNT coding sequence is placed in-frame with the amino terminal β- galactosidase coding sequences in lambda gtll. Lysogens infected with gtll/IFNτ are used to inoculate 500 ml of NZYDT broth. The culture is incubated at 32°C with aeration to an O.D. of about 0.2 to 0.4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gtll peptide synthesis, and incubated further at 37°C for 1 hour. The cells are pelleted by centrifuga¬ tion, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use. The resuspended cells are frozen in liquid nitrogen then thawed, resulting in substantially complete cell lysis. The lysate iε treated with DNasel to digest bacterial and phage DNA, as evidenced by a gradual loss of viscosity in the lysate. Non-solubilized material is removed by centrifugation.
The clarified lysate material is loaded on the Sepharose column, the ends of the column closed, and the column placed on a rotary shaker for 2 hrs. at room temperature and 16 hours at 4°C. After the column set¬ tles, it is washed with 10 ml of TX buffer. The fused protein iε eluted with 0.1 M carbonate/bicarbonate buff¬ er, pHlO. Typically, 14 ml of the elution buffer is passed through the column, and the fusion protein is eluted in the first 4-6 ml of eluate.
The eluate containing the fusion protein is concen¬ trated in "CENTRICON-30" cartridges (Amicon, Danvers, Mass.). The final protein concentrate is resuspended in, for example, 400 μl PBS buffer. Protein purity is ana¬ lyzed by SDS-PAGE.
For polyclonal antibodies, the purified fused pro¬ tein is injected subcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum is typically collected at 6 and 8 weeks.
EXAMPLE 20
Preparation of Anti-IFNτ Antibody A. Expression of Glutathione-S-Transferase Fusion Proteins.
The IFNT coding sequence (e . g. , Figure 7) is cloned into the pGEX vector (Boyer, et al . ; Frangioni, et al . ;
Guan, et al . ; Hakes, et al . ; Smith, et al . , 1988). The pGEX vector (Smith, et al . ) was modified by inεertion of a thrombin cleavage sequence in-frame with the glutathi- one-S-transferase protein (GST — sj26 coding sequence) . This vector is designated pGEXthr. The IFNT coding sequence is placed in-frame with the εj26-thrombin coding sequences (Guan, et a . ; Hakes, et al . ) . The IFNT coding sequence insert can be generated by the polymerase chain reaction using PCR primers specific for the insert. The IFNT fragment is ligated to the linearized pGEXthr vector. The ligation mixture is transformed into E. coli and ampicillin resistant colonies are selected. Plasmids are isolated from the ampicillin resistant colonies and analyzed by restriction enzyme digestion to identify clones containing the IFNT insert (vector desig¬ nated pGEXthr-IFNT) . E. coli strain XL-I Blue is transformed with pGEXthr-IFNT and is grown at 37°C overnight. DNA iε prepared from randomly-picked colonies. The presence of the insert coding sequence is typically confirmed by (i) restriction digest mapping, (ii) hybridization screening uεing labelled IFNT probes (i.e.. Southern analysis), or (iii) direct DNA sequence analysis.
B. Partial Purification of Fusion Proteins. A pGEXthr-IFNT clone is grown overnight. The over- night culture is diluted 1:10 with LB medium containing ampicillin and grown for one hour at 37°C. Alternative¬ ly, the overnight culture is diluted 1:100 and gro-- to OD of 0.5-1.0 before addition of IPTG (isopropylth -β- galactoεide) . IPTG (GIBCO-BRL, Gaithersburg MD) is added to a final concentration of 0.2-0.5 mM for the induction of protein expreεεion and the incubation iε typically continued for 2-5 hourε, preferably 3.5 hours.
Bacterial cells are harvested by centrifugation and resuspended in 1/100 culture volume of MTPBS (150 mM NaCl, 16 mM Na2HP04, 4 mM NaH2P04) . Cells are lysed by lysozyme, sonication or French press, and lysateε cleared of cellular debriε by centrifugation.
An aliquot of the supernatant obtained from IPTG- induced cultures of pGEXthr-IFNτ-containing cells and an 86 aliquot of the supernatant obtained from IPTG-induced cultures of pGEXthr-vector alone are analyzed by SDS- polyacrylamide gel electrophoresis followed by Western blotting, as described below. If necesεary, the extractε can be concentrated by ultrafiltration uεing, for example, a "CENTRICON 10" filter.
Alternatively, the fusion proteins are partially purified over a glutathione agarose affinity column as deεcribed in detail by Smith, et a . In thiε method, 100 ml cultureε are grown overnight. The cultureε are dilut¬ ed to 1 liter, and the cellε grown another hour at 37°C. Expression of the fusion proteins is induced using IPTG. The induced cultures are grown at 37°C for 3.5 hours. Cells are harvested and a εonicator uεed to lyse the cells. Cellular debris is pelleted and the clear lysate loaded onto a glutathione "SEPHAROSE" column. The column is washed with several column volumes. The fusion pro¬ tein is eluted from the affinity column with reduced glutathione and dialyzed. The IFNT can be liberated from the hybrid protein by treatment with thrombin. The εj26 and IFNT fragmentε of the hybrid protein can then be εeparated by εize fractionation over columnε or on gelε. Alternatively, the IFNT portion of the hybrid pro- tein is released from the column by treatment with throm¬ bin (Guan, et al . ; Hakes, et al . ) .
C. Antibodies Against the Fusion Protein. The purified Sj26/IFNτ fused protein iε injected subcutaneously in Freund's adjuvant in a rabbit. Approx¬ imately 1 mg of fused protein is injected at dayε 0 and 21, and rabbit εerum is typically collected at 6 and 8 weeks. A second rabbit is similarly immunized with purified Sj26 protein obtained from control bacterial lysate. Minilysates from the following bacterial cultures are prepared: (1) KM392 cells infected with pGEXthr and pGEXthr containing the IFNT insert; and (2) cells infect¬ ed with lambda gtll containing the IFNT insert. The minilysates and a commercial source 8-galactosidase are fractionated by SDS-PAGE, and the bands transferred to nitrocellulose filters for Western blotting (Sambrook, et al . ; Ausubel, et al . ) .
Summarizing the expected results, serum from control (Sj26) rabbits is immunoreactive with each of the Sj.26 and Sj26 fused protein antigens. Serum from the animal immunized with Sj26/IFNτ fused protein is reactive with all Sj-26 and beta-gal fusion proteins containing IFNT coding εequenceε, indicating the presence of specific immunoreaction with the IFNT antigen. None of the sera are expected to be immunoreactive with beta-galactosi- dase.
Anti-IFNT antibody present in the sera from the ani¬ mal immunized with the Sj26/IFNτ is purified by affinity chromatography (uεing immobilized recombinantly produced IFNT aε ligand, eεsentially as described above in Example 12 for the anti-beta-galactosidase antibody) .
While the invention has been described with refer- ence to specific methods and embodiments, it will be appreciated that various modificationε and changes may be made without departing from the invention. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: University of Florida
The Women's Research Institute
(ii) TITLE OF INVENTION: Interferon Tau Compositions and Methods of Use
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Law Offices of Peter J. Dehlinger (B) STREET: 350 Cambridge Ave., Suite 300
(C) CITY: Palo Alto
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94306
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/969,890
(B) FILING DATE: 30-OCT-1992
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Fabian, Gary R.
(B) REGISTRATION NUMBER: 33,875
(C) REFERENCE/DOCKET NUMBER: 5600-0001.41
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-324-0880 (B) TELEFAX: 415-324-0960
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 518 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Ovis aries
(B) STRAIN: Domestic (D) DEVELOPMENTAL STAGE: Blastula (blastocyst)
(F) TISSUE TYPE: Trophectoderm
(G) CELL TYPE: Mononuclear trophectoderm cells
(vii) IMMEDIATE SOURCE: (B) CLONE: oTP-la
(viii) POSITION IN GENOME:
(C) UNITS: bp
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..518
(x) PUBLICATION INFORMATION: (A) AUTHORS: Ott, Troy L
Van Heeke, Gino Johnson, Howard M Bazer, Fuller W (B) TITLE: Cloning and Expression in Saccharomyces cerevisiae of a Synthetic Gene for the Type I
Trophoblast Interferon Ovine Trophoblast Protein-1:Purification and Antiviral Activity
(C) JOURNAL: J. Interferon Res.
(D) VOLUME: 11
(F) PAGES: 357-364
(G) DATE: 1991
(K) RELEVANT RESIDUES IN SEQ ID NO:l: FROM 1 TO 518
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TGC TAC CTG TCG CGA AAA CTG ATG CTG GAC GCT CGA GAA AAT TTA AAA Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Ala Arg Glu Asn Leu Lys 1 5 10 15
CTG CTG GAC CGT ATG AAT CGA TTG TCT CCG CAC AGC TGC CTG CAA GAC Leu Leu Asp Arg Met Asn Arg Leu Ser Pro His Ser Cys Leu Gin Asp 20 25 30
CGG AAA GAC TTC GGT CTG CCG CAG GAA ATG GTT GAA GGT GAC CAA CTG 1 Arg Lys Asp Phe Gly Leu Pro Gin Glu Met Val Glu Gly Asp Gin Leu 35 40 45
CAA AAA GAC CAA GCT TTC CCG GTA CTG TAT GAA ATG CTG CAG CAG TCT 1 Gin Lys Asp Gin Ala Phe Pro Val Leu Tyr Glu Met Leu Gin Gin Ser 50 55 60
TTC AAC CTG TTC TAC ACT GAA CAT TCT TCG GCC GCT TGG GAC ACT ACT 2
Phe Asn Leu Phe Tyr Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr
65 70 75 80
CTT CTA GAA CAA CTG TGC ACT GGT CTG CAA CAG CAA CTG GAC CAT CTG 2
Leu Leu Glu Gin Leu Cys Thr Gly Leu Gin Gin Gin Leu Asp His Leu 85 90 95
GAC ACT TGC CGT GGC CAG GTT ATG GGT GAA GAA GAC TCT GAA CTG GGT 3 Asp Thr Cys Arg Gly Gin Val Met Gly Glu Glu Asp Ser Glu Leu Gly 100 105 110
AAC ATG GAT CCG ATC GTT ACT GTT AAA AAA TAT TTC CAG GGT ATC TAC 3 Asn Met Asp Pro lie Val Thr Val Lys Lys Tyr Phe Gin Gly lie Tyr 115 120 125 GAC TAC CTG CAG GAA AAA GGT TAC TCT GAC TGC GCT TGG GAA ATC GTA 4 Asp Tyr Leu Gin Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu lie Val 130 135 140
CGC GTT GAA ATG ATG CGG GCC CTG ACT GTG TCG ACT ACT CTG CAA AAA 4 Arg Val Glu Met Met Arg Ala Leu Thr Val Ser Thr Thr Leu Gin Lys 145 150 155 160
CGG TTA ACT AAA ATG GGT GGT GAC CTG AAT TCT CCG 5 Arg Leu Thr Lys Met Gly Gly Asp Leu Asn Ser Pro
165 170
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 172 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Ala Arg Glu Asn Leu Lys 1 5 10 15
Leu Leu Asp Arg Met Asn Arg Leu Ser Pro His Ser Cys Leu Gin Asp 20 25 30
Arg Lys Asp Phe Gly Leu Pro Gin Glu Met Val Glu Gly Asp Gin Leu 35 40 45
Gin Lys Asp Gin Ala Phe Pro Val Leu Tyr Glu Met Leu Gin Gin Ser 50 55 60
Phe Asn Leu Phe Tyr Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80 Leu Leu Glu Gin Leu Cys Thr Gly Leu Gin Gin Gin Leu Asp His Leu 85 90 95
Asp Thr Cys Arg Gly Gin Val Met Gly Glu Glu Asp Ser Glu Leu Gly 100 105 110
Asn Met Asp Pro lie Val Thr Val Lys Lys Tyr Phe Gin Gly lie Tyr 115 120 125
Asp Tyr Leu Gin Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu lie Val 130 135 140
Arg Val Glu Met Met Arg Ala Leu Thr Val Ser Thr Thr Leu Gin Lys 145 150 155 160
Arg Leu Thr Lys Met Gly Gly Asp Leu Asn Ser Pro 165 170
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 516 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
TGTGACTTGT CTCAAAACCA CGTTTTGGTT GGTAGAAAGA ACTTAAGACT ACTAGACGAA 6
ATGAGACGTC TATCTCCACA CTTCTGTCTA CAAGACAGAA AGGACTTCGC TTTGCCTCAG 12
GAAATGGTTG AAGGTGGCCA ACTACAAGAA GCTCAAGCGA TATCTGTTTT GCACGAAATG 18
TTGCAACAAA GCTTCAACTT GTTCCACACC GAACACTCTT CGGCCGCTTG GGACACCACC 24
TTGTTGGAAC CATGTAGAAC CGGTTTGCAC CAACAATTGG ACAACTTGGA TGCATGTTTG 30 GGTCAAGTTA TGGGTGAAGA AGACTCTGCT CTCGGGAGAA CCGGTCCAAC GCTAGCTTTG
AAGAGATACT TCCAAGGTAT CCACGTTTAC TTGAAGGAAA AGGGTTACTC TGACTGTGCT
TGGGAAACCG TGCGTCTAGA AATCATGCGT AGCTTCTCTT CTTTGATCAG CTTGCAAGAA 4
AGATTACGTA TGATGGACGG TGACTTGTCG AGCCCA 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 172 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Cys Asp Leu Ser Gin Asn His Val Leu Val Gly Arg Lys Asn Leu Arg 1 5 10 15
Leu Leu Asp Glu Met Arg Arg Leu Ser Pro His Phe Cys Leu Gin Asp 20 25 30
Arg Lys Asp Phe Ala Leu Pro Gin Glu Met Val Glu Gly Gly Gin Leu 35 40 45
Gin Glu Ala Gin Ala lie Ser Val Leu His Glu Met Leu Gin Gin Ser 50 55 60
Phe Asn Leu Phe His Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80
Leu Leu Glu Pro Cys Arg Thr Gly Leu His Gin Gin Leu Asp Asn Leu 85 90 95 Asp Ala Cys Leu Gly Gin Val Met Gly Glu Glu Asp Ser Ala Leu Gly 100 105 110
Arg Thr Gly Pro Thr Leu Ala Leu Lys Arg Tyr Phe Gin Gly lie His 115 120 125
Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val 130 135 140
Arg Leu Glu lie Met Arg Ser Phe Ser Ser Leu lie Ser Leu Gin Glu 145 150 155 160
Arg Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Ala Arg Glu Asn Leu Lys 5 10 15
Leu Leu Asp Arg Met Asn Arg Leu Ser Pro His Ser Cys Leu Gin Asp 20 25 30
Arg Lys Asp Phe Gly 35
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Lys Asp Phe Gly Leu Pro Gin Glu Met Val Glu Gly Asp Gin Leu Gin 5 10 15
Lys Asp Gin Ala Phe Pro Val Leu Tyr Glu Met Leu Gin Gin Ser 20 25 30
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gin Gin Ser Phe Asn Leu Phe Tyr Thr Glu His Ser Ser Ala Ala Trp 5 10 15
sp Thr Thr Leu Leu Glu Gin Leu Cys Thr Gly Leu Gin Gin Gin 20 25 30
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino acids
(B) TYPE amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gin Gin Gin Leu Asp His Leu Asp Thr Cys Arg Gly Gin Val Met Gly 5 10 15
Glu Glu Asp Ser Glu Leu Gly Asn Met Asp Pro lie Val Thr Val Lys 20 25 30
Lys
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Thr Val Lys Lys Tyr Phe Gin Gly lie Tyr Asp Tyr Leu Gin Glu Lys 5 10 15
Gly Tyr Ser Asp Cys Ala Trp Glu lie Val Arg Val Glu Met Met Arg 20 25 30
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Cys Ala Trp Glu lie Val Arg Val Glu Met Met Arg Ala Leu Thr Val 5 10 15
Ser Thr Thr Leu Gin Lys Arg Leu Thr Lys Met Gly Gly Asp Leu Asn 20 25 30
Ser Pro
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 588 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Human Interferon Tau Coding
Sequences
(i ) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..585
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATG GCC TTC GTG CTC TCT CTA CTC ATG GCC CTG GTG CTG GTC AGC TAC
Met Ala Phe Val Leu Ser Leu Leu Met Ala Leu Val Leu Val Ser Tyr 1 5 10 15
GGC CCA GGA GGA TCC CTG GGT TGT GAC CTG TCT CAG AAC CAC GTG CTG Gly Pro Gly Gly Ser Leu Gly Cys Asp Leu Ser Gin Asn His Val Leu
20 25 30 GTT GGC AGG AAG AAC CTC AGG CTC CTG GAC GAA ATG AGG AGA CTC TCC Val Gly Arg Lys Asn Leu Arg Leu Leu Asp Glu Met Arg Arg Leu Ser 35 40 45
CCT CAC TTT TGT CTG CAG GAC AGA AAA GAC TTC GCT TTA CCC CAG GAA Pro His Phe Cys Leu Gin Asp Arg Lys Asp Phe Ala Leu Pro Gin Glu 50 55 60
ATG GTG GAG GGC GGC CAG CTC CAG GAG GCC CAG GCC ATC TCT GTG CTC Met Val Glu Gly Gly Gin Leu Gin Glu Ala Gin Ala He Ser Val Leu 65 70 75 80
CAT GAG ATG CTC CAG CAG AGC TTC AAC CTC TTC CAC ACA GAG CAC TCC His Glu Met Leu Gin Gin Ser Phe Asn Leu Phe His Thr Glu His Ser 85 90 95
TCT GCT GCC TGG GAC ACC ACC CTC CTG GAG CCA TGC CGC ACT GGA CTC Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Pro Cys Arg Thr Gly Leu 100 105 110
CAT CAG CAG CTG GAC AAC CTG GAT GCC TGC CTG GGG CAG GTG ATG GGA His Gin Gin Leu Asp Asn Leu Asp Ala Cys Leu Gly Gin Val Met Gly 115 120 125
GAG GAA GAC TCT GCC CTG GGA AGG ACG GGC CCC ACC CTG GCT CTG AAG Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu Ala Leu Lys 130 135 140
AGG TAC TTC CAG GGC ATC CAT GTC TAC CTG AAA GAG AAG GGA TAC AGC Arg Tyr Phe Gin Gly He His Val Tyr Leu Lys Glu Lys Gly Tyr Ser 145 150 155 160
GAC TGC GCC TGG GAA ACC GTC AGA CTG GAA ATC ATG AGA TCC TTC TCT Asp Cys Ala Trp Glu Thr Val Arg Leu Glu He Met Arg Ser Phe Ser 165 170 175
TCA TTA ATC AGC TTG CAA GAA AGG TTA AGA ATG ATG GAT GGA GAC CTG 5 Ser Leu He Ser Leu Gin Glu Arg Leu Arg Met Met Asp Gly Asp Leu 180 185 190 AGC TCA CCT TGA
Ser Ser Pro 195
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 195 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Ala Phe Val Leu Ser Leu Leu Met Ala Leu Val Leu Val Ser Tyr 1 5 10 15
Gly Pro Gly Gly Ser Leu Gly Cys Asp Leu Ser Gin Asn His Val Leu
20 25 30
Val Gly Arg Lys Asn Leu Arg Leu Leu Asp Glu Met Arg Arg Leu Ser 35 40 45
Pro His Phe Cys Leu Gin Asp Arg Lys Asp Phe Ala Leu Pro Gin Glu 50 55 60
Met Val Glu Gly Gly Gin Leu Gin Glu Ala Gin Ala He Ser Val Leu 65 70 75 80
His Glu Met Leu Gin Gin Ser Phe Asn Leu Phe His Thr Glu His Ser 85 90 95
Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Pro Cys Arg Thr Gly Leu 100 105 110
His Gin Gin Leu Asp Asn Leu Asp Ala Cys Leu Gly Gin Val Met Gly 115 120 125 Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu Ala Leu Lys 130 135 140
Arg Tyr Phe Gin Gly He His Val Tyr Leu Lys Glu Lys Gly Tyr Ser 145 150 155 160
Asp Cys Ala Trp Glu Thr Val Arg Leu Glu He Met Arg Ser Phe Ser 165 170 175
Ser Leu He Ser Leu Gin Glu Arg Leu Arg Met Met Asp Gly Asp Leu 180 185 190
Ser Ser Pro 195
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 bases (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CCTGTCTGCA GGACAGAAAA GACTT
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 bases
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TCTGAATTCT GACGATTTCC CAGGC (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment 1-37, Human Tau-IFN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Cys Asp Leu Ser Gin Asn His Val Leu Val Gly Arg Lys Asn Leu Arg 1 5 10 15
Leu Leu Asp Glu Met Arg Arg Leu Ser Pro His Phe Cys Leu Gin Asp 20 25 30
Arg Lys Asp Phe Ala 35
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment 34-64, Human Tau-IFN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Lys Asp Phe Ala Leu Pro Gin Glu Met Val Glu Gly Gly Gin Leu Gin 1 5 10 15
Glu Ala Gin Ala He Ser Val Leu His Glu Met Leu Gin Gin Ser 20 25 30
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment 62-92, Human Tau-IFN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Gin Gin Ser Phe Asn Leu Phe His Thr Glu His Ser Ser Ala Ala Trp 1 5 10 15
Asp Thr Thr Leu Leu Glu Pro Cys Arg Thr Gly Leu His Gin Gin 20 25 30
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment 90-122, Human Tau-IFN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
His Gin Gin Leu Asp Asn Leu Asp Ala Cys Leu Gly Gin Val Met Gly 1 5 10 15
Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu Ala Leu' Lys
20 25 30
Arg
(2) INFORMATION FOR SEQ ID NO:19i
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment 119-150, Human Tau-IFN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Ala Leu Lys Arg Tyr Phe Gin Gly He His Val Tyr Leu Lys Glu Lys 1 5 10 15 Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val Arg Leu Glu He Met Arg 20 25 30
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment 139-172, Human Tau-IFN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Cys Ala Trp Glu Thr Val Arg Leu Glu He Met Arg Ser Phe Ser Ser 1 5 10 15
Leu He Ser Leu Gin Glu Arg Leu Arg Met Met Asp Gly Asp Leu Ser 20 25 30
Ser Pro

Claims

IT IS CLAIMED:
1. A method of inhibiting tumor cell growth, com¬ prising contacting the cells with interferonτ at a con- centration effective to inhibit growth of the tumor cells.
2. The method of claim l, where said interferon-τ is that which can be obtained from a mammal of the group consisting of cows, sheep, and humans.
3. The method of claim 1, wherein the interferon-τ comprises a sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
4. The method of claim 1, wherein the interferon-T has the sequence presented as SEQ ID NO:4.
5. The method of claim 4, wherein said cells are human carcinoma cells, human leukemia cells, human T- lymphoma cellε, and human melanoma cells.
6. The method of claim 5, wherein said cells are steroid-sensitive tumor cells.
7. The method of claim 6, wherein said cells are mammary tumor cells.
8. A method of inhibiting viral replication in cells, comprising contacting cellε infected with a virus with inter¬ feron-T at a concentration effective to inhibit viral replication within said cells.
9. The method of claim 8, where said interferon-T is obtained from a mammal of the group consiεting of cowε, εheep, and humans.
10. The method of claim 8, wherein the interferon-τ comprises a εequence selected from the group consisting of: SEQ ID NO:2 or SEQ ID NO:4.
11. The method of claim 8, where said virus is an RNA virus.
12. The method of claim 11, where said virus is selected from the group consisting of feline leukemia virus, human immunodeficiency virus, or hepatitis c virus.
13. The method of claim 8, where said virus is hepatitis B virus.
14. A method of enhancing fertility in a female mamma1, comprising administering to said mammal an effective mammalian fertility enhancing amount of human interferon-T in a pharmaceutically acceptable carrier.
15. The method of claim 14, where said interferon-T has the protein εequence presented as SEQ ID NO:4.
16. An isolated nucleic acid which encodeε a human interferon-T.
17. The nucleic acid of claim 16, wherein said nucleic acid molecule has the sequence presented as SEQ ID NO:11.
18. The nucleic acid of claim 16, wherein said nucleic acid molecule has the sequence presented aε SEQ ID NO:3.
19. An expression vector comprising
(a) a nucleic acid containing an open reading frame that encodes human interferon-T; and
(b) regulatory sequences effective to express said open reading frame in a host cell.
20. The expression vector of claim 19, wherein said regulatory sequences include, 5' to said nucleic acid sequence, a promoter region and an ATG start codon in- frame with the interferon-T coding sequence, and 3' to said coding sequence, a translation termination signal followed by a transcription termination signal.
21. The expression vector of claim 19, wherein said nucleic acid is included in the sequence is εelected from the group consisting of SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:11.
22. A recombinantly produced human interferon-τ protein.
23. The recombinantly produced protein of claim 22, which includes the sequence preεented aε SEQ ID NO:4.
24. The recombinantly produced protein of claim 23, which further includeε an amino terminal extension, where said protein has the sequence presented as SEQ ID NO:12.
25. A method of recombinantly producing interferon- T, comprising introducing into suitable host cells, a recombinant expresεion system containing an open reading frame (ORF) having a polynucleotide sequence which encodes a human interferon-T polypeptide, where the vector iε deεigned to expreεε the ORF in said host, and culturing said host under conditions resulting in the expression of the ORF εequence.
26. The method of claim 25, wherein the interferon- T polypeptide has the sequence presented as SEQ ID NO:4.
27. The method of claim 25, wherein the expression vector is a lambda gtll phage vector and the host cells are E. coli .
28. The method of claim 25, wherein the polynucleo¬ tide sequence has the sequence presented as SEQ ID NO:3.
29. The method of claim 28, wherein the hoεt cellε are yeast.
30. The method of claim 28, wherein the host cells are insect cells.
31. The method of claim 25, wherein the polypeptide has the sequence presented aε SEQ ID NO:4, and the poly¬ nucleotide has the sequence presented in SEQ ID NO:11.
32. An expresεion εystem for expressing a interfer- on-τ polypeptide, comprising a hoεt capable of supporting expression of an open reading frame in a selected expression vector, and the selected expression vector containing an open reading frame (ORF) having a polynucleotide sequence which encodes a human interferon-T polypeptide.
33. The expression system of claim 32, wherein the polypeptide is selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.
34. The expression system of claim 32, wherein the polypeptide is the polypeptide having the sequence pre¬ sented as SEQ ID NO:4.
35. An isolated interferon-T polypeptide, where said polypeptide is (i) derived from the interferon-T amino acid coding sequence, and (ii) between 15 and 172 amino acids long.
36. The polypeptide of claim 35, where said inter¬ feron-T sequence is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
37. The polypeptides of claim 35, where said poly- peptide is selected from the group consiεting of SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:10.
38. The polypeptides of claim 35, where said poly¬ peptide is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20.
39. A method of blocking the binding of an alpha- interferon to a cell having an alpha-interferon receptor, comprising contacting the cell with an interferon-T polypeptide at a concentration effective to allow the binding of interferon-T polypeptide to each alpha-interferon recep¬ tor, and exposing the cells having interferon-T polypep¬ tide bound to said receptor to alpha-interferon.
40. The method of claim 39, where the interferon-T polypeptide is selected from the group consisting of SEQ ID N0:2, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:10.
41. The method of claim 39, where the interferon-τ polypeptide is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:20.
42. A method of blocking the binding of interferon- T to a cell having a interferon-T receptor, compriεing contacting the cell with an interferon-T polypeptide εelected from the group conεiεting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:20, where said polypeptide is at a concentration effective to allow the binding of the polypeptide to each interferon-T-receptor, and exposing the cells having interferon-T polypeptides bound to said receptor to interferon-τ.
43. Purified antibodies that are immunoreactive wit human interferon-T.
44. The antibodies of claim 43 which are polyclonal antibodies.
45. The antibodies of claim 43 which are monoclonal antibodies.
46. The antibodies of claim 43, where said antibod- ies are reactive with a polypeptide selected from the group consiεting of SEQ ID NO: , SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:20.
47. A fuεed polypeptide, comprising: (a) an interferon-T polypeptide, where said polypep¬ tide is (i) derived from an interferon-T amino acid coding sequence, and (ii) between 15 and 172 amino acids long; and
(b) a second soluble polypeptide.
48. A fused polypeptide of claim 47, where said interferon-T sequence is selected from the group consist¬ ing of SEQ ID NO:2 and SEQ ID NO:4.
49. A fused polypeptide of claim 47, where said polypeptide is selected from the group consiεting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:10.
50. A fused polypeptides of claim 47, where said polypeptide is selected from the group consisting of SEQ ID NO:15, SEQ ID N0:17, SEQ ID N0:19 and SEQ ID NO:20.
51. A fused polypeptide of claim 47, where said second soluble polypeptide is serum albumin.
52. A fused polypeptide of claim 47, where said second soluble polypeptide is interferon-α.
EP93924958A 1992-10-30 1993-10-19 Interferon tau compositions and methods of use Withdrawn EP0669981A1 (en)

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JPH08505047A (en) 1996-06-04
KR100357768B1 (en) 2003-04-07
CN1090510A (en) 1994-08-10
WO1994010313A3 (en) 1994-10-27
CA2148119A1 (en) 1994-05-11
EP1360962A3 (en) 2004-02-04
AU689450B2 (en) 1998-04-02
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TW585911B (en) 2004-05-01
AU5444994A (en) 1994-05-24

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