WO1997033607A1 - Orally-administered interferon-tau compositions and methods - Google Patents

Orally-administered interferon-tau compositions and methods Download PDF

Info

Publication number
WO1997033607A1
WO1997033607A1 PCT/US1997/003794 US9703794W WO9733607A1 WO 1997033607 A1 WO1997033607 A1 WO 1997033607A1 US 9703794 W US9703794 W US 9703794W WO 9733607 A1 WO9733607 A1 WO 9733607A1
Authority
WO
WIPO (PCT)
Prior art keywords
ifnr
leu
orally
ovifnr
administered
Prior art date
Application number
PCT/US1997/003794
Other languages
French (fr)
Other versions
WO1997033607A9 (en
Inventor
Jeanne M. Soos
Joel Schiffenbauer
Howard M. Johnson
Original Assignee
University Of Florida
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Florida filed Critical University Of Florida
Priority to AU20759/97A priority Critical patent/AU2075997A/en
Publication of WO1997033607A1 publication Critical patent/WO1997033607A1/en
Publication of WO1997033607A9 publication Critical patent/WO1997033607A9/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00—Peptide or protein sequence
    • Y10S930/01—Peptide or protein sequence
    • Y10S930/14—Lymphokine; related peptides
    • Y10S930/142—Interferon

Definitions

  • the present invention relates to orally-administered pharmaceutical compositions containing interferon-tau and methods of uses thereof.
  • Mullis, K.B. U.S. Patent No. 4,683,202, issued 28 Jul ⁇ 987. Mullis, K.B., et al., U. S. Patent No. 4,683,195, issued 28 July 1987.
  • 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, etal, 1979; Bazer, et al , 1986). 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 biological 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 various 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 lymphocyte 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).
  • IFNr interferon-t u
  • the Greek letter r stands for trophoblast.
  • interferons have been classified into two distinct groups: type I interferons, including IFN ⁇ , IFN/3, and IFN ⁇ (also known as IFN ⁇ ll); and type II interferons, represented by IFN ⁇ (reviewed by DeMaeyer, et al , 1988).
  • type I interferons including IFN ⁇ , IFN/3, and IFN ⁇ (also known as IFN ⁇ ll); and type II interferons, represented by IFN ⁇ (reviewed by DeMaeyer, et al , 1988).
  • IFN ⁇ In humans, it is estimated that there are at least 17 IFN ⁇ non-allelic genes, at least about 2 or 3 IFNjS non-allelic genes, and a single IFN ⁇ gene.
  • IFN ⁇ 's have 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).
  • these proteins 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 cancers (Bonnem, etal, 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).
  • IFN ⁇ 's are also useful against various types of viral infections (Finter, et al, 1991).
  • Alpha interferons 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).
  • type I interferons are useful in treating autoimmune diseases such as multiple sclerosis (MS).
  • MS multiple sclerosis
  • IFNjS has been tested and approved by the U.S. Food and Drug Administration (FDA) as an MS therapy.
  • IFN ⁇ 's have been limited by their toxicity: use of interferons in the treatment of cancer, autoimmune disorders 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 treatment. 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 provides methods of treatment for cancer, autoimmune diseases (such as MS) and for inhibiting cellular proliferation and viral infection. These methods do not have the toxic side effects associated with currently-used therapies, and employ a convenient route of administration. Summary of the Invention
  • the present invention includes an improvement in a me ⁇ od of treating a disease condition in a mammal (e.g., mouse, dog or human) responsive to treatment by interferon- tau (IFNr).
  • the improvement comprises orally administering a therapeutically-effective amount of IFN ⁇ .
  • the orally-administered IFNr is preferably ingested by the mammal.
  • the IFN ⁇ is orally-administered at a dosage of between about 1x10 s and about 1x10* units per day, preferably at a dosage of between about lxlO 6 and about lxlO 7 units per day.
  • the IFNr may be, for example, ovine IFNr (OvIFNr), e.g., a polypeptide having the sequence represented as SEQ ID NO:2, or a human IFNr (HuIFNr), e.g., a polypeptide having the sequence represented as SEQ ID NO:4 or SEQ ID NO:6.
  • OvIFNr ovine IFNr
  • Human IFNr e.g., a polypeptide having the sequence represented as SEQ ID NO:4 or SEQ ID NO:6.
  • die disease condition is an immune system disorder, such as an autoimmune disorder (e.g., multiple sclerosis (MS), type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, allergies or psoriasis).
  • an autoimmune disorder e.g., multiple sclerosis (MS), type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, allergies or psoriasis.
  • MS is particularly amenable to treatment using the methods of the present invention.
  • the disease condition is a cell proliferation disorder, such as a cancer (e.g., hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous T cell lymphoma, and glioma).
  • a cancer e.g., hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous T cell lymphoma, and glioma).
  • a cancer e.g., hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer
  • the disease condition is a viral disease (e.g., hepatitis A, hepatitis
  • hepatitis C non-A, non-B, non-C hepatitis, Epstein-Barr viral infection, HIV infection, herpes virus (EB, CML, herpes simplex), papilloma, poxvirus, picorna virus, adeno virus, rhino virus,
  • EB herpes virus
  • CML herpes simplex
  • papilloma poxvirus
  • picorna virus picorna virus
  • adeno virus rhino virus
  • the invention includes a me ⁇ hod of treating an autoimmune disorder in a subject (e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNr) to the subject.
  • a subject e.g., a human subject
  • the orally-administered IFNT is preferably ingested by the subject. Examples of autoimmune conditions amenable to treatment, dosages, and sources of IFNT are as presented above.
  • the invention also includes a method of decreasing the severity or frequency of a relapse of multiple sclerosis (MS) in a human suffering from MS, by orally administering a therapeutically- effective amount of interferon-tau (IFNT) to the human.
  • MS multiple sclerosis
  • the invention includes a method of treating a cell proliferation disorder in a subject (e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNr) to the subject.
  • IFNr interferon-tau
  • the orally-administered IFNr is preferably ingested by the subject.
  • cell proliferation disorders amenable to treatment, dosages, and sources of IFNT are as presented above.
  • the invention includes a method of treating a viral disease in a subject
  • a further aspect of the invention includes a metiiod of enhancing fertility in a female mammal (e.g., a human female), by orally administering a therapeutically-effective amount of interferon-tau (IFNT) to the mammal. Examples of dosages and sources of IFNT are as presented above.
  • Figure 1 shows the amount of OvIFNr in NZW mouse sera after administration by either oral feeding (filled bars) or i.p. injection (open bars) as measured using an anti-viral assay.
  • Figures 2A, 2B and 2C show the prevention of chronic-relapsing experimental allergic encephalomyelitis (EAE) in SJL mice by orally-administered (Fig. 2C) and i.p.-injected (Fig. 2B) IFNT as compared with mice receiving no treatment (Fig. 2A).
  • EAE allergic encephalomyelitis
  • Figures 3A, 3B and 3C show sections of mouse spinal cord stained with cresyl violet for detection of lymphocyte infiltration from EAE-induced animals receiving either no IFNT treatment (Fig. 3A), OvIFNr treatment by i.p. injection (Fig. 3B) or OvIFNr treatment by oral feeding (Fig. 3C).
  • Figure 4 shows induction of IL-10 by either single-dose or prolonged IFNr treatment administered by i.p. injection or oral feeding.
  • Figure 5 shows relapses of EAE in SJL mice following removal of IFNr treatment.
  • Figure 6 shows ELISA detection of anti-OvIFNr antibodies in the sera of OvIFNr-treated mice following i.p. injection or oral feeding of OVIFNT. Brief Description of the Sequences
  • SEQ ID NO:l is the nucleotide sequence of a synthetic gene encoding ovine interferon-r (OvIFNr). Also shown is the encoded amino acid sequence.
  • SEQ ID NO:2 is an amino acid sequence of a mature OvIFNr protein.
  • SEQ ID NO: 3 is a synthetic nucleotide sequence encoding a mature human interferon- ⁇ (HUIFNT) protein.
  • SEQ ID NO:4 is an amino acid sequence for a mature HuIFNrl protein.
  • SEQ ID NO:5 is the nucleotide sequence, excluding leader sequence, of genomic DNA clone
  • HuIFN ⁇ 3 a natural HuIFNr gene.
  • SEQ ID NO:6 is the predicted amino acid sequence of a mature human LFNr protein encoded by HuIFN ⁇ 3, encoded by the sequence represented as SEQ ID NO:5.
  • Interferon- ⁇ refers to any one of a family of interferon proteins having at least one characteristic from each of the following two groups of characteristics: (i) (a) anti-luteolytic properties, (b) anti-viral properties, (c) anti-cellular proliferation properties; and (ii) about 45 to 68% amino acid homology with ⁇ -Interferons and greater than 70% amino acid homology to known IFNr sequences (e.g., Ott, et al., 1991; Helmer, et al, 1987; Imakawa, et al, 1989; Whaley, et al, 1994; Bazer, et al, 1994). Amino acid homology can be determined using, for example, the LALIGN program with default parameters.
  • IFNr can be obtained from a number of sources including cows, sheep, ox, and humans.
  • An interferon-r polypeptide is a polypeptide having between about 15 and 172 amino acids derived from an interferon-r amino acid coding sequence, where said 15 to 172 amino acids are contiguous in native interferon-r. Such 15-172 amino acid regions can also be assembled into polypeptides where two or more such interferon-r regions are joined that are normally discontinuous in the native protein.
  • Treating a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
  • IFNr Interferon-tau
  • OvIFNr ovine IFNr
  • conceptus the embryo and surrounding membranes
  • oTP-1 ovine trophoblast protein-one
  • Bovine IFNr Bovine IFNr (BoIFNr) and OvIFNr have (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%.
  • IFNr displays many of the activities classically associated with type I IFNs (see Table 1, above), considerable differences exist between it and the other type I IFNs. The most prominent difference is its role in pregnancy, detailed above. Also different is viral induction. All type I IFNs, except IFNr, are induced readily by virus and dsRNA (Roberts, et al., 1992). Induced IFN ⁇ and IFN/3 expression is transient, lasting approximately a few hours. In contrast, IFNr synthesis, once induced, is maintained over a period of days (Godkin, et al, 1982). On a per-cell basis, 300-fold more IFNr is produced than other type I IFNs (Cross and Roberts, 1991).
  • IFNr expression may also differ between species. For example, although IFNr expression is restricted to a particular stage (primarily days 13-21) of conceptus development in ruminants (Godkin, et al, 1982), preliminary studies suggest that the human form of IFNr is constitutively expressed throughout pregnancy (Whaley, et al, 1994).
  • IFNr polypeptides suitable for use in the methods of the present invention may produced in any of a number of ways. For example, they may be purified from animal tissues in which they are expressed, synthesized using a peptide synthesizer or produced recombinantly.
  • Recombinant IFNT protein may be produced from any selected IFNr polynucleotide fragment using a suitable expression system, such as bacterial or yeast cells.
  • a suitable expression system such as bacterial or yeast cells.
  • the isolation of IFNr nucleotide and polypeptide sequences is described in Bazer, et al (1994). For example, Bazer, et al., describe the identification and isolation of a human IFNr gene.
  • a synthetic nucleotide sequence encoding a mature human interferon-r (HuIFNr) protein is presented herein as SEQ ID NO:3.
  • SEQ ID NO:4 is the corresponding amino acid sequence for a mature HuIFNrl protein.
  • SEQ ID NO:5 is the nucleotide sequence, excluding leader sequence, of genomic DNA clone HuIFN ⁇ 3, a natural HuIFNr gene, and SEQ ID NO:6 is the predicted amino acid sequence of a mature human IFNr protein encoded by the sequence represented as SEQ ID NO:5.
  • an IFNr coding sequence (e.g, SEQ ID NO: 1) is placed in an expression vector, e.g., a bacterial expression vector, and expressed according to standard methods.
  • suitable vectors include lambda gtl 1 (Promega, Madison WI); pGEX (Smith, et ⁇ l , 1985); pGEMEX (Promega); and pBS (Stratagene, La Jolla CA) vectors.
  • Other bacterial expression vectors containing suitable promoters, such as the T7 RNA polymerase promoter or the tac promoter, may also be used.
  • OVIFNT synthetic polynucleotide into a modified pIN III omp-A expression vector is described in the Materials and Methods.
  • OvIFNr coding sequence present in SEQ ID NO: 1 was cloned into a vector, suitable for transformation of yeast cells, containing the methanol- regulated alcohol oxidase (AOX) promoter and a Phol signal sequence.
  • the vector was used to transform P. pastoris host cells and transformed cells were used to express the protein according to the manufacturer's instructions.
  • yeast vectors suitable for expressing IFNr for use with methods of the present invention include 2 micron plasmid vectors (Ludwig, et al, 1993), yeast integrating plasmids (Yips; e.g., Shaw, et al, 1988), YEP vectors (Shen, et al, 1986), yeast centromere plasmids (YCps; e.g., Ernst, 1986), and other vectors with regulatable expression (Hitzeman, et al. , 1988; Rutter, et al, 1988; Oeda, et al, 1988).
  • the vectors include an expression cassette containing an effective yeast promoter, such as the MF ⁇ l promoter (Ernst, 1986; Bayne, et al, 1988, GADPH promoter (glyceraldehyde -3-phosphate-dehydrogenase; Wu, et al, 1991) or the galactose-inducible GAL10 promoter (Ludwig, et al, 1993; Feher, et al., 1989; Shen, et al., 1986).
  • an effective yeast promoter such as the MF ⁇ l promoter (Ernst, 1986; Bayne, et al, 1988, GADPH promoter (glyceraldehyde -3-phosphate-dehydrogenase; Wu, et al, 1991) or the galactose-inducible GAL10 promoter (Ludwig, et al, 1993; Feher, et al., 1989; Shen, et al., 1986).
  • the yeast transformation host is typically Saccharomyces cerevisiae, however, as illustrated above, other yeast suitable for transformation can be used as well (e.g., Schizosac- charomycespom.be, Pichia pastoris and the like). Further, a DNA encoding an IFNr polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system.
  • baculovirus expression Reilly, et al, 1992; Beames, et al, 1991; Clontech, Palo Alto CA
  • plant cell expression e.g., Gelvin and Schilperoot, 1988
  • expression in mammalian cells e.g., Gelvin and Schilperoot, 1988
  • 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 chromato ⁇ graphy, and affinity chromatography. Immunoaffinity chromatography can be employed, as described above, using antibodies generated based on the IFNT polypeptides. In addition to recombinant methods, IFNr proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies. Further, IFNT peptides may be chemically syndiesized using methods known to those skilled in the art.
  • orally-administered IFNr resultsed in unexpected advantages relative to treatment with injected IFNr compositions.
  • orally-administered IFNr resulted in a significantly lower level of anti-IFNr antibodies in the serum of treated individuals (see Example 7).
  • EAE allergic encephalomyelitis
  • MS multiple sclerosis
  • mice having the H-2 U haplotype are susceptible to EAE.
  • Susceptible mouse strains include PL/J mice (Klein, et al, 1983), (PL/J x SJL)Fj mice (Zamvil and Steinman, 1990; Wraith, et al. , 1989), BIO.PL mice (Figuero, et al. , 1982), NZW mice (Kotzin, et al. , 1987), and (NZB x NZW)F1 (Kotzin, et al, 1987) mice.
  • IFN ⁇ Gamma-interferon
  • IFN beta-interferon
  • IFNjS has been approved by the FDA as a therapeutic for multiple sclerosis.
  • ⁇ - IFN is effective against MS, it has relatively high toxicity, and as a result, has a variety of undesirable side effects.
  • IFNr has significantly lower toxicity that other interferons and may therefore exhibit fewer undesirable side effects.
  • orally-administered and injected IFN-r was tested for its ability to prevent the induction of EAE.
  • EAE was induced in New Zealand White (NZW) mice by immunization with bovine myelin basic protein (bMBP).
  • BMBP bovine myelin basic protein
  • Recipient NZW mice received OVIFNT by either i.p. injection or oral feeding 48 hours prior to, on the day of, and 48 hours after immunization with bovine myelin basic protein (bMBP) for induction of experimental allergic encephalomyelitis (EAE).
  • mice that received IFNr by i.p injection or by oral administration were tested for the presence of IFNT using a cytopathic effect (antiviral) assay (Familetti, et al, 1981) as described in Example 2.
  • Fig. 1 Specific activities are expressed in antiviral units/mg protein obtained from antiviral assays using MDBK cells. OvIFNr was detected for up to two 2 hours following oral feeding (filled bars) at levels of 200 U/ml. These data indicate that orally- administered IFNT enters the circulation and remains in serum for about two hours after being administered.
  • OvIFNr exhibits a remarkable lack of toxicity both in vitro and in vivo.
  • Experiments performed in support of the present invention compared OvIFNr with IFNs ⁇ and ⁇ for induction of toxicity as measured by lymphocyte depression in peripheral blood when given via oral feeding. Blood was obtained from the tail and white blood cells (WBC) counts were enumerated using a hemocytometer. Differential WBC counts were performed on Wright-Giemsa-stained blood smears.
  • orally-administered OVIFNT prevents paralysis in a chronic-relapsing model of EAE, as detailed in Example 3.
  • 5/5 mice immunized with MBP (to induce EAE) which did not receive OVIFNT treatment developed chronic relapsing paralysis
  • 4/5 animals treated with OvIFNr either i.p. injection or oral feeding, administered every 48 hours
  • IFNr can block the development of chronic relapsing EAE.
  • the experiments also suggest that or ally-administration of IFNr as infrequently as once every 48 hours, over an extended period of time, is as effective as i.p. injection at treating a disease condition responsive to treatment by interferon-tau.
  • mice from each treatment group were bled and sera were examined for the presence of anti-OvIFN ⁇ antibodies by ELISA.
  • mice receiving IFNr by i.p. injection exhibited elevated levels of anti-IFNr antibodies
  • animals receiving IFNr by oral feeding exhibited much lower anti-IFNr antibody titers (typically 3 to 5 -fold lower).
  • mice which received no OvIFNr treatment displayed no anti-OvIFNr antibodies.
  • IFNT as a Treatment for Immune System Disorders Diseases which may be treated using methods of the present invention include autoimmune, inflammatory, proliferative and hyperproliferative diseases, as well as cutaneous manifestations of immunologically mediated diseases.
  • methods of the present invention are advantageous for treating conditions relating to immune system hypersensitivity.
  • immune system hypersensitivity There are four types of immune system hypersensitivity (Clayman, 1991).
  • Type I or immediate/anaphylactic hypersensitivity, is due to mast cell degranulation in response to an allergen (e.g., pollen), and includes asthma, allergic rhinitis (hay fever), urticaria (hives), anaphylactic shock, and other illnesses of an allergic nature.
  • Type II or autoimmune hypersensitivity
  • Type III hypersensitivity is due to the formation of antigen/antibody immune complexes which lodge in various tissues and activate further immune responses, and is responsible for conditions such as serum sickness, allergic alveolitis, and the large swellings mat sometimes form after booster vaccinations.
  • Type IV hypersensitivity is due to the release of lymphokines from sensitized T-cells, which results in an inflammatory reaction. Examples include contact dermatitis, the rash of measles, and "allergic" reactions to certain drugs.
  • Autoimmune disorders may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body.
  • organ-specific disorders include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thyroiditis (thyroid gland), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junction), rheumatoid arthritis G° mt lining), uveitis (eye), psoriasis (skin), Guillain-Barr ⁇ Syndrome (nerve cells) and Grave's disease (thyroid).
  • Systemic autoimmune diseases include systemic lupus erythematosus and dermatomyositis.
  • hypersensitivity disorders include asthma, eczema, atopical dermatitis, contact dermatitis, other ecze atous dermatitides, seborrheic dermatitis, rhinitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, uritcaris, angioedemas, vasculitides, ery ⁇ iemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis, primary biliary cirrhosis and nephrotic syndrome.
  • Autoimmune diseases particularly amenable for treatment using the methods of the present invention include multiple sclerosis, type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, uveitis, allergies and psoriasis.
  • Methods of the present invention may be used to therapeutically treat and thereby alleviate autoimmune disorders such as those discussed above. These treatments are exemplified herein with respect to the treatment of EAE, an animal model for multiple sclerosis.
  • IFNr bears some similarity to the IFN ⁇ family based on structure and its potent antiviral properties, the IFN ⁇ s do not possess the reproductive properties associated with IFNr. For example, recombinant human IFN ⁇ had no effect on interestrous interval compared to IFNr, even when administered at twice the dose (Davis, et al. , 1992).
  • IFNr has some structural similarities 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 IFNr compositions 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. (1991). According to the teachings herein, such methods of enhancing fertility include oral administration of IFNr in a therapeutically-effective amount. Further, the compositions may be similarly employed to regulate growth and development of uterine and/or fetal-placental tissues. Compositions containing human IFNr are particularly useful for treatment of humans, since potential antigenic responses are less likely using a same-species protein.
  • IFNr The antiviral activity of IFNr has broad therapeutic applications without the toxic effects that are usually associated with IFN ⁇ s. As described above, IFNr exerts its therapeutic activity without adverse effects on the cells. The relative lack of cytotoxicity of IFNr makes it extremely valuable as an in vivo therapeutic agent and sets IFNr apart from most other known antiviral agents and all other known interferons.
  • Formulations containing IFNr can be orally-administered to inhibit viral replication. Further, the compositions can be 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. Compositions containing a human interferon-r are particularly useful for treatment of humans, since potential antigenic responses are less likely using a homologous protein.
  • Examples of specific viral diseases which may be treated by orally-administered IFNr include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, non-A, non-B, non-C hepatitis, Epstein-Barr viral infection, HIV infection, herpes virus (EB, CML, herpes simplex), papilloma, poxvirus, picorna virus, adeno virus, rhino virus, HTLV I, HTLV II, and human rotavirus.
  • hepatitis A hepatitis B, hepatitis C, non-A, non-B, non-C hepatitis
  • Epstein-Barr viral infection HIV infection
  • herpes virus EB, CML, herpes simplex
  • papilloma poxvirus
  • picorna virus picorna virus
  • adeno virus rhino virus
  • HTLV I HTLV II
  • human rotavirus human rotavirus
  • compositions containing IFNT suitable for oral administration, can be used to inhibit cellular growth without the negative side effects associated with other interferons which are currently known. Such compositions or formulations can be used to inhibit, prevent, or slow tumor growth.
  • IFNr include, but are not limited to, hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous
  • T cell lymphoma T cell lymphoma
  • glioma T cell lymphoma
  • the development of certain tumors is mediated by estrogen.
  • IFNr can suppress estrogen receptor numbers. Therefore, the IFNr-containing compositions may be particularly useful in the treatment or prevention of estrogen-dependent tumors.
  • hypothyroidism in dogs typically results from a progressive destruction of the thyroid, which may be associated with Lymphocytic thyroiditis (Kemppainen and Clark, 1994). Lymphocytic thyroiditis, which resembles Hashimoto's thyroiditis in humans, is thought to be an autoimmune disorder. According to the guidance presented herein, hypothyroidism due to Lymphocytic thyroiditis in dogs may be treated with IFNr as described above.
  • autoimmune disorder in dogs that may be alleviated by treatment with IFNr is characterized by antinuclear antibody (ANA) positivity, pyrexia and seronegative arthritis Pay, et al, 1985).
  • Immune-mediated thrombocytopenia TTP; Kristensen, et al, 1994; Werner, et al., 1985), systemic lupus erythematosus (Kristensen, et al., 1994), and leukopenia and Coomb's positive hemolytic anemia (Werner, et al, 1985), may also be amenable to treatment using mediods of the present invention.
  • Formulation Therapeutic preparations containing IFNr or related polypeptides or proteins can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations comprising polypeptides like interferons have been previously described (e.g., Martin, 1976). In general, the IFNr therapeutic compositions are formulated such that an effective amount of the IFNr is combined with a suitable additive, carrier and/or excipient in order to facilitate effective oral administration of the composition.
  • tablets and capsules containing IFNr may be prepared by combining IFNr (e.g., lyophilized IFNr protein) with additives such as pharmaceutically acceptable carriers (e.g., lactose, corn starch, light silicic anhydride, microcrystalline cellulose, sucrose), binders (e.g., alpha-form starch, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyr- rolidone), disintegrating agents (e.g., carboxymethylcellulose calcium, starch, low substituted hydroxy-propylcellulose), surfactants (e.g., Tween 80, polyoxyemylene-polyoxypropylene copolymer), antioxidants (e.g., L-cysteine, sodium sulfite, sodium ascorbate), lubricants (e.g., magnesium stearate, talc), or the like.
  • pharmaceutically acceptable carriers e.g., lactose,
  • IFNr polypeptides of the present invention can be mixed with a solid, pulverulent or other carrier, for example lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, millopectine, cellulose derivative or gelatine, and may also include lubricants, such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets.
  • a solid, pulverulent or other carrier for example lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, millopectine, cellulose derivative or gelatine
  • lubricants such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets.
  • Liquid preparations for oral administration can be made in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1% to about 30% by weight of IFNr, sugar and a mixture of etiianol, water, glycerol, propylene, glycol and possibly other additives of a conventional nature.
  • An orally-active IFNr pharmaceutical composition is administered in a merapeutically effective amount to an individual in need of treatment.
  • the dose may vary considerably and is dependent on factors such as the seriousness of the disorder, the age and the weight of the patient, omer medications that me patient may be taking and the like. This amount or dosage is typically determined by the attending physician.
  • the dosage will typically be between about 1 x IO 3 and 1 x IO 8 units/day, preferably between about 1 x IO 6 and 1 x IO 7 units/day. It will be appreciated that because of its lower toxicity, IFNT can be administered at higher doses than, for example, IFN/3.
  • Disorders requiring a steady elevated level of IFNT in plasma will benefit from administration as often as about every two to four hours, while other disorders, such as MS, may be effectively treated by administering a therapeutically-effective dose at less frequent intervals, e.g., once every 48 hours.
  • the rate of administration of individual doses is typically adjusted by an attending physician to enable administration of the lowest total dosage while alleviating the severity of the disease being treated.
  • compositions and mediods of mis invention may be used in combination with other erapies.
  • MS patients that do not show improvement at IFNjSlb's low dosage or could not tolerate IFNjS lb due to toxicity may benefit from subsequent or simultaneous treatment with higher dosages of IFNr or peptides derived therefrom.
  • IFNr may be an important alternative therapy, since antibody cross-reactivity is unlikely to occur, and IFNr is unlikely to generate neutralizing antibodies (see Example 7). Orally-administered IFNr is particularly advantageous in mis respect, since it causes a significantly lower anti-IFNr antibody response than injected IFNr.
  • Another type of combination therapy enabled by the present invention is the oral administration of an antigen against which an autoimmune response is directed in combination with IFNr. Oral administration of such an antigen can result in tolerization, reducing the severity of the autoimmune disease (for review, see, e.g., Weiner, et al, 1994). It is contemplated that the IFNr has a synergistic effect with the tolerization induced by the antigen, thereby alleviating the severity of the autoimmune disease.
  • MBP has been shown to suppress EAE (Lider, et al, 1989). According to the methods of me present invention, MBP may be administered in combination with IFNr to treat multiple sclerosis.
  • Other examples include administration of IFNr with collagen to treat rheumatoid arthritis, and with acetylcholine receptor polypeptides to treat myasthenia gravis.
  • IFNr may be orally administered with known immunosuppressants, such as steroids, to treat autoimmune diseases such a multiple sclerosis.
  • immunosuppressants such as steroids
  • the immunosuppressants may act synergistically with IFNr and result in a more effective treatment that could be obtained widi an equivalent dose of IFNr or the immunosuppressant alone.
  • IFNr may be administered in conjunction with, e.g., a dierapeutically effective amount of one or more chemotherapy agents such as busulfan, 5-fluoro-uracil (5-FU), zidovudine (AZT), leucovorin, melphalan, prednisone, cyclophosphamide, dacarbazine, cisplatin, and dipyridamole.
  • chemotherapy agents such as busulfan, 5-fluoro-uracil (5-FU), zidovudine (AZT), leucovorin, melphalan, prednisone, cyclophosphamide, dacarbazine, cisplatin, and dipyridamole.
  • chemotherapy agents such as busulfan, 5-fluoro-uracil (5-FU), zidovudine (AZT), leucovorin, melphalan, prednisone, cyclophosphamide, dacarbazine, cisplatin, and dip
  • PBS Phosphate-buffered saline
  • the plates were aspirated and blocked with 300 ⁇ L 10% NGS and incubated at 37°C for 1 hr. The plates were men 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 plates incubated 1 hour at 37 C C. The plates were then washed 5 times with PBS 0.5% "TWEEN-20".
  • 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 (w/v). The relative absorbance is determined at 410 nm.
  • a synthetic OvIFNr gene was generated using standard molecular methods (Ausubel, et al. , 1988) by Hgating oligonucloetides containing contiguous portions of a DNA sequence encoding the OVIFNT amino acid sequence (Imakawa, et al, 1987). The resulting IFNr polynucleotide coding sequence spans position 16 through 531: a coding sequence of 172 amino acids.
  • the full length synthetic gene StuVSstl fragment (540 bp) was cloned into a modified pIN UI omp-A expression vector and transformed : nto a competent SB221 strain of E. coli.
  • me IFNr protein cells carrying the expression vector were grown in L-broth containing ampicillin to an OD (550 nm) of 0.1-1, induced witii IPTG (isopropyl- 1-thio-b-D- galactoside) for 3 hours and harvested by centrifugation. Soluble recombinant IFNr was liberated from the cells by sonication or osmotic fractionation.
  • the IFNr gene was amplified using polymerase chain reaction (PCR; Mullis, 1987; Mullis, et al. , 1987) with PCR primers containing StuI and Sad restriction sites at the 5' and 3' ends, respectively.
  • the amplified fragments were digested with Stul and S ⁇ cII and ligated into the Sac ⁇ . and Smal sites of ⁇ pBLUESCRIPT+(KS)", generating pBSY-IFN ⁇ .
  • Plasmid pBSY-IFNr was digested with S ⁇ cII and Ec ⁇ KV and the fragment containing the synthetic IFNr gene was isolated.
  • the yeast expression vector pBS24Ub (Sabin, et al, 1989; Ecker, et al, 1989) was digested with Sail. Blunt ends were generated using T4 DNA polymerase.
  • the vector DNA was extracted with phenol and ethanol precipitated (Sambrook, et al, 1989).
  • the recovered plasmid was digested with S ⁇ cII, purified by agarose gel electro ⁇ phoresis, and ligated to the Sacll-Ec ⁇ RW fragment isolated from pBSY-IFNr.
  • 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.
  • IFNr coding sequences were isolated form pBS24Ub-IFN ⁇ and cloned into a Pichia pastoris expression vector containing the alcohol oxidase (AOX1) promoter (Invitrogen, San Diego, CA). The vector was then used to transform Pichia pastoris GS115 His " host cells and protein was expressed following the manufacturer's instructions.
  • AOX1 alcohol oxidase
  • the protein was secreted into the medium and purified by successive DEAE-cellulose and hydroxyapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining.
  • the purified protein had a specific activity of about 0.29 to about 0.44 x IO 8 U/mg as measured by anti-viral activity on Madin-Darby bovine kidney (MDBK) cells.
  • Orally-administered and injected IFN-r was tested for its ability to prevent the induction of
  • EAE Recipient New Zealand White mice received OvIFNr (IO 5 U/ml) by either i.p. injection or oral feeding 48 hours prior to, on the day of, and 48 hours after immunization with bovine myelin basic protein (bMBP) for induction of experimental allergic encephalomyelitis (EAE). 10' U of IFNr were mixed with PBS to a total volume of 100 ⁇ l and administered using a feeding tube placed down the esophagus and into the stomach. The dilution of the IFNT in PBS was done immediately before administration.
  • OvIFNr IO 5 U/ml
  • bMBP bovine myelin basic protein
  • bovine myelin basic protein (bMBP) was emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml of H37Ra (Mycobacterium tuberculosis, Difco, Detroit, MI) and injected on either side of the base of the tail.
  • CFA complete Freund's adjuvant
  • H37Ra Mycobacterium tuberculosis, Difco, Detroit, MI
  • 400 ng of Pertussis toxin List Biologicals, Campbell, CA
  • mice were immunized again 7 days after the initial immunization. Mice were examined daily for signs of EAE and severity of disease was graded on the following scale: 1, loss of tail tone; 2, hind limb weakness; 3, paraparesis; 4, paraplegia; 5, moribund/death.
  • an anti-OvIFNr monoclonal antibody HL127
  • mAb monoclonal antibody
  • HL127 was used to neutralize OvIFNr ability to block EAE (antibody HL127, directed against aa 139-172 of SEQ ID NO:2, neutralizes the antiviral activity of OvIFNr in an antiviral assay using the MDBK cell line).
  • a 1:10 dilution of HL127 was incubated for 2 hours with OvIFNr prior to administration by either i.p. injection or oral feeding.
  • Antibodies directed against IFNr antigens may be generated using the information herein combined with known techniques for antibody production (e.g., Harlow, et al, 1988).
  • OVIFNT (10 U) was administered 48 hours prior to MBP immunization, on the day of MBP immunization and 48 hours after MBP immunization by either i.p. injection or oral feeding.
  • HL127 a monoclonal antibody specific for OvIFNr, was incubated with OVIFNT for two hours prior to administration.
  • mice The amount of OvIFNr detectable in the sera of mice (treated as above) was compared over time after oral feeding or i.p. injection of OvIFNr. Mice were administered 3 x 10' U of
  • OVIFNT OVIFNT and bled at 0.5, 2, 4, 6, 24 and 48 hours following IFNT administration.
  • Sera were tested in a cytopathic effect (viral plaque) assay (Familetti, et al, 1981) to determine the amount of IFNr in the samples.
  • VSV Vesicular stomatatosis virus
  • OvIFNr was detected at 0.5 hour and 2 hours after oral feeding (filled bars) at levels of 200 U/ml. By comparison, somewhat higher levels of OvIFNr were detected for over a 24 hour period of time after i.p. injection (open bars). These data show that die above dose of IFNT can be detected in serum for about two hours following oral administration.
  • OvIFNTr Prevention of Chronic Relapse of Experimental Allergic Encephalomyelitis by Orally-Administered OVIFNT
  • a chronic-relapsing model of EAE in which SJL mice immunized with MBP develop a chronic form of the disease where the appearance of symptoms occurs in a relapsing-remitting manner (Zamvil and Steinman, 1990).
  • EAE was induced in SJL mice essentially as described above. The mice were treated with
  • Histological analyses were performed to determine the extent of lymphocyte infiltration into the CNS of MBP-immunized mice treated with OVIFNT by oral and i.p. routes.
  • mice were perfused with 4% paraformaldehyde, vertebral columns were removed and treated with formalin for 2 to 3 days.
  • Spinal cords were dissected out and soaked in 0.5 % sucrose overnight at 4 °C.
  • Spinal cord sections were embedded and sections cut in a microtome. Sections were fixed to slides in 4 % paraformaldehyde and stained with cresyl violet for visualization of inflammatory infiltrates.
  • mice were bled and sera were examined for the presence of interleukin 10 (IL10).
  • Sera from mice which received either a single IFNr (10 5 U) treatment (by i.p. injection or oral feeding), prolonged IFNr (IO 5 U) treatment (by i.p. injection or oral treatment for greater than 20 days) or no treatment were examined for IL10 by enzyme-linked immunosorbent assay (ELISA) using IL10 ELISA kits (Genzyme, Cambridge, MA) following the manufacturer's instructions. All sera samples were tested in duplicate.
  • ELISA enzyme-linked immunosorbent assay
  • Example 6 Cessation of Treatment with OvIFNr Results in Relapsing Paralysis SJL mice which were protected from EAE by OvIFNr treatment via i.p. injection or oral feeding (every 48 hours) were followed for 58 days, during which time no disease development was observed. Treatment with OvIFNr was then removed and the mice were observed for an additional 22 days for symptoms of disease.
  • IFNr treatment is denoted as plus signs and removal of IFNT treatment is denoted as minus signs beneath the graph.
  • the antigen, OvIFNr was adsorbed to the flat bottoms of plastic tissue culture wells overnight at a concentration of 600 ng/well, and subsequently evaporated to dryness.
  • the plates were treated with 5% milk (Carnation) in PBS for 2 hours in order to block nonspecific binding and then washed 3 times with PBS containing 0.05% Tween 20.
  • Various dilutions of sera from mice which were untreated, OvIFNr treated by i.p. injection and OvIFNr treated by oral feeding were added and incubated for 3 hours. Binding was assessed with goat anti-mouse immuno- globulin coupled to horseradish peroxidase. Color development was monitored at 492 nm in an
  • HuIFNtau protein HuIFNtaul.
  • Gin Glu Ala Gin Ala lie Ser Val Leu His Glu Met Leu Gin Gin Ser 50 55 60
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • CAG GAG GCC CAG GCC ATC TCT GTG CTC CAC GAG ATG CTC CAG CAG AGC 192 Gin Glu Ala Gin Ala lie Ser Val Leu His Glu Met Leu Gin Gin Ser 50 55 60
  • 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

Abstract

The present invention includes interferon-tau (IFNτ) pharmaceutical compositions useful for oral administration to treat cancers, autoimmune disorders (particularly multiple sclerosis), cell proliferative disorders and viral disease.

Description

ORALLY-ADMINISTERED INTERFERON-TAU COMPOSITIONS AND METHODS
Field of the Invention
The present invention relates to orally-administered pharmaceutical compositions containing interferon-tau and methods of uses thereof.
References
Ausubel, F.M., et al, in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. John Wiley and Sons, Inc., Media, PA (1988).
Bartol, F.F., et al., Biol. Reprod. 32:681-693 (1985). Bayne, M.L., et al., Gene 66:235 (1988).
Bazer, F.W., et al., J. Animal Sci. 5_7(Supp. 2):425 (1983).
Bazer, F.W., et al., J. Reproduction and Fertility 76:841 (1986).
Bazer, F.W., et al., PCT Application publication No. WO 94/10313, published 11 May, 1994. Beames, et al., Biotechniques H.:378 (1991).
Benoit, P., et al., J. Immunol. 150ι3):707 (1993).
Blatt, L.M., et al., U.S. Patent No. 5,372,808, issued 13 December 1994.
Bonnem, E.M., et al, J. Bio. Response Modifiers 3:580 (1984). dayman, C.B., Ed., AMERICAN MEDICAL ASSOCIATION ENCYCLOPEDIA OF MEDICINE (Random House, New York, NY), 1991.
Cross, J.C, and Roberts, R.M., Proc. Natl. Acad. Sci. USA 88:3817-3821 (1991).
Davis, G.L., et al, N. England J. Med. 321:1501 (1989).
Davis, G.L., et al, Theriogenology 38:867 (1992).
Day, M.J., et al, Clin. Immunol Immunopathol. 35(1):85-91 (1985). Degre, M., Int. J. Cancer 14:699 (1974).
DeMaeyer, E., et al, in INTERFERONS AND OTHER REGULATORY CYTOKINES. John Wiley and Sons, New York (1988).
Dusheiko, G.M., et al, J. Hematology 3(Suppl. 2):S199 (1986).
Ecker, D.J., et al, J. Biol. Chem. 264:7715-7719 (1989). Ernst, J.F., DNA 5:483 (1986).
Familetti, P.C., et al, Meth. Enzymol 78:387 (1981).
Feher, Z., et al , Curr. Genet. 16:461 (1989). Fent, K. and G. Zbinden, Trnds. Pharm. Sci. 5_6_:l-26 (1987).
Figuero, F., et al, Immunogenetics 15(4):399-404 (1982).
Finter, N.B., et al. Drugs 4215): 749 (1991).
Fritz, R.B., et al, J. Immunol 130J3): 1024-1026 (1983). Gelvin, S.B. and R.A. Schilperoot, Plant Molecular Biology (1988).
Gnatek, G.G., et al, Biol. Reprod. 41:655-664 (1989).
Godkin, J.D., et al, J. Reprod. Fertil. 65:141-150 (1982).
Hansen, P.J., et al , U.S. Patent No. 4,997,646, issued 5 March 1991.
Harlow, E., et al, in ANTIBODIES: A LABORATORY MANUAL. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).
Hitzeman, R.A., et al, U.S. Patent No. 4,775,622, issued October 4, 1988.
Helmer, S.D., et al, J. Reprod. Pert. 79:83-91 (1987).
IFN/S Multiple Sclerosis Study Group, Neurology 43_(4):655 (1993).
Imakawa, K., et al, Nature 33Q-.377-379 (1987). Imakawa, K., et al, Mol Endocrinol 3:127 (1989).
Johnson, H.M., et al, Sci. Am. 270(51:40-47 (1994).
Kashima, H., et al, Laryngoscope 9 £:334 (1988).
Kemppainen, R.J., and Clark, T.P., Vet. Clin. N. Am. Small Anim. Pract. 24_(3):467-476 (1994). Klein, J., et al, Immunogenetics 17:553 (1983).
Kotzin, B.L., et al, J. Exp. Med. 265:1237 (1987).
Kristensen, A.T., et al, J. Vet. Intern. Med. 8_(l):36-39 (1994).
Lider, et al, J. Immunol, 142:148-752 (1989).
Ludwig, D.L., et al, Gene 132:33 (1993). Martal, J., et al, J. Reprod. Fertil. 5_6.:63-73 (1979).
Martin, E.W., in DISPENSING OF MEDICATION: A PRACTICAL MANUAL ON THE FOR¬ MULATION AND DISPENSING OF PHARMACEUTICAL PRODUCTS Mack Publishing Co., Easton, PA (1976).
Mullis, K.B., U.S. Patent No. 4,683,202, issued 28 Jul ι987. Mullis, K.B., et al., U. S. Patent No. 4,683,195, issued 28 July 1987.
Oeda, K., et al, U.S. Patent No. 4,766,068, issued August 23, 1988.
Oldham, R.K., Hospital Practice 2 :71 (1985).
Pearson, W.R. and Lipman, D.J., PNAS g5_:2444-2448 (1988).
Pearson, W.R., Methods in Enzymology 1S3_:63- 98 (1990).
Pontzer, C.H., et al, Cancer Res. 5.1:5304 (1991).
Quesada, J.R., et al, N. England J. Med. 3J0:15 (1984).
Reilly, P.R., et al, in BACULOVTRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).
Roberts, R.M., et al, Endocrin. Rev. 11:432-452 (1992).
Rutter, W.J., et al, U.S. Patent No. 4,769,238, issued September 6, 1988.
Sabin, E., et al, Bio/Technology 7:705-709 (1989).
Sambrook, ]., et al, in MOLECULAR CLONING: A LABORATORY MANUAL. Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
Shaw, K.J., et al, DNA 7:117 (1988).
Shen, L.P., et al, Sci. Sin. 29:856 (1986).
Smith, P.K., et al, Anal Biochem. 150:76 (1985).
Stewart, H.J., et al, J. Endocrinol. 1 5:R13 (1987). Weiner, H., et al, Ann. Rev. Immunol. 12:809-837 (1994).
Weinstock-Guttman, B., et al, Ann. Neurol. 22:7-15 (1995).
Werner, L.L., et al, Vet. Immunol. Immunopathol £(1-2): 183-192 (1985).
Whaley, A.E., et al, J. Biol. Chem. 269(14): 10864-10868 (1994).
Wilson, et al., Biology of Reproduction 20(Supρ. 1):101A, Abstract (1979). Wraith, D.C., et al, Cell 59:247 (1989).
Wu, D.A., et al, DNA 10:201 (1991).
Zamvil, S.S., and Steinman, L., Ann. Rev. Immunol. 8:579-621 (1990).
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, etal, 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 biological 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 various 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 IFNα 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 (IFNα is derived from lymphocyte cells), oTP- l's size - 172 amino acids (IFNα is typically about 166 amino acids), and oTP-1 is weakly inducible by viruses (IFNα is highly inducible by viruses). The International Interferon Society recognizes oTP-1 as belonging to an entirely new class of interferons which have been named interferon-t u (IFNr). The Greek letter r stands for trophoblast.
The interferons have been classified into two distinct groups: type I interferons, including IFNα, IFN/3, and IFNω (also known as IFNαll); and type II interferons, represented by IFNγ (reviewed by DeMaeyer, et al , 1988). In humans, it is estimated that there are at least 17 IFNα non-allelic genes, at least about 2 or 3 IFNjS non-allelic genes, and a single IFNγ gene. IFNα's have 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 proteins 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 cancers (Bonnem, etal, 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 interferons 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).
In addition, type I interferons are useful in treating autoimmune diseases such as multiple sclerosis (MS). In fact, IFNjS has been tested and approved by the U.S. Food and Drug Administration (FDA) as an MS therapy.
Significantly, however, the usefulness of IFNα's has been limited by their toxicity: use of interferons in the treatment of cancer, autoimmune disorders 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 treatment. 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 provides methods of treatment for cancer, autoimmune diseases (such as MS) and for inhibiting cellular proliferation and viral infection. These methods do not have the toxic side effects associated with currently-used therapies, and employ a convenient route of administration. Summary of the Invention
In one aspect, the present invention includes an improvement in a meϋ od of treating a disease condition in a mammal (e.g., mouse, dog or human) responsive to treatment by interferon- tau (IFNr). The improvement comprises orally administering a therapeutically-effective amount of IFNτ. The orally-administered IFNr is preferably ingested by the mammal. In a general embodiment, the IFNτ is orally-administered at a dosage of between about 1x10s and about 1x10* units per day, preferably at a dosage of between about lxlO6 and about lxlO7 units per day. The IFNr may be, for example, ovine IFNr (OvIFNr), e.g., a polypeptide having the sequence represented as SEQ ID NO:2, or a human IFNr (HuIFNr), e.g., a polypeptide having the sequence represented as SEQ ID NO:4 or SEQ ID NO:6.
In one embodiment, die disease condition is an immune system disorder, such as an autoimmune disorder (e.g., multiple sclerosis (MS), type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, allergies or psoriasis). MS is particularly amenable to treatment using the methods of the present invention.
In another embodiment, the disease condition is a cell proliferation disorder, such as a cancer (e.g., hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous T cell lymphoma, and glioma).
In yet another embodiment, the disease condition is a viral disease (e.g., hepatitis A, hepatitis
B, hepatitis C, non-A, non-B, non-C hepatitis, Epstein-Barr viral infection, HIV infection, herpes virus (EB, CML, herpes simplex), papilloma, poxvirus, picorna virus, adeno virus, rhino virus,
HTLV I, HTLV II, and human rotavirus). In another aspect, the invention includes a meϋhod of treating an autoimmune disorder in a subject (e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNr) to the subject. The orally-administered IFNT is preferably ingested by the subject. Examples of autoimmune conditions amenable to treatment, dosages, and sources of IFNT are as presented above. The invention also includes a method of decreasing the severity or frequency of a relapse of multiple sclerosis (MS) in a human suffering from MS, by orally administering a therapeutically- effective amount of interferon-tau (IFNT) to the human. Examples of dosages and sources of IFNT are as presented above. In another aspect, the invention includes a method of treating a cell proliferation disorder in a subject (e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNr) to the subject. The orally-administered IFNr is preferably ingested by the subject. Examples of cell proliferation disorders amenable to treatment, dosages, and sources of IFNT are as presented above. In still another aspect, the invention includes a method of treating a viral disease in a subject
(e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNr) to the subject. The orally-administered IFNT is preferably ingested by the subject. Examples of viral diseases amenable to treatment, dosages, and sources of IFNT are as presented above. A further aspect of the invention includes a metiiod of enhancing fertility in a female mammal (e.g., a human female), by orally administering a therapeutically-effective amount of interferon-tau (IFNT) to the mammal. Examples of dosages and sources of IFNT are as presented above.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings. Brief Description of the Figures
Figure 1 shows the amount of OvIFNr in NZW mouse sera after administration by either oral feeding (filled bars) or i.p. injection (open bars) as measured using an anti-viral assay.
Figures 2A, 2B and 2C show the prevention of chronic-relapsing experimental allergic encephalomyelitis (EAE) in SJL mice by orally-administered (Fig. 2C) and i.p.-injected (Fig. 2B) IFNT as compared with mice receiving no treatment (Fig. 2A).
Figures 3A, 3B and 3C show sections of mouse spinal cord stained with cresyl violet for detection of lymphocyte infiltration from EAE-induced animals receiving either no IFNT treatment (Fig. 3A), OvIFNr treatment by i.p. injection (Fig. 3B) or OvIFNr treatment by oral feeding (Fig. 3C).
Figure 4 shows induction of IL-10 by either single-dose or prolonged IFNr treatment administered by i.p. injection or oral feeding.
Figure 5 shows relapses of EAE in SJL mice following removal of IFNr treatment. Figure 6 shows ELISA detection of anti-OvIFNr antibodies in the sera of OvIFNr-treated mice following i.p. injection or oral feeding of OVIFNT. Brief Description of the Sequences
SEQ ID NO:l is the nucleotide sequence of a synthetic gene encoding ovine interferon-r (OvIFNr). Also shown is the encoded amino acid sequence.
SEQ ID NO:2 is an amino acid sequence of a mature OvIFNr protein.
SEQ ID NO: 3 is a synthetic nucleotide sequence encoding a mature human interferon-τ (HUIFNT) protein.
SEQ ID NO:4 is an amino acid sequence for a mature HuIFNrl protein. SEQ ID NO:5 is the nucleotide sequence, excluding leader sequence, of genomic DNA clone
HuIFNτ3, a natural HuIFNr gene.
SEQ ID NO:6 is the predicted amino acid sequence of a mature human LFNr protein encoded by HuIFNτ3, encoded by the sequence represented as SEQ ID NO:5.
Detailed Description of the Invention I. Definitions
Interferon-τ refers to any one of a family of interferon proteins having at least one characteristic from each of the following two groups of characteristics: (i) (a) anti-luteolytic properties, (b) anti-viral properties, (c) anti-cellular proliferation properties; and (ii) about 45 to 68% amino acid homology with α-Interferons and greater than 70% amino acid homology to known IFNr sequences (e.g., Ott, et al., 1991; Helmer, et al, 1987; Imakawa, et al, 1989; Whaley, et al, 1994; Bazer, et al, 1994). Amino acid homology can be determined using, for example, the LALIGN program with default parameters. This program is found in the FASTA version 1.7 suite of sequence comparison programs (Pearson and Lipman, 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, VA). IFNr can be obtained from a number of sources including cows, sheep, ox, and humans.
An interferon-r polypeptide is a polypeptide having between about 15 and 172 amino acids derived from an interferon-r amino acid coding sequence, where said 15 to 172 amino acids are contiguous in native interferon-r. Such 15-172 amino acid regions can also be assembled into polypeptides where two or more such interferon-r regions are joined that are normally discontinuous in the native protein.
Treating a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease. II. Interferon-tau (IFNr)
A. Introduction
The first IFNr to be identified was ovine IFNr (OvIFNr). Several isoforms of the 18-19 kDa protein were identified in conceptus (the embryo and surrounding membranes) homogenates (Martal, et al., 1979). Subsequently, a low molecular weight protein released into conceptus culture medium was purified and shown to be both heat labile and susceptible to proteases (Godkin, et al, 1982). OvIFNr was originally called ovine trophoblast protein-one (oTP-1) because it was the primary secretory protein initially produced by trophectoderm of the sheep conceptus during the critical period of maternal recognition in sheep. One isolate of mature OVIFNT is 172 amino acids in length (SEQ ID NO:2).
IFNrs with similar characteristics and activities have been isolated from other ruminant species including cows and goats (Bartol, etal, 1985; Gnatek, etal, 1989; Helmer, et al, 1987; and Imakawa, et al, 1989). Bovine IFNr (BoIFNr) and OvIFNr have (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%.
Antisera to all the IFNrs cross-react. This is not unexpected since the species specific forms of IFNr are more closely homologous to each other than to the IFNsα from the identical species (Roberts, et al, 1992). Relative to other interferons, OvIFNr shares about 45 to 68% amino acid homology with Interferon-α and the greatest sequence similarity with the interferon-ωs (IFNωs) of about 68%.
Table 1
Overview of the Interferons
j Aspects Type 1 Type II
[Types a & ω β T y
Produced by: leukocyte fibioblast trophoblast lymphocyte
Effects:
Antiviral + + + + Antiproliferative + + + + Pregnancy Signally +
While IFNr displays many of the activities classically associated with type I IFNs (see Table 1, above), considerable differences exist between it and the other type I IFNs. The most prominent difference is its role in pregnancy, detailed above. Also different is viral induction. All type I IFNs, except IFNr, are induced readily by virus and dsRNA (Roberts, et al., 1992). Induced IFNα and IFN/3 expression is transient, lasting approximately a few hours. In contrast, IFNr synthesis, once induced, is maintained over a period of days (Godkin, et al, 1982). On a per-cell basis, 300-fold more IFNr is produced than other type I IFNs (Cross and Roberts, 1991).
Other differences may exist in the regulatory regions of the IFNr gene. For example, transfection of the human trophoblast cell line JAR wiui the gene for bovine IFNr resulted in antiviral activity while transfection witii the bovine IFN/J gene did not. This implies unique transacting factors involved in IFNr gene expression. Consistent with this is the observation that while the proximal promoter region (from 126 to the transcriptional start site) of IFNr is highly homologous to that of IFNα and IFNS; the region from -126 to -450 is not homologous and enhances only IFNT expression (Cross and Roberts, 1991). Thus, different regulatory factors appear to be involved in IFNT expression as compared wiui the other type I IFNs.
IFNr expression may also differ between species. For example, although IFNr expression is restricted to a particular stage (primarily days 13-21) of conceptus development in ruminants (Godkin, et al, 1982), preliminary studies suggest that the human form of IFNr is constitutively expressed throughout pregnancy (Whaley, et al, 1994).
B. Production of IFNT
IFNr polypeptides suitable for use in the methods of the present invention may produced in any of a number of ways. For example, they may be purified from animal tissues in which they are expressed, synthesized using a peptide synthesizer or produced recombinantly.
Recombinant IFNT protein may be produced from any selected IFNr polynucleotide fragment using a suitable expression system, such as bacterial or yeast cells. The isolation of IFNr nucleotide and polypeptide sequences is described in Bazer, et al (1994). For example, Bazer, et al., describe the identification and isolation of a human IFNr gene. A synthetic nucleotide sequence encoding a mature human interferon-r (HuIFNr) protein is presented herein as SEQ ID NO:3. SEQ ID NO:4 is the corresponding amino acid sequence for a mature HuIFNrl protein. SEQ ID NO:5 is the nucleotide sequence, excluding leader sequence, of genomic DNA clone HuIFNτ3, a natural HuIFNr gene, and SEQ ID NO:6 is the predicted amino acid sequence of a mature human IFNr protein encoded by the sequence represented as SEQ ID NO:5.
To make an IFNr expression vector, an IFNr coding sequence (e.g, SEQ ID NO: 1) is placed in an expression vector, e.g., a bacterial expression vector, and expressed according to standard methods. Examples of suitable vectors include lambda gtl 1 (Promega, Madison WI); pGEX (Smith, et αl , 1985); pGEMEX (Promega); and pBS (Stratagene, La Jolla CA) vectors. Other bacterial expression vectors containing suitable promoters, such as the T7 RNA polymerase promoter or the tac promoter, may also be used. Cloning of the OVIFNT synthetic polynucleotide into a modified pIN III omp-A expression vector is described in the Materials and Methods. For the experiments described herein, the OvIFNr coding sequence present in SEQ ID NO: 1 was cloned into a vector, suitable for transformation of yeast cells, containing the methanol- regulated alcohol oxidase (AOX) promoter and a Phol signal sequence. The vector was used to transform P. pastoris host cells and transformed cells were used to express the protein according to the manufacturer's instructions.
Other yeast vectors suitable for expressing IFNr for use with methods of the present invention include 2 micron plasmid vectors (Ludwig, et al, 1993), yeast integrating plasmids (Yips; e.g., Shaw, et al, 1988), YEP vectors (Shen, et al, 1986), yeast centromere plasmids (YCps; e.g., Ernst, 1986), and other vectors with regulatable expression (Hitzeman, et al. , 1988; Rutter, et al, 1988; Oeda, et al, 1988). Preferably, the vectors include an expression cassette containing an effective yeast promoter, such as the MFαl promoter (Ernst, 1986; Bayne, et al, 1988, GADPH promoter (glyceraldehyde -3-phosphate-dehydrogenase; Wu, et al, 1991) or the galactose-inducible GAL10 promoter (Ludwig, et al, 1993; Feher, et al., 1989; Shen, et al., 1986). The yeast transformation host is typically Saccharomyces cerevisiae, however, as illustrated above, other yeast suitable for transformation can be used as well (e.g., Schizosac- charomycespom.be, Pichia pastoris and the like). Further, a DNA encoding an IFNr 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, 1992; Beames, et al, 1991; Clontech, Palo Alto CA); plant cell expression, transgenic plant expression (e.g., Gelvin and Schilperoot, 1988), 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 chromato¬ graphy, and affinity chromatography. Immunoaffinity chromatography can be employed, as described above, using antibodies generated based on the IFNT polypeptides. In addition to recombinant methods, IFNr proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies. Further, IFNT peptides may be chemically syndiesized using methods known to those skilled in the art.
III. Effectiveness of Orally-Administered IFNr Experiments performed in support of the present invention and detailed below demonstrate that orally-administered IFNr polypeptide compositions are comparable in efficacy to injected IFNr compositions with respect to the treatment of diseases or disease conditions which benefit from treatment with IFNr.
Not only was orally-administered IFNr effective at treating a disease benefiting from EFNT treatment (EAE), but the oral route of administration resulted in unexpected advantages relative to treatment with injected IFNr compositions. For example, orally-administered IFNr resulted in a significantly lower level of anti-IFNr antibodies in the serum of treated individuals (see Example 7). This is beneficial because the orally-administered IFNr is therefore less likely to be rendered ineffective by a host immune response (i.e., desensitization to the treatment and/or dose level is significantly decreased), and the individual receiving the treatment is less likely to suffer adverse side effects as a result of such an immune response.
Results of experiments demonstrating these and related findings are presented below.
A. Orallv-Administered IFNr Inhibits Development of EAE
The efficacy of IFNr in treating autoimmune disorders may be evaluated in rodents with experimental allergic encephalomyelitis (EAE; Zamvil and Steinman, 1990), an animal model of antigen-induced autoimmunity. EAE resembles human multiple sclerosis (MS) both in its clinical and pathological manifestations and can thus be used to assess treatments for human autoimmune diseases such as MS. EAE is a T-cell-mediated inflammatory autoimmune demyelinating disease induced by immunizing susceptible mouse, rat or guinea pig strains with myelin basic protein (MBP) or with encephalitogenic peptide fragments. Genetic susceptibility in the model animal strains is based in part on the capacity of encephalitogenic peptides to bind to particular class II major histocompatibility complex (MHC-II) molecules (Fritz, et al., 1983; Wraith, et al, 1989). In particular, mice having the H-2U haplotype are susceptible to EAE. Susceptible mouse strains include PL/J mice (Klein, et al, 1983), (PL/J x SJL)Fj mice (Zamvil and Steinman, 1990; Wraith, et al. , 1989), BIO.PL mice (Figuero, et al. , 1982), NZW mice (Kotzin, et al. , 1987), and (NZB x NZW)F1 (Kotzin, et al, 1987) mice.
Gamma-interferon (IFNγ) and beta-interferon (IFN?) have been demonstrated to be effective in treating multiple sclerosis (Johnson, et al, 1994; IFN/J Multiple Sclerosis Study Group, 1993). In fact, IFNjS has been approved by the FDA as a therapeutic for multiple sclerosis. Although β- IFN is effective against MS, it has relatively high toxicity, and as a result, has a variety of undesirable side effects. As described herein, however, IFNr has significantly lower toxicity that other interferons and may therefore exhibit fewer undesirable side effects. In experiments performed in support of the present invention and detailed in Example 1 , orally-administered and injected IFN-r was tested for its ability to prevent the induction of EAE. EAE was induced in New Zealand White (NZW) mice by immunization with bovine myelin basic protein (bMBP). Recipient NZW mice received OVIFNT by either i.p. injection or oral feeding 48 hours prior to, on the day of, and 48 hours after immunization with bovine myelin basic protein (bMBP) for induction of experimental allergic encephalomyelitis (EAE).
Both oral feeding and i.p. injection of OvIFNr protected against EAE (Example 1, Table 3). All animals that received IFNr via i.p. injection, and 7 of 9 animals that received IFNr orally, were protected from symptoms of EAE. Furthermore, anti-OvIFNr monoclonal antibody HL127 was effective at partially neutralizing the ability of the OvIFNr to block EAE. These experiments demonstrate that orally-administered IFNr is effective in treating symptoms of EAE, an animal model of multiple sclerosis.
B. OvIFNr is Present in Sera Following Oral Administration.
To confirm that orally-administered IFNr enters the circulation, the sera of mice that received IFNr by i.p injection or by oral administration were tested for the presence of IFNT using a cytopathic effect (antiviral) assay (Familetti, et al, 1981) as described in Example 2.
The results are shown in Fig. 1. Specific activities are expressed in antiviral units/mg protein obtained from antiviral assays using MDBK cells. OvIFNr was detected for up to two 2 hours following oral feeding (filled bars) at levels of 200 U/ml. These data indicate that orally- administered IFNT enters the circulation and remains in serum for about two hours after being administered.
C. Lack of Toxicity from Orally-administered OVIFNT
It has been previously demonstrated that the type I IFNs IFNα and IFN/3 induced toxic side effects manifested as flu like symptoms, fever, nausea and malaise when used as therapeutics in humans (Degre, 1974; Fent and Zbinden, 1987). In contrast, OvIFNr exhibits a remarkable lack of toxicity both in vitro and in vivo. Experiments performed in support of the present invention compared OvIFNr with IFNs α and β for induction of toxicity as measured by lymphocyte depression in peripheral blood when given via oral feeding. Blood was obtained from the tail and white blood cells (WBC) counts were enumerated using a hemocytometer. Differential WBC counts were performed on Wright-Giemsa-stained blood smears. The results are shown in Tables 2a, 2b and 2c, below. Significant levels of toxicity were detected in mice fed either IFN α and β while no significant lymphocyte depression was detected in mice fed 10', 2x10' or 5x10' U of OvIFNr or PBS alone. These data suggest that orally- administered OvIFNr has significantly-reduced toxicity with respect to other type I IFNs.
Tables 2a-2c Comparison of IFNs r. β and α for Toxicity After Oral Feeding
Table 2a
Figure imgf000015_0001
Table 2b
CELL COUNT (CELL NO. x IO3)
IFN (DOSE) 18 H AFTER ORAL FEEDING
% LYMPHOCYTE
TOTAL WBC LYMPHOCYTES DEPRESSION
PBS — — — τ(105) 7.0 ± 1.4 6.0 ± 1.3 6.2
T^ X lO5) 7.0 + 2.8 5.9 ± 2.4 0 τ(5x l05) 7.5 ± 2.1 6.3 ± 1.8 3.1 (10J) 6.5 ± 0.7 5.1 + 0.6 13.6 β(2 lO5) 6.5 ± 0.7 4.1 ± 0.4+ 37.0 α(K)5) 6.5 ± 2.1 4.7 ± 1.6 28.8
tp<0.05 Table 2c
CELL COUNT (CELL NO. x IO3)
IFN (DOSE) 24 H AFTER ORAL FEEDING
% LYMPHOCYTE
TOTAL WBC LYMPHOCYTES DEPRESSION
PBS 7.5 ± 0.7 6.4 ± 0.6 0 rOO5) 8.0 ± 2.8 6.9 ± 2.4 0
TC X IO5) 7.0 ± 1.4 6.0 ± 1.1 0 τ(5 l05) 8.0 ± 4.2 7.0 + 3.6 0 β(Hf) 6.5 ± 3.5 5.1 ± 2.8 13.6
/S(2 x l05) 6.5 ± 0.7 4.0 + 0.4* 38.5 α(105) 7.0 ± 0 5.0 ± 0* 24.2
tp<0.05 *p <0.03
D. OVIFNT Prevents Chronic Relapse of EAE
In addition to preventing the onset of symptoms associated with EAE, orally-administered OVIFNT prevents paralysis in a chronic-relapsing model of EAE, as detailed in Example 3. Whereas 5/5 mice immunized with MBP (to induce EAE) which did not receive OVIFNT treatment developed chronic relapsing paralysis, 4/5 animals treated with OvIFNr (either i.p. injection or oral feeding, administered every 48 hours) were fully protected from the disease (Figs. 2B and 2C). These data further support the results described above, and indicate that oral administration of IFNr can block the development of chronic relapsing EAE. The experiments also suggest that or ally-administration of IFNr as infrequently as once every 48 hours, over an extended period of time, is as effective as i.p. injection at treating a disease condition responsive to treatment by interferon-tau.
E. Histological Analyses of Spinal Chord from EAE Mice following Oral Administration of IFNT. The ability of OVIFNT to prevent EAE was also assayed by analyzing the effect of OvIFNr treatment on cellular consequences of the disease, manifested in the central nervous system (CNS) as lymphocytic lesions in spinal cord white matter. The lesions are indicative of the extent of lymphocyte infiltration into the CNS. MBP-immunized mice were either not treated (control) or treated with OvIFNr by oral or i.p. routes, and sections of the spinal cord lumbar region were stained and evaluated for lymphocytes as described in Example 4. Lymphocytic lesions were present in spinal cord white matter of control animals (Fig. 3A), but not in mice treated wi OvIFNr by i.p. injection (Fig. 3B) or oral feeding (Fig. 3C). These data indicate that the protective effect of IFNr is associated with inhibition of lymphocyte infiltration of the CNS. Further, the data demonstrate that IFNT treatment inhibits cellular manifestation of the autoimmune disease, rather than simply masking symptoms.
F. Cessation of Treatment with OvIFNr Results in Relapsing Paralysis. Experiments detailed in Example 6 were performed to determine the type and duration of treatment effective to prevent EAE in mice injected with MBP. The mice were protected from EAE by OvIFNr treatment via i.p. injection or oral feeding (every 48 hours) as long as the treatment persisted (58 days in Example 6), but developed symptoms of the disease after OvIFNr treatment was stopped (Figure 5). These results suggest that while IFNr may not cure an autoimmune condition like EAE (e.g., MS), it is an effective treatment that inhibits die pathological manifestations of the condition so long as treatment is continued.
G. Oral Administration of OvIFNr Reduces Anti-OvIFNr Antibody Response. As detailed in Example 7, one advantage of orally-administered (as opposed to injected) IFNr treatment is a reduction in the anti-IFNr antibody titer in individuals receiving the oral treatment. After removal of OvIFNr treatment, mice from each treatment group were bled and sera were examined for the presence of anti-OvIFNτ antibodies by ELISA. Whereas mice receiving IFNr by i.p. injection exhibited elevated levels of anti-IFNr antibodies, animals receiving IFNr by oral feeding exhibited much lower anti-IFNr antibody titers (typically 3 to 5 -fold lower). As expected mice which received no OvIFNr treatment displayed no anti-OvIFNr antibodies.
The sera were also examined for their ability to neutralize OvIFNr antiviral activity on the MDBK cell line. None of the sera from eid er i.p. injected or orally fed mice possessed neutralizing activity O^able 4). These results suggest that oral feeding of OvIFNr largely circumvents an antibody response directed against the OvIFNr protein. Such a reduced antibody response in orally-treated subjects reduces the chance of undesirable immune system-related side effects of IFNr treatment.
IV. Applications
A. IFNT as a Treatment for Immune System Disorders Diseases which may be treated using methods of the present invention include autoimmune, inflammatory, proliferative and hyperproliferative diseases, as well as cutaneous manifestations of immunologically mediated diseases. In particular, methods of the present invention are advantageous for treating conditions relating to immune system hypersensitivity. There are four types of immune system hypersensitivity (Clayman, 1991). Type I, or immediate/anaphylactic hypersensitivity, is due to mast cell degranulation in response to an allergen (e.g., pollen), and includes asthma, allergic rhinitis (hay fever), urticaria (hives), anaphylactic shock, and other illnesses of an allergic nature. Type II, or autoimmune hypersensitivity, is due to antibodies mat are directed against perceived "antigens" on the body's own cells. Type III hypersensitivity is due to the formation of antigen/antibody immune complexes which lodge in various tissues and activate further immune responses, and is responsible for conditions such as serum sickness, allergic alveolitis, and the large swellings mat sometimes form after booster vaccinations. Type IV hypersensitivity is due to the release of lymphokines from sensitized T-cells, which results in an inflammatory reaction. Examples include contact dermatitis, the rash of measles, and "allergic" reactions to certain drugs.
The mechanisms by which certain conditions may result in hypersensitivity in some individuals are generally not well understood, but may involve both genetic and extrinsic factors. For example, bacteria, viruses or drugs may play a role in triggering an autoimmune response in an individual who already has a genetic predisposition to me autoimmune disorder. It has been suggested that the incidence of some types of hypersensitivity may be correlated with otiiers. For example, it has been proposed that individuals with certain common allergies are more susceptible to autoimmune disorders.
Autoimmune disorders may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body. Examples of organ-specific disorders (with the organ affected) include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thyroiditis (thyroid gland), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junction), rheumatoid arthritis G°mt lining), uveitis (eye), psoriasis (skin), Guillain-Barrέ Syndrome (nerve cells) and Grave's disease (thyroid). Systemic autoimmune diseases include systemic lupus erythematosus and dermatomyositis.
Other examples of hypersensitivity disorders include asthma, eczema, atopical dermatitis, contact dermatitis, other ecze atous dermatitides, seborrheic dermatitis, rhinitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, uritcaris, angioedemas, vasculitides, eryύiemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis, primary biliary cirrhosis and nephrotic syndrome. Related diseases include intestinal inflammations, such as Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis, inflammatory bowel disease, Chrohn's disease and ulcerative colitis, as well as food-related allergies. Autoimmune diseases particularly amenable for treatment using the methods of the present invention include multiple sclerosis, type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, uveitis, allergies and psoriasis. Methods of the present invention may be used to therapeutically treat and thereby alleviate autoimmune disorders such as those discussed above. These treatments are exemplified herein with respect to the treatment of EAE, an animal model for multiple sclerosis.
B. IFNr as Treatment for Reproductive Disorders.
Although IFNr bears some similarity to the IFNα family based on structure and its potent antiviral properties, the IFNαs do not possess the reproductive properties associated with IFNr. For example, recombinant human IFNα had no effect on interestrous interval compared to IFNr, even when administered at twice the dose (Davis, et al. , 1992).
Therefore, although IFNr has some structural similarities 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 IFNr compositions 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. (1991). According to the teachings herein, such methods of enhancing fertility include oral administration of IFNr in a therapeutically-effective amount. Further, the compositions may be similarly employed to regulate growth and development of uterine and/or fetal-placental tissues. Compositions containing human IFNr are particularly useful for treatment of humans, since potential antigenic responses are less likely using a same-species protein.
C. IFNT as an Antiviral Treatment
The antiviral activity of IFNr has broad therapeutic applications without the toxic effects that are usually associated with IFNαs. As described above, IFNr exerts its therapeutic activity without adverse effects on the cells. The relative lack of cytotoxicity of IFNr makes it extremely valuable as an in vivo therapeutic agent and sets IFNr apart from most other known antiviral agents and all other known interferons.
Formulations containing IFNr can be orally-administered to inhibit viral replication. Further, the compositions can be 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. Compositions containing a human interferon-r are particularly useful for treatment of humans, since potential antigenic responses are less likely using a homologous protein.
Examples of specific viral diseases which may be treated by orally-administered IFNr include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, non-A, non-B, non-C hepatitis, Epstein-Barr viral infection, HIV infection, herpes virus (EB, CML, herpes simplex), papilloma, poxvirus, picorna virus, adeno virus, rhino virus, HTLV I, HTLV II, and human rotavirus.
D. IFNT as an Antiproliferative Treatment
IFNr exhibits potent anticellular proliferation activity. Accordingly, pharmaceutical compositions containing IFNT, suitable for oral administration, can be used to inhibit cellular growth without the negative side effects associated with other interferons which are currently known. Such compositions or formulations can be used to inhibit, prevent, or slow tumor growth.
Examples of specific cell proliferation disorders which may be treated by orally-administered
IFNr include, but are not limited to, hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous
T cell lymphoma, and glioma.
Furthermore, the development of certain tumors is mediated by estrogen. Experiments performed in support of the present invention indicate that IFNr can suppress estrogen receptor numbers. Therefore, the IFNr-containing compositions may be particularly useful in the treatment or prevention of estrogen-dependent tumors.
E. Veterinary Applications
In addition to the uses of the methods of the present invention detailed above, it will be appreciated that the methods may be applied to the treatment of a variety of immune system disorders suffered by domesticated and wild animals. For example, hypothyroidism in dogs typically results from a progressive destruction of the thyroid, which may be associated with Lymphocytic thyroiditis (Kemppainen and Clark, 1994). Lymphocytic thyroiditis, which resembles Hashimoto's thyroiditis in humans, is thought to be an autoimmune disorder. According to the guidance presented herein, hypothyroidism due to Lymphocytic thyroiditis in dogs may be treated with IFNr as described above. Another type of autoimmune disorder in dogs that may be alleviated by treatment with IFNr is characterized by antinuclear antibody (ANA) positivity, pyrexia and seronegative arthritis Pay, et al, 1985). Immune-mediated thrombocytopenia (TTP; Kristensen, et al, 1994; Werner, et al., 1985), systemic lupus erythematosus (Kristensen, et al., 1994), and leukopenia and Coomb's positive hemolytic anemia (Werner, et al, 1985), may also be amenable to treatment using mediods of the present invention.
V. IFN Pharmaceutical Composition Useful for Oral Administration
A. Formulation Therapeutic preparations containing IFNr or related polypeptides or proteins can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations comprising polypeptides like interferons have been previously described (e.g., Martin, 1976). In general, the IFNr therapeutic compositions are formulated such that an effective amount of the IFNr is combined with a suitable additive, carrier and/or excipient in order to facilitate effective oral administration of the composition. For example, tablets and capsules containing IFNr may be prepared by combining IFNr (e.g., lyophilized IFNr protein) with additives such as pharmaceutically acceptable carriers (e.g., lactose, corn starch, light silicic anhydride, microcrystalline cellulose, sucrose), binders (e.g., alpha-form starch, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyr- rolidone), disintegrating agents (e.g., carboxymethylcellulose calcium, starch, low substituted hydroxy-propylcellulose), surfactants (e.g., Tween 80, polyoxyemylene-polyoxypropylene copolymer), antioxidants (e.g., L-cysteine, sodium sulfite, sodium ascorbate), lubricants (e.g., magnesium stearate, talc), or the like.
Further, IFNr polypeptides of the present invention can be mixed with a solid, pulverulent or other carrier, for example lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, millopectine, cellulose derivative or gelatine, and may also include lubricants, such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets. By using several layers of the carrier or diluent, tablets operating with slow release can be prepared. Liquid preparations for oral administration can be made in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1% to about 30% by weight of IFNr, sugar and a mixture of etiianol, water, glycerol, propylene, glycol and possibly other additives of a conventional nature.
B. Dosage An orally-active IFNr pharmaceutical composition is administered in a merapeutically effective amount to an individual in need of treatment. The dose may vary considerably and is dependent on factors such as the seriousness of the disorder, the age and the weight of the patient, omer medications that me patient may be taking and the like. This amount or dosage is typically determined by the attending physician. The dosage will typically be between about 1 x IO3 and 1 x IO8 units/day, preferably between about 1 x IO6 and 1 x IO7 units/day. It will be appreciated that because of its lower toxicity, IFNT can be administered at higher doses than, for example, IFN/3. By way of comparison, patients witii multiple sclerosis (MS) were treated with IO6 U and 8 x 10* U of IFN/3. Patients receiving 8 x 10* U suffered fewer relapses of disease than did patients receiving 10* U. However, patients receiving the higher dose of IFN/S (8 x IO6 U) also exhibited more side-effects associated with IFN/S's toxicity. In view of the lower toxicity of IFNT, these higher effective dosages could be administered without the associated toxic side- effects. Disorders requiring a steady elevated level of IFNT in plasma will benefit from administration as often as about every two to four hours, while other disorders, such as MS, may be effectively treated by administering a therapeutically-effective dose at less frequent intervals, e.g., once every 48 hours. The rate of administration of individual doses is typically adjusted by an attending physician to enable administration of the lowest total dosage while alleviating the severity of the disease being treated.
Once improvement of a patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. C. Combination Therapies It will, of course, be understood that the compositions and mediods of mis invention may be used in combination with other erapies. For example, in view of IFNr's relative lack of toxicity at high dosages, MS patients that do not show improvement at IFNjSlb's low dosage or could not tolerate IFNjS lb due to toxicity may benefit from subsequent or simultaneous treatment with higher dosages of IFNr or peptides derived therefrom. Further, development of neutralizing antibodies has been demonstrated in IFNj31b treated patients (Weinstock-Guttman, et al, 1995). In cases where such neutralizing antibodies prove to impede the effectiveness of IFNjS lb, IFNr may be an important alternative therapy, since antibody cross-reactivity is unlikely to occur, and IFNr is unlikely to generate neutralizing antibodies (see Example 7). Orally-administered IFNr is particularly advantageous in mis respect, since it causes a significantly lower anti-IFNr antibody response than injected IFNr.
Another type of combination therapy enabled by the present invention is the oral administration of an antigen against which an autoimmune response is directed in combination with IFNr. Oral administration of such an antigen can result in tolerization, reducing the severity of the autoimmune disease (for review, see, e.g., Weiner, et al, 1994). It is contemplated that the IFNr has a synergistic effect with the tolerization induced by the antigen, thereby alleviating the severity of the autoimmune disease. For example, MBP has been shown to suppress EAE (Lider, et al, 1989). According to the methods of me present invention, MBP may be administered in combination with IFNr to treat multiple sclerosis. Other examples include administration of IFNr with collagen to treat rheumatoid arthritis, and with acetylcholine receptor polypeptides to treat myasthenia gravis.
Furthermore, IFNr may be orally administered with known immunosuppressants, such as steroids, to treat autoimmune diseases such a multiple sclerosis. The immunosuppressants may act synergistically with IFNr and result in a more effective treatment that could be obtained widi an equivalent dose of IFNr or the immunosuppressant alone.
Similarly, in a treatment for a cancer or viral disease, IFNr may be administered in conjunction with, e.g., a dierapeutically effective amount of one or more chemotherapy agents such as busulfan, 5-fluoro-uracil (5-FU), zidovudine (AZT), leucovorin, melphalan, prednisone, cyclophosphamide, dacarbazine, cisplatin, and dipyridamole. The following examples illustrate but in no way are intended to limit the present invention.
VI. Materials and Methods
A. Buffers Phosphate-buffered saline (PBS)
10 x stock solution, 1 liter: 80 g NaCl
Figure imgf000023_0001
Working solution, pH 7.3: 137 mM NaCl
2.7 mM KC1
4.3 mM Na2HPO4-7H2O
1.4 mM KH2PO4
B. 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 carbonate/bicarbonate buffer, pH 9.5. The plates were sealed with parafilm and stored at 4°C overnight.
Following incubation, the plates were aspirated and blocked with 300 μL 10% NGS and incubated at 37°C for 1 hr. The plates were men 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 plates incubated 1 hour at 37 CC. The plates were then washed 5 times with PBS 0.5% "TWEEN-20".
Horseradish peroxidase (HRP) conjugated goat anti-human antiserum (Cappel, Durham, NC) was diluted 1/5,000 in PBS. 0.1 mL of mis 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 me 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 (w/v). The relative absorbance is determined at 410 nm.
C. Production of OvIFN-τ
A synthetic OvIFNr gene was generated using standard molecular methods (Ausubel, et al. , 1988) by Hgating oligonucloetides containing contiguous portions of a DNA sequence encoding the OVIFNT amino acid sequence (Imakawa, et al, 1987). The resulting IFNr polynucleotide coding sequence spans position 16 through 531: a coding sequence of 172 amino acids.
The full length synthetic gene StuVSstl fragment (540 bp) was cloned into a modified pIN UI omp-A expression vector and transformed :nto a competent SB221 strain of E. coli. For expression of me IFNr protein, cells carrying the expression vector were grown in L-broth containing ampicillin to an OD (550 nm) of 0.1-1, induced witii IPTG (isopropyl- 1-thio-b-D- galactoside) for 3 hours and harvested by centrifugation. Soluble recombinant IFNr was liberated from the cells by sonication or osmotic fractionation.
For expression in yeast, the IFNr gene was amplified using polymerase chain reaction (PCR; Mullis, 1987; Mullis, et al. , 1987) with PCR primers containing StuI and Sad restriction sites at the 5' and 3' ends, respectively. The amplified fragments were digested with Stul and SαcII and ligated into the SacΩ. and Smal sites of κpBLUESCRIPT+(KS)", generating pBSY-IFNτ.
Plasmid pBSY-IFNr was digested with SαcII and EcόKV and the fragment containing the synthetic IFNr gene was isolated. The yeast expression vector pBS24Ub (Sabin, et al, 1989; Ecker, et al, 1989) was digested with Sail. Blunt ends were generated using T4 DNA polymerase. The vector DNA was extracted with phenol and ethanol precipitated (Sambrook, et al, 1989). The recovered plasmid was digested with SαcII, purified by agarose gel electro¬ phoresis, and ligated to the Sacll-EcόRW fragment isolated from pBSY-IFNr. 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. IFNr coding sequences were isolated form pBS24Ub-IFNτ and cloned into a Pichia pastoris expression vector containing the alcohol oxidase (AOX1) promoter (Invitrogen, San Diego, CA). The vector was then used to transform Pichia pastoris GS115 His" host cells and protein was expressed following the manufacturer's instructions. The protein was secreted into the medium and purified by successive DEAE-cellulose and hydroxyapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining. The purified protein had a specific activity of about 0.29 to about 0.44 x IO8 U/mg as measured by anti-viral activity on Madin-Darby bovine kidney (MDBK) cells.
Example 1 Orally- Administered OvIFNr Blocks
Development of Experimental Allergic Encephalomyelitis
Orally-administered and injected IFN-r was tested for its ability to prevent the induction of
EAE. Recipient New Zealand White (NZW) mice received OvIFNr (IO5 U/ml) by either i.p. injection or oral feeding 48 hours prior to, on the day of, and 48 hours after immunization with bovine myelin basic protein (bMBP) for induction of experimental allergic encephalomyelitis (EAE). 10' U of IFNr were mixed with PBS to a total volume of 100 μl and administered using a feeding tube placed down the esophagus and into the stomach. The dilution of the IFNT in PBS was done immediately before administration.
For induction of EAE in NZW mice, 300 μg of bovine myelin basic protein (bMBP) was emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml of H37Ra (Mycobacterium tuberculosis, Difco, Detroit, MI) and injected on either side of the base of the tail. On the day of immunization and 48 hours later, 400 ng of Pertussis toxin (List Biologicals, Campbell, CA) was also injected. For induction of EAE in SJL/J mice, the same protocol was used as described except mice were immunized again 7 days after the initial immunization. Mice were examined daily for signs of EAE and severity of disease was graded on the following scale: 1, loss of tail tone; 2, hind limb weakness; 3, paraparesis; 4, paraplegia; 5, moribund/death.
To determine whether prevention of EAE was specific to OvIFNr treatment, an anti-OvIFNr monoclonal antibody (mAb), HL127, was used to neutralize OvIFNr ability to block EAE (antibody HL127, directed against aa 139-172 of SEQ ID NO:2, neutralizes the antiviral activity of OvIFNr in an antiviral assay using the MDBK cell line). A 1:10 dilution of HL127 was incubated for 2 hours with OvIFNr prior to administration by either i.p. injection or oral feeding. Antibodies directed against IFNr antigens, may be generated using the information herein combined with known techniques for antibody production (e.g., Harlow, et al, 1988). The results are shown in Table 3, below. Both oral feeding and i.p. injection of OvIFNr protected against acute induction of EAE. None of the animals that received IFNT via i.p. injection developed symptoms of EAE, while of the animals that received IFNr orally, 7 of 9 (78%) were protected. Anti-OvIFNr antibody HL127 was effective at partially neutralizing the ability of the OvIFNr to block EAE. These data indicate that orally-administered IFNr is effective as a treatment in an animal model of multiple sclerosis.
Table 3
Oral Feeding of OVIFNT Blocks Acute EAE and Can Be Reversed bv an OvIFNr Specific Monoclonal Antibody in NZW Mice
ROUTE OF AD¬ DISEASE MEAN DAY MEAN MINISTRATION TREATMENT INCIDENCE OF ONSET SEVERITY i.p. PBS 4/4 24.8 ± 2.1 2.5 ± 0 i.p. OvIFNr 0/4 — — i.p. OVIFNT + HL127 3/4 20.7 ± 1.2 2.3 ± 0.6 oral PBS 7/9 22.0 ± 1.0 2.7 ± 0.6 oral OVIFNT 2/9 19 3 oral OVIFNT + HL127 5/8 20.7 ± 0.6 3 ± 0
OVIFNT (10 U) was administered 48 hours prior to MBP immunization, on the day of MBP immunization and 48 hours after MBP immunization by either i.p. injection or oral feeding. HL127, a monoclonal antibody specific for OvIFNr, was incubated with OVIFNT for two hours prior to administration.
Example 2
Detection of OVIFNT in Sera Following Oral Administration
The amount of OvIFNr detectable in the sera of mice (treated as above) was compared over time after oral feeding or i.p. injection of OvIFNr. Mice were administered 3 x 10' U of
OVIFNT and bled at 0.5, 2, 4, 6, 24 and 48 hours following IFNT administration. Sera were tested in a cytopathic effect (viral plaque) assay (Familetti, et al, 1981) to determine the amount of IFNr in the samples.
Briefly, dilutions of IFNr were added to MDBK cells grown to confluency in a flat bottom 96 well plate and incubated for 18 to 24 hours at 37CC. Vesicular stomatatosis virus (VSV) was added to the plate for 45 minutes at room temperature. Virus was removed and methyl cellulose was added and the plate incubated for 48 hours at 37°C. After removal of methyl cellulose, the plate was stained with crystal violet for visualization of plaques. For measurement of IFN neutralization, OVIFNT at a concentration of 500 U/ml was incubated for 1 hour at 37 °C with either sera or HL127 (a monoclonal specific of OvIFNr). One antiviral unit caused a 50% reduction in destruction of the monolayer, relative to untreated MDBK cells infected with VSV
(control plates). All samples were assayed simultaneously to eliminate interassay variability.
As shown in Fig. 1, OvIFNr was detected at 0.5 hour and 2 hours after oral feeding (filled bars) at levels of 200 U/ml. By comparison, somewhat higher levels of OvIFNr were detected for over a 24 hour period of time after i.p. injection (open bars). These data show that die above dose of IFNT can be detected in serum for about two hours following oral administration.
Example 3
Prevention of Chronic Relapse of Experimental Allergic Encephalomyelitis by Orally-Administered OVIFNT The ability of OvIFNr to prevent paralysis was examined using a chronic-relapsing model of EAE, in which SJL mice immunized with MBP develop a chronic form of the disease where the appearance of symptoms occurs in a relapsing-remitting manner (Zamvil and Steinman, 1990).
EAE was induced in SJL mice essentially as described above. The mice were treated with
105 U of OvIFNr by either i.p. injection or oral feeding on the day of immunization (day 0) and every 48 hours thereafter for the duration of die experiment. As presented in Figure 2A, SJL mice which were immunized with MBP but did not receive OvIFNr treatment developed chronic relapsing paralysis witii a 5/5 incidence of disease, with a peak mean severity of ~ 2.5 occurring 14 days after the start of the experiment. In contrast, treatment with OvIFNr by either i.p. injection or oral feeding (Figures 2B and 2C, respectively) resulted in protection from EAE. Incidence of disease in both OvIFNr treatment groups was reduced to 1/5 animals, with a mean severity of ~ 1.0. These data indicate that oral administration of IFNr can block the development of chronic relapsing EAE, and suggest that orally-administered IFNr may be as effective as i.p. injection when the IFNr is fed about every 48 hours over an extended period of time.
Example 4 Histological Analysis
Histological analyses were performed to determine the extent of lymphocyte infiltration into the CNS of MBP-immunized mice treated with OVIFNT by oral and i.p. routes.
Mice were perfused with 4% paraformaldehyde, vertebral columns were removed and treated with formalin for 2 to 3 days. Spinal cords were dissected out and soaked in 0.5 % sucrose overnight at 4 °C. Spinal cord sections were embedded and sections cut in a microtome. Sections were fixed to slides in 4 % paraformaldehyde and stained with cresyl violet for visualization of inflammatory infiltrates.
The results are shown in Figures 3A, 3B and 3C at a final magnification of 222 x . Lymphocytic lesions were present in control spinal cord white matter (Fig. 3A). In contrast, no lymphocytic infiltrates were detected in mice treated with OvIFNr by i.p. injection (Fig. 3B) or oral feeding (Fig. 3C). These data suggest that the protective effect of IFNr is associated with inhibition of lymphocyte infiltration of the CNS.
Example 5 Induction of IL10 by Treatment with OvIFNr During the course of OvIFNr treatment of SJL for prevention of chronic relapsing EAE, mice were bled and sera were examined for the presence of interleukin 10 (IL10). Sera from mice which received either a single IFNr (105 U) treatment (by i.p. injection or oral feeding), prolonged IFNr (IO5 U) treatment (by i.p. injection or oral treatment for greater than 20 days) or no treatment were examined for IL10 by enzyme-linked immunosorbent assay (ELISA) using IL10 ELISA kits (Genzyme, Cambridge, MA) following the manufacturer's instructions. All sera samples were tested in duplicate.
No IL10 was detected in control mice or in mice which received a single treatment of
OvIFNr by either i.p. injection or oral feeding. In contrast, SJL mice which received OVIFNT by either i.p. injection or oral feeding every 48 hours for greater than 20 days had detectable levels of IL10 in their sera (Figure 4). These data suggest that IFNr-induced production of IL10 may be a contributing mechanism by which OvIFNr prevents development of EAE.
Example 6 Cessation of Treatment with OvIFNr Results in Relapsing Paralysis SJL mice which were protected from EAE by OvIFNr treatment via i.p. injection or oral feeding (every 48 hours) were followed for 58 days, during which time no disease development was observed. Treatment with OvIFNr was then removed and the mice were observed for an additional 22 days for symptoms of disease.
The results are shown in Figure 5. IFNr treatment is denoted as plus signs and removal of IFNT treatment is denoted as minus signs beneath the graph. Disease incidence in each treatment group was as follows: PBS control = 3/4 (square); i.p. injection=3/3 (triangle); oral feeding^ :3/4 (circle).
Both groups of mice which had previously been protected from EAE by OvIFNr treatment developed signs of paralysis 6 to 12 days after removal of the OvIFNr treatment. These data indicate that ongoing administration of IFNr, by either i.p. injection or oral feeding, is desirable for continued protection from EAE in the chronic-relapsing model of EAE. Example 7 Oral Administration of OvIFNr Reduces Anti-OvIFNr Antibody Response After removal of OvIFNr treatment in the experiments described in Example 6, above, mice from each treatment group were bled and sera were examined for the presence of anti-OvIFNr antibodies (Ab).
The antigen, OvIFNr, was adsorbed to the flat bottoms of plastic tissue culture wells overnight at a concentration of 600 ng/well, and subsequently evaporated to dryness. The plates were treated with 5% milk (Carnation) in PBS for 2 hours in order to block nonspecific binding and then washed 3 times with PBS containing 0.05% Tween 20. Various dilutions of sera from mice which were untreated, OvIFNr treated by i.p. injection and OvIFNr treated by oral feeding were added and incubated for 3 hours. Binding was assessed with goat anti-mouse immuno- globulin coupled to horseradish peroxidase. Color development was monitored at 492 nm in an
ELISA plate reader (Bio-Rad, Richmond, CA) after o-phenylenediamine and H2O2 were added and the reaction terminated with 2M H2SO4. Exemplary results are shown in Figure 6. Sera from untreated, OvIFNr treated-i.p. injected and OvIFNr treated-orally fed (2 mice/group) were examined by ELISA using multiple dilutions, including 1:30 (open bars) and 1:120 (filled bars). Mice which received OvIFNr by oral feeding exhibited minimal Ab levels while mice which received OvIFNr by i.p. injection exhibited elevated levels of anti-OvIFNr Ab. As expected, mice which received no OvIFNr treatment displayed no anti-OvIFNr Ab.
Sera were also examined for their ability to neutralize OvIFNr antiviral activity on MDBK cells as described above. The results are shown in Table 4, below. None of the sera from either i.p. injected or orally fed mice possessed neutralizing activity. These data suggest that oral treatment with IFNr circumvents the Ab response directed against OvIFNr protein observed in i.p. injection-treated individuals, and that neither treatment typically results in the generation of neutralizing antibodies.
Table 4
Sera from Mice Treated with OvIFNr by i.p. Injection or Oral Feeding do Not Possess Neutralizing Activity
500 U/ML OF OVIFNT COCULTURED OVIFNT TrTER WITH SERA FROM: (U/ML) untreated 500 i.p. injected 500 orally fed 500
HL127 <50
While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: UNIVERSITY OF FLORIDA (ii) TITLE OF INVENTION: Orally-Administered Interferon-Tau
Compositions and Methods
(iii) NUMBER OF SEQUENCES: 6
<iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Dehlinger & Associates
(B) STREET: 350 Cambridge Ave., Suite 250
(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: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 12-MAR-1997
(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/616,904
(B) FILING DATE: 15-MAR-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sholtz, Charles K. (B) REGISTRATION NUMBER: 38,615
(C) REFERENCE/DOCKET NUMBER: 5600-0003.41
(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: 516 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..516
(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 516 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TGC TAC CTG TCG CGA AAA CTG ATG CTG GAC GCT CGA GAA AAT TTA AAA 48
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
96
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 144 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 192 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 240 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 288 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 336 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 384 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 432 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 4B0 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 516 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
(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: amino acid sequence of a mature
OvIFNtau 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
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: synthetic nucleotide sequence encoding a mature human interferon-tau protein, HuIFNtaul. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TGTGACTTGT CTCAAAACCA CGTTTTGGTT GGTAGAAAGA ACTTAAGACT ACTAGACGAA 60
ATGAGACGTC TATCTCCACG CTTCTGTCTA CAAGACAGAA AGGACTTCGC TTTGCCTCAG 120 GAAATGGTTG AAGGTGGCCA ACTACAAGAA GCTCAAGCGA TATCTGTTTT GCACGAAATG 180
TTGCAACAAA GCTTCAACTT GTTCCACACC GAACACTCTT CGGCCGCTTG GGACACCACC 240
TTGTTGGAAC AGCTCAGAAC CGGTTTGCAC CAACAATTGG ACAACTTGGA TGCATGTTTG 300
GGTCAAGTTA TGGGTGAAGA AGACTCTGCT CTCGGGAGAA CCGGTCCAAC GCTAGCTTTG 360
AAGAGATACT TCCAAGGTAT CCACGTTTAC TTGAAGGAAA AGGGTTACTC TGACTGTGCT 420 TGGGAAACCG TGCGTCTAGA AATCATGCGT AGCTTCTCTT CTTTGATCAG CTTGCAAGAA 480
AGATTACGTA TGATGGACGG TGACTTGTCG AGCCCA 516
(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
(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: amino acid sequence for a mature
HuIFNtau protein, HuIFNtaul.
(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 Arg 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 Gin Leu 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: 516 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: HuIFNtau3, mature no leader sequence
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..516
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :
TGT GAC CTG TCT CAG AAC CAC GTG CTG GTT GGC AGC CAG AAC CTC AGG 48 Cys Asp Leu Ser Gin Asn His Val Leu Val Gly Ser Gin Asn Leu Arg 1 5 10 15
CTC CTG GGC CAA ATG AGG AGA CTC TCC CTT CGC TTC TGT CTG CAG GAC 96
Leu Leu Gly Gin Met Arg Arg Leu Ser Leu Arg Phe Cys Leu Gin Asp 20 25 30
AGA AAA GAC TTC GCT TTC CCC CAG GAG ATG GTG GAG GGT GGC CAG CTC 144 Arg Lys Asp Phe Ala Phe Pro Gin Glu Met Val Glu Gly Gly Gin Leu 35 40 45
CAG GAG GCC CAG GCC ATC TCT GTG CTC CAC GAG ATG CTC CAG CAG AGC 192 Gin Glu Ala Gin Ala lie Ser Val Leu His Glu Met Leu Gin Gin Ser 50 55 60
TTC AAC CTC TTC CAC ACA GAG CAC TCC TCT GCT GCC TGG GAC ACC ACC 240 Phe Asn Leu Phe His Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80 CTC CTG GAG CAG CTC CGC ACT GGA CTC CAT CAG CAG CTG GAT GAC CTG 288 Leu Leu Glu Gin Leu Arg Thr Gly Leu His Gin Gin Leu Asp Asp Leu 85 90 95
GAT GCC TGC CTG GGG CAG GTG ACG GGA GAG GAA GAC TCT GCC CTG GGA 336 Asp Ala Cys Leu Gly Gin Val Thr Gly Glu Glu Asp Ser Ala Leu Gly 100 105 110
AGA ACG GGC CCC ACC CTG GCC ATG AAG AGG TAT TTC CAG GGC ATC CAT 384 Arg Thr Gly Pro Thr Leu Ala Met Lys Arg Tyr Phe Gin Gly lie His 115 120 125
GTC TAC CTG AAA GAG AAG GGA TAT AGT GAC TGC GCC TGG GAA ATT GTC 432 Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu lie Val 130 135 140
AGA CTG GAA ATC ATG AGA TCC TTG TCT TCA TCA ACC AGC TTG CAC AAA 480 Arg Leu Glu lie Met Arg Ser Leu Ser Ser Ser Thr Ser Leu His Lys 145 150 155 160
AGG TTA AGA ATG ATG GAT GGA GAC CTG AGC TCA CCT 516
Arg Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170
(2) INFORMATION FOR SEQ ID NO:6:
(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:6:
Cys Asp Leu Ser Gin Asn His Val Leu Val Gly Ser Gin Asn Leu Arg 1 5 10 15
Leu Leu Gly Gin Met Arg Arg Leu Ser Leu Arg Phe Cys Leu Gin Asp 20 25 30
Arg Lys Asp Phe Ala Phe 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 Gin Leu Arg Thr Gly Leu His Gin Gin Leu Asp Asp Leu 85 90 95
Asp Ala Cys Leu Gly Gin Val Thr Gly Glu Glu Asp Ser Ala Leu Gly 100 105 110
Arg Thr Gly Pro Thr Leu Ala Met 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 lie Val 130 135 140 Arg Leu Glu lie Met Arg Ser Leu Ser Ser Ser Thr Ser Leu His Lys 145 150 155 160
Arg Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170

Claims

IT IS CLAIMED:
I . In a method of treating a disease condition in a mammal responsive to treatment by interferon- tau (IFNr), an improvement comprising orally administering a therapeutically-effective amount of IFNr.
2. The method of claim 1, wherein IFNr is orally-administered at a dosage of between about lxlO5 and about 1x10* units per day.
3. The method of claim 2, wherein IFNr is orally-administered at a dosage of between about lxlO6 and about lxlO7 units per day.
4. The method of claim 1, wherein the orally-administered IFNr is ovine IFNT (OVIFNT).
5. The method of claim 1 , wherein said OvIFNr has the sequence represented as SEQ ID NO:2.
6. The method of claim 1, wherein the orally-administered IFNr is human IFNr (HuIFNr).
7. The method of claim 1 , wherein said HuIFNr has the sequence represented as SEQ ID NO:4.
8. The method of claim 1, wherein said mammal is a human.
9. The method of claim 1, wherein said mammal is a dog.
10. The method of claim 1, wherein said disease condition is an immune system disorder.
II. The method of claim 10, wherein said disease condition is an autoimmune disorder.
12. The method of claim 11, wherein said autoimmune disorder is selected from d e group consisting of multiple sclerosis, type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, allergies and psoriasis.
13. The method of claim 12, wherein said autoimmune disorder is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, lupus erythematosus and type I diabetes mellitus.
14. The method of claim 13, wherein said autoimmune disorder is multiple sclerosis.
15. A method of treating an autoimmune disorder in a subject, comprising orally administering a therapeutically-effective amount of interferon-tau (IFNT) to said subject.
16. The method of claim 15, wherein said autoimmune disorder is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, lupus erythematosus and type I diabetes mellitus.
17. A method of claim 16, wherein said autoimmune disorder is multiple sclerosis.
18. The method of claim 15, wherein IFNr is orally-administered at a dosage of between about lxlO3 and about lxlO8 units per day.
19. The method of claim 18, wherein IFNr is orally-administered at a dosage of between about lxlO6 and about lxlO7 units per day.
PCT/US1997/003794 1996-03-15 1997-03-12 Orally-administered interferon-tau compositions and methods WO1997033607A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU20759/97A AU2075997A (en) 1996-03-15 1997-03-12 Orally-administered interferon-tau compositions and methods

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/616,904 1996-03-15
US08/616,904 US6372206B1 (en) 1989-03-02 1996-03-15 Orally-administered interferon-TAU compositions and methods

Publications (2)

Publication Number Publication Date
WO1997033607A1 true WO1997033607A1 (en) 1997-09-18
WO1997033607A9 WO1997033607A9 (en) 1998-04-16

Family

ID=24471451

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/003794 WO1997033607A1 (en) 1996-03-15 1997-03-12 Orally-administered interferon-tau compositions and methods

Country Status (4)

Country Link
US (3) US6372206B1 (en)
AU (1) AU2075997A (en)
TW (1) TW542722B (en)
WO (1) WO1997033607A1 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078266A2 (en) * 1999-06-22 2000-12-28 University Of Maryland College Park Interferon tau mutants and methods for making them
WO2001016178A1 (en) * 1999-08-27 2001-03-08 University Of Florida MATERIALS AND METHODS FOR INHIBITION OF IgE PRODUCTION
US6833256B1 (en) 1999-06-22 2004-12-21 University Of Maryland Interferon tau mutants and methods for making them
WO2005087255A2 (en) * 2004-03-10 2005-09-22 Pepgen Corporation Method of optimizing treatment with interferon-tau
US7083782B2 (en) 2000-07-19 2006-08-01 Pepgen Corporation Method of treatment using interferon-tau
WO2006108128A2 (en) * 2005-04-05 2006-10-12 Pepgen Corporation Ocular delivery of interferon-tau
WO2007070385A2 (en) * 2005-12-09 2007-06-21 Pepgen Corporation Treatment of multiple sclerosis using interferon-tau
WO2008021487A1 (en) * 2006-08-18 2008-02-21 Pepgen Corporation Combination treatment method with interferon-tau
US7431920B2 (en) 2000-07-19 2008-10-07 Pepgen Corporation Method of treating IL-10 deficiency

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372206B1 (en) * 1989-03-02 2002-04-16 University Of Florida Orally-administered interferon-TAU compositions and methods
US20020025304A1 (en) * 2000-06-16 2002-02-28 Croze Edward M. Novel interferon for the treatment of multiple sclerosis
US20040247565A1 (en) * 2000-07-19 2004-12-09 Chih-Ping Liu Method of treatment using interferon-tau
US20050118137A1 (en) * 2000-07-19 2005-06-02 Chih-Ping Liu Method of treatment using interferon-tau
US20050084478A1 (en) * 2000-10-17 2005-04-21 Chih-Ping Liu Combination therapy using interferon-tau
US20050201981A1 (en) * 2004-03-10 2005-09-15 Chih-Ping Liu Method of optimizing treatment with interferon-tau
JP2006213597A (en) * 2000-07-19 2006-08-17 Pepgen Corp Composition for treating hepatitis c virus by using interferon tau and method of monitoring
US7105154B2 (en) * 2000-07-19 2006-09-12 Pepgen Corporation Method of treatment using interferon-tau
JP2006501137A (en) * 2002-01-16 2006-01-12 ペプジェン コーポレイション Oral administration of interferon-τ
US20040126360A1 (en) * 2002-10-09 2004-07-01 Manning Mark C. Oral formulations for proteins and polypeptides
US8778880B2 (en) 2004-02-02 2014-07-15 Ambrx, Inc. Human growth hormone modified at position 35
US20080025948A1 (en) * 2004-03-10 2008-01-31 Chih-Ping Liu Methods of Treatment Using Interferon-Tau
US20060078942A1 (en) * 2004-03-10 2006-04-13 Pepgen Corporation Method of treatment using interferon-tau
US20070025963A1 (en) * 2005-07-27 2007-02-01 Chih-Ping Liu Methods for reduction of scar tissue formation
US20070243163A1 (en) * 2006-02-17 2007-10-18 Chih-Ping Liu Respiratory tract delivery of interferon-tau
WO2008021536A2 (en) * 2006-08-18 2008-02-21 Pepgen Corporation Use of interferon tau for the manufacture of a medicament in combination with interferon-alpha or interferon-beta for reduction of their adverse effects
CA2685596A1 (en) 2007-05-02 2008-11-13 Ambrx, Inc. Modified interferon beta polypeptides and their uses
NZ620606A (en) 2008-02-08 2015-08-28 Ambrx Inc Modified leptin polypeptides and their uses
BG66517B1 (en) 2008-04-08 2016-02-29 Tigo Gmbh Inhibitor of endogenous human interferon - gamma
KR101857825B1 (en) 2010-03-04 2018-05-14 피페넥스 인크. Method for producing soluble recombinant interferon protein without denaturing
US8906384B2 (en) * 2010-12-22 2014-12-09 The United States Of America As Represented By The Secretary Of Agriculture Antiviral activity of bovine type III interferon against foot-and-mouth disease virus
US9526694B2 (en) 2012-05-03 2016-12-27 Amarillo Biosciences, Inc. Treatment of thrombocytopenia using orally administered interferon

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009806A2 (en) * 1989-03-02 1990-09-07 University Of Florida Composition for the inhibition of tumors and for the non-cytotoxic inhibition of replication of viruses
WO1996028183A1 (en) * 1995-03-16 1996-09-19 University Of Florida Method for treatment of autoimmune diseases using interferon-tau

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA878295B (en) * 1986-11-06 1988-05-03 Amarillo Cell Culture Co. Inc. Treatment of immuno-resistant disease
US4997646A (en) 1989-02-23 1991-03-05 University Of Florida Research Foundation, Inc. Use of interferons of the alpha family to enhance fertility in mammals
US5705363A (en) * 1989-03-02 1998-01-06 The Women's Research Institute Recombinant production of human interferon τ polypeptides and nucleic acids
US6372206B1 (en) * 1989-03-02 2002-04-16 University Of Florida Orally-administered interferon-TAU compositions and methods
US5372808A (en) 1990-10-17 1994-12-13 Amgen Inc. Methods and compositions for the treatment of diseases with consensus interferon while reducing side effect
US5206219A (en) 1991-11-25 1993-04-27 Applied Analytical Industries, Inc. Oral compositions of proteinaceous medicaments
TW391983B (en) * 1992-10-30 2000-06-01 Univ Florida Human interferon TAU, processes thereof and pharmaceutical uses thereof
WO1995027499A1 (en) * 1994-04-08 1995-10-19 Brigham And Women's Hospital Treatment of autoimmune disease using oral tolerization and/or type i interferon
IL113280A (en) * 1994-04-12 2004-12-15 Res Dev Foundation Composition for treating auto-immune diseases using type one interferons
HUP0303415A2 (en) * 2001-03-09 2004-01-28 Pfizer Products Inc. Triazolopyridines as anti-inflammatory agents and pharmaceutical compositions containing them

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009806A2 (en) * 1989-03-02 1990-09-07 University Of Florida Composition for the inhibition of tumors and for the non-cytotoxic inhibition of replication of viruses
WO1996028183A1 (en) * 1995-03-16 1996-09-19 University Of Florida Method for treatment of autoimmune diseases using interferon-tau

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J.M. SOOS ET AL.: "THE IFN PREGNANCY RECOGNITION HORMONE IFN-TAU BLOCKS BOTH DEVELOPMENT AND SUPERANTIGEN REACTIVATION OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS WITHOUT ASSOCIATED TOXICITY.", JOURNAL OF IMMUNOLOGY, vol. 155, no. 5, 1 September 1995 (1995-09-01), BALTIMORE US, pages 2747 - 2753, XP002010954 *
J.M. SOOS ET AL.: "THE NOVEL TYPE I INTERFERON TAU PREVENTS DEVELOPMENT AND SUPERANTIGEN REACTIVATION OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS IN MICE WITHOUT ASSOCIATED TOXICITY.", FASEB JOURNAL FOR EXPERIMENTAL BIOLOGY, vol. 9, no. 4, 10 March 1995 (1995-03-10), BETHESDA, MD US, pages A1025, XP002010951 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078266A2 (en) * 1999-06-22 2000-12-28 University Of Maryland College Park Interferon tau mutants and methods for making them
WO2000078266A3 (en) * 1999-06-22 2001-08-23 Univ Maryland Interferon tau mutants and methods for making them
US6833256B1 (en) 1999-06-22 2004-12-21 University Of Maryland Interferon tau mutants and methods for making them
WO2001016178A1 (en) * 1999-08-27 2001-03-08 University Of Florida MATERIALS AND METHODS FOR INHIBITION OF IgE PRODUCTION
AU781377B2 (en) * 1999-08-27 2005-05-19 University Of Florida Research Foundation, Inc. Materials and methods for inhibition of IgE production
US7431920B2 (en) 2000-07-19 2008-10-07 Pepgen Corporation Method of treating IL-10 deficiency
US7083782B2 (en) 2000-07-19 2006-08-01 Pepgen Corporation Method of treatment using interferon-tau
WO2005087255A3 (en) * 2004-03-10 2006-04-20 Pepgen Corp Method of optimizing treatment with interferon-tau
WO2005087254A3 (en) * 2004-03-10 2006-02-02 Pepgen Corp Pharmaceutical compositions comprising interferon-tau
WO2005087254A2 (en) * 2004-03-10 2005-09-22 Pepgen Corporation Pharmaceutical compositions comprising interferon-tau
JP2007528407A (en) * 2004-03-10 2007-10-11 ペプジェン コーポレイション Pharmaceutical composition comprising interferon-tau
WO2005087255A2 (en) * 2004-03-10 2005-09-22 Pepgen Corporation Method of optimizing treatment with interferon-tau
WO2006108128A2 (en) * 2005-04-05 2006-10-12 Pepgen Corporation Ocular delivery of interferon-tau
WO2006108128A3 (en) * 2005-04-05 2007-03-29 Pepgen Corp Ocular delivery of interferon-tau
WO2007070385A2 (en) * 2005-12-09 2007-06-21 Pepgen Corporation Treatment of multiple sclerosis using interferon-tau
WO2007070385A3 (en) * 2005-12-09 2008-04-10 Pepgen Corp Treatment of multiple sclerosis using interferon-tau
WO2008021487A1 (en) * 2006-08-18 2008-02-21 Pepgen Corporation Combination treatment method with interferon-tau

Also Published As

Publication number Publication date
US20040086534A1 (en) 2004-05-06
US6372206B1 (en) 2002-04-16
US6942854B2 (en) 2005-09-13
AU2075997A (en) 1997-10-01
US7214367B2 (en) 2007-05-08
TW542722B (en) 2003-07-21
US20030012766A1 (en) 2003-01-16

Similar Documents

Publication Publication Date Title
US6372206B1 (en) Orally-administered interferon-TAU compositions and methods
AU688214B2 (en) Method for treatment of autoimmune diseases using interferon-TAU
WO1997033607A9 (en) Orally-administered interferon-tau compositions and methods
AU718864B2 (en) Hybrid interferon compositions and methods of use
US6174996B1 (en) Hybrid interferon τ/type I interferon polypeptides
KR100223255B1 (en) Improved alpha interferon composition and method for its production from human peripheral blood leukocytes
EP1360962A2 (en) Interferon tau composition and methods of use
US7083782B2 (en) Method of treatment using interferon-tau
US20030049277A1 (en) Composition for treatment of and method of monitoring hepatitis C virus using interferon-TAU
US20040247565A1 (en) Method of treatment using interferon-tau
AU2006326688A1 (en) Treatment of multiple sclerosis using interferon-tau
US20030219405A1 (en) Oral administration of interferon-tau
US7105154B2 (en) Method of treatment using interferon-tau
US20040131589A1 (en) Materials and methods for inhibition of IgE production
US20060257363A1 (en) Treatment using an interferon
AU706762B2 (en) Interferon tau compositions and methods of use
US20050118138A1 (en) Method of treatment using interferon-tau
AU2003210568A1 (en) Oral administration of interferon-tau

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN YU AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
COP Corrected version of pamphlet

Free format text: PAGES 1/6-6/6, DRAWINGS, REPLACED BY NEW PAGES BEARING THE SAME NUMBER; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97532763

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase